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Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis., (© 2022. The Author(s).)

Aims: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM., Methods and Results: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy., Conclusion: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Normal cardiac contractile and relaxation functions are critically dependent on a continuous energy supply. Accordingly, metabolic perturbations and impaired mitochondrial bioenergetics with subsequent disruption of ATP production underpin a wide variety of cardiac diseases, including diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, anthracycline cardiomyopathy, peripartum cardiomyopathy, and mitochondrial cardiomyopathies. Crucially, there are no specific treatments for preventing the onset or progression of these cardiomyopathies to heart failure, one of the leading causes of death and disability worldwide. Therefore, new treatments are needed to target the metabolic disturbances and impaired mitochondrial bioenergetics underlying these cardiomyopathies in order to improve health outcomes in these patients. However, investigation of the underlying mechanisms and the identification of novel therapeutic targets have been hampered by the lack of appropriate animal disease models. Furthermore, interspecies variation precludes the use of animal models for studying certain disorders, whereas patient-derived primary cell lines have limited lifespan and availability. Fortunately, the discovery of human-induced pluripotent stem cells has provided a promising tool for modelling cardiomyopathies via human heart tissue in a dish. In this review article, we highlight the use of patient-derived iPSCs for studying the pathogenesis underlying cardiomyopathies associated with metabolic perturbations and impaired mitochondrial bioenergetics, as the ability of iPSCs for self-renewal and differentiation makes them an ideal platform for investigating disease pathogenesis in a controlled in vitro environment. Continuing progress will help elucidate novel mechanistic pathways, and discover novel therapies for preventing the onset and progression of heart failure, thereby advancing a new era of personalized therapeutics for improving health outcomes in patients with cardiomyopathy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Significance: Myeloperoxidase (MPO) is a heme peroxidase that is primarily expressed by neutrophils. It has the capacity to generate several reactive species, essential for its inherent antimicrobial activity and innate host defense. Dysregulated MPO release, however, can lead to tissue damage, as seen in several diseases. Increased MPO levels in circulation are therefore widely associated with conditions of increased oxidative stress and inflammation. Recent Advances: Several studies have shown a strong correlation between MPO and cardiovascular disease (CVD), through which elevated levels of circulating MPO are linked to poor prognosis with increased risk of CVD-related mortality. Accordingly, circulating MPO is considered a "high-risk" biomarker for patients with acute coronary syndrome, atherosclerosis, heart failure, hypertension, and stroke, thereby implicating MPO as a multifaceted target for cardiovascular protection. Consistently, recent studies that target MPO in animal models of CVD have demonstrated favorable outcomes with regard to disease progression. Critical Issues: Although most of these studies have established a critical link between circulating MPO and worsening cardiac outcomes, the mechanisms by which MPO exerts its detrimental effects in CVD remain unclear. Future Directions: Elucidating the mechanisms by which elevated MPO leads to poor prognosis and, conversely, investigating the beneficial effects of therapeutic MPO inhibition on alleviating disease phenotype will facilitate future MPO-targeted clinical trials for improving CVD-related outcomes.

Diabetic cardiomyopathy (DC) is defined as a clinical condition of cardiac dysfunction that occurs in the absence of coronary atherosclerosis, valvular disease, and hypertension in patients with diabetes mellitus (DM). Despite the increasing worldwide prevalence of DC, due to the global epidemic of DM, the underlying pathophysiology of DC has not been fully elucidated. In addition, the clinical criteria for diagnosing DC have not been established, and specific therapeutic options are not currently available. The current paradigm suggests the impaired cardiomyocyte function arises due to a number of DM-related metabolic disturbances including hyperglycemia, hyperinsulinemia, and hyperlipidemia, which lead to diastolic dysfunction and signs and symptoms of heart failure. Other factors, which have been implicated in the progression of DC, include mitochondrial dysfunction, increased oxidative stress, impaired calcium handling, inflammation, and cardiomyocyte apoptosis. Herein, we review the current theories surrounding the occurrence and progression of DC, and discuss the recent advances in diagnostic methodologies and therapeutic strategies. Moreover, apart from conventional animal DC models, we highlight alternative disease models for studying DC such as the use of patient-derived human induced pluripotent stem cells (hiPSCs) for studying the mechanisms underlying DC. The ability to obtain hiPSC-derived cardiomyocytes from DM patients with a DC phenotype could help identify novel therapeutic targets for preventing and delaying the progression of DC, and for improving clinical outcomes in DM patients., Competing Interests: Conflicts and disclosures All authors have no conflicts or disclosures.

Background : New treatments are needed to reduce myocardial infarct size (MI) and prevent heart failure (HF) following acute myocardial infarction (AMI), which are the leading causes of death and disability worldwide. Studies in rodent AMI models showed that genetic and pharmacological inhibition of mitochondrial fission, induced by acute ischemia and reperfusion, reduced MI size. Whether targeting mitochondrial fission at the onset of reperfusion is also cardioprotective in a clinically-relevant large animal AMI model remains to be determined. Methods : Adult pigs (30-40 kg) were subjected to closed-chest 90-min left anterior descending artery ischemia followed by 72 h of reperfusion and were randomized to receive an intracoronary bolus of either mdivi-1 (1.2 mg/kg, a small molecule inhibitor of the mitochondrial fission protein, Drp1) or vehicle control, 10-min prior to reperfusion. The left ventricular (LV) size and function were both assessed by transthoracic echocardiography prior to AMI and after 72 h of reperfusion. MI size and the area-at-risk (AAR) were determined using dual staining with Tetrazolium and Evans blue. Heart samples were collected for histological determination of fibrosis and for electron microscopic analysis of mitochondrial morphology. Results : A total of 14 pigs underwent the treatment protocols (eight control and six mdivi-1). Administration of mdivi-1 immediately prior to the onset of reperfusion did not reduce MI size (MI size as % of AAR: Control 49.2 ± 8.6 vs. mdivi-1 50.5 ± 11.4; p = 0.815) or preserve LV systolic function (LV ejection fraction %: Control 67.5 ± 0.4 vs. mdivi-1 59.6 ± 0.6; p = 0.420), when compared to vehicle control. Similarly, there were no differences in mitochondrial morphology or myocardial fibrosis between mdivi-1 and vehicle control groups. Conclusion : Our pilot study has shown that treatment with mdivi-1 (1.2 mg/kg) at the onset of reperfusion did not reduce MI size or preserve LV function in the clinically-relevant closed-chest pig AMI model. A larger study, testing different doses of mdivi-1 or using a more specific Drp1 inhibitor are required to confirm these findings.

