Journal of Molecular and Cellular Cardiology - Articles in Press

Beta1-Adrenergic Receptors Promote Focal Adhesion Signaling Downregulation And Myocyte Apoptosis in Acute Volume Overload - Accepted Manuscript

2012-05-17

Abstract: Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (β1-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk2 and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. β1-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with β1-AR agonists or adenoviral expression of β1-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by β1-ARs. Collectively, these data show that ß1-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.Highlights: ► Acute VO induces extracellular matrix loss and FA signaling alterations ► β1-AR blockade attenuates VO-induced FA signaling alterations and myocyte apoptosis ► PTEN signaling mediates β1-AR-induced FA signaling alterations and myocyte apoptosis

PKCβII Modulation of Myocyte Contractile Performance - Accepted Manuscript

2012-05-15

Abstract: Significant up-regulation of the protein kinase CβII (PKCβII) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβII modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβII protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβII was distributed in a perinuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβII (PKCβDN). Similar decreases were observed in the Ca2+ transient and the Ca2+ decay rate slowed in response to caffeine in PKCβII-expressing myocytes. Parallel phosphorylation studies indicated PKCβII targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβII expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβII increased Ca2+-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβII modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.Highlights: ► This study provides a detailed analysis of PKCβII modulation of cardiac myocyte contractile function ► We examined the distribution of PKCβII and phosphorylation of Ca2+ cycling and myofilament proteins. ► PKCβII signaling involves intermediate downstream phosphatase and kinase activation

Combined deep microRNA and mRNA sequencing identifies protective transcriptomal signature of enhanced PI3Kα signaling in cardiac hypertrophy - Uncorrected Proof

Kai-Chien Yang, Yuan-Chieh Ku, Michael Lovett, Jeanne M. Nerbonne — 2012-05-14

Abstract: The perturbation of myocardial transcriptome homeostasis is the hallmark of pathological hypertrophy, underlying the maladaptive myocardial remodeling secondary to pathological stresses. Classic and novel therapeutics that provide beneficial effects against pathological remodeling likely impact myocardial transcriptome architecture, including miRNA and mRNA expression profiles. Microarray and PCR-based technologies, although employed extensively, cannot provide adequate sequence coverage or quantitative accuracy to test this hypothesis directly. The goal of this study was to develop and exploit next-generation sequencing approaches for comprehensive and quantitative analyses of myocardial miRNAs and mRNAs to test the hypothesis that augmented phosphoinositide-3-kinase-p110α (PI3Kα) signaling in the setting of pathological hypertrophy provides beneficial effects through remodeling of the myocardial transcriptome signature. In these studies, a molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kα signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored. These analyses identified multiple miRNAs and mRNAs that were abnormally expressed in pathological hypertrophy and partially or completely normalized with increased PI3Kα signaling. Additionally, several novel miRNAs potentially linked to remodeling in cardiac hypertrophy were identified. Additional experiments revealed that increased PI3Kα signaling reduces cardiac fibrosis in pathological hypertrophy through modulating TGF-β signaling and miR-21 expression. In conclusion, using the approach of combined miRNA and mRNA sequencing, we identify the protective transcriptome signature of enhanced PI3Kα signaling in the context of pathological hypertrophy, and demonstrate the regulation of TGF-β/miR-21 by which enhanced PI3Kα signaling protects against cardiac fibrosis.Highlights: ► We exploited next-gen sequencing for a comprehensive cardiac mRNA and miRNA profiling. ► We revealed the transcriptome signature of the cardiac protective effects of PI3Kα. ► Increased PI3Kα signaling normalizes miRNA/mRNA expression with pathological LVH. ► PI3Kα reduces cardiac fibrosis through modulating TGF-β pathway and miR-21 expression. ► We identified multiple novel miRNAs linked to remodeling in cardiac hypertrophy.

AAV micro-dystrophin gene therapy alleviates stress-induced cardiac death but not myocardial fibrosis in > 21-m-old mdx mice, an end-stage model of Duchenne muscular dystrophy cardiomyopathy - Accepted Manuscript

2012-05-14

Abstract: Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by the absence of the sarcolemmal protein dystrophin. Dilated cardiomyopathy leading to heart failure is a significant source of morbidity and mortality in DMD. We recently demonstrated amelioration of DMD heart disease in 16 to 20-m-old dystrophin-null mdx mice using adeno-associated virus (AAV) mediated micro-dystrophin gene therapy. Like DMD patients, mdx mice exhibit profoundly worsening heart disease when they reach beyond 21months of age. To more rigorously test micro-dystrophin therapy, we treated mdx mice that were between 21.2 to 22.7-m-old (average, 22.1±0.2months; N=8). The ∆R4-23/∆C micro-dystrophin gene was packaged in the cardiotropic AAV-9 virus. 5 x 1012 viral genome particles/mouse were delivered to mdx mice via the tail vein. AAV transduction, myocardial fibrosis and heart function were examined 1.7±0.2months after gene therapy. Efficient micro-dystrophin expression was observed in the myocardium of treated mice. Despite the robust dystrophin expression, myocardial fibrosis was not mitigated. Most hemodynamic parameters were not improved either. However, ECG abnormalities were partially corrected. Importantly, treated mice became more resistant to dobutamine-induced cardiac death. In summary, we have revealed for the first time the potential benefits and limitations of AAV micro-dystrophin therapy in end-stage Duchenne dilated cardiomyopathy. Our findings have important implications for the use of AAV gene therapy in dilated cardiomyopathy and heart failure.Highlights: ► AAV-9 efficiently transduced end-stage dystrophic myocardium. ► Micro-dystrophin gene therapy in end-stage Duchenne cardiomyopathy mouse model. ► Micro-dystrophin improved ECG but not heart fibrosis in terminal aged mdx mice. ► AAV-9 Micro-dystrophin did not correct hemodynamic defects in >21-m-old mdx mice. ► AAV-9 micro-dystrophin prevented death from dobutamine stress in end-stage mdx mice.

Non-neuronal cholinergic machinery present in cardiomyocytes offsets hypertrophic signals - Accepted Manuscript

2012-05-14

Abstract: Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counter-act or partially neutralize hypertrophic adrenergic effects.Highlights: ► Neuronal proteins required for ACh synthesis and release are found in cardiomyocytes ► In cardiomyocytes VAChT positive vesicles are mainly found in the perinuclear area ► ACh secretion in cardiomyocytes is dependent on VAChT activity and choline reuptake ► Cardiomyocyte secreted ACh prevents adrenergic induced hypertrophic signaling ► Adrenergic stimulation increases expression of cholinergic machinery in cardiac cells

Automated imaging reveals a concentration dependent delay in reversibility of cardiac myocyte hypertrophy - Uncorrected Proof

Karen A. Ryall, Jeffrey J. Saucerman — 2012-05-10

Abstract: Cardiac hypertrophy is controlled by a dense signaling network with many pathways associated with cardiac myocyte growth. New large scale methodology is required to quantitatively characterize the pathways that distinguish reversible forms of hypertrophy from irreversible forms that lead to heart failure. Our automated image acquisition method records 5×5 mosaic images of fluorescent protein-labeled cardiac myocytes within each well of a 96-well plate using an automated stage and focus. Post-processing algorithms automatically identify cell edges, quantify cell phenotypes, and track cells. We uniquely applied our imaging platform to study hypertrophy reversibility in a scalable cell model. Cell area changes after washout of a dose response to the α-adrenergic receptor (αAR) agonist phenylephrine (PE) showed that hypertrophy reverses at low but not high levels of α-adrenergic signaling: a reversibility delay. Perturbations with specialized αAR antagonists, a mathematical model, and live imaging of αAR localization identify the mechanism for this reversibility delay: ligand trapping with internalized PE acting on intracellular αAR's.Highlights: ► Automated image acquisition and analysis approach for cardiac myocyte hypertrophy. ► Tracks changes in measurements of individual myocytes over time. ► Reversibility of hypertrophy shown in a scalable cell culture model. ► Hypertrophy reverses at low and persists at high levels of α-adrenergic signaling. ► Delay in reversal explained by a ligand trapping model.

Syndecan-4 over-expression preserves cardiac function in a rat model of myocardial infarction - Uncorrected Proof

2012-05-07

Abstract: Syndecan-4 (synd4) is a heparan sulfate proteoglycan, involved in repair following tissue damage, through modulating neovascularization and inflammation. In acute myocardial infarction its myocardial expression is up-regulated in a time-dependent manner, and in synd4-deficient mice severe cardiac dysfunction and abnormal remodeling are observed following induction of myocardial infarction. Here we explored the therapeutic potential of sustained synd4 over-expression in the context of myocardial infarction. Adenovirus containing the synd4 gene (Ad-synd4), or corresponding control adenovirus (Ad-null), was administered intramyocardially in rats immediately after induction of myocardial infarction. Cardiac function was ascertained by echocardiography, hemodynamic assessment and brain natriuretic peptide level 28days post-intervention. Hearts were excised for molecular and histological analyses at predetermined time points. We observed reduced mortality and improved cardiac function post-myocardial infarction in the Ad-synd4 as compared to the Ad-null group, with associated attenuation of cardiac remodeling, less myocyte loss and reduced fibrosis. Additionally, the Ad-synd4 group exhibited endothelial cell activation and increased angiogenesis and arteriogenesis in the myocardium. The Ad-synd4 group also showed evidence of reduced myocardial inflammation as compared with the Ad-null group, with reduced inflammatory cell (CD45+) and myofibroblast (α-SMA+) infiltration as well as suppressed collagen III deposition and iNOS expression. Our results suggest that sustained synd4 over-expression in the myocardium is of therapeutic benefit following experimental myocardial infarction, through inducing neovascularization, suppressing tissue inflammation and fibrosis, with resultant improvements in cardiac function and remodeling.Highlights: ► We explored the therapeutic potential of synd4 in myocardial infarction. ► The synd4 induced endothelial cell activation and neovascularization in the myocardium. ► The synd4 suppressed myocardial inflammation and collagen deposition after infarction. ► The synd4 protected cardiac myocytes against apoptosis.

