Journal of Molecular and Cellular Cardiology - Articles in Press

Une cellule type? - Accepted Manuscript

Leighton T. Izu, Ye Chen-Izu — 2012-02-06

The function is called the Gaussian distribution or normal distribution with about equal frequency. We have used the two names interchangeably. However, these names reflect the two fundamentally different interpretations of f(x). Karl Frederick Gauss (1777-1855) recognized that f(x)could describe the probability distribution of errors in the measurement of a single entity, say an angle of a star or that of a geodetic triangle . Adolphe Quetelet (1796-1874) found that anthropometric measures such as the height of men or the girth of soldiers’ chests could also be described by f(x). From this population distribution Quetelet defined in 1844 “un homme type”, the average man . In Quetelet's interpretation of f(x), differences from the mean μ were not errors but reflected the natural variation in the population. f(x)quantifies what we mean by normal. “Deviation” in standard deviation (σ) connotes no malevolence but “deviant” has no such neutrality.

MyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction - Accepted Manuscript

Madhu V. Singh, Swaminathan, E.D. Luczak, W. Kutschke, R.M. Weiss, M.E. Anderson — 2012-02-06

Abstract: The toll-like receptors (TLR) and myocardial infarction (MI) promote NF-κB-dependent inflammatory transcription and oxidative injury in myocardium. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by oxidation and contributes to NF-κB-dependent transcription, myocardial hypertrophy and post-MI death. The myeloid differentiation protein 88 (MyD88) is an adapter protein critical for many TLR functions, but downstream targets for TLR/MyD88 signaling in MI are not well understood. We asked if CaMKII and TLR/MyD88 pathways are interconnected and if TLR/MyD88 contributes to adverse outcomes after MI. Here we show that TLR-4 activation by lipopolysaccharide (LPS) induces CaMKII oxidation (ox-CaMKII) in cardiomyocytes. MI enhances ox-CaMKII in wild type (WT) hearts but not in MyD88−/− hearts that are defective in MyD88-dependent TLR signaling. In post-MI WT hearts expression of pro-inflammatory genes TNF-α (Tnfa), complement factor B (Cfb), myocyte death and fibrosis were significatly increased, but increases were significantly less in MyD88−/− hearts after MI. MyD88−/− cardiomyocytes were defective in NF-κB activation by LPS but not by the MyD88-independent TLR agonist poly(I:C). In contrast, TNF-α induced Cfb gene expression was not deficient in MyD88−/− cardiomyocytes. Several hypertrophy marker genes were upregulated in both WT and MyD88−/− hearts after MI, but Acta1 was significantly attenuated in MyD88−/− hearts, suggesting that MyD88 selectively affects expression of hypertrophic genes. Post-MI cardiac hypertrophy, inflammation, apoptosis, ox-CaMKII expression and mortality were significantly reduced in MyD88−/− compared to WT littermates. These data suggest that MyD88 contributes to CaMKII oxidation and is important for adverse hypertrophic and inflammatory responses to LPS and MI.Highlights: ► MyD88−/− mice have improved post-MI survival. ► MI induces TLR/MyD88 dependent inflammatory gene expression. ► MyD88−/− mice have reduced cardiac hypertrophy and fibrosis. ► MyD88−/− mice have reduced post-MI oxidation-dependent CaMKII activation.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes - Accepted Manuscript

2012-02-06

Abstract: This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1 stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.Highlights: ► Doxo leads to HIF1α-stabilization and VEGF-A release in CMEC. ► Doxo-induced VEGF-A release is regulated by MEK1/2 and p38 signaling pathways. ► Sunitinib shows cytotoxic effects in CMEC and ARVM at nM concentrations. ► In ARVM recombinant VEGF-A and VEGF-B have only minor effects on signaling. ► ARVM and CMEC differ in their response to anthracyclines.

Regulation of microRNA Expression in the Heart by the ATF6 Branch of the ER Stress Response - Accepted Manuscript

Peter J. Belmont, Wenqiong J. Chen, Donna J. Thuerauf, Christopher C. Glembotski — 2012-02-03

Abstract: A nodal regulator of endoplasmic reticulum stress is the transcription factor, ATF6, which is activated by ischemia and protects the heart from ischemic damage, in vivo. To explore mechanisms of ATF6-mediated protection in the heart, a whole-genome microRNA (miRNA) array analysis of RNA from the hearts of ATF6 transgenic (TG) mice was performed. The array identified 13 ATF6-regulated miRNAs, eight of which were downregulated, suggesting that they could contribute to increasing levels of their mRNAs. The down-regulated miRNAs, including miR-455, were predicted to target 45 mRNAs that we had previously shown by microarray analysis to be up-regulated by ATF6 in the heart. One of the miR-455 targets was calreticulin (Calr), which is up-regulated in the pathologic heart, where it modulates hypertrophic growth, potentially reducing the impact of the pathology. To validate the effects of miR-455, we showed that Calr protein was increased by ATF6 in mouse hearts, in vivo. In cultured cardiac myocytes, treatment with the ER stressor, tunicamycin, or with adenovirus encoding activated ATF6 decreased miR-455 and increased Calr levels, consistent with the effects of ATF6 on miR-455 and Calr, in vivo. Moreover, transfection of cultured cardiac myocytes with a synthetic precursor, premiR-455, decreased Calr levels, while transfection with an antisense, antimiR-455, increased Calr levels. The results of this study suggest that ER stress can regulate gene expression via ATF6-mediated changes in micro-RNA levels. Moreover, these findings support the hypothesis that ATF6-mediated down-regulation of miR-455 augments Calr expression, which may contribute to the protective effects of ATF6 in the heart.Highlights: ► The ER stress-activated transcription factor, ATF6, is cardioprotective. ► ATF6 increases expression of calreticulin in the heart. ► We found 13 miRNAs to be regulated by ATF6 in the heart, in vivo. ► MicroR-455, which targets the calreticulin mRNA, was downregulated by ATF6. ► Thus, ATF6 may induce calreticulin, partly, by decreasing miR-455.

The natriuretic peptides BNP and CNP increase heart rate and electrical conduction by stimulating ionic currents in the sinoatrial node and atrial myocardium following activation of guanylyl cyclase-linked natriuretic peptide receptors - Accepted Manuscript

2012-02-03

Abstract: Natriuretic peptides (NPs) are best known for their ability to regulate blood vessel tone and kidney function whereas their electrophysiological effects on the heart are less clear. Here, we measured the effects of BNP and CNP on sinoatrial node (SAN) and atrial electrophysiology in isolated hearts as well as isolated SAN and right atrial myocytes from mice. BNP and CNP dose-dependently increased heart rate and conduction through the heart as indicated by reductions in R-R interval, P wave duration and P-R interval on ECGs. In conjunction with these ECG changes BNP and CNP (100 nM) increased spontaneous action potential frequency in isolated SAN myocytes by increasing L-type Ca2+ current (ICa,L) and the hyperpolarization activated current (If). BNP had no effect on right atrial myocyte APs in basal conditions; however, in the presence of isoproterenol (10 nM), BNP increased atrial AP duration and ICa,L. Quantitative gene expression and immunocytochemistry data show that all three NP receptors (NPR-A, NPR-B and NPR-C) are expressed in the SAN and atrium. The effects of BNP and CNP on SAN and right atrial myocytes were maintained in mutant mice lacking functional NPR-C receptors and blocked by the NPR-A antagonist A71915 indicating that BNP and CNP function through their guanylyl cyclase-linked receptors. Our data also show that the effects of BNP and CNP are completely absent in the presence of the phosphodiesterase 3 inhibitor milrinone. Based on these data we conclude that NPs can increase heart rate and electrical conduction by activating the guanylyl cyclase-linked NPR-A and NPR-B receptors and inhibiting PDE3 activity.Highlights: ► BNP and CNP increase heart rate and speed conduction across the atrial myocardium. ► BNP and CNP increase spontaneous AP frequency in SAN myocytes by increasing ICa,L and If. ► BNP also increases AP duration and ICa,L in right atrial myocytes in the presence of ISO. ► The BNP effects are maintained in NPR-C−/− mice and blocked by an NPR-A antagonist. ► The effects of BNP are lost following phosphodiesterase 3 inhibition with milrinone

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells - Accepted Manuscript

2012-02-03

Abstract: Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.Highlights: ► Shear stress enhances the migratory, adhesive and proliferatory activities of EPCs. ► Shear stress accelerates EPCs-mediated reendothelialization after vascular injury. ► Tie2/PI3K/Akt/eNOS signaling of EPCs is activated by shear stress. ► Tie2-dependent signaling contributes to shear stress-upregulated EPC function.

Alternatively Spliced Tissue Factor and Full-length Tissue Factor Protect Cardiomyocytes against TNF-α-induced Apoptosis - Accepted Manuscript

2012-02-02

Abstract: Tissue Factor (TF) is expressed in various cell types of the heart, such as cardiomyocytes. In addition to its role in the initiation of blood coagulation, the TF:FVIIa complex protects cells from apoptosis. There are two isoforms of Tissue Factor (TF): “full length” (fl)TF – an integral membrane protein; and alternatively spliced (as)TF – a protein that lacks a transmembrane domain and can thus be secreted in a soluble form. Whether asTF or flTF affect apoptosis of cardiomyocytes is unknown.In this study, we examined whether asTF or flTF protect murine cardiomyocytes from TNF-α-induced apoptosis. We used murine cardiomyocytic HL-1 cells and primary murine embryonic cardiomyocytes that overexpressed either murine asTF or murine flTF, and stimulated them with TNF-α to initiate cell death. Apoptosis was assessed by Annexin-V assay,propidium iodide assay, as well as activation of caspase-3 and −9. In addition, signaling via integrins, Akt, NFκB and Erk1/2, and gene-expression of Bcl-2 family members were analyzed.We here report that overexpression of asTF reduced phosphatidylserine exposure upon TNF-α-stimulation. asTF overexpression led to an increased expression and phosphorylation of Akt, as well as up-regulation of the anti-apoptotic protein Bcl-xL. The anti-apoptotic effects of asTF overexpression were mediated via αVβ3/Akt/NFκB signaling and were dependent on Bcl-xL expression in HL-1 cells. The anti-apoptotic activity of asTF was also observed using primary cardiomyocytes. Analogous yet less pronounced anti-apoptotic sequelae were observed due to overexpression of flTF. Importantly, cardiomyocytes deficient in TF exhibited increased apoptosis compared to wild type cells.We propose that asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis via activation of specific signaling pathways, and up-regulation of anti-apoptotic members of the Bcl-2 protein family.Highlights: ► asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis. ► asTF exhibits a greater anti-apoptotic potential than flTF. ► The anti-apoptotic effects of asTF are mediated via Akt, NFκB, Integrin-αVβ3 signaling and Bcl-xL expression. ► TF-deficient cardiomyocytes exhibit increased apoptosis compared with wild type cells.