Hypertrophic cardiomyopathy (HCM) is one of the most commonly inherited cardiac disorders that manifests with increased ventricular wall thickening, cardiomyocyte hypertrophy, disarrayed myofibers and interstitial fibrosis. The major pathophysiological features include, diastolic dysfunction, obstruction of the left ventricular outflow tract and cardiac arrhythmias. Mutations in genes that encode mostly for sarcomeric proteins have been associated with HCM but, despite the abundant research conducted to decipher the molecular mechanisms underlying the disease, it remains unclear as to how a primary defect in the sarcomere could lead to secondary phenotypes such as cellular hypertrophy. Mounting evidence suggests energy deficiency could be an important contributor of disease pathogenesis as well. Various animal models of HCM have been generated for gaining deeper insight into disease pathogenesis, however species variation between animals and humans, as well as the limited availability of human myocardial samples, has encouraged researchers to seek alternative 'humanized' models. Using induced pluripotent stem cells (iPSCs), human cardiomyocytes (CMs) have been generated from patients with HCM for investigating disease mechanisms. While these HCM-iPSC models demonstrate most of the phenotypic traits, it is important to ascertain if they recapitulate all pathophysiological features, especially that of energy deficiency. In this review we discuss the currently established HCM-iPSC models with emphasis on altered energetics.

Background: Preferential utilization of fatty acids for ATP production represents an advanced metabolic phenotype in developing cardiomyocytes. We investigated whether this phenotype could be attained in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and assessed its influence on mitochondrial morphology, bioenergetics, respiratory capacity and ultra-structural architecture., Methods and Results: Whole-cell proteome analysis of day 14 and day 30-CMs maintained in glucose media revealed a positive influence of extended culture on mitochondria-related processes that primed the day 30-CMs for fatty acid metabolism. Supplementing the day 30-CMs with palmitate/oleate (fatty acids) significantly enhanced mitochondrial remodeling, oxygen consumption rates and ATP production. Metabolomic analysis upon fatty acid supplementation revealed a β-oxidation fueled ATP elevation that coincided with presence of junctional complexes, intercalated discs, t-tubule-like structures and adult isoform of cardiac troponin T. In contrast, glucose-maintained day 30-CMs continued to harbor underdeveloped ultra-structural architecture and more subdued bioenergetics, constrained by suboptimal mitochondria development., Conclusion: The advanced metabolic phenotype of preferential fatty acid utilization was attained in hiPSC-CMs, whereby fatty acid driven β-oxidation sustained cardiac bioenergetics and respiratory capacity resulting in ultra-structural and functional characteristics similar to those of developmentally advanced cardiomyocytes. Better understanding of mitochondrial bioenergetics and ultra-structural adaptation associated with fatty acid metabolism has important implications in the study of cardiac physiology that are associated with late-onset mitochondrial and metabolic adaptations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Importance: The genetic variant MYBPC3Δ25bp occurs in 4% of South Asian descendants, with an estimated 100 million carriers worldwide. MYBPC3 Δ25bp has been linked to cardiomyopathy and heart failure. However, the high prevalence of MYBPC3Δ25bp suggests that other stressors act in concert with MYBPC3Δ25bp., Objective: To determine whether there are additional genetic factors that contribute to the cardiomyopathic expression of MYBPC3Δ25bp., Design, Setting, Andparticipants: South Asian individuals living in the United States were screened for MYBPC3Δ25bp, and a subgroup was clinically evaluated using electrocardiograms and echocardiograms at Loyola University, Chicago, Illinois, between January 2015 and July 2016., Main Outcomes and Measures: Next-generation sequencing of 174 cardiovascular disease genes was applied to identify additional modifying gene mutations and correlate genotype-phenotype parameters. Cardiomyocytes derived from human-induced pluripotent stem cells were established and examined to assess the role of MYBPC3Δ25bp., Results: In this genotype-phenotype study, individuals of South Asian descent living in the United States from both sexes (36.23% female) with a mean population age of 48.92 years (range, 18-84 years) were recruited. Genetic screening of 2401 US South Asian individuals found an MYBPC3Δ25bpcarrier frequency of 6%. A higher frequency of missense TTN variation was found in MYBPC3Δ25bp carriers compared with noncarriers, identifying distinct genetic backgrounds within the MYBPC3Δ25bp carrier group. Strikingly, 9.6% of MYBPC3Δ25bp carriers also had a novel MYBPC3 variant, D389V. Family studies documented D389V was in tandem on the same allele as MYBPC3Δ25bp, and D389V was only seen in the presence of MYBPC3Δ25bp. In contrast to MYBPC3Δ25bp, MYBPC3Δ25bp/D389V was associated with hyperdynamic left ventricular performance (mean [SEM] left ventricular ejection fraction, 66.7 [0.7%]; left ventricular fractional shortening, 36.6 [0.6%]; P < .03) and stem cell-derived cardiomyocytes exhibited cellular hypertrophy with abnormal Ca2+ transients., Conclusions and Relevance: MYBPC3Δ25bp/D389V is associated with hyperdynamic features, which are an early finding in hypertrophic cardiomyopathy and thought to reflect an unfavorable energetic state. These findings support that a subset of MYBPC3Δ25bp carriers, those with D389V, account for the increased risk attributed to MYBPC3Δ25bp.