A novel player in cellular hypertrophy: Giβγ/PI3K-dependent activation of the RacGEF TIAM-1 is required for α1-adrenoceptor induced hypertrophy in neonatal rat cardiomyocytes - Uncorrected Proof

2012-05-07

Abstract: Activation of α1-adrenoceptors (α1-AR) by high catecholamine levels, e.g. in heart failure, is thought to be a driving force of cardiac hypertrophy. In this context several downstream mediators and cascades have been identified to potentially play a role in cardiomyocyte hypertrophy. One of these proteins is the monomeric G protein Rac1. However, until now it is unclear how this essential G protein is activated by α1-AR agonists and what are the downstream targets inducing cellular growth. By using protein-based as well as pharmacological inhibitors and the shRNA technique, we demonstrate that in neonatal rat cardiomyocytes (NRCM) Rac1 is activated via a cascade involving the α1A-AR subtype, Giβγ, the phosphoinositide-3′-kinase and the guanine nucleotide exchange factor Tiam1. We further demonstrate that this signaling induces an increase in protein synthesis, cell size and atrial natriuretic peptide expression. We identified the p21-activated kinase 2 (PAK2) as a downstream effector of Rac1 and were able to link this cascade to the activation of the pro-hypertrophic kinases ERK1/2 and p90RSK. Our data thus reveal a prominent role of the α1A-AR/Giβγ/Tiam1-mediated activation of Rac1 and its effector PAK2 in the induction of hypertrophy in NRCM.

Role of PI3Kα and sarcolemmal ATP-sensitive potassium channels in epoxyeicosatrienoic acid mediated cardioprotection - Uncorrected Proof

2012-04-30

Abstract: Aims: Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have known cardioprotective properties. While the mechanism(s) remains unknown, evidence suggests that phosphoinositide 3-kinase (PI3K) and sarcolemmal ATP-sensitive potassium channels (pmKATP) are important. However the role of specific PI3K isoforms and corresponding intracellular mechanisms remains unknown.Methods and results: To study this, mice hearts were perfused in Langendorff mode for 40min of baseline and subjected to 20 or 30min of global no-flow ischemia followed by 40min of reperfusion. C57BL6 mice perfused with 11,12-EET (1μM) had improved postischemic recovery, whereas co-perfusion with PI3Kα inhibitor, PI-103 (0.1μM), abolished the EET-mediated effect. In contrast, blocking of PI3Kβ or PI3Kγ isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmKATP) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmKATP currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia–reoxygenation triggered Ca2+ accumulation and maintains mitochondrial ΔΨm compared to controls. Both PI-103 and glibenclamide (10μM, pmKATP inhibitor) abolished EET cytoprotection.Conclusion: Together our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmKATP, which prevents Ca2+ overload and maintains mitochondrial function.Highlights: ► Involves activation of the PI3Kα isoform ► PI3Kα is upstream of ATP-sensitive K+(KATP) channel activation. ► Limits calcineurin activity and subsequent apoptotic events. ► Preserves mitochondrial function.

CIKS (Act1 or TRAF3IP2) mediates Angiotensin-II-induced Interleukin-18 expression, and Nox2-dependent cardiomyocyte hypertrophy - Uncorrected Proof