AMPK isoform expression in the normal and failing hearts - Accepted Manuscript

2012-02-02

Abstract: AMP-activated protein kinase (AMPK) is a master metabolic switch that plays an important role in energy homeostasis at the cellular and whole body level, hence a promising drug target. AMPK is a heterotrimeric complex composed of catalytic α-subunit and regulatory β- and γ-subunits with multiple isoforms for each subunit. It has been shown that AMPK activity is increased in cardiac hypertrophy and failure but it is unknown whether changes in subunit composition of AMPK contribute to the altered AMPK activity. In this study, we determined the protein expression pattern of AMPK subunit isoforms during cardiac development as well as during cardiac hypertrophy and heart failure in mouse heart. We also compared the findings in failing mouse heart to that of the human failing hearts in order to determine whether the mouse heart is a good model of AMPK in human diseases. In mouse developmental hearts, AMPK was highly expressed in the fetal stages and fell back to the adult level after birth. In the failing mouse heart, there was a significant increase in α2, β2, and γ2 subunits both at the mRNA and protein levels. In contrary, we found significant increases in the protein level of α1, β1 and γ2c subunits in human failing hearts with no change in the mRNA level. We also compared isoform-specific AMPK activity in the mouse and human failing hearts. Consistent with the literature, in the failing mouse heart, the α2 complexes accounted for ~2/3 of total AMPK activity while the α1 complexes accounted for the remaining 30-35%. In the human hearts, however, the contribution of α1-AMPK activity were significantly higher (>40%) in the non-failing hearts, and it further increased to 50% in the failing hearts. Thus, the human hearts have a greater amount of α1-AMPK activity compared to the rodent hearts. In summary, the protein level and the isoform distribution of AMPK in the heart change significantly during normal development as well as in heart failure. These observations provide a basis for future development of therapeutic strategies for targeting AMPK.Highlights: ► AMPK expression is higher in the fetal heart than the adult. ► Part of the fetal expression profile of the AMPK isoforms reappears in the failing heart. ► The protein expression and the activity of AMPK both increased significantly in the failing heart. ► Changes in the specific isoforms are different in human failing heart compared to the mouse. ► Keyword ► AMPK, human, mice, isoform expression, heart failure

microRNAs in cardiovascular development - Uncorrected Proof

Jinghai Chen, Da-Zhi Wang — 2012-01-27

Abstract: Heart development requires precise temporal-spatial regulation of gene expression, in which the highly conserved modulation networks of transcription factors accurately control the signaling pathways required for normal cardiovascular development. Even slight perturbation of such programming during cardiogenesis can cause congenital heart defects and late neonatal or adult heart disease. microRNAs (miRNAs), a class of “small” non-coding RNAs, have recently drawn a lot of attention for their “big” impact on cardiovascular development and diseases. miRNAs negatively regulate the expression of their target genes in most biological organisms through post-transcriptional processes. Here, we review the roles of miRNAs in cardiovascular development and function, looking inside the molecular mechanisms by which miRNAs act as “fine tuners” and/or “safeguards” to maintain the homeostasis of cardiovascular system. We also propose new directions for therapeutic potential of these tiny molecules.Highlights: ► Current knowledge on miRNA function during cardiovascular development is reviewed. ► We briefly describe the discovery of miRNAs and their mechanism of action. ► miRNAs in cardiovascular development and physiology pathway is highlighted. ► The potential of miRNAs as therapeutic targets is discussed.

The secondary heart field is a new site of calcineurin/Nfatc1 signaling for semilunar valve development - Uncorrected Proof

2012-01-27

Abstract: Semilunar valve malformations are common human congenital heart defects. Bicuspid aortic valves occur in 2–3% of the population, and pulmonic valve stenosis constitutes 10% of all congenital heart disease in adults (Brickner et al., 2000) [1]. Semilunar valve defects cause valve regurgitation, stenosis, or calcification, leading to endocarditis or congestive heart failure. These complications often require prolonged medical treatment or surgical intervention. Despite the medical importance of valve disease, the regulatory pathways governing semilunar valve development are not entirely clear. In this report we investigated the spatiotemporal role of calcineurin/Nfatc1 signaling in semilunar valve development. We generated conditional knockout mice with calcineurin gene disrupted in various tissues during semilunar valve development. Our studies showed that calcineurin/Nfatc1 pathway signals in the secondary heart field (SHF) but not in the outflow tract myocardium or neural crest cells to regulate semilunar valve morphogenesis. Without SHF calcineurin/Nfatc1 signaling, the conal endocardial cushion—the site of prospective semilunar valve formation—first develop and then regress due to apoptosis, resulting in a striking phenotype with complete absence of the aortic and pulmonic valves, severe valve regurgitation, and perinatal lethality. This role of calcineurin/Nfatc1 signaling in the SHF is different from the requirement of calcineurin/Nfatc1 in the endocardium for semilunar valve formation (Chang et al., 2004) [2], indicating that calcineurin/Nfatc1 signals in multiple tissues to organize semilunar valve development. Also, our studies suggest distinct mechanisms of calcineurin/Nfat signaling for semilunar and atrioventricular valve morphogenesis. Therefore, we demonstrate a novel developmental mechanism in which calcineurin signals through Nfatc1 in the secondary heart field to promote semilunar valve morphogenesis, revealing a new supportive role of the secondary heart field for semilunar valve formation.

Phospholemman is a negative feed-forward regulator of Ca2+ in β-adrenergic signaling, accelerating β-adrenergic inotropy - Uncorrected Proof

Jason H. Yang, Jeffrey J. Saucerman — 2012-01-20

Abstract: Sympathetic stimulation enhances cardiac contractility by stimulating β-adrenergic signaling and protein kinase A (PKA). Recently, phospholemman (PLM) has emerged as an important PKA substrate capable of regulating cytosolic Ca2+ transients. However, it remains unclear how PLM contributes to β-adrenergic inotropy. Here we developed a computational model to clarify PLM's role in the β-adrenergic signaling response. Simulating Na+ and sarcoplasmic reticulum (SR) Ca2+ clamps, we identify an effect of PLM phosphorylation on SR unloading as the key mechanism by which PLM confers cytosolic Ca2+ adaptation to long-term β-adrenergic receptor (β-AR) stimulation. Moreover, we show that phospholamban (PLB) opposes and overtakes these actions on SR load, forming a negative feed-forward loop in the β-adrenergic signaling cascade. This network motif dominates the negative feedback conferred by β-AR desensitization and accelerates β-AR-induced inotropy. Model analysis therefore unmasks key actions of PLM phosphorylation during β-adrenergic signaling, indicating that PLM is a critical component of the fight-or-flight response.Graphical abstract: Highlights: ► PLM confers cytosolic Ca2+ adaptation via manipulation of SR load. ► PLM and PLB form a negative feed-forward loop in β-adrenergic signaling. ► PLM phosphorylation accelerates β-adrenergic stimulation enhanced contractility. ► PLM is therefore a fundamentally important part of the fight-or-flight response.

Arrhythmogenic coupling between the Na+–Ca2+ exchanger and inositol 1,4,5-triphosphate receptor in rat pulmonary vein cardiomyocytes - Corrected Proof

Yosuke Okamoto, Makoto Takano, Takayoshi Ohba, Kyoichi Ono — 2012-01-20

Abstract: Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10μM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca2+ concentrations. The Ca2+ transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current–clamp to voltage–clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na+–Ca2+ exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the β1- or α1-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP3R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP3R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP3R causes arrhythmic excitability of the PV during the presence of combined β1- and α1-adrenoceptor stimulation.Graphical abstract: Highlights: ► NE induces spontaneous activity in isolated PV cardiomyocytes. ► Morphological and electrophysiological properties differ between PV and LA myocytes. ► Both a1 and b1-receptor stimulations are required for NE-induced automaticity. ► Release of Ca2+ via IP3R activates NCX to cause automaticity. ► T-tubule system is enriched in PV cardiomyocytes, in contrast to atrial cells.

New insights into microRNA-29 regulation: A new key player in cardiovascular disease - Corrected Proof

Yajaira Suárez, Carlos Fernández-Hernando — 2012-01-20

MicroRNAs (miRNAs) are a class of endogenously expressed small non-coding RNAs (22 nucleotides) that represent a new elegant layer of post-transcriptional control of gene expression . They are thought to negatively regulate target mRNAs by imperfect binding to their 3′UTR, causing translational repression, mRNA destabilization, or both. Since their discovery as important regulators for the timing of larval development in Caenorhabditis elegans, miRNAs have emerged as critical fine tuners of many biological processes . The crucial role of miRNAs in the cardiovascular system is supported by the finding that conditional depletion of the miRNA-processing enzyme, Dicer, in the heart or endothelium leads to defects in cardiac development, vessel formation and angiogenesis . Interestingly, deletion of Dicer in vascular smooth muscle cells (VSMC) results in late embryonic lethality at embryonic days 16 to 17, suggesting that miRNAs are necessary for vascular smooth muscle growth, differentiation and function . Moreover, inducible depletion of Dicer in VSMCs produces a dramatic reduction in blood pressure, suggesting that miRNAs are critical for postnatal regulation of VSMC functions . Together, these results strongly suggest that miRNAs are essential, not only for the differentiation of VSMCs during early development, but also for the maintenance of VSMCs in adult.