Aims: Loss-of-function mutations in the hERG gene causes long-QT syndrome type 2 (LQT2), a condition associated with reduced IKr current. Four different mutation classes define the molecular mechanisms impairing hERG. Among them, Class 2 mutations determine hERG trafficking defects. Lumacaftor (LUM) is a drug acting on channel trafficking already successfully tested for cystic fibrosis and its safety profile is well known. We hypothesize that LUM might rescue also hERG trafficking defects in LQT2 and exert anti-arrhythmic effects., Methods and Results: From five LQT2 patients, we generated lines of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harbouring Class 1 and 2 mutations. The effects of LUM on corrected field potential durations (cFPD) and calcium-handling irregularities were verified by multi electrode array and by calcium transients imaging, respectively. Molecular analysis was performed to clarify the mechanism of action of LUM on hERG trafficking and calcium handling. Long-QT syndrome type 2 induced pluripotent stem cell-derived cardiomyocytes mimicked the clinical phenotypes and showed both prolonged cFPD (grossly equivalent to the QT interval) and increased arrhythmias. Lumacaftor significantly shortened cFPD in Class 2 iPSC-CMs by correcting the hERG trafficking defect. Furthermore, LUM seemed to act also on calcium handling by reducing RyR2S2808 phosphorylation in both Class 1 and 2 iPSC-CMs., Conclusion: Lumacaftor, a drug already in clinical use, can rescue the pathological phenotype of LQT2 iPSC-CMs, particularly those derived from Class 2 mutated patients. Our results suggest that the use of LUM in LQT2 patients not protected by β-blockers is feasible and may represent a novel therapeutic option.

Replacing cardiac tissues lost to myocardial infarction remains a therapeutic goal for regenerative therapy in recovering cardiac function. We assessed the feasibility of constructing a macrosized human cardiac tissue construct using pluripotent stem cell-derived cardiomyocytes or control fibroblasts infused fibrin/collagen hydrogel and performed ectopic implantation in peripheral vascular system of a porcine model for 3 weeks. Finally, an optimized vascularized cardiac construct was explanted and grafted onto porcine myocardium for 2 weeks. Myocardial-grafted human cardiac constructs showed a nascent tissue-like organization with aligned cardiomyocytes within the remodelled collagen matrix. Nevertheless, no significant changes in intraconstruct density of cardiomyocytes were observed in the myocardial-grafted constructs (human embryonic stem cell [hESC]-derived cardiomyocyte [n = 4]: 70.5 ± 22.8 troponin I + cardiomyocytes/high power field [HPF]) as compared to peripherally implanted constructs (hESC-derived cardiomyocyte [n = 4]: 59.0 ± 19.6 troponin I + cardiomyocytes/HPF; human induced pluripotent stem cell-derived cardiomyocyte [n = 3]: 50.9 ± 8.5 troponin I + cardiomyocytes/HPF, p = ns). However, the myocardial-grafted constructs showed an increased in neovascularization (194.4 ± 24.7 microvessels/mm 2 tissue, p < .05), microvascular maturation (82.8 ± 24.7 mature microvessels/mm 2 , p < .05), and tissue-like formation whereas the peripherally implanted constructs of hESC-derived cardiomyocyte (168.3 ± 98.2 microvessels/mm 2 tissue and 68.1 ± 33.4 mature microvessels/mm 2 ) and human induced pluripotent stem cell-derived cardiomyocyte (86.8 ± 57.4 microvessels/mm 2 tissue and 22.0 ± 32.7 mature microvessels/mm 2 ) were not significantly different in vascularized response when compared to the control human fibroblasts (n = 3) constructs (65.6 ± 34.1 microvessels/mm 2 tissue and 30.7 ± 20.7 mature microvessels/mm 2 ). We presented results on technical feasibility and challenges of grafting vascularized centimetre-sized human cardiac construct that may spur novel approaches in cardiac tissue replacement strategy., (Copyright © 2017 John Wiley & Sons, Ltd.)

Activation of signal transducer and activator of transcription 3 (STAT3) is imperative for mammalian development, specifically cardiogenesis. STAT3 phosphorylation and acetylation are key post-translational modifications that regulate its transcriptional activity. Significance of such modifications during human cardiogenesis remains elusive. Using human pluripotent stem cells to recapitulate cardiogenesis, two independently modified STAT3α (92 kDa) isoforms (phosphorylated and acetylated), which perform divergent functions were identified during cardiomyocyte (CM) formation. Phosphorylated STAT3α functioned as the canonical transcriptional activator, while acetylated STAT3α underwent caspase-3-mediated cleavage to generate a novel STAT3ζ fragment (∼45 kDa), which acted as a molecular adaptor integral to the ErbB4-p38γ signaling cascade in driving CM formation. While STAT3α knockdown perturbed cardiogenesis by eliminating both post-translationally modified STAT3α isoforms, caspase-3 knockdown specifically abrogates the function of acetylated STAT3α, resulting in limited STAT3ζ formation thereby preventing nuclear translocation of key cardiac transcription factor Nkx2-5 that disrupted CM formation. Our findings show the coexistence of two post-translationally modified STAT3α isoforms with distinct functions and define a new role for STAT3 as a molecular adaptor that functions independently of its canonical transcriptional activity during human cardiogenesis. Stem Cells 2017;35:2129-2137., (© 2017 AlphaMed Press.)

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Background: Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (I Kr ). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown., Methods and Results: We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG 1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG 1aUSO /ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN., Conclusion: Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The current study explored the Na + /K + -ATPase (NKA) inhibition-independent proarrhythmic mechanisms of cardiac glycosides (CGs) which are well-known NKA inhibitors. With the cytosolic Ca 2+ chelated by EGTA and BAPTA or extracellular Ca 2+ replaced by Ba 2+ , effects of bufadienolides (bufalin (BF) and cinobufagin (CBG)) and cardenolides (ouabain (Oua) and pecilocerin A (PEA)) on the L-type calcium current (I Ca,L ) were recorded in heterologous expression Cav1.2-CHO cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). BF and CBG demonstrated a concentration-dependent (0.1 to100 µM) I Ca,L inhibition (maximal ≥50%) without and with the NKA activity blocked by 10 µM Oua. BF significantly shortened the action potential duration at 1.0 µM and shortened the extracellular field potential duration at 0.01~1.0 µM. On the other hand, BF and CBG at 100 µM demonstrated a strong inhibition (≥40%) of the rapidly activating component of the delayed rectifier K + current (I Kr ) in heterologous expression HEK293 cells and prolonged the APD of the heart of day-3 Zebrafish larva with disrupted rhythmic contractions. Moreover, hESC-CMs treated with BF (10 nM) for 24 hours showed moderate yet significant prolongation in APD90. In conclusion, our data indicate that CGs particularly bufadienolides possess cytosolic [Ca 2+ ] i - and NKA inhibition- independent proarrhythmic potential through I Ca,L and I Kr inhibitions.

