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Introduction DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Treatments for DCM in DMD are limited to steroids and standard heart failure medications such as β-blockers and ACE-inhibitors, and therefore novel therapeutic modalities are urgently needed. Purpose We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) were generated from healthy volunteer and 3 DMD patients: young male (YM), adult male (AM) and adult female (AF). We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of healthy and DMD iPSC-CMs using the Seahorse Flux analyzer, patch clamp, confocal fluorescence microscopy and Liquid chromatography mass spectrometry (LC-MS) technologies, respectively. Results To test the hypothesis, we measured respiration and glycolytic rates of healthy and DMD iPSC-CMs. Compared to healthy iPSC-CMs, in both AM and AF DMD, but not in YM DMD cardiomyocytes, there was a 75% decrease in ATP production, and 80% and 45% decrease in basal respiration, respectively. In agreement with the healthy-like bioenergetic status of YM, the iPSC-CMs showed no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. To determine whether the impairment in the phosphorylation pathway (OXPHOS) affects glycolysis, we measured the cardiomyocytes' response to glycolytic stress test. These experiments showed that the glycolytic rates were similar in healthy and DMD iPSC-CMs. In agreement with impaired OXPHOS, mitochondrial activity measured by 3D life confocal microscopy was attenuated in the DMD male by 35%, compared to healthy cardiomyocytes. Furthermore, the metabolomic LC-MS analyses demonstrated significant differences in metabolite levels in YM, AM and AF DMD iPSC-CMs relative to healthy iPSC-CMs. For example, compared to healthy iPSC-CMs, there was a dramatic fall to undetected levels in phosphocreatine in both AM and AF, but not in YM DMD, indicating a dysfunctional phosphocreatine energy system. Conclusions DMD iPSC-CMs exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): ISF - Israel Science Foundation

Introduction Familial hypertrophic cardiomyopathy (HCM) is caused by over 400 mutations affecting mostly key sarcomere components, such as titin and myosin. However, HCM also results from mutations in non-structural genes such as PRKAG2 which is involved directly in a variety of bioenergetic and metabolic pathways. When the metabolic processes fail to work properly or effectively, structural and functional aberrations resulting in cardiac dysfunction can occur. Thus, mutations in the human PRKAG2 gene lead to HCM, autosomal dominant ventricular pre-excitation - Wolff-Parkinson-White syndrome (WPW), a progressive conduction system disease and vacuolar glycogen accumulation in cardiomyocytes. Purpose To investigate the hypothesis that intervening with the bioenergetic and metabolic consequences of the R302Q mutation in the PRKAG2 gene causing inherited cardiomyopathy, will attenuate the cardiac impairments. Methods We generated mutated and isogenic induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) from a WPW patient carrying the R302Q mutation. As additional control, we used healthy volunteers' iPSC-CMs. Bioenergetic (Oxygen Consumption Rate, OCR) and metabolic status were measured using the Seahorse Flux Analyzer and LC-MS, respectively. To decipher the molecular basis underlying the bioenergetic and metabolic deficits, RNA-seq analysis was performed. Results The OCR results demonstrated in PRKAG2-mutated compared to isogenic and healthy iPSC-CMs, a 2-fold increase in maximal respiration rate and a 3.75-fold increase in spare respiratory capacity, while basal OCR parameters were similar in all groups. Importantly, when treated with the AMPK activator metformin (2.5 [mM]), all the abovementioned OCR parameters were similar in the three groups. RNA-seq analysis demonstrated that of the 553 differently expressed genes (DEGs), and of the 99 DEGs mutually differentially expressed, compared to isogenic and healthy cells, the most relevant altered pathways were glycolysis, carbon metabolism, biosynthesis of amino acids, HIF-1 signaling and fructose and mannose metabolism. These findings are consistent with the LC-MS results demonstrating in PRKAG2-mutated versus isogenic and healthy iPSC-CMs, at least a 3-fold decrease in metabolites linked to the abovementioned pathways: butyryl-carnitine, creatine, docosahexaenoic acid, GMP, IMP, myristoyl-carnitine, palmitoyl-carnitine, succinyl-Cys, UMP, UTP, UDP-GlcNAc. Conclusion PRKAG2-mutated iPSC-CMs exhibit bioenergetic and metabolic aberrations, which contribute to the cardiac pathological aspects of WPW syndrome. Importantly, treatment with the AMPK activator metformin eliminated the bioenergetic abnormalities in the mutated cells, while isogenic and healthy control cells remained unaffected. Based on these novel findings, a new therapeutic modality in WPW patients may be considered. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation (ISF)

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca2+-induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP3)-operated intracellular Ca2+ ([Ca2+]i) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP3 pathway, on [Ca2+]i transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10−9 to 10−7 M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10−9 to 10−7 M) increased the [Ca2+]i transient amplitude. The involvement of 1,4,5-IP3-dependent intracellular Ca2+ release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP3 receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 μM 2-aminoethoxyphenyl borate (a 1,4,5-IP3 receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP3-operated releasable Ca2+ stores. Disclosure of potential conflicts of interest is found at the end of this article.

Objective: Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas→phospholipase C (PLC)→1,4,5-inositol trisphosphate (1,4,5-IP3)→sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy. Methods: The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence. Results: Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122, xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002. Conclusions: Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3β pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.

Objectives : Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. Methods : Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O2) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. Results : After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a ∼20% decrease in conduction velocity compared to normoxic cultures. Conclusions : Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate.