Search

Background: Transcriptional reconfiguration is central to heart failure, the most common cause of which is dilated cardiomyopathy (DCM). The effect of 3-dimensional chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. Methods: We generated a compendium of 3-dimensional epigenome and transcriptome maps from 101 biobanked human DCM and nonfailing heart tissues through highly integrative chromatin immunoprecipitation (H3K27ac [acetylation of lysine 27 on histone H3]), in situ high–throughput chromosome conformation capture, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin using sequencing, and RNA sequencing. We used human induced pluripotent stem cell–derived cardiomyocytes and mouse models to interrogate the key transcription factor implicated in 3-dimensional chromatin organization and transcriptional regulation in DCM pathogenesis. Results: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated in DCM development. For example, we identified that nontranscribing NPPA-AS1 (natriuretic peptide A antisense RNA 1) promoter functions as an enhancer and physically interacts with the NPPA (natriuretic peptide A) and NPPB (natriuretic peptide B) promoters, leading to the cotranscription of NPPA and NPPB in DCM hearts. We revealed that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (compartments, topologically associating domains) and their anchors unexpectedly had equivalent chromatin accessibility. We discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for HAND1 (heart and neural crest derivatives expressed 1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer–promoter connectome in DCM hearts, we performed comprehensive 3-dimensional epigenome mappings in human induced pluripotent stem cell–derived cardiomyocytes. We found that forced overexpression of HAND1 in human induced pluripotent stem cell–derived cardiomyocytes induced a distinct gain of enhancer–promoter connectivity and correspondingly increased the expression of their connected genes implicated in DCM pathogenesis, thus recapitulating the transcriptional signature in human DCM hearts. Electrophysiology analysis demonstrated that forced overexpression of HAND1 in human induced pluripotent stem cell–derived cardiomyocytes induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in dilated cardiac remodeling with impaired contractility/Ca 2+ handling in cardiomyocytes, increased ratio of heart weight/body weight, and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. Conclusions: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer–promoter connectome to drive DCM pathogenesis.

Introduction: Heat shock protein 20 (Hsp20) is the only small heat shock protein that can be phosphorylated by PKA upon β-adrenergic stimulation. Interestingly, phosphorylation of Hsp20 is highly elevated following I/R injury and in human heart failure. However, the functional significance of this phosphorylation in vivo is currently unclear. Results: Transgenic lines with cardiac overexpression of constitutively phosphorylated Hsp20 (S16D) were generated and we chose one with 10-fold overexpression to mimic the levels observed in human failing hearts. Longitudinal assessment of cardiac function via echocardiography indicated significantly reduced LVEF in S16D mice starting at 3 months of age which progressed to heart failure and early lethality (50% dead by six months). Histological examination revealed interstitial fibrosis as early as 2 months with no evidence of apoptosis. To elucidate the mechanisms underlying fibrosis in S16D hearts, we performed cardiomyocyte-fibroblast crosstalk experiments. Adult mouse cardiomyocytes were infected with GFP, S16D or S16A-Hsp20, cultured for 48 hours, and their supernatant was then used to culture adult mouse fibroblasts. S16D-cultured fibroblasts exhibited enhanced proliferation as well as pro-fibrotic signaling (increased expression of α-SMA, TGF-β and collagen as well as cytokines IL-6 & IL-1β), while GFP or S16A-cultured fibroblasts showed no alterations. At the sub-cellular level, Stat3 phosphorylation and nuclear translocation was increased in S16D-cultured fibroblasts. These pro-fibrotic effects were associated with increased expression and secretion of IL-6 from cardiomyocytes, which were inhibited by inclusion of an IL-6 neutralizing antibody to the S16D-myocyte supernatant upon fibroblast exposure. Consistent with our ex-vivo results, the cardiac levels of IL-6 as well as Stat3 activation were upregulated in S16D hearts at two months, concurrent with increased IL-6 levels in the serum. Conclusion: Phosphorylation of Hsp20 (P-Hsp20) in cardiomyocytes appears to contribute to fibrosis and ventricular remodeling via upregulation of IL-6. Thus, targeting the observed increases in failing hearts may hold therapeutic potential.

Background— Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied. Methods and Results— We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes. Conclusions— Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases.

Introduction: Exosomes, a group of nano-vesicles secreted from living cells, are documented to increase in the circulation and are believed to promote cardiac dysfunction in sepsis patients and animal models. However, whether inhibition of exosome release could exert a cardio-protective effect in polymicrobial sepsis remains unexplored. Methods and Results: C57BL/6 mice (male, 8-week old) were pre-treated with GW4869 (dissolved in DMSO, injection i.p. at a dose of 2.5μg/g body weight), a known inhibitor of exosome secretion. Same volume of DMSO was used as controls. One hour later, cecal ligation and puncture (CLP) surgery was performed to induce polymicrobial sepsis. We found that the concentration of serum exosomes, measured by membrane markers CD63 and CD81with detection ELISA kits, was increased by 3.5-fold in DMSO-CLP mice, but no increase was detected in GW4869-CLP mice or in sham operated mice (n=4-6, p Conclusions: Together, this study indicates that blockade of exosome secretion could attenuate the inflammatory cytokine response as well as the consequent cardiac dysfunction and mortality in polymicrobial sepsis. Thus, our study may provide a new approach to the treatment of sepsis.