Glioblastoma is the most aggressive cancer of the brain. The dismal prognosis is largely attributed to the heterogeneous nature of the tumor, which in addition to intrinsic molecular and genetic changes is also influenced by the microenvironmental niche in which the glioma cells reside. The cancer stem cells (CSCs) hypothesis suggests that all cancers arise from CSCs that possess the ability to self-renew and initiate tumor formation. CSCs reside in specialized niches where interaction with the microenvironment regulates their stem cell behavior. The reciprocal interaction between glioma stem cells (GSCs) and cells from the microenvironment, such as endothelial cells, immune cells, and other parenchymal cells, may also promote angiogenesis, invasion, proliferation, and stemness of the GSCs and be likely to have an underappreciated role in their responsiveness to therapy. This crosstalk may also promote molecular transition of GSCs. Hence the inherent plasticity of GSCs can be seen as an adaptive response, changing according to the signaling cue from the niche. Given the association of GSCs with tumor recurrence and treatment sensitivity, understanding this bidirectional crosstalk between GSCs and its niche may provide a framework to identify more effective therapeutic targets and improve treatment outcome.

In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients' cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia-reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients' outcome., Competing Interests: HEB is a shareholder of CellAegis Inc.

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and Epidemal growth factor receptor (EGFR or ErbB1) in determining cardiac differentiation in vitro as these receptor tyrosine kinases are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation, cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment toward neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (>twofold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/noncanonical Wnt signaling determines cardiogenic fate in hPSCs. Stem Cells 2016;34:2461-2470., (© 2016 AlphaMed Press.)

Aims: Previous studies have associated issues such as incomplete stent apposition with delayed healing and adverse events (stent thrombosis). The aim of this study was to evaluate the impact of strut apposition and stent type on the progression of stent strut coverage., Methods and Results: We evaluated in vivo in porcine models the follow-up response and coverage characteristics of well-apposed and malapposed segments of drug-eluting stents (DES) (CYPHER, PROMUS Element and Orsiro) and the Absorb bioresorbable vascular scaffold (BVS) by optical coherence tomography (OCT) sequentially, at baseline, and at one week and four weeks of follow-up. Supporting results were provided by histological analysis performed at four-week follow-up and computer simulation describing the shear characteristics around apposed and non-apposed struts. A total of 325 cross-sections containing 3,166 struts were analysed. The extent of malapposition decreased over time as a result of neointimal healing (from 7.1% at baseline to 0% at four weeks; p=0.03). At one week, 13.6% of struts in well-apposed segments were still uncovered versus 19.2% of struts in malapposed cross-sections and 77.8% of NASB struts (p<0.01). At four-week follow-up, 3.1% of struts were uncovered in well-apposed cross-sections vs. 1.6% in malapposed cross-sections and 35.7% of NASB struts (p<0.01). A comparison of the apposed segments revealed that the thin-strut Orsiro had only 1.3% of uncovered struts at one week while PROMUS Element, CYPHER and BVS had 6.6%, 48.4% and 16.2% of struts still uncovered, respectively., Conclusions: This study shows that early coverage is influenced by stent apposition as well as platform strut characteristics (stent type). At four weeks, NASB struts remained a focus of delayed endothelialisation.

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.

Activation of ErbB4 receptor signaling is instrumental in heart development, lack of which results in embryonic lethality. However, mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells, we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38γ MAPK isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38γ/NKx2.5 downregulation is followed by p38α/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38γ MAPK similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38γ signaling hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38γ to the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development., (© 2015 AlphaMed Press.)

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors, cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 μl, P < 0.05) and cardiomyocyte (19.52 ± 3.97 μl, P < 0.05) groups, but not in progenitor group (25.65 ± 3.61 μl), significantly deteriorated as compared to sham control group (28.41 ± 4.41 μl). Consistently, pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 μl, P < 0.05; ESV: 17.08 ± 5.82 μl, P < 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 μl, P < 0.05; ESV: 18.03 ± 6.58 μl, P < 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 μl; ESV: 8.41 ± 2.94 μl). In contrast, cardiac progenitors (EDV: 20.09 ± 7.76 μl; ESV: 13.98 ± 6.74 μl) persistently protected chamber geometry against negative cardiac remodelling. Similarly, as compared to sham control (54.64 ± 11.37%), LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%, P < 0.05) and saline (35.34 ± 11.86%, P < 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue, P < 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Efficient generation of cardiomyocytes from pluripotent stem cells (PSCs) for multiple downstream applications such as regenerative medicine, disease modeling, and drug screening remains a challenge. Cardiomyogenesis may be regulated in vitro by a controlled differentiation process, which involves various signaling molecules and extracellular environment. Here, we describe a simple method to efficiently generate cardiomyocytes from human embryonic stem cells and human induced pluripotent stem cells.

The heart is an electrically conducting organ with networked bioelectric currents that transverse a large segment of interstitial space interspersed with the muscular parenchyma. Non-excitable connective cells in the interstitial space contributed importantly to many structural, biochemical, and physiological activities of cardiac homeostasis. However, contribution of interstitial cells in the cardiac niche has long been neglected. Telocyte is recently recognized as a distinct class of interstitial cell that resides in a wide array of tissues including in the epicardium, myocardium, and endocardium of the heart. They are increasingly described to conduct ionic currents that may have significant implications in bioelectric signaling. In this review, we highlight the significance of telocytes in such connectivity and conductivity within the interstitial bioelectric network in tissue homeostasis.