Role of regulatory T cells in atheroprotective effects of granulocyte colony-stimulating factor - Corrected Proof

2012-01-19

Abstract: We and others have previously reported that granulocyte colony-stimulating factor (G-CSF) prevents left ventricular remodeling and dysfunction after myocardial infarction in animal models and human. We have also reported that G-CSF inhibits the progression of atherosclerosis in animal models, but its precise mechanism is still elusive. So, we examined the effects of G-CSF on atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Twelve-week-old male ApoE−/− mice were subcutaneously administrated with 200μg/kg of G-CSF or saline once a day for 5 consecutive days per a week for 4weeks. Atherosclerotic lesion of aortic sinus was significantly reduced in the G-CSF-treated mice compared with the saline-treated mice (35% reduction, P<0.05). G-CSF significantly reduced the expression level of interferon-γ by 31% and increased the expression level of interleukin-10 by 20% in atherosclerotic lesions of aortic sinus. G-CSF increased the number of CD4+CD25+ regulatory T cells in lymph nodes and spleen, and enhanced the suppressive function of regulatory T cells in vitro. G-CSF markedly increased the number of Foxp3-positive regulatory T cells in atherosclerotic lesions of aortic sinus. Administration of anti-CD25 antibody (PC61) that depletes regulatory T cells abrogated these atheroprotective effects of G-CSF. Moreover, in ApoE−/−/CD28−/− mice, that lack regulatory T cells, the protective effects of G-CSF on atherosclerosis were not recognized. These findings suggest that regulatory T cells play an important role in the atheroprotective effects of G-CSF.Highlights: ► G-CSF inhibited the progression of atherosclerosis in ApoE−/− mice. ► G-CSF increased the number of regulatory T cells in atherosclerotic lesions. ► The atheroprotective effects of G-CSF were not recognized in ApoE−/−/CD28−/− mice. ► Regulatory T cells play an important role in the atheroprotective effects of G-CSF.

Partial restoration of cardio-vascular defects in a rescued severe model of spinal muscular atrophy - Corrected Proof

2012-01-19

Abstract: Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death. Loss of a gene called Survival Motor Neuron 1 (SMN1) and, as a result, reduced levels of the Survival Motor Neuron (SMN) protein leads to SMA development. SMA is characterized by the loss of functional motor neurons in the spinal cord. However, accumulating evidence suggests the contribution of other organs to the composite SMA phenotype and disease progression. A growing number of congenital heart defects have been identified in severe SMA patients. Consistent with the clinical cases, we have recently identified developmental and functional heart defects in two SMA mouse models, occurring at embryonic stage in a severe SMA model and shortly after birth in a less severe model (SMN∆7). Our goal was to examine the late stage cardiac abnormalities in untreated SMN∆7 mice and to determine whether gene replacement therapy restores cardiac structure/function in rescued SMN∆7 model. To reveal the extent of the cardiac structural/functional repair in the rescued mice, we analyzed the heart of untreated and treated SMN∆7 model using self-complementary Adeno-associated virus (serotype 9) expressing the full-length SMN cDNA. We examined the characteristics of the heart failure such as remodeling, fibrosis, oxidative stress, and vascular integrity in both groups. Our results clearly indicate that fibrosis, oxidative stress activation, vascular remodeling, and a significant decrease in the number of capillaries exist in the SMA heart. The cardiac structural defects were improved drastically in the rescued animals, however, the level of impairment was still significant compared to the age-matched wildtype littermates. Furthermore, functional analysis by in vivo cardiac magnetic resonance imaging (MRI) revealed that the heart of the treated SMA mice still exhibits functional defects. In conclusion, cardiac abnormalities are only partially rescued in post-birth treated SMA animals and these abnormalities may contribute to the premature death of vector-treated SMA animals with seemingly rescued motor function but an average life span of less than 70days as reported in several studies.Highlights: ► Rescued SMA mice demonstrate improvements in cardiac structural and cellular pathology. ► Vascular and capillary defects are improved in rescued mice. ► Cine MRI indicates cardiac functional impairments in rescued SMA mice. ► Functional defects are most likely contributing to the premature death of treated SMA mice.

The dynamic role of cardiac myosin binding protein-C during ischemia - Uncorrected Proof

2012-01-19

Abstract: Cardiac myosin binding protein C (cMyBP-C) is a myofibrillar protein important for normal myocardial contractility and stability. In mutated form it can cause cardiomyopathy and heart failure. cMyBP-C appears to have separate regions for different functions. Three phosphorylation sites near the N terminus modulate contractility by their effect on both the kinetics of contraction and the binding site of the N-terminus. The C terminal region binds to myosin rods and stabilizes thick filament structure.The aim of the study reported here was to test whether cMyBPC is important in producing the structural and functional changes that result from ischemia/reperfusion. In this study the sequential changes in cMyBP-C, contractility, and thick filament structure following dephosphorylation of cMyBP-C associated with ischemia and reperfusion have been studied in biopsied specimens from chronically instrumented dogs. One and two dimensional electrophoresis, electron microscopy and immunocytochemistry with multiple antibodies generated against different domains in cMyBP-C have been used to follow structural changes in cMyBP-C. Ischemia produced dephosphorylation of cMyBP-C. Subsequent reperfusion released the dephosphorylated cMyBP-C from myofibrils and activated proteolysis of the cytoplasmic cMyBP-C. This in turn leads to increased vulnerability of cMyBP-C to proteolysis and increased degradation of thick filaments. The state of cMyBP-C appears to be closely related to phosphorylation and dephosphorylation of serine 282. In the absence of the stabilizing action of cMyBP-C either as a consequence of genetic mutation or dephosphorylation, premature degradation of thick filaments occurs and is accompanied by persistent contractile dysfunction.Highlights: ► Ischemia of the heart dephosphorylates cMyBP-C bound to myofibrils. ► Reperfusion releases dephosphorylated cMyBP-C from myofibrils. ► Reperfusion activates proteolysis of the cytoplasmic cMyBP-C. ► Increased degradation of thick filaments follows proteolysis of cMyBP-C. ► Phosphorylation of serine 282 and the state of cMyBP-C change in parallel.

Preserved cardiomyocyte function and altered desmin pattern in transgenic mouse model of dilated cardiomyopathy - Corrected Proof

2012-01-19

Abstract: Taking advantage of the unique model of slowly developing dilated cardiomyopathy in mice with cardiomyocyte-specific transgenic overexpression of activated Gαq protein (Tgαq*44 mice) we analyzed the contribution of the cardiomyocyte malfunction, fibrosis and cytoskeleton remodeling to the development of heart failure in this model. Left ventricular (LV) in vivo function, myocardial fibrosis, cytoskeletal proteins expression and distribution, Ca2+ handling and contractile function of isolated cardiomyocytes were evaluated at the stages of the early, compensated, and late, decompensated heart failure in 4-, 12- and 14-month-old Tgαq*44 mice, respectively, and compared to age-matched wild-type FVB mice. In the 4-month-old Tgαq*44 mice significant myocardial fibrosis, moderate myocyte hypertrophy and increased expression of regularly arranged and homogenously distributed desmin accompanied by increased phosphorylation of desmin chaperone protein, αB-crystallin, were found. Cardiomyocyte shortening, Ca2+ handling and LV function were not altered. At 12 and 14months of age, Tgαq*44 mice displayed progressive deterioration of the LV function. The contractile performance of isolated myocytes was still preserved, and the amplitude of Ca2+ transients was even increased probably due to impairment of Na+/Ca2+ exchanger function, while fibrosis was more extensive than in younger mice. Moreover, substantial disarrangement of desmin distribution accompanied by decreasing phosphorylation of αB-crystallin appeared. In Tgαq*44 mice disarrangement of desmin, at least partly related to inadequate phosphorylation of αB-crystallin seems to be importantly involved in the progressive deterioration of contractile heart function.Highlights: ► In Tgαq*44 mice increase of desmin content and fibrosis precede LV failure onset. ► In Tgαq*44 mice desmin disarrangement coincides with LV failure onset. ► Transient increase of αB-crystallin phosphorylation contributes to desmin remodeling. ► Myocyte contractile performance is not depressed in failing LV of Tgαq*44 mice.

Myocardial edema: A translational view - Uncorrected Proof

2012-01-19

Abstract: Myocardial edema occurs in a large number of myocardial pathologies particularly during ischemia–reperfusion, and may contribute to cell dysfunction and death occurring in these conditions. Cardiomyocyte cell volume is tightly regulated by modifications in cytosolic osmolality. Changes in membrane water permeability through aquaporin and connexin hemichannels also contribute to cell volume changes while caveolae may be important in sensing cell volume changes sensing and associated signaling. Ischemia–reperfusion alters these mechanisms and increases microvascular permeability by endothelial hypercontracture-induced gap formation, endothelial cell death and basal membrane disruption. Detection of myocardial edema by MRI has many useful diagnostic applications in acute myocardial infarction and other conditions. However, discrimination between intra and extracellular myocardial edema is presently difficult at the bench and impossible at the bedside. Developing methods to differentiate intra from extracellular myocardial water should allow a better understanding of the mechanisms and consequences of myocardial edema and, as a consequence lead to new diagnostic and therapeutic applications.Highlights: ►Pathophysiologic, diagnostic and therapeutic importance of edema discussed. ►Roles of aquaporins, connexin hemichannels and caveolae in myocardial edema. ►Analysis of intracellular and mitochondrial water distribution. ►The importance of discriminating intracellular from extracellular edema stressed.