2012-04-30

Abstract: Chronic elevation of angiotensin (Ang)-II can lead to myocardial inflammation, hypertrophy and cardiac failure. The adaptor molecule CIKS (connection to IKK and SAPK/JNK) activates the IκB kinase/nuclear factor (NF)-κB and JNK/activator protein (AP)-1 pathways in autoimmune and inflammatory diseases. Since Ang-II is a potent activator of NF-κB and AP-1, we investigated whether CIKS is critical in Ang-II-mediated cardiac hypertrophy. Here we report that Ang-II induced CIKS mRNA and protein expression, CIKS binding to IKK and JNK perhaps functioning as a scaffold protein, CIKS-dependent IKK/NF-κB and JNK/AP-1 activation, p65 and c-Jun phosphorylation and nuclear translocation, NF-κB- and AP-1-dependent IL-18 and MMP-9 induction, and hypertrophy of adult cardiomyocytes isolated from WT, but not CIKS-null mice. These results were recapitulated in WT-cardiomyocytes following CIKS knockdown. Infusion of Ang-II for 7days induced cardiac hypertrophy, increased collagen content, and upregulated CIKS mRNA and protein expression in WT mice, whereas cardiac hypertrophy and collagen deposition were markedly attenuated in the CIKS-null mice, despite a similar increase in systolic blood pressure and DPI-inhibitable superoxide generation in both types of animals. Further, Ang-II-induced IKK/p65 and JNK/c-Jun phosphorylation, NF-κB and AP-1 activation, and IL-18 and MMP-9 expression were also markedly attenuated in CIKS-null mice. These results demonstrate that CIKS is critical in Ang-II-induced cardiomyocyte hypertrophy and fibrosis, and that CIKS is an important intermediate in Ang-II induced redox signaling. CIKS is a potential therapeutic target in cardiac hypertrophy, fibrosis, and congestive heart failure.Highlights: ► Ang-II induces cardiomyocyte hypertrophy and fibrosis via CIKS. ► Ang-II induces CIKS expression via AT1/Nox2-mediated ROS generation. ► Ang-II enhances CIKS binding to IKKβ and JNK. ► CIKS acts as a scaffold in IKKγ/JNK interaction. ► CIKS is a potential therapeutic target in cardiac hypertrophy and adverse remodeling.

A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells - Uncorrected Proof

2012-04-30

Abstract: The simultaneous overexpression of several transcription factors has emerged as a successful strategy to convert fibroblasts into other cell types including pluripotent cells, neurons, and cardiomyocytes. The selection and screening of factors are critical, and have often involved testing a large pool of transcription factors, followed by successive removal of single factors. Here, to identify a cardiac transcription factor combination facilitating mouse fibroblast reprogramming into cardiomyocytes, we directly screened all triplet combinations of 10 candidate factors combined with a Q-PCR assay reporting induction of multiple cardiac-specific genes. Through this screening method the combination of Tbx5, Mef2c, and Myocd was identified to upregulate a broader spectrum of cardiac genes compared to the combination of Tbx5, Mef2c, and Gata4 that was recently shown to induce reprogramming of fibroblasts into cardiomyocytes. Cells cotransduced with Tbx5, Mef2c, Myocd expressed cardiac contractile proteins, had cardiac-like potassium and sodium currents and action potentials could be elicited. In summary the alternative screening approach that is presented here avoided the elimination of transcription factors whose potency is masked in complex transcription factor mixes. Furthermore, our results point to the importance of verifying multiple lineage specific genes when assessing reprogramming.

Probenecid: Novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation - Corrected Proof

2012-04-30

Abstract: Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2−/− mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca2+ handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca2+.Highlights: ► We describe transient receptor potential vanilloid 2 (TRPV2) in the murine heart. ► We have identified a novel property of probenecid as a positive inotrope. ► Probenecid does not cause a positive inotropic response in TRPV2−/− mice. ► Probenecid increases myocyte cytosolic calcium concentration. ► Probenecid does not cause electrophysiologic changes in the murine heart or myocyte.

Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFβ1-induced apoptosis by stabilization of the mitochondrial permeability transition pore - Corrected Proof

2012-04-30

Abstract: Aims: Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin–angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ1-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected.Methods and results: Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ1 (1ng/ml). TGFβ1 treatment of WT cells enhanced the number of apoptotic cells by 31.8±11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ1‐induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ1 decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0±3.7% in WT cardiomyocytes, but only by 7.2±2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ1 in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ1 stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ1 treatment, SMAD signaling was still responsive to TGFβ1 indicated by an upregulation of SMAD7, a TGFβ1 target protein.Conclusion: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ1 by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.Highlights: ► TGFβ1 does not induce necrosis but apoptosis in treated cardiomyocytes. ► TGFβ1-induced apoptosis is associated with a decline in Δψm and MPTP opening. ► ANT1 overexpression attenuates TGFβ1-induced apoptosis and delays MPTP-opening in ANT1-transgenic cardiomyocytes. ► TGFβRII expression is downregulated and SMAD signaling affected but not restricted for TGFβ1 stimulation in ANT1-transgenic cells.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest - Uncorrected Proof

Thomas J. O'Neill, Christopher P. Mack, Joan M. Taylor — 2012-04-26

Abstract: The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK−/− but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium.Highlights: ► Focal adhesion kinase-related non-kinase (FRNK) limits perinatal cell cycling. ► Myocytes from FRNK-null mice exhibit sustained DNA synthesis and transient polyploidy. ► FRNK-null hearts exhibit hyperplastic growth.