Introduction: Type 1 long QT syndrome (LQT1) is a common type of cardiac channelopathy associated with loss-of-function mutations of KCNQ1. Currently there is a lack of drugs that target the defected slowly activating delayed rectifier potassium channel (IKs). With LQT1 patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs), we tested the effects of a selective IKs activator ML277 on reversing the disease phenotypes., Methods: A LQT1 family with a novel heterozygous exon 7 deletion in the KCNQ1 gene was identified. Dermal fibroblasts from the proband and her healthy father were reprogrammed to hiPSCs and subsequently differentiated into hiPSC-CMs., Results: Compared with the control, LQT1 patient hiPSC-CMs showed reduced levels of wild type KCNQ1 mRNA accompanied by multiple exon skipping mRNAs and a ~50% reduction of the full length Kv7.1 protein. Patient hiPSC-CMs showed reduced IKs current (tail current density at 30 mV: 0.33±0.02 vs. 0.92±0.21, P<0.05) and prolonged action potential duration (APD) (APD 50 and APD90: 603.9±39.2 vs. 319.3±13.8 ms, P<0.005; and 671.0±41.1 vs. 372.9±14.2 ms, P<0.005). ML277, a small molecule recently identified to selectively activate KV7.1, reversed the decreased IKs and partially restored APDs in patient hiPSC-CMs., Conclusions: From a LQT1 patient carrying a novel heterozygous exon7 deletion mutation of KCNQ1, we generated hiPSC-CMs that faithfully recapitulated the LQT1 phenotypes that are likely associated with haploinsufficiency and trafficking defect of KCNQ1/Kv7.1. The small molecule ML277 restored IKs function in hiPSC-CMs and could have therapeutic value for LQT1 patients.

Cardiomyocytes isolated from chick and rodent are widely used in studying cardiac physiology. However, contaminating non-cardiomyocytes are an inherent problem that hinders downstream analysis. Here, we report a novel electrical stimulation coupled with metabolic selection method using cytosine arabinoside (AraC) to efficiently eliminate contaminating cells in isolating chick embryonic cardiomyocytes. Compared with conventional methods of pre-plating or AraC alone, electrical stimulation coupled with AraC increased the percentage purity of cardiomyocytes by 2-6-fold with added effect of improved contractile function and maturation. This simple method could be useful in isolating and maintaining purified cardiomyocytes for long-term studies of cardiac physiology.

High-resolution optical imaging provides valuable window in examining transitional and systemic changes in cellular processes. The relative spatial relationship of structural, transport, and signaling proteins, surface antigens, and transcription factors may reveal developmental state of the cellular system. Here, we describe the use of confocal microscopy to evaluate the organization of sarcomeric structural proteins, sarcoplasmic channel proteins, and cardiac transcription factors in human pluripotent stem cell (PSC)-derived cardiomyocytes.

Background: Speckle tracking echocardiography (STE) using dedicated high-resolution ultrasound is a relatively new technique that is useful in assessing myocardial deformation in 3 myocardial layers in small animals. However, comparative studies of STE parameters acquired from murine are limited., Methods: A high-resolution rodent ultrasound machine (VSI Vevo 2100) and a clinically validated ultrasound machine (GE Vivid 7) were used to consecutively acquire echocardiography images from standardized parasternal long axis and short axis at midpapillary muscle level from 13 BALB/c mice. Speckle tracking strain (longitudinal, circumferential, and radial) from endocardial, myocardial, and epicardial layers was analyzed using vendor-specific offline analysis software., Results: Intersystem differences were not statistically significant in the global peak longitudinal strain (-16.8 ± 1.7% versus -18.7 ± 3.1%) and radial strain (46.8 ± 14.2% versus 41.0 ± 9.5%), except in the global peak circumferential strain (-16.9 ± 3.1% versus 27.0 ± 5.2%, P < 0.05). This was corroborated by Bland Altman analysis that revealed a weak agreement in circumferential strain (mean bias ± 1.96 SD of -10.12 ± 6.06%) between endocardium and midmyocardium. However, a good agreement was observed in longitudinal strain between midmyocardium/endocardium (mean bias ± 1.96 SD of -1.88 ± 3.93%) and between midmyocardium/epicardium (mean bias ± 1.96 SD of 3.63 ± 3.91%). Radial strain (mean bias ± 1.96 SD of -5.84 ± 17.70%) had wide limits of agreement between the two systems that indicated an increased variability., Conclusions: Our study shows that there is good reproducibility and agreement in longitudinal deformation of the 3 myocardial layers between the two ultrasound systems. Directional deformation gradients at endocardium, myocardium, and epicardium observed in mice were consistent to those reported in human subjects, thus attesting the clinical relevance of STE findings in murine cardiovascular disease models.

Marfan syndrome (MFS) is a genetic disorder that affects multiple organs. Mortality imposed by aortic aneurysm and dissections represent the most serious clinical manifestation of MFS. Progressive pathological aortic root enlargement as the result of degeneration of microfibril architecture and consequential loss of extracellular matrix integrity due to fibrillin-1 (FBN1) mutations are commonly diagnosed clinical manifestations of MFS. However, overlapping clinical manifestations with other aneurysmal disorders present a significant challenge in early and accurate diagnosis of MFS. While FBN1 mutations, abnormal transforming growth factor-β signaling and dysregulated matrix metalloproteinases have been implicated in MFS, clinically accepted risk-stratifying biomarkers have yet to be reliably identified. In this review, we summarize current consensus and recent insights in the understanding of MFS pathogenesis. Finally, we introduce the application of induced pluripotent stem cells (iPSCs) as cellular models for MFS and its potential as a novel platform into providing better appreciation of mechanisms underlying MFS diverse manifestations in the cardiovascular system., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Introduction: Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)., Methods and Results: The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine., Conclusions: Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5(+) (up to 98%) populations. These Nkx2.5(+) populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: The induced pluripotent stem cells (iPSCs) have characteristics similar to embryonic stem cells, including the capability of self-renewal and large-scale expansion and the ability to differentiate into all types of cells including germ cells, which defines pluripotency. Using iPSC avoids problems of immunological rejection and ethical controversy. The possible future uses of iPSC are diverse and go beyond the differentiation into somatic cells for regeneration of damaged tissues., Areas Covered: A unique feature of iPSC is the potential to generate patient disease-specific tissues. Thus, cells from patients can be differentiated into relevant cells of interest for drug screening, characterization of drug effects and cytotoxic assays. This review presents key aspects related to iPSC, such as their generation, potential for disease modeling, treatment, drug development and future contributions to the craniofacial complex., Expert Opinion: It is undisputable that the evolution in iPSC knowledge will improve the approaches for drug screening and development, help to understand and treat disease origins and mechanisms and provide new strategies to clinical treatment. However, it is necessary to fine-tune protocols to establish iPSCs that are cost-effective and safe for clinical use.

We investigated global and regional effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) in infarcted myocardium. Acute myocardial infarction (MI) was induced by ligation of left coronary artery of severe combined immunodeficient mice before 2 × 10(5) iMSCs or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Global and regional myocardial function was assessed serially at 1-week and 8-week by segmental strain analysis by using two dimensional (2D) speckle tracking echocardiography. Early myocardial remodelling was observed at 1-week and persisted to 8-week with global contractility of ejection fraction and fractional area change in saline- (32.96 ± 14.23%; 21.50 ± 10.07%) and iMSC-injected (32.95 ± 10.31%; 21.00 ± 7.11%) groups significantly depressed as compared to sham control (51.17 ± 11.69%, P < 0.05; 34.86 ± 9.82%, P < 0.05). However, myocardial dilatation was observed in saline-injected animals (4.40 ± 0.62 mm, P < 0.05), but not iMSCs (4.29 ± 0.57 mm), when compared to sham control (3.74 ± 0.32 mm). Furthermore, strain analysis showed significant improved basal anterior wall strain (28.86 ± 8.16%, P < 0.05) in the iMSC group, but not saline-injected (15.81 ± 13.92%), when compared to sham control (22.18 ± 4.13%). This was corroborated by multi-segments deterioration of radial strain only in saline-injected (21.50 ± 5.31%, P < 0.05), but not iMSC (25.67 ± 12.53%), when compared to sham control (34.88 ± 5.77%). Improvements of the myocardial strain coincided with the presence of interconnecting telocytes in interstitial space of the infarcted anterior segment of the heart. Our results show that localized injection of iMSCs alleviates ventricular remodelling, sustains global and regional myocardial strain by paracrine-driven effect on neoangiogenesis and myocardial deformation/compliance via parenchymal and interstitial cell interactions in the infarcted myocardium., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by "foot-print free" reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

Aims: Long QT syndrome 2 (LQTS2) caused by missense mutations in hERG channel is clinically associated with abnormally prolonged ventricular repolarization and sudden cardiac deaths. Modelling monogenic arrhythmogenic diseases using human-induced pluripotent stem cells (hiPSCs) offers unprecedented mechanistic insights into disease pathogenesis. We utilized LQTS2-hiPSC-derived cardiomyocytes (CMs) to elucidate pathological changes and to demonstrate reversal of LQTS2 phenotype in a therapeutic intervention using a pharmacological agent, (N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine) (ALLN)., Methods and Results: We generated LQTS2-specific CMs (A561V missense mutation in KCNH2) from iPSCs using the virus-free reprogramming method. These CMs recapitulate dysfunction of hERG potassium channel with diminished IKr currents, prolonged repolarization durations, and elevated arrhythmogenesis due to reduced membrane localization of glycosylated/mature hERG. Dysregulated expression of folding chaperones and processing proteasomes coupled with sequestered hERG in the endoplasmic reticulum confirmed trafficking-induced disease manifestation. Treatment with ALLN, not only increased membrane localization of mature hERG but also reduced repolarization, increased IKr currents and reduced arrhythmogenic events. Diverged from biophysical interference of hERG channel, our results show that modulation of chaperone proteins could be therapeutic in LQTS2 treatment., Conclusion: Our in vitro study shows an alternative approach to rescue diseased LQTS2 phenotype via corrective re-trafficking therapy using a small chemical molecule, such as ALLN. This potentially novel approach may have ramifications in other clinically relevant trafficking disorders., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber-specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole-cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c-Kit and PDGFR-β were identified. These cells expressed large conductance Ca²⁺-activated K⁺ current (BK(Ca)) and inwardly rectifying K⁺ current (IK(ir)), but not transient outward K⁺ current (I(to)) and ATP-sensitive potassium current (K(ATP)). The active channels were functionally competent with demonstrated modulatory response to H2 S and transforming growth factor (TGF)-β1 whereby H₂S significantly inhibited the stimulatory effect of TGF-β1 on current density of both BKCa and IK(ir). Furthermore, H₂S attenuated TGF-β1-stimulated KCa1.1/Kv1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)) expression in TCs. Our results show that functionally competent K⁺ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Hydrogen sulfide (H2S) is an endogenously generated gaseous transmitter known for its cytoprotective effect mediated by the PI3K-Akt signaling pathway. Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stromal cells (MSCs), or hiPSC-MSCs, represent an alternative source of MSCs for autologous cell therapy. The big-conductance Ca(2+)-activated outward K(+) currents (BKCa), known to mediate cell proliferation, have been detected in >80% of hiPSC-MSCs. The present study aimed to explore the effect of H2S on survival and proliferation of hiPSC-MSCs and investigate the mediatory role of BKCa., Methods: Effects of H2S on proliferation and survival of hiPSC-MSCs were measured by 5-bromo-2-deoxyuridine incorporation, population doubling and cell cycle assays, and by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay and 4'-6-diamidino-2-phenylindole staining, respectively. BKCa was recorded by means of the whole-cell patch-clamp technique. The expressions of KCa 1.1 (encoding BKCa) and apoptosis-related genes were measured by reverse transcriptase-polymerase chain reaction. The phosphorylation of Akt was assessed by Western blot analysis., Results: Exogenously administered NaHS (an H2S donor, 50-300 μmol/L) significantly promoted proliferation of hiPSC-MSCs. NaHS prevented the hypoxia-induced apoptosis and suppressed BKCa currents without altering the expression levels of α- and β-KCa 1.1. In addition, NaHS increased the phosphorylation of Akt and decreased the expression of Caspase 8 and Bax in hiPSC-MSCs. Paxilline (1 μmol/L), a BKCa blocker, showed similar effects on promoting cell proliferation and phosphorylation of Akt and suppression of apoptotic genes in hiPSC-MSCs., Conclusions: Our data confirmed that H2S arguments the proliferation and survival of hiPSC-MSCs through activation of the PI3K-Akt pathway and that such effects could be mediated through inhibition of BKCa., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Type 3 long QT syndrome (LQT3) is the third most common form of LQT syndrome and is characterized by QT-interval prolongation resulting from a gain-of-function mutation in SCN5A. We aimed to establish a patient-specific human induced pluripotent stem cell (hiPSC) model of LQT3, which could be used for future drug testing and development of novel treatments for this inherited disorder., Methods and Results: Dermal fibroblasts obtained from a patient with LQT3 harboring a SCN5A mutation (c.5287G>A; p.V1763M) were reprogrammed to hiPSCs via repeated transfection of mRNA encoding OCT-4, SOX-2, KLF-4, C-MYC and LIN-28. hiPSC-derived cardiomyocytes (hiPSC-CMs) were obtained via cardiac differentiation. hiPSC-CMs derived from the patient's healthy sister were used as a control. Compared to the control, patient hiPSC-CMs exhibited dominant mutant SCN5A allele gene expression, significantly prolonged action potential duration or APD (paced CMs of control vs. patient: 226.50 ± 17.89 ms vs. 536.59 ± 37.1 ms; mean ± SEM, p < 0.005), an increased tetrodotoxin (TTX)-sensitive late or persistent Na(+) current (control vs. patient: 0.65 ± 0.11 vs. 3.16 ± 0.27 pA/pF; n = 9, p < 0.01), a positive shift of steady state inactivation and a faster recovery from inactivation. Mexiletine, a NaV1.5 blocker, reversed the elevated late Na(+) current and prolonged APD in LQT3 hiPSC-CMs., Conclusions: We demonstrate that hiPSC-CMs derived from a LQT3 patient recapitulate the biophysical abnormalities that define LQT3. The clinical significance of such an in vitro model is in the development of novel therapeutic strategies and a more personalized approach in testing drugs on patients with LQT3., (© 2013.)