Transgenic swine: Expression of human CD39 protects against myocardial injury: Wheeler, CD39 reduces myocardial injury in swine - Uncorrected Proof

2012-01-16

Abstract: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60min followed by 3hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2±4.3% vs. Control: 44.7±5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature - Uncorrected Proof

2012-01-16

Abstract: ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5′-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A1, and A2 but not by A3 receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.Highlights: ► P2Y and adenosine A2 receptor activation exert opposite effect on coronary vessels. ► ATP reduces coronary microvascular endothelial permeability via P2Y receptors. ► Adenosine increases coronary microvascular endothelial permeability via A2 receptors. ► ATP functionally antagonises adenosine-induced coronary microvascular leakage.

Delayed endosome-dependent CamKII and p38 kinase signaling in cardiomyocytes destabilizes Kv4.3 mRNA - Corrected Proof

Chaoming Zhou, Samantha L. Cavolo, Edwin S. Levitan — 2012-01-13

Abstract: The Kv4.3 transient outward current (Ito) channel, which produces early repolarization in human cardiomyocytes, is downregulated with cardiac pathology. This is evident in cultured neonatal rat cardiomyocytes in which Angiotensin II (Ang II) acts via p38 mitogen-activated protein kinase (p38K) to increase apoptosis and induce Kv4.3 mRNA destabilization to downregulate the channel protein. However, it is not understood how p38K activation, which is activated transiently for minutes, induces downstream effects hours later. Here we show that there is a second phase of p38K activation. Inhibiting this delayed p38K activation eliminated Kv4.3 mRNA destabilization. Furthermore, inhibiting endosome generation left the transient activation of p38K intact, but blocked delayed p38K activation and the Kv4.3 effect. CamKII was also found to be required for delayed p38K activation and Kv4.3 mRNA destabilization. Finally, CamKII methionine oxidation and activation are biphasic, with the delayed phase requiring endosomes. Hence, in addition to participating in channel traffic, cardiomyocyte endosomes control channel mRNA expression by mediating delayed oxidative CamKII–p38K signaling.Highlights: ► Angiotensin II induces biphasic CamKII and p38 kinase activation in cardiomyocytes. ► Delayed oxidative activation of these kinases requires endosomes. ► Endosome-dependent kinase signaling regulates K+ channel mRNA expression.

The expanding world of post-translational modifications - Corrected Proof

Elizabeth Murphy, Cam Patterson — 2012-01-13

Phosphorylation has long been known to regulate protein activity and has played a central role in most signal transduction pathways. Indeed 20years ago, in 1992 the Nobel Prize in Medicine was awarded to Drs. Edmond Fischer and Edwin Krebs for “their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism” . However, in the past 10years it has become apparent that phosphorylation is only one of many post-translational modifications (PTMs). Acetylation, ubiquitination, O-GlcNAc, S-nitrosylation, and glutathiolation have also been reported. In recent years a number of novel PTMs have been reported and JMCC has published several studies showing how these novel PTMs modulate cell function . This mini-special issue will highlight some of the newly identified PTMs.

Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease - Corrected Proof

2012-01-11

Abstract: Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.Highlights: ► Osteogenesis imperfecta mice have myxomatous valve disease. ► Klotho null mice have calcific aortic valve disease without inflammation. ► Mouse models have differential expression of valvulogenic, cartilage and bone genes. ► Valve disease in mice can occur without myofibroblast activation.

Common genetic variation modulating cardiac ECG parameters and susceptibility to sudden cardiac death - Corrected Proof

Iris C.R.M. Kolder, Michael W.T. Tanck, Connie R. Bezzina — 2012-01-09

Abstract: Sudden cardiac death (SCD) is a prevalent cause of death in Western societies. Genome-wide association studies (GWAS) conducted over the last few years have uncovered common genetic variants modulating risk of SCD. Furthermore, GWAS studies uncovered several loci impacting on heart rate and ECG indices of conduction and repolarization, as measures of cardiac electrophysiological function and likely intermediate phenotypes of SCD risk. We here review these recent developments and their implications for the identification of novel molecular pathways underlying normal electrophysiological function and susceptibility to SCD.Highlights: ► A heritable component modulates electrophysiological traits and susceptibility to SCD. ► GWAS uncovered common genetic variants at multiple loci modulating ECG indices. ► GWAS has started to uncover common genetic variants impacting on SCD risk. ► Further work is required to understand the mechanisms associated with these loci. ► Much of the heritability of these traits remains unexplained.

Introduction to the Special Issue on Coronary Blood Flow - Corrected Proof

William M. Chilian, Gerd Heusch — 2012-01-09

Some 25years ago, the coronary circulation was at the center stage of the basic science program at the annual Scientific Sessions of the American Heart Association meetings, with 4–5 oral sessions plus additional poster sessions at what was then the biggest and most important cardiovascular meeting worldwide. Nowadays, there is hardly one session on the coronary circulation left, and other topics such as cell therapy, micro RNA, and growth signaling appear more attractive and prevalent. However, the coronary circulation has not only lost ground to the success of other rising topics, but also became a victim of its own success. Many of the issues which we addressed in complex large animal experiments and discussed in great detail then, e.g. pressure–flow and flow–function relationships at rest and during exercise in the absence or presence of coronary stenosis, can now be measured in humans by modern imaging technologies and have become issues of daily clinical routine in the cath lab. The success of coronary interventions has not only made prior more esoteric and theoretic considerations on the functional relevance of a coronary stenosis truly practically relevant for clinical decisions and patients' prognosis, but also brought upon new issues, such as restenosis, stent thrombosis, coronary microembolization, impairment of vasomotion after mechanical coronary manipulation and with drug-eluting stents etc. We would like to also emphasize that not all issues regarding coronary control are really settled: the role that microvascular disease plays in conditions like heart failure and diabetic cardiomyopathy are beginning to be appreciated; fundamental issues such as how metabolism is connected to flow are yet not fully understood, but critical to our understanding of how coronary microvascular dysfunction leads to ischemic heart disease.

Measuring and evaluating the role of ATP-Sensitive K+ channels in cardiac muscle - Corrected Proof

2012-01-06

Abstract: Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the KATP channel, but many of the techniques described are also used to study other ion channels.Graphical abstract: Highlights: ► This review focuses on techniques commonly employed to examine KATP channel function. ► Electrophysiological, molecular, biochemical and genetic approaches are reviewed. ► These methods may further apply in other channel/integral membrane protein studies.

The mitochondrial KATP channel—Fact or fiction? - Corrected Proof

Keith D. Garlid, Andrew P. Halestrap — 2012-01-04

Abstract: The mitochondrial ATP-dependent K+ channel (mitoKATP) is widely considered by many to play a central role in cardioprotection by ischemic and pharmacological preconditioning and by ischemic postconditioning. Nevertheless, several laboratories have questioned the existence of mitoKATP. This article summarizes the evidence for and against and addresses two key questions: How strong is the evidence for the presence of a KATP channel in mitochondria? Are the pharmacological agents used to modulate mitoKATP activity sufficiently specific to allow the role of these channels in cardioprotection to be established?

Homocysteine induces cardiomyocyte dysfunction and apoptosis through p38 MAPK-mediated increase in oxidant stress - Corrected Proof

2012-01-03

Abstract: Elevated plasma homocysteine (Hcy) is a risk factor for cardiovascular disease. While Hcy has been shown to promote endothelial dysfunction by decreasing the bioavailability of nitric oxide and increasing oxidative stress in the vasculature, the effects of Hcy on cardiomyocytes remain less understood. In this study we explored the effects of hyperhomocysteinemia (HHcy) on myocardial function ex vivo and examined the direct effects of Hcy on cardiomyocyte function and survival in vitro. Studies with isolated hearts from wild type and HHcy mice (heterozygous cystathionine-beta synthase deficient mice) demonstrated that HHcy mouse hearts had more severely impaired cardiac relaxation and contractile function and increased cell death following ischemia reperfusion (I/R). In isolated cultured adult rat ventricular myocytes, exposure to Hcy for 24h impaired cardiomyocyte contractility in a concentration-dependent manner, and promoted apoptosis as revealed by terminal dUTP nick-end labeling and cleaved caspase-3 immunoblotting. These effects were associated with activation of p38 MAPK, decreased expression of thioredoxin (TRX) protein, and increased production of reactive oxygen species (ROS). Inhibition of p38 MAPK by the selective inhibitor SB203580 (5μM) prevented all of these Hcy-induced changes. Furthermore, adenovirus-mediated overexpression of TRX in cardiomyocytes significantly attenuated Hcy-induced ROS generation, apoptosis, and impairment of myocyte contractility. Thus, Hcy may increase the risk for CVD not only by causing endothelial dysfunction, but also by directly exerting detrimental effects on cardiomyocytes.Highlights: ► Effects of hyperhomocysteinemia on myocardium are not well understood. ► Ischemia–reperfusion caused worse injury in hyperhomocysteinemic mice. ► Homocysteine impaired contractility and promoted apoptosis in cardiomyocytes. ► Mechanism is via activation of p38MAPK and increase in oxidant stress. ► Overexpression of thioredoxin decreased oxidant stress and rescued cardiomyocytes.