Background— MicroRNAs (miRs) participate in many cardiac pathophysiological processes, including ischemia/reperfusion (I/R)-induced cardiac injury. Recently, we and others observed that miR-494 was downregulated in murine I/R-injured and human infarcted hearts. However, the functional consequence of miR-494 in response to I/R remains unknown. Methods and Results— We generated a mouse model with cardiac-specific overexpression of miR-494. Transgenic hearts and wild-type hearts from multiple lines were subjected to global no-flow I/R with the Langendorff system. Transgenic hearts exhibited improved recovery of contractile performance over the reperfusion period. This improvement was accompanied by remarkable decreases in both lactate dehydrogenase release and the extent of apoptosis in transgenic hearts compared with wild-type hearts. In addition, myocardial infarction size was significantly reduced in transgenic hearts on I/R in vivo compared with wild-type hearts. Similarly, short-term overexpression of miR-494 in cultured adult cardiomyocytes demonstrated an inhibition of caspase-3 activity and reduced cell death on simulated I/R. In vivo treatment with antisense oligonucleotide miR-494 increased I/R-triggered cardiac injury relative to the administration of mutant antisense oligonucleotide miR-494 and saline controls. We further identified that 3 proapoptotic proteins (PTEN, ROCK1, and CaMKIIδ) and 2 antiapoptotic proteins (FGFR2 and LIF) were authentic targets for miR-494. Importantly, the Akt-mitochondrial signaling pathway was activated in miR-494–overexpressing myocytes. Conclusions— Our findings suggest that although miR-494 targets both proapoptotic and antiapoptotic proteins, the ultimate consequence is activation of the Akt pathway, leading to cardioprotective effects against I/R-induced injury. Thus, miR-494 may constitute a new therapeutic agent for the treatment of ischemic heart disease.

Junctin (JCN) and triadin (TRD) share similar structures and they both function to anchor calsequestrin (CSQ) to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR) quaternary Ca-release complex. In failing human hearts, JCN and TRD protein levels are markedly decreased, implicating alterations in SR Ca-cycling and contractility. To address the role of combined JCN and TRD down-regulation in cardiac function, we generated and characterized a murine model deficient in both JCN and TRD. The double-knockout (DKO) mice presented lower body weight, poor fertility, and an apparent cardiac hypertrophy. In addition, deficiency of both JCN/TRD was associated with decreased peak Ca transient amplitude (34%), prolonged transient decay time (48%) and reduced caffeine-induced SR Ca-release. This depressed contractility was also confirmed at the intact organ (Langendorff perfusion) and whole animal (by echocardiography and catheterization) levels. Furthermore, examination of the properties of Ca sparks, which are informative of ryanodine receptor (RyR) gating , revealed increased frequency in the DKO myocytes, which indicated a larger opening probability of RyR. These findings suggest that the combined JCN/TRD-deficiency is associated with leaky RyR and depressed cardiac Ca-cycling, which may contribute to the progression of heart failure. This research has received full or partial funding support from the American Heart Association, AHA National Center. Table 1. Hemodynamic parameters from echocardiography and catheterization

Recent studies show that electro-stimulation from upper extremities or spinal cord elicits cardioprotective effects against I/R injury. In previous studies, we have identified a novel cardioprotective phenomenon that occurs subsequent to remote trauma (abdominal skin incision), termed RPCT. The purpose of this study was to determine whether an abdominal nociceptive stimulation (ANS), either by electrical or by chemical stimulation of remote peripheral sensory nerves along the same area of skin incision can produce cardioprotection similar to that of RPCT against MI. An in vivo mouse I/R model was used. ANS was performed 15 minutes prior to I/R or at the beginning of reperfusion with the needles placed along the line of abdominal incision. Infarct size was used to evaluate the results of I/R injury (45 min coronary occlusion, 24h reperfusion) with or without prior ANS treatments. Histological, immunochemical, cellular and functional studies were performed at appropriate timepoints. The results of demonstrate that both pre and post-conditioning produce an 85% reduction in infarct size (6.3 ± 2.2 vs. 52.6 ± 2.8 in preconditioning, P < 0.01; 10.6 ± 3.1 vs . 60.2 ± 3.8 in post-conditioning, P < 0.01, n = 6). Hematoxylin and Eosin staining showed that there was decreased infiltration after ANS compared to sham. We further show that ANS-preconditioning reduces apoptosis as evidenced by TUNEL staining and DNA fragmentation and reduces post-ischemic ventricular dysfunction. In conclusion, our results support 1) ANS is as protective as early phase of RPCT and 2) ANS elicited pre- and post-conditioning provides cardioprotection against myocardial infarction and dysfunction, at least in part, by reducing cardiomyocyte apoptosis after I/R. These results may suggest a novel therapeutic strategy for cardioprotection against I/R injury and have important implications for surgical patients at risk for myocardial infarction.

Background— Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown. Methods and Results— The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts. Conclusions— Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.

Background— Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against β-agonist–induced apoptosis. Methods and Results— To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region–to–risk region ratio was 8.1±1.1% in TG hearts (n=7), compared with 19.5±2.1% in WT hearts (n=11, P Conclusions— Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.