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca(2+)-activated K(+) current (BK(Ca)), transient outward K(+) current (I(to)) and inwardly rectifying K(+) current (IK(ir)) were found in human atrial fibroblasts. Current density of BK(Ca) (IC50 = 69.4 μM; n = 6), I(to) (IC50 = 55.1 μM; n = 6) and IK(ir) (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100-500 μM) inhibited fibroblast proliferation induced by transforming growth factor-β1 (TGF-β1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode I(to)), but not KCa1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)). Furthermore, H2S significantly attenuated TGF-β1-stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-β-induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K(+) channel activity and moderates their differentiation towards myofibroblasts., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Cellular morphogenesis in response to biophysical and topographical cues provides insights into cytoskeletal status, biointerface communications, and phenotypic adaptations in an incessant signaling feedback that governs cellular fate. Morphometric characterization is an important element in the study of the dynamic cellular behaviors, in their interactive response to environmental influence exerted by culture system. They collectively serve to reflect cellular proliferation, migration, and differentiation, which may serve as prognostic indices for clinical and pathological diagnosis. Various parameters are proposed to categorize morphological adaptations in relation to cellular function. In this review, the underlying principles, assumptions, and limitations of morphological characterizations are discussed. The significance, challenges, and implications of quantitative morphometric characterization of cell shapes and sizes in determining cellular functions are discussed., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vascular homeostasis is an integral reflection of quiescent, but competent endothelium. Endothelial microparticles (EMPs) are circulating submicron-sized membranous vesicles released by endothelium that are increasingly recognized and play a multitude of biological roles reflecting competency of endothelial function and vascular biology. EMPs have been shown to act as primary and secondary messengers of vascular inflammation, thrombosis, vasomotor response, angiogenesis, and endothelial survival. These often-neglected vesicles are emerging as potentially useful indicators of dysfunctioning endothelium, whereby differential presence of EMPs is linked to disease manifestation. In this review, we outline the evolving understanding of EMPs from mere passive vesiculation to active messenger in pathophysiological responses. Current evidence implicating EMPs in cardiopulmonary, renal, cerebral, and metabolic disorders warrants vigorous clinical validation. While studies of EMPs face significant technical challenges that await standardization and consolidation, unravelling its biological significance in endothelial dysfunction could herald the advent of novel non-invasive surveillance platforms for vascular health, risk stratification, and disease prognostication.