Stimulation of the AT2 receptor reduced atherogenesis in ApoE−/−/AT1A−/− double knock out mice - Corrected Proof

2011-12-26

Abstract: AT1 receptor blockers (ARB) and in part ACE inhibitors (ACI) potentially exert beneficial effects on atherogenesis independent of AT1 receptor inhibition. These pleiotropic effects might be related to angiotensin II mediated activation of the AT2 receptor. To analyze this hypothesis we investigated the development of atherosclerosis and the role of ACIs and ARBs in apolipoprotein E-deficient (ApoE−/−) mice and in ApoE/AT1A receptor double knockout mice (ApoE−/−/AT1A−/−). ApoE−/− mice and ApoE−/−/AT1A−/− mice were fed cholesterol-rich diet for 7weeks. Vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation were evident in ApoE−/− mice, but were markedly reduced in ApoE−/−/AT1A−/− mice. Concomitant treatment of ApoE−/−/AT1A−/− mice with either telmisartan or ramipril had no additional effect on blood pressure, vascular oxidative stress, AT2 receptor expression, and endothelial function. Remarkably, atherosclerotic lesion formation was increased in ramipril treated ApoE−/−/AT1A−/− mice compared to untreated ApoE−/−/AT1A−/− mice whereas pharmacological AT1 receptor inhibition with telmisartan had no additional effect on atherogenesis. Moreover, chronic AT2 receptor inhibition with PD123,319 significantly increased plaque development in ApoE−/−/AT1A−/− mice. In additional experiments, direct AT2 receptor stimulation reduced atherogenesis in ApoE−/−/AT1A−/− mice. Taken together, our data demonstrate a relevant antiatherosclerotic role of the AT2 receptor in atherosclerotic mice and provide novel insight in RAS-physiology.Highlights: ► Atherogenesis was increased in ramipril treated ApoE−/−/AT1A−/− mice. ► PD123,319 increased plaque development in ApoE−/−/AT1A−/− mice. ► AT2 receptor confers atheroprotective effects when AT1 receptor action is inhibited. ► Relevant antiatherosclerotic role of the AT2 receptor in atherosclerotic mice.

Carbonic anhydrase XIV in the normal and hypertrophic myocardium - Corrected Proof

Lorena A. Vargas, Bernardo V. Alvarez — 2011-12-26

Abstract: Two AE3 transcripts, full-length (AE3fl) and cardiac (AE3c) are expressed in the heart. AE3 catalyzes electroneutral Cl−/HCO3− exchange across cardiomyocyte sarcolemma. AE proteins associate with carbonic anhydrases (CA), including CAII and CAIV, forming a HCO3− transport metabolon (BTM), increasing HCO3− fluxes and regulating cardiomyocytes pH. CAXIV, which is also expressed in the heart's sarcolemma, is a transmembrane enzyme with an extracellular catalytic domain. Herein, AE3/CAXIV physical association was examined by coimmunoprecipitation using rodent heart lysates. CAXIV immunoprecipitated with anti-AE3 antibody and both AE3fl and AE3c were reciprocally immunoprecipitated using anti-CAXIV antibody, indicating AE3fl–AE3c/CAXIV interaction in the myocardium. Coimmunoprecipitation experiments on heart lysates from a mouse with targeted disruption of the ae3 gene, failed to pull down AE3 with the CAXIV antibody. Confocal images demonstrated colocalization of CAXIV and AE3 in mouse ventricular myocytes. Functional association of AE3fl and CAXIV was examined in isolated hypertrophic rat cardiomyocytes, using fluorescence measurements of BCECF to monitor cytosolic pH. Hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHR) presented elevated myocardial AE-mediated Cl−/HCO3− exchange activity (JHCO3− mM.min−1) compared to normal (Wistar) rats (7.5±1.3, n=4 versus 2.9±0.1, n=6, respectively). AE3fl, AE3c, CAII, CAIV, and CAIX protein expressions were similar in SHR and Wistar rat hearts. However, immunoblots revealed a twofold increase of CAXIV protein expression in the SHR myocardium compared to normal hearts (n=11). Furthermore, the CA-inhibitor, benzolamide, neutralized the stimulatory effect of extracellular CA on AE3 transport activity (3.7±1.5, n=3), normalizing AE3-dependent HCO3− fluxes in SHR. CAXIV/AE3 interaction constitutes an extracellular component of a BTM which potentiates AE3-mediated HCO3− transport in the heart. Increased CAXIV expression and consequent AE3/CAXIV complex formation would render AE3 hyperactive in the SHR heart.Highlights: ► AE3 Cl−/HCO3−– exchanger interacts with the CAXIV enzyme in the heart. ► CAXIV bound to and raised AE3 transport, establishing a physical/functional complex. ► Hypertrophic rat hearts showed elevated CAXIV protein expression. ► AE-mediated HCO3− fluxes increased in the hypertrophic myocardium compared to normal. ► AE3/CAXIV complex formation would render AE3 hyperactive in the hypertrophic heart.

OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: Novel mechanisms for cardiovascular diseases - Corrected Proof

2011-12-22

Abstract: MicroRNA-29b has been reported to epigenetically regulate proatherogenic genes in response to oxLDL. Since transcription factors and epigenetic regulations are important mechanisms to regulate gene expression, we investigated whether these mechanisms are involved in oxLDL-induced microRNA-29b upregulation. First, we confirmed that microRNA-29b expression was increased in the aorta of mice fed with a high-fat diet, which was consistent with our previous in vitro findings. Next, we found that oxLDL only activated the microRNA-29b-1/microRNA-29a cluster gene on chromosome 7 but not the other distinct microRNA-29b gene located on chromosome 1. Using the promoter reporter assay and chromatin immunoprecipitation, activator protein-1 (AP-1) was shown to bind to the microRNA-29b-1 promoter. We further identified the signaling pathway of LOX-1/Ca2+/ROS/ERK/c-Fos was involved in oxLDL-mediated microRNA-29b overexpression after treating with the MAPTAM (Ca2+ chelator), NAC (ROS scavenger), U0126 (ERK inhibitor) and c-Fos (one of the AP-1 proteins) shRNA, respectively. To investigate epigenetic regulations, we found that microRNA-29b promoter contained no CpG islands for DNA methylation. Therefore we investigated whether histone modifications influence microRNA-29b promoter activity. We showed that down-regulation of HDAC1 and the modifications on histone 3 lysine 4 (H3K4) and H3K9 significantly affected microRNA-29b expression. Furthermore, knockdown of c-Fos expression attenuated the effect of oxLDL-induced histone modifications on the microRNA-29b gene expression. Taken together, our data suggest that both transcription factor activation and histone modifications are important regulatory mechanisms of oxLDL-induced atherogenic process. This article is part of a Special Issue entitled OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: Novel mechanisms for cardiovascular diseases.Highlights: ► In this study we studied mechanisms of oxLDL-upregulated miR-29b. ► OxLDL activated miR-29b from microRNA-29b-1/microRNA-29a cluster gene. ► The LOX-1/Ca2+/ROS/ERK/c-Fos involved in oxLDL-mediated miR-29b overexpression. ► OxLDL-downregulated HDAC1 affected miR-29b expression. ► OxLDL-mediated histone 3 lysine 4 (H3K4) and H3K9 regulated miR-29b expression.

Increased neointimal formation in cystathionine gamma-lyase deficient mice: Role of hydrogen sulfide in α5β1-integrin and matrix metalloproteinase-2 expression in smooth muscle cells - Corrected Proof

2011-12-21

Abstract: The physiological and pathological roles of hydrogen sulfide (H2S) in the regulation of cardiovacular functions have been recognized. Vascular smooth muscle cells (SMCs) express cystathionine gamma-lyase (CSE) and produce significant amount of H2S. Although growing evidence demonstated the anti-atherosclerotic effect of H2S, less is known about the contribution of the endogenous CSE/H2S pathway to the development of vascular remodeling. This study investigated the roles of the CSE/H2S pathway on SMC migration and neoimtimal formation by using CSE knockout (KO) mice. SMCs and aortic explants isolated from CSE KO mice exhibited more migration and outgrowth compared with that from wild-type (WT) mice, and exogenously applied NaHS (a H2S donor) at 100μM significantly inhibited SMC migration and outgrowth. SMCs became more elongated and spread in the absence of CSE, and fibronectin significantly stimulated adhesion and migration of SMCs from CSE KO mice (KO–SMCs) in comparison with SMCs from WT mice (WT–SMCs). The expressions of α5- and β1-integrins were significantly higher in KO–SMCs, and functional blocking of α5β1-integrin effectively abrogated KO–SMC migration. CSE deficiency also enhanced matrix metalloproteinase-2 (MMP-2) expression, and the selective blocking of MMP-2 decreased KO–SMC migration. NaHS treatment decreased both the expressions of α5- and β1-integrins and MMP-2. We further found that the expressions of α5- and β1-integrins as well as MMP-2, were stimulated by fibronectin, and that the blockage of α5β1-integrin reduced but overexpression of α5β1-integrin induced MMP-2 expression in both WT–SMCs and KO–SMCs. We also noticed that CSE deficiency in mice led to increased neointima formation in carotid arteries 4weeks after ligation, which were attenuated by NaHS administration. In conclusion, inhibition of SMC migration by H2S may be a novel target for the treatment of vascular occlusive disorder.Highlights: ► Cystathionine gamma-lyase deficiency induces smooth muscle cell migration. ► H2S inhibits the expressions of α5β1-integrin and MMP-2. ► Cystathionine gamma-lyase deficiency induces neointima formation in mice. ► H2S attenuates neointima formation in mice.

Coronary collateral growth—Back to the future - Corrected Proof

2011-12-21

Abstract: The coronary collateral circulation is critically important as an adaptation of the heart to prevent the damage from ischemic insults. In their native state, collaterals in the heart would be classified as part of the microcirculation, existing as arterial–arterial anastomotic connections in the range of 30 to 100μM in diameter. However, these vessels also show a propensity to remodel into components of the macrocirculation and can become arteries larger than 1000μM in diameter. This process of outward remodeling is critically important in the adaptation of the heart to ischemia because the resistance to blood flow is inversely related to the fourth power of the diameter of the vessel. Thus, an expansion of a vessel from 100 to 1000μM would reduce resistance (in this part of the circuit) to a negligible amount and enable delivery of flow to the region at risk. Our goal in this review is to highlight the voids in understanding this adaptation to ischemia—the growth of the coronary collateral circulation. In doing so we discuss the controversies and unknown aspects of the causal factors that stimulate growth of the collateral circulation, the role of genetics, and the role of endogenous stem and progenitor cells in the context of the normal, physiological situation and under more pathological conditions of ischemic heart disease or with some of the underlying risk factors, e.g., diabetes. The major conclusion of this review is that there are many gaps in our knowledge of coronary collateral growth and this knowledge is critical before the potential of stimulating collateralization in the hearts of patients can be realized. This article is part of a Special Issue entitled Coronary Blood Flow.Highlights: ► This review focuses on what is not known about coronary collateral growth. ► We raise fundamental questions about the regulation of coronary collateral growth. ► We present a schematic to highlight strategies of therapeutic collateral growth.