Normalizing the metabolic phenotype after myocardial infarction: Impact of subchronic high fat feeding - Corrected Proof

2012-04-23

Abstract: The normal heart relies primarily on the oxidation of fatty acids (FA) for ATP production, whereas during heart failure (HF) glucose utilization increases, implying pathological changes to cardiac energy metabolism. Despite the noted lipotoxic effects of elevating FA, our work has demonstrated a cardioprotective effect of a high fat diet (SAT) when fed after myocardial infarction (MI), as compared to normal chow (NC) fed cohorts. This data has suggested a mechanistic link to energy metabolism. The goal of this study was to determine the impact of SAT on the metabolic phenotype of the heart after MI. Male Wistar rats underwent coronary ligation surgery (MI) and were evaluated after 8weeks of SAT. Induction of MI was verified by echocardiography. LV function assessed by in vivo hemodynamic measurements revealed improvements in the MI-SAT group as compared to MI-NC. Perfused working hearts revealed a decrease in cardiac work in MI-NC that was improved in MI-SAT. Glucose oxidation was increased and FA oxidation decreased in MI-NC compared to shams suggesting an alteration in the metabolic profile that was ameliorated by SAT. 31P NMR analysis of Langendorff perfused hearts revealed no differences in PCr:ATP indicating no overt energy deficit in MI groups. Phospho-PDH and PDK4 were increased in MI-SAT, consistent with a shift towards fatty acid oxidation (FAO). Overall, these results support the hypothesis that SAT post-infarction promotes a normal metabolic phenotype that may serve a cardioprotective role in the injured heart.Highlights: ► Previous work has shown high dietary fat (SAT) is cardioprotective post-infarction. ► Energy metabolism changes were evaluated as the potential cardioprotective mechanism. ► SAT increases fatty acid oxidation and reduces glucose oxidation post-MI. ► Pyruvate dehydrogenase regulation is also affected, distinct from SAT or MI alone. ► We conclude SAT after MI improves lipid metabolism to confer cardioprotection.

Extracellular cyclophilin A as a humoral factor modulating cardiovascular inflammatory responses - Uncorrected Proof

Fumiyuki Hattori — 2012-04-23

Peptidyl prolylisomerases (PPIase) catalyze the cis-trans isomerization of peptidyl-prolyl bonds . In spite of their simple molecular function, they comprise a large and mysterious protein family that elicits various physiological and pathophysiological functions . Cyclophilin A (CyPA) is a PPIase family member and a well-known target of the immunosuppressive drug cyclosporine A (CsA), which binds into the catalytic cavity of CyPA. The formed CyPA–CsA complex can then bind to calcineurin and inhibit its phosphatase activity, which eventually inhibits T-cell activation () .

Postnatal development of transmural gradients in expression of ion channels and Ca2+-handling proteins in the ventricle - Uncorrected Proof

2012-04-19

Abstract: Transmural gradients in myocyte action potential duration (APD) and Ca2+-handling proteins are argued to be important for both the normal functioning of the ventricle and arrhythmogenesis. In rabbit, the transmural gradient in APD (left ventricular wedge preparation) is minimal in the neonate. During postnatal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in the expression of ion channels and also Ca2+-handling proteins in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2–7days of age) and adult male (~6months of age) New Zealand White rabbits using quantitative PCR and also, when possible, in situ hybridisation and immunohistochemistry. In the adult, there were significant and substantial transmural gradients in Cav1.2, KChIP2, ERG, KvLQT1, Kir2.1, NCX1, SERCA2a and RyR2 at the mRNA and, in some cases, protein level—in every case the mRNA or protein was more abundant in the epicardium than the endocardium. Of the eight transmural gradients seen in the adult, only three were observed in the neonate and, in two of these cases, the gradients were smaller than those in the adult. However, in the neonate there were also transmural gradients not observed in the adult: in HCN4, Nav1.5, minK, Kir3.1 and Cx40 mRNAs — in every case the mRNA was more abundant in the endocardium than the epicardium. If the postnatal changes in ion channel mRNAs are used to predict changes in ionic conductances, mathematical modelling predicts the changes in APD observed experimentally. It is concluded that many of the well known transmural gradients in the ventricle develop postnatally.Highlights: ► In adult heart, transmural gradients in ventricle are crucial for arrhythmogenesis. ► Transmural gradients in neonatal and young adult rabbits were measured. ► In adult, there were gradients in 8 key ion channels and Ca2+-handling proteins. ► In neonate, only 3 of these were observed and 2 were smaller. ► Postnatal development of gradient in action potential duration is predicted.

Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1 - Corrected Proof

S. Meng, J.-T. Cao, B. Zhang, Q. Zhou, C.-X. Shen, C.-Q. Wang — 2012-04-18

Abstract: Diabetes mellitus (DM) adversely affects the number and function of circulating endothelial progenitor cells (EPCs). Consequently, there is also a reduction in the repair mechanism of these cells, which is a critical and initiating factor in the development of diabetic vascular disease. The aim of the present study was to analyze miR expression profiles in EPCs from patients with DM and choose the most significantly regulated miR to study its possible role on EPC dysfunction and elucidate its mechanism of action. EPCs were collected from subjects with Type II DM and non-diabetic control subjects. Total RNA was harvested from EPCs, and a total of 5 candidate miRNAs were identified by microarray screening and were quantified by TaqMan real-time PCR. Lentiviral vectors expressing miR-126 and miR-126 inhibitor (anti-miR-126) were transfected into EPCs, and the EPC colony-forming capacity, proliferation activity, migratory activity, differentiation capacity, and apoptotic susceptibility were determined and Western Blotting and mRNA real-time PCR analyses were performed. To study the mechanisms, lentiviral vectors expressing Spred-1 and a short interfering RNA (siRNA) targeting Spred-1 were prepared. Five miRs were aberrantly downregulated in EPCs from DM patients. These miRs included miR-126, miR-21, miR-27a, miR-27b and miR-130a. Anti-miR-126 inhibited EPC proliferation, migration, and enhanced apoptosis. Restored miR-126 expression in EPCs from DM promoted EPC proliferation, migration, and inhibited EPC apoptosis ability. Despite this, miR-126 had no effect on EPC differentiation. miR-126 overexpression significantly downregulated Spred-1 in EPCs. The knockdown of Spred-1 expression in EPCs from DM promoted proliferation, migration, and inhibited apoptosis of the cells. The signal pathway of miR-126 effecting on EPCs is partially mediated through Ras/ERK/VEGF and PI3K/Akt/eNOS regulation. This study provides the first evidence that miR-126 is downregulated in EPCs from diabetic patients, and impairs EPCs-mediated function via its target, Spred-1, and through Ras/ERK/VEGF and PI3K/Akt/eNOS signal pathway.

Treatment of the ventricular tachycardia with engraftment of pluripotent stem cells-derived cardiomyocytes - Corrected Proof

Jun Fujita, Motoaki Sano — 2012-04-16

Over the past few decades, advances in medical science have improved the prognosis of patients with cardiovascular diseases . However, the mortality rates for heart failure remain as high as those for advanced cancer . Patients with severe chronic heart failure due to ischemic cardiomyopathy or dilated cardiomyopathy are often resistant to conventional therapy, and cardiac transplantation is the only radical treatment. Unfortunately, only approximately 4,000 patients every year worldwide have the chance of cardiac transplantation . Thus, an innovative treatment substitute for cardiac transplantation is an important unmet medical need.

Conditioning the whole heart—not just the cardiomyocyte - Uncorrected Proof

Robert M. Bell, Derek M. Yellon — 2012-04-13

Abstract: Conditioning, the recruitment of endogenous cytoprotective pathways that protect the myocardium against injurious ischaemia/reperfusion injury, has developed into a range of modalities that can be applied before (preconditioning), during (perconditioning) or after the injurious ischaemic insult (postconditioning), either directly to the heart or in a distal tissue (remote preconditioning). A wide range of triggers, signaling pathways and potential end-effector mechanisms have been identified, which appear common to all forms of conditioning. Interestingly, conditioning applies to not only the cardiac myocyte, but to all the constitutive cell types within the myocardium. As our understanding of conditioning mechanisms continue to develop and we start to realise some of the difficulties in translating these phenomena to clinical treatments, it may be time to take a more integrative approach to conditioning, considering the many cellular and tissue types within the heart, and how they contribute to cytoprotective adaptations. In this review, we shall look at the conditioning phenomena, how different cell types contribute to the conditioned phenotype, and where novel cardioprotective modalities may be developed.Highlights: ► We review whether cardiac conditioning maybe protective beyond the cardiomyocyte. ► Autocrine and paracrine signalling are examined in the context of conditioning. ► Potential paracrine mechanisms are identified between myocardial cell compartments. ► Conditioning may be a complex interaction between myocardial cell populations. ► We propose cellular interaction be studied as part of future conditioning study.