In humans injured myocardium cannot avert the onset and progression of ventricular dysfunction because of limited regenerative ability of myocytes. Although limited renaissance of cardiomyocytes has been reported in human infarcted hearts, it is generally accredited that non-functional fibrous tissue replaces the dead myocardium. High cardiovascular morbidity and dearth of donor hearts warrant a constant hunt for radically different approach to treat heart failure. Pluripotent stem (PS) cells possess the ability to produce functional cardiomyocytes for clinical applications and drug development, which may provide the answer to this problem. Although progress has been made in differentiating human PS cells into cardiomyocytes, however, the in vitro differentiation of pluripotent cells into cardiomyocytes involves a poorly defined, inefficient and relatively non-selective process. A thorough understanding of signaling pathways would tender a roadmap for the streamlined development of in vitro cardiac differentiation strategies. The ability to obtain unlimited numbers of human cardiomyocytes would improve development of cell-based therapies for cardiovascular diseases, facilitate the study of cardiovascular biology and improve the early stages of drug discovery. Here in this review, we highlight the interacting endogenous cellular signals and their modulators involved in directing the human PSCs towards cardiac differentiation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Despite optimal medical therapy, cardiovascular disease remains a leading cause of morbidity and mortality worldwide. One emerging therapeutic approach for treatment of cardiomyopathies is stem cell therapy. Use of stem cells for regenerative medicine has quickly evolved over the last decade. On the basis of encouraging pre-clinical results, stem cell therapy has developed rapidly into a potentially promising treatment for ischemic heart disease, myocardial infarction and congestive heart failure. In this review, we summarize the current state-of-the-art of stem cell therapy and compare collective experiences gleaned from trials using intravenous, intra-coronary and intra-myocardial delivery in exacting credible benefits. We discuss implications of clinical outcomes reported in relation to the delivered stem cells as probable destiny therapy for cardiovascular repair., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder associated with sudden cardiac death. Its pathophysiology is still poorly understood. We aimed to produce an in vitro cellular model of ARVC using patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and determine whether the model could recapitulate key features of the disease phenotype., Methods and Results: Dermal fibroblasts were obtained from a 30-year-old man with a clinical diagnosis of ARVC, harbouring a plakophilin 2 (PKP2) gene mutation. Four stable iPSC lines were generated using retroviral reprogramming, and functional cardiomyocytes were derived. Gene expression levels of desmosomal proteins (PKP2 and plakoglobin) in cardiomyocytes from ARVC-iPSCs were significantly lower compared with cardiomyocytes from control iPSCs (P< 0.01); there were no significant differences in the expression of desmoplakin, N-cadherin, and connexin 43 between the two groups. Cardiomyocytes derived from ARVC-iPSCs exhibited markedly reduced immunofluorescence signals when stained for PKP2 and plakoglobin, but similar levels of staining for desmoplakin, N-cadherin, and connexin 43 compared with control cardiomyocytes. Transmission electron microscopy showed that ARVC-iPSC cardiomyocytes were larger and contained darker lipid droplets compared with control cardiomyocytes. After 2 weeks of cell exposure to adiopgenic differentiation medium, ARVC-iPSC cardiomyocytes were found to contain a significantly greater amount of lipid, calculated using Oil Red O staining, compared with controls (734 ± 35.6 vs. 8.1 ± 0.49 a.u., respectively; n = 7, P = 0.001)., Conclusion: Patient-specific iPSC-derived cardiomyocytes display key features of ARVC, including reduced cell surface localization of desmosomal proteins and a more adipogenic phenotype.

Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study, we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide), class III (sotalol and amiodarone), and class IV (verapamil), whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability, class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs, verapamil shortened cFPD and FPmin by 25 and 50%, respectively. Furthermore, verapamil reduced beating frequencies drastically. Importantly, the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes, drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine, flecainide, and sotalol was demonstrated at supraphysiological concentrations. Furthermore, off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine.

Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study, we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs., Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR., Key Findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers, generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers, followed by early cardiac committed markers, structural and ion channel genes. Furthermore, our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin, actinin, myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally, microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol, reduced Na(+) influx with quinidine, prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil., Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes., Methods and Results: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks., Conclusions: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

Cardiac stem cell therapy to promote engraftment of de novo beating cardiac muscle cells in cardiomyopathies could potentially improve clinical outcomes for many patients with congestive heart failure. Clinical trials carried out over the last decade for cardiac regeneration have revealed inadequacy of current approaches in cell therapy. Chief among them is the choice of stem cells to achieve the desired outcomes. Initial enthusiasm of adult bone marrow stems cells for myocyte regeneration has largely been relegated to paracrine-driven, donor cell-independent, endogenous cardiac repair. However, true functional restoration in heart failure is likely to require considerable myocyte replacement. In order to match stem cell application to various clinical scenarios, we review the necessity to preprime stem cells towards cardiac fate before myocardial transplantation and if these differentiated stem cells could confer added advantage over current choice of undifferentiated stem cells. We explore differentiation ability of various stem cells to cardiac progenitors/cardiomyocytes and compare their applicability in providing targeted recovery in light of current clinical challenges of cell therapy.

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tissue-engineered scaffolds may improve experimental outcomes in cardiac cell therapy by targeted delivery of stem cells and mechanically support an infarcted left ventricular (LV) wall. We transplanted cardiomyocyte-like cells (5×10(5)) with scaffolding via epicardial patching (cell patch, n=17) or a low-dose intramyocardial hydrogel (LD hydrogel, n=18), a high-dose (5×10(6)) intramyocardial hydrogel (HD hydrogel, n=18) or transplanting a serum-free medium control (control, n=13), a blank patch (n=14), and a blank gel (n=16) for targeted cardiomyoplasty in a myocardial infarcted rat model. LV real-time hemodynamics were assessed using a 1.9-F pressure-volume catheter 7 weeks after stem cell transplantation. All mode of scaffold transplantation protected diastolic function by preserving LV wall integrity that resulted in a lower end diastolic pressure-volume relationship (EDPVR) as compared to a control medium-injected group. Moreover, epicardial patching, but not hydrogel injection, reduced ventricular wall stress with a significantly better LV end diastolic pressure (EDP: 5.3±2.4 mmHg vs. 9.6±6.9 mmHg, p<0.05) as compared to control. Furthermore, epicardial patching additionally preserved systolic function by modulating negative remodeling through restricting dilatation of the LV chamber. In comparison to control, an improved ejection fraction in the cell patch group (80.1%±5.9% vs. 67.9%±3.2%, p<0.01) was corroborated by load-independent enhancement of the end systolic pressure-volume relationship (ESPVR: 0.88±0.61 mmHg/uL vs. 0.29±0.19 mmHg/uL, p<0.05) and preload recruitable stroke work (PRSW: 68.7±26.4 mmHg vs. 15.6±16.2 mmHg, p<0.05) in systolic function. Moreover, the cell patch group (14.2±1.7 cells/high-power field vs. 7.4±1.6 cells/high power field, p<0.05) was significantly better