Pathophysiology of the cardiac late Na current and its potential as a drug target - Corrected Proof

Jonathan D. Moreno, Colleen E. Clancy — 2011-12-19

Abstract: A pathological increase in the late component of the cardiac Na+ current, INaL, has been linked to disease manifestation in inherited and acquired cardiac diseases including the long QT variant 3 (LQT3) syndrome and heart failure. Disruption in INaL leads to action potential prolongation, disruption of normal cellular repolarization, development of arrhythmia triggers, and propensity to ventricular arrhythmia. Attempts to treat arrhythmogenic sequelae from inherited and acquired syndromes pharmacologically with common Na+ channel blockers (e.g. flecainide, lidocaine, and amiodarone) have been largely unsuccessful. This is due to drug toxicity and the failure of most current drugs to discriminate between the peak current component, chiefly responsible for single cell excitability and propagation in coupled tissue, and the late component (INaL) of the Na+ current. Although small in magnitude as compared to the peak Na+ current (~1–3%), INaL alters action potential properties and increases Na+ loading in cardiac cells. With the increasing recognition that multiple cardiac pathological conditions share phenotypic manifestations of INaL upregulation, there has been renewed interest in specific pharmacological inhibition of INa. The novel antianginal agent ranolazine, which shows a marked selectivity for late versus peak Na+ current, may represent a novel drug archetype for targeted reduction of INaL. This article aims to review common pathophysiological mechanisms leading to enhanced INaL in LQT3 and heart failure as prototypical disease conditions. Also reviewed are promising therapeutic strategies tailored to alter the molecular mechanisms underlying INa mediated arrhythmia triggers.Highlights: ► INaL can disrupt cellular repolarization and increase propensity to ventricular arrhythmia. ► Although small compared to peak Na+ current, INaL increases Na+ loading in cardiac cells. ► Multiple cardiac pathological conditions share phenotypic manifestation of INaL upregulation. ► Specific pharmacological inhibition of INa is desired.

Transcription coactivator Eya2 is a critical regulator of physiological hypertrophy - Corrected Proof

2011-12-16

Abstract: Despite its significant clinical implications, physiological hypertrophy remains poorly understood. In this study, the transcription coactivator Eya2 was shown to be up-regulated during physiological hypertrophy. Transgene- or adenovirus-mediated overexpression of Eya2 led to up-regulation of mTOR, a critical mediator of physiological hypertrophy. Luciferase reporter and chromatin immunoprecipitation assays revealed that Eya2 directly binds to and activates mTOR expression. The phosphorylation of mTOR downstream molecules was significantly enhanced in Eya2 transgenic (TG) hearts, implying that the Eya2-mediated induction of mTOR expression leads to an elevated mTOR activity. The transcription factor Six1 was also up-regulated during physiological hypertrophy and formed a complex with Eya2. Luciferase reporter and electrophoretic mobility shift assays revealed that the Eya2-Six1 complex binds to and enhances the expression of mTOR in a synergistic manner. Under pressure overload, Eya2 transgenic hearts developed hypertrophy which exhibited important molecular signatures of physiological hypertrophy, as assessed by gene expression profiling and measurements of expression levels of physiological hypertrophy-related genes by quantitative (q) RT-PCR. Examination of heart sections under electron microscopy revealed that the mitochondrial integrity remained largely intact in Eya2 transgenic mice, but not in wild-type littermates, under pressure overload. This finding was confirmed by measurements of mitochondrial DNA contents and the expression levels of mitochondrial function-related genes by qRT-PCR. These data suggest that Eya2 in a physical complex with Six1 plays a critical role in physiological hypertrophy. The cardioprotective effect of Eya2 appears to be due, at least in part, to its preservation of mitochondrial integrity upon pressure overload.Highlights: ► Eya2 directly regulates the expression of mTOR during physiological hypertrophy. ► Eya2 and Six1 synergistically up-regulate mTOR expression. ► Eya2 TG exhibits characteristics of physiological cardiac hypertrophy. ► Mitochondrial integrity is preserved in Eya2 TG hearts under pressure overload. ► Activation of Eya2 and Six1 transcriptional complex is cardioprotective.

MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3 - Corrected Proof

2011-12-12

Abstract: Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress.To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17–92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/“reperfusion” model. Given the potential apoptosis-modulating properties of the miR17–92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3.Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.Highlights: ► Excessive stress is a potent inductor of cardiomyocyte apoptosis and remodelling. ► miR-20a is upregulated in acute biomechanical stress as well as hypoxia. ► miR-20a significantly inhibited hypoxia-mediated apoptosis. ► miR-20a’s antiapoptotic effect appears to be mediated through targeting of Egln3. ► This study reveals miR-20a as a novel cardioprotective microRNA.

The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: Implications for neural control of cardiac excitability - Corrected Proof

2011-12-09

Abstract: The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10Hz, 2min, in the presence of 10μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p<0.05) reduction in the magnitude of vagal bradycardia (5Hz) maintained over the subsequent 20min (n=6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n=6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10min (1μM, n=5), and completely reversed with the NPY Y2 receptor antagonist BIIE 0246 at all time points (1μM, n=6). Exogenous galanin (n=6, 50–500nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n=6). Galanin (500nM) also significantly attenuated the release of 3H-acetylcholine from isolated atria during field stimulation (5Hz, n=5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n=5). Importantly the GalR1 receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100nM, n=6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500nM, n=6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR1 receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.Highlights: ► Galanin is found in guinea pig stellate neurons and GalR1 on cardiac vagal neurons. ► Stellate galanin expression increases following 3days of cell culture. ► High level sympathetic stimulation releases galanin which reduces vagal bradycardia. ► Galanin reduces acetylcholine release and bradycardia via a GalR1 dependent pathway. ► Galanin signals via protein kinase C rather than protein kinase A dependent pathways.

Soluble epoxide hydrolase inhibition improves myocardial perfusion and function in experimental heart failure - Corrected Proof

2011-12-07

Abstract: The study addressed the hypothesis that soluble epoxide hydrolase (sEH) inhibition, which increases cardiovascular protective epoxyeicosatrienoic acids (EETs), exerts beneficial effects in an established chronic heart failure (CHF) model. In CHF rats, left ventricular (LV) function, perfusion and remodeling were assessed using MRI and invasive hemodynamics after 42-day (starting 8days after coronary ligation) and delayed 3-day (starting 47days after coronary ligation) treatments with the sEH inhibitor AUDA (twice 0.25mg/day). Delayed 3-day and 42-day AUDA increased plasma EETs demonstrating the effective inhibition of sEH. Delayed 3-day and 42-day AUDA enhanced cardiac output without change in arterial pressure, thus reducing total peripheral resistance. Both treatment periods increased the slope of the LV end-systolic pressure–volume relation, but only 42-day AUDA decreased LV end-diastolic pressure, relaxation constant Tau and the slope of the LV end-diastolic pressure–volume relation, associated with a reduced LV diastolic volume and collagen density. Delayed 3-day and, to a larger extent, 42-day AUDA increased LV perfusion associated with a decreased LV hypoxia-inducible factor-1alpha. Both treatment periods decreased reactive oxygen species level and increased reduced-oxidized glutathione ratio. Finally, MSPPOH, an inhibitor of the EET-synthesizing enzyme cytochrome epoxygenases, abolished the beneficial effects of 3-day AUDA on LV function and perfusion. Augmentation of EET availability by pharmacological inhibition of sEH increases LV diastolic and systolic functions in established CHF. This notably results from short-term processes, i.e. increased LV perfusion, reduced LV oxidative stress and peripheral vasodilatation, but also from long-term effects, i.e. reduced LV remodeling.Highlights: ► We studied the effects of a soluble epoxide hydrolase inhibitor in heart failure rats. ► The inhibitor induced an increase in epoxyeicosatrienoic acid bioavailability. ► This was associated with an improvement in systolic and diastolic cardiac function. ► Enhanced cardiac perfusion and reduced oxidative stress contributed to this effect. ► This pharmacological strategy represents a promising approach to treat heart failure.