Restoration of glucose metabolism in leptin-resistant mouse hearts after acute myocardial infarction through the activation of survival kinase pathways - Corrected Proof

William Witham, Keith Yester, Christopher P. O'Donnell, Kenneth R. McGaffin — 2012-04-09

Abstract: In the normal heart, leptin modulates cardiac metabolism. It is unknown, however, what effect leptin has on cardiac metabolism and outcomes in acute myocardial infarction (MI). This study was performed to test the hypothesis that leptin signaling increases glucose metabolism and attenuates injury in the acutely infarcted heart. Mice with (ObR+/+) and without (ObR−/−) cardiomyocyte specific expression of leptin receptor (ObR) were randomly assigned to experimental MI or sham procedure, and studied 3days later. ObR+/+ and ObR−/− sham mice were not significantly different in any measured outcome. However, after MI, ObR−/− mice had greater cardiac dysfunction, left ventricular dilation, and levels of oxidative stress. These worse indices of cardiac injury in ObR−/− mice were associated with attenuated signal transducer and activator of transcription (STAT) 3, phosphatidylinositol-3-kinase (PI3K), and Akt signaling, decreased malonyl CoA content, and reduced mitochondrial pyruvate dehydrogenase and electron transport Complex I, II and IV activities. Furthermore, ObR−/− mice maintained high rates of cardiac fatty acid oxidation after MI, whereas ObR+/+ mice demonstrated a switch away from fatty acid oxidation to glucose metabolism. Restoration of cardiac STAT3, PI3K and Akt activity and mitochondrial function in ObR−/− mice post-MI was accomplished by ciliary neurotrophic factor (CNTF), an established STAT3 activator, administered immediately after MI. Moreover, CNTF therapy resulted in mitigation of cardiac structural and functional injury, attenuated levels of oxidative stress, and rescued glucose metabolism in the infarcted ObR−/− heart. These data demonstrate that impaired cardiac leptin signaling results in metabolic inflexibility for glucose utilization in the face of cardiac stress, and greater morbidity after MI. Further, these studies show that cardiac glucose metabolism can be restored in leptin-resistant hearts by CNTF-mediated activation of survival kinases, resulting in multiple improved structural and functional outcomes post-MI.Highlights: ► Impaired cardiac leptin signaling exacerbates morbidity after myocardial infarction. ► Increased morbidity is linked to high rates of cardiac fatty acid oxidation. ► CNTF rescues cardiac survival kinase activity and glucose metabolism. ► CNTF is a potentially valuable therapy for myocardial infarction in obesity.

Mitochondrial aldehyde dehydrogenase 2 activation and cardioprotection - Corrected Proof

Dingxu Gong, Hao Zhang, Shengshou Hu — 2012-04-09

Abstract: Cardiac ischemia and reperfusion promote oxidative stress, leading to the accumulation of reactive aldehydes that cause cardiac damage. Mitochondrial aldehyde dehydrogenase 2 is emerging as a key cardioprotective enzyme for its central role in the detoxification of reactive aldehydes. Mitochondrial aldehyde dehydrogenase 2 activity strongly correlates to a better cardioprotective effect, and mitochondrial aldehyde dehydrogenase 2 can be activated by several pathways. After phosphorylation, the active mitochondrial aldehyde dehydrogenase 2 can reduce the build-up of aldehydes, inhibit autophagy, inhibit opening of the mitochondrial permeability transition pore, and prevent reperfusion arrhythmias. Therefore, mitochondrial aldehyde dehydrogenase 2 activation by small molecule activators suggests a promising new direction in cardiovascular research and the development of novel cardioprotective strategies. This review will discuss the cardioprotective effects of mitochondrial aldehyde dehydrogenase 2 activation in detail. This article is part of a Special Issue entitled ‘Focus on Cardiac Metabolism SI’.Highlights: ► PKCε activates ALDH2 and confers cardioprotection. ► ALDH2 overexpression antagonizes cardiac hypertrophy and failure. ► ALDH2 activation inhibits autophagy. ► ALDH2 activation reduces mitochondrial permeability. ► ALDH2 activation prevents arrhythmias.