Reduced cardiac CapZ protein protects hearts against acute ischemia–reperfusion injury and enhances preconditioning - Corrected Proof

Feng Hua Yang, W. Glen Pyle — 2011-12-05

Abstract: The Z-disc protein CapZ has historically been classified as a structural element, anchoring sarcomeric actin. Our previous work expanded its role to include signal transduction, as CapZ transgenic myofilaments are less sensitive to protein kinase C (PKC). Myocardial PKC has paradoxical effects, mediating both preconditioning and ischemia–reperfusion (IR) injury. Our objective was to determine how decreased CapZ affects IR injury and cardiac preconditioning. Mouse hearts were subjected to 20min global ischemia and 60min reperfusion. Some hearts were preconditioned with intermittent IR (IPC). Left ventricular function was assessed and myocardial tissue collected post-IR for molecular analysis and tissue staining. Post-ischemic function was significantly better and infarct size smaller in CapZ transgenic hearts, as compared to wildtype. IPC decreased IR damage in both wildtype and CapZ transgenic hearts, although CapZ transgenic hearts performed significantly better than wildtype. Immunoblotting revealed increased myofilament-associated PKC-α and -ε following IR in wildtype hearts, but no change in PKC-δ or -ζ. By contrast, post-IR myofilament-associated PKC-α was significantly higher in CapZ transgenic mice but the rise in PKC-ε was attenuated. Both PKC-δ and PKC-ζ decreased in CapZ transgenic myofilaments following IR. IPC increased myofilament-associated PKC-α and -ε, while decreasing PKC-δ in wildtype hearts. Preconditioned CapZ IPC hearts showed attenuated increases in myofilament PKC-α and -ε, but also a significant decrease in myofilament PKC-δ and -ζ. These data demonstrate significant differences in post-IR myofilament PKC in untreated and preconditioned CapZ transgenic mice. CapZ reduction did not dramatically affect post-IR myofilament function, nor did preconditioning. These results demonstrate that CapZ deficiency decreases IR injury, while providing enhanced cardioprotection with IPC. The cardioprotected phenotype of CapZ transgenic mice is associated with an altered translocation of PKC-isoforms to cardiac myofilaments.Highlights: ► We examined post-ischemic heart function in CapZ transgenic mice. ► CapZ transgenic mice were subjected to acute ischemia–reperfusion. ► Reduced CapZ protein increased post-ischemic function compared to wildtype. ► Protein kinase C activation was altered in CapZ transgenic mice.

Sexual dimorphism in cardiac triacylglyceride dynamics in mice on long term caloric restriction - Corrected Proof

2011-12-05

Abstract: Human studies indicate augmented myocardial lipid metabolism in females, and that sex and obesity interact to predict myocardial fatty acid oxidation and storage. Altered lipid dynamics precede cardiomyopathies, and many studies now address high fat diets. Conversely, caloric restriction (CR), is the most studied model for longevity and stress resistance, including protection against myocardial ischemia. However, no information exists on the effects of long-term caloric restriction (CR) on triacylglyceride (TAG) content and dynamics in the heart. This study explored the effects of CR, sex and age on TAG dynamics in mouse hearts. Male and female SVJ129 mice were fed either normal (ND) or CR diet for 3 or 10months. In 5-month-old mice, CR similarly decreased cardiac TAG in males (ND: 25.5±4.5nmol/mg protein; CR: 12.6±2.7, P<0.05) and females (ND: 30.1±4.4; CR: 13.7±1.2) (no significant differences in TAG content were seen between sexes). CR reduced the contribution of exogenous palmitate to oxidative metabolism in males and females, by 15% and 11% respectively, versus ND, without affecting cardiac workload. CR also induced a larger reduction in TAG turnover in male (68%) than female hearts (38%). Interestingly, in 5month old male mice, CR reproduced the lower TAG turnover rates of middle-aged males (ND 13-month-old male=423±76nmol/mgprotein/min). Thus, long term CR reduces TAG pool dynamics. Despite reduced content, hearts of female mice subjected to CR retained a more dynamic TAG pool than males, while males respond with greater metabolic remodeling of cardiac lipid dynamics.Highlights: ► The effects of gender and age on TAG dynamics after CR are reported. ► Long-term CR results in a reduction of endogenous lipid stores. ► Long-term CR results in a significant decrease in TAG turnover. ► The female myocardium preserves TAG involvement in lipid metabolism.

Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy - Corrected Proof

2011-12-02

Abstract: Cardiac hypertrophy is controlled by a complex signal transduction and gene regulatory network, containing multiple layers of crosstalk and feedback. While numerous individual components of this network have been identified, understanding how these elements are coordinated to regulate heart growth remains a challenge. Past approaches to measure cardiac myocyte hypertrophy have been manual and often qualitative, hindering the ability to systematically characterize the network's higher-order control structure and identify therapeutic targets. Here, we develop and validate an automated image analysis approach for objectively quantifying multiple hypertrophic phenotypes from immunofluorescence images. This approach incorporates cardiac myocyte-specific optimizations and provides quantitative measures of myocyte size, elongation, circularity, sarcomeric organization, and cell–cell contact. As a proof-of-concept, we examined the hypertrophic response to α-adrenergic, β-adrenergic, tumor necrosis factor (TNFα), insulin-like growth factor-1 (IGF-1), and fetal bovine serum pathways. While all five hypertrophic pathways increased myocyte size, other hypertrophic metrics were differentially regulated, forming a distinct phenotype signature for each pathway. Sarcomeric organization was uniquely enhanced by α-adrenergic signaling. TNFα and α-adrenergic pathways markedly decreased cell circularity due to increased myocyte protrusion. Surprisingly, adrenergic and IGF-1 pathways differentially regulated myocyte–myocyte contact, potentially forming a feed-forward loop that regulates hypertrophy. Automated image analysis unlocks a range of new quantitative phenotypic data, aiding dissection of the complex hypertrophic signaling network and enabling myocyte-based high-content drug screening.Highlights: ► Automated image analysis method for cardiac myocyte hypertrophy. ► New quantitative measure of sarcomeric organization. ► TNFα and α-adrenergic signaling have opposite effects on myocyte elongation. ► Sarcomeric organization is uniquely enhanced by α-adrenergic signaling. ► α- and β-adrenergic pathways enhance myocyte–myocyte contact.

Wnt2 accelerates cardiac myocyte differentiation from ES-cell derived mesodermal cells via non-canonical pathway - Corrected Proof

2011-12-02

Abstract: The efficient induction of cardiomyocyte differentiation from embryonic stem (ES) cells is crucial for cardiac regenerative medicine. Although Wnts play important roles in cardiac development, complex questions remain as to when, how and what types of Wnts are involved in cardiogenesis. We found that Wnt2 was strongly up-regulated during cardiomyocyte differentiation from ES cells. Therefore, we investigated when and how Wnt2 acts in cardiogenesis during ES cell differentiation. Wnt2 was strongly expressed in the early developing murine heart. We applied this embryonic Wnt2 expression pattern to ES cell differentiation, to elucidate Wnt2 function in cardiomyocyte differentiation. Wnt2 knockdown revealed that intrinsic Wnt2 was essential for efficient cardiomyocyte differentiation from ES cells. Moreover, exogenous Wnt2 increased cardiomyocyte differentiation from ES cells. Interestingly, the effects on cardiogenesis of intrinsic Wnt2 knockdown and exogenous Wnt2 addition were temporally restricted. During cardiomyocyte differentiation from ES cells, Wnt2 didn't activate canonical Wnt pathway but utilizes JNK/AP-1 pathway which is required for cardiomyocyte differentiation from ES cells. Therefore we conclude that Wnt2 plays strong positive stage-specific role in cardiogenesis through non-canonical Wnt pathway in murine ES cells.Highlights: ► Wnt2 is highly expressed during cardiac differentiation from ES cells. ► Wnt2 is necessary for efficient cardiac induction from ES cells. ► Wnt2 promotes cardiac differentiation from ES cells through JNK/AP-1 pathway.

Enhanced desumoylation in murine hearts by overexpressed SENP2 leads to congenital heart defects and cardiac dysfunction - Corrected Proof

2011-12-02

Abstract: Sumoylation is a posttranslational modification implicated in a variety of cellular activities, and its role in a number of human pathogeneses such as cleft lip/palate has been well documented. However, the importance of the SUMO conjugation pathway in cardiac development and functional disorders is newly emerging. We previously reported that knockout of SUMO-1 in mice led to congenital heart diseases (CHDs). To further investigate the effects of imbalanced SUMO conjugation on heart development and function and its underlying mechanisms, we generated transgenic (Tg) mice with cardiac-specific expression of SENP2, a SUMO-specific protease that deconjugates sumoylated proteins, to evaluate the impact of desumoylation on heart development and function. Overexpression of SENP2 resulted in premature death of mice with CHDs—atrial septal defects (ASDs) and/or ventricular septal defects (VSDs). Immunobiochemistry revealed diminished cardiomyocyte proliferation in SENP2-Tg mouse hearts compared with that in wild type (WT) hearts. Surviving SENP2-Tg mice showed growth retardation, and developed cardiomyopathy with impaired cardiac function with aging. Cardiac-specific overexpression of the SUMO-1 transgene reduced the incidence of cardiac structural phenotypes in the sumoylation defective mice. Moreover, cardiac overexpression of SENP2 in the mice with Nkx2.5 haploinsufficiency promoted embryonic lethality and severity of CHDs, indicating the functional interaction between SENP2 and Nkx2.5 in vivo. Our findings indicate the indispensability of a balanced SUMO pathway for proper cardiac development and function. This article is part of a Special Issue entitled ‘Post-translational Modification SI’.Highlights: ► We studied the effect of de-sumoylation on heart development in a transgenic model. ► Increased desumoylation by overexpressed SENP2 in heart caused cardiac defects and dysfunction. ► SENP2 overexpression also caused defect in cardiomyocyte proliferation. ► SUMO-1 overexpression rescued cardiac structural phenotypes in SENP2-Tg mice. ► Balanced SUMO conjugation is essential for normal heart development and function.

The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase - Corrected Proof

2011-12-02

Abstract: Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

Guanylyl cyclase (GC)-A and GC-B activities in ventricles and cardiomyocytes from failed and non-failed human hearts: GC-A is inactive in the failed cardiomyocyte - Corrected Proof

Deborah M. Dickey, Daniel L. Dries, Kenneth B. Margulies, Lincoln R. Potter — 2011-11-23

Abstract: Cardiomyocytes release atrial natriuretic peptide (ANP) and B-type natriuretic peptide to stimulate processes that compensate for the failing heart by activating guanylyl cyclase (GC)-A. C-type natriuretic peptide is also elevated in the failing heart and inhibits cardiac remodeling by activating the homologous receptor, GC-B. We previously reported that GC-A is the most active membrane GC in normal mouse ventricles while GC-B is the most active membrane GC in failing ventricles due to increased GC-B and decreased GC-A activities. Here, we examined ANP and CNP-specific GC activity in membranes obtained from non-failing and failing human left ventricles and in membranes from matched cardiomyocyte-enriched pellet preparations. Similar to our findings in the murine study, we found that CNP-dependent GC activity was about half of the ANP-dependent GC activity in the non-failing ventricular and was increased in the failing ventricle. ANP and CNP increased GC activity 9- and 5-fold in non-failing ventricles, respectively. In contrast to the mouse study, in failing human ventricles, ANP-dependent activity was unchanged compared to non-failing values whereas CNP-dependent activity increased 35% (p=0.005). Compared with ventricular membranes, basal GC activity was reduced an order of magnitude in membranes derived from myocyte-enriched pellets from non-failing ventricles. ANP increased GC activity 2.4-fold but CNP only increased GC activity 1.3-fold. In contrast, neither ANP nor CNP increased GC activity in equivalent preparations from failing ventricles. We conclude that: 1) GC-B activity is increased in non-myocytes from failing human ventricles, possibly as a result of increased fibrosis, 2) human ventricular cardiomyocytes express low levels of GC-A and much lower levels or possibly no GC-B, and 3) GC-A in cardiomyocytes from failing human hearts is refractory to ANP stimulation.Highlights: ► Membranes from human heart left ventricles express half as much GC-B activity as GC-A. ► In failing human ventricles, CNP-GC-B-dependent activity was significantly increased. ► Human cardiomyocytes express GC-A but little or no GC-B. ► GC-A is expressed at reduced levels compared to expression in other cells in the ventricle. ► GC-A in cardiomyocyte-enriched samples from failing human hearts is refractory to ANP.

The role of SIRT3 in mitochondrial homeostasis and cardiac adaptation to hypertrophy and aging - Corrected Proof

Michael N. Sack — 2011-11-21

Abstract: Although acetyl-modification of protein lysine residues has been recognized for many decades, the appreciation that this post-translational modification is highly prevalent in mitochondria and plays a pivotal regulatory role in mitochondrial function has only become apparent since 2006. The classical biological stressors that modulate mitochondrial protein acetylation include alterations in caloric levels and redox signaling and the major enzyme orchestrating deacetylation is the mitochondrial enriched sirtuin SIRT3. Overall the action of SIRT3 modulates mitochondrial homeostasis and SIRT3 target proteins include mediators of energy metabolism and mitochondrial redox stress adaptive program proteins. Given these effects, it is not surprising that the role of SIRT3 has begun to be implicated in cardiac biology. This review gives a brief overview of sirtuin biology and then focuses on the role of the SIRT3 regulatory program in the control of cardiac hypertrophy and aging. This article is part of a Special Issue entitled Post-translational Modification.Highlights: ► SIRT3 is a mitochondrial enriched protein lysine residue deacetylase. ► Deacetylation of mitochondrial proteins modifies target protein function. ► SIRT3 target proteins control mitochondrial energetics and ROS levels. ► SIRT3 ameliorates pressure-overload and aging associated cardiac dysfunction.

Back to your heart: Ubiquitin proteasome system-regulated signal transduction - Corrected Proof

2011-11-07

Abstract: Awareness of the regulation of cell signaling by post-translational ubiquitination has emerged over the past 2 decades. Like phosphorylation, post-translational modification of proteins with ubiquitin can result in the regulation of numerous cellular functions, for example, the DNA damage response, apoptosis, cell growth, and the innate immune response. In this review, we discuss recently published mechanisms by which the ubiquitin proteasome system regulates key signal transduction pathways in the heart, including MAPK JNK, calcineurin, FOXO, p53, and estrogen receptors α and β. We then explore how ubiquitin proteasome system-specific regulation of these signal transduction pathways plays a role in the pathophysiology of common cardiac diseases, such as cardiac hypertrophy, heart failure, ischemia reperfusion injury, and diabetes. This article is part of a Special Issue entitled ‘Post-translational Modification SI’.Highlights: ► The ubiquitin proteasome system (UPS) regulates signal transduction in the heart ► The UPS regulates JNK, calcineurin, FOXO, p53, and estrogen receptor signaling in relevant models of cardiac disease ► Regulation of the UPS can influence outcomes in cardiac hypertrophy, heart failure, ischemia, and diabetic cardiomyopathy ► The UPS affects cardiac signal transduction pathways significant to cardiac health and disease.

The transcriptional coactivators, PGC-1α and β, cooperate to maintain cardiac mitochondrial function during the early stages of insulin resistance - Corrected Proof

2011-10-24

Abstract: We previously demonstrated a cardiac mitochondrial biogenic response in insulin resistant mice that requires the nuclear receptor transcription factor PPARα. We hypothesized that the PPARα coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is necessary for mitochondrial biogenesis in insulin resistant hearts and that this response was adaptive. Mitochondrial phenotype was assessed in insulin resistant mouse models in wild-type (WT) versus PGC-1α deficient (PGC-1α−/−) backgrounds. Both high fat-fed (HFD) WT and 6week-old Ob/Ob animals exhibited a significant increase in myocardial mitochondrial volume density compared to standard chow fed or WT controls. In contrast, HFD PGC-1α−/− and Ob/Ob-PGC-1α−/− hearts lacked a mitochondrial biogenic response. PGC-1α gene expression was increased in 6week-old Ob/Ob animals, followed by a decline in 8week-old Ob/Ob animals with more severe glucose intolerance. Mitochondrial respiratory function was increased in 6week-old Ob/Ob animals, but not in Ob/Ob-PGC-1α−/− mice and not in 8week-old Ob/Ob animals, suggesting a loss of the early adaptive response, consistent with the loss of PGC-1α upregulation. Animals that were deficient for PGC-1α and heterozygous for the related coactivator PGC-1β (PGC-1α−/−β+/−) were bred to the Ob/Ob mice. Ob/Ob-PGC-1α−/−β+/− hearts exhibited dramatically reduced mitochondrial respiratory capacity. Finally, the mitochondrial biogenic response was triggered in H9C2 myotubes by exposure to oleate, an effect that was blunted with shRNA-mediated PGC-1 “knockdown”. We conclude that PGC-1 signaling is important for the adaptive cardiac mitochondrial biogenic response that occurs during the early stages of insulin resistance. This response occurs in a cell autonomous manner and likely involves exposure to high levels of free fatty acids.Highlights: ► PGC-1α is necessary for mitochondrial biogenesis in the insulin resistant heart. ► PGC-1β is responsive to insulin resistance and has an overlapping role with PGC-1α. ► Deficiency of both PGC-1 isoforms impairs mitochondrial function.

Regulation of coronary resistance vessel tone in response to exercise - Corrected Proof

Dirk J. Duncker, Robert J. Bache, Daphne Merkus — 2011-10-17

Abstract: Exercise is a primary stimulus for increased myocardial oxygen demand. The ~6-fold increase in oxygen demand of the left ventricle during heavy exercise is met principally by augmenting coronary blood flow (~5-fold), as hemoglobin concentration and oxygen extraction (which is already ~70% at rest) increase only modestly in most species. As a result, coronary blood flow is tightly coupled to myocardial oxygen consumption over a wide range of physical activity. This tight coupling has been proposed to depend on periarteriolar oxygen tension, signals released from cardiomyocytes and the endothelium as well as neurohumoral influences, but the contribution of each of these regulatory pathways, and their interactions, to exercise hyperemia in the heart remain incompletely understood. In humans, nitric oxide, adenosine and KATP channels each appear to contribute to resting coronary resistance vessel tone, but evidence for a critical contribution to exercise hyperemia is lacking. In dogs KATP-channel activation together with adenosine and nitric oxide contribute to exercise hyperemia in a non-linear redundant fashion. In contrast, in swine nitric oxide, adenosine and KATP channels contribute to resting coronary resistance vessel tone control in a linear additive manner, but do not appear to be mandatory for exercise hyperemia. Rather, exercise hyperemia in swine appears to involve β-adrenergic activation in conjunction with exercise-induced blunting of an endothelin-mediated vasoconstrictor influence. In view of these remarkable species differences in coronary vasomotor control during exercise, future studies are required to determine the system of vasodilator components that mediate exercise hyperemia in humans. This article is part of a Special Issue entitled ‘Coronary Blood Flow SI’.Highlights: ► Mechanisms of exercise hyperemia in the heart remain incompletely understood. ► Mechanisms of exercise hyperemia appear highly species dependent. ► Hyperemia in dogs involves adenosine, nitric oxide, and KATP channel activation. ► Hyperemia in swine involves β-adrenergic activation and loss of endothelin influence. ► The mechanisms of exercise hyperemia in the human heart await further elucidation.

Regulation of the human coronary microcirculation - Corrected Proof

Andreas M. Beyer, David D. Gutterman — 2011-10-14

Abstract: Atherosclerosis of conduit epicardial arteries is the principal culprit behind the complications of coronary heart disease, but a growing body of literature indicates that the coronary microcirculation also contributes substantially to the pathophysiology of cardiovascular disease. An understanding of mechanisms regulating microvascular function in humans is an essential foundation for understanding the role in disease, especially since these regulatory mechanisms vary substantially across species and vascular beds. In fact all subjects whose coronary tissue was used in the studies described have medical conditions that warrant cardiac surgery, thus relevance to the normal human must be inferential and is based on tissue from subjects without known arteriosclerotic disease. This review will focus on recent advances in the physiological and pathological mechanisms of coronary microcirculatory control, describing a robust plasticity in maintaining endothelial control over dilation, including mechanisms that are most relevant to the human heart.Highlights: ► Coronary blood flow is regulated by arterioles between 50–200μm in diameter. ► Shear-induced dilation amplifies metabolic dilation by reducing upstream vascular resistance. ► Shear-induced endothelial hydrogen peroxide originates from mitochondria. ► This shear-released hydrogen peroxide dilates coronary arterioles from humans with CAD. ► Transient receptor potential vanilloid 4 channels are required for the dilation to shear.