Age-related divergent remodeling of the cardiac extracellular matrix in heart failure: Collagen accumulation in the young and loss in the aged - Corrected Proof

2012-04-05

Abstract: The incidence of heart failure (HF) increases with age. This study sought to determine whether aging exacerbates structural and functional remodeling of the myocardium in HF. HF was induced in young (~18months) and aged sheep (>8years) by right ventricular tachypacing. In non-paced animals, aging was associated with increased left ventricular (LV) end diastolic internal dimensions (EDID, P<0.001), reduced fractional shortening (P<0.01) and an increase in myocardial collagen content (P<0.01). HF increased EDID and reduced fractional shortening in both young and aged animals, although these changes were more pronounced in the aged (P<0.05). Age-associated differences in cardiac extracellular matrix (ECM) remodeling occurred in HF with collagen accumulation in young HF (P<0.001) and depletion in aged HF (P<0.05). MMP-2 activity increased in the aged control and young HF groups (P<0.05). Reduced tissue inhibitor of metalloproteinase (TIMP) expression (TIMPs 3 and 4, P<0.05) was present only in the aged HF group. Secreted protein acidic and rich in cysteine (SPARC) was increased in aged hearts compared to young controls (P<0.05) while serum procollagen type I C-pro peptide (PICP) was increased in both young failing (P<0.05) and aged failing (P<0.01) animals. In conclusion, collagen content of the cardiac ECM changes in both aging and HF although; whether collagen accumulation or depletion occurs depends on age. Changes in TIMP expression in aged failing hearts alongside augmented collagen synthesis in HF provide a potential mechanism for the age-dependent ECM remodeling. Aging should therefore be considered an important factor when elucidating cardiac disease mechanisms.Highlights: ► Extracellular matrix remodeling in an aged HF animal model remains unexplored. ► Gross functional and morphological LV remodeling is greatest in aged HF sheep. ► Collagen content is increased in young HF but decreased in aged HF. ► MMP activity and collagen fibrillogenesis increases in both young HF and aged HF. ► TIMP protein levels are diminished only in aged HF hearts.

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity - Corrected Proof

Nina D. Ullrich, Héctor H. Valdivia, Ernst Niggli — 2012-04-03

Abstract: During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation–contraction coupling and Ca2+ signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca2+ channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca2+ imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca2+ waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca2+ sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca2+ ([Ca2+]SR). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca2+ release synchronization, Ca2+ wave propagation and functional adaptation of RyR2 to variable [Ca2+]SR. These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca2+ sensing.Highlights: ► PKA-phosphorylation of RyR2-S2808 is involved in Ca2+ release synchronization. ► RyR2-S2808 phosphorylation promotes Ca2+ wave acceleration. ► SR luminal Ca2+ sensing is reduced in unphosphorylatable RyR2-S2808A channels.

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis - Corrected Proof

2012-03-19

Abstract: Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA−/− mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8days p.i. At 28days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.Highlights: ► In virus myocarditis treatment with a CyPA-inhibitor affects lesion area and collagen. ► Macrophage, T cell infiltration and MMP9 expression were reduced by CyPA inhibition. ► Similar effects are found using CyPA−/− mice in comparison to WT mice. ► CyPA may represent a target to modulate myocardial remodeling in myocarditis.

PICOT increases cardiac contractility by inhibiting PKCζ activity - Corrected Proof

2012-03-19

Abstract: Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα activity, which led to the elevation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKCζ activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility.Highlights: ►PICOT increases cardiac contractility through inhibition of PKCζ activity. ►PICOT-mediated inhibition of PKCζ down-regulates PKCα activity. ►PICOT-mediated inhibition of PKCζ activity down–regulates PP2A activity. ►PICOT exerts significant functional benefits in failing hearts.

Engraftment of human embryonic stem cell derived cardiomyocytes improves conduction in an arrhythmogenic in vitro model - Corrected Proof

2012-02-13

Abstract: In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275±36ms and conduction velocity (CV) of 10.6±4.2cm/s (n=3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4±0.9cm/s, n=35 vs. 30.1±3.2cm/s, n=20 in the longitudinal direction and 4.3±0.3cm/s vs. 9.3±0.9cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6±1.3cm/s and 3.1±0.5cm/s, n=11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.Highlights: ► We show electrophysiological benefits of cells from beating embryoid bodies (EBs). ► An in vitro model with abnormal conduction is used in which the cells are engrafted. ► Cells from beating EBs help to normalize conduction and reduce reentrant arrhythmia. ► Cells from non-beating EBs do not improve conduction. ► The mechanism for improvement appears to be via heterocellular electrical coupling.

WITHDRAWN: Novel approaches for gene-specific interference of HCN2 and HCN4 channels via manipulating actions of microRNAs - Corrected Proof

Jiening Xiao, Baofeng Yang, Huixian Lin, Yanjie Lu, Xiaobin Luo, Zhiguo Wang — 2009-10-12

This abstract has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause.The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy

WITHDRAWN: Is EPAC (exchange protein directly activated by cyclic AMP) involved in preconditioning? - Corrected Proof

E. Marais, A. Lochner — 2009-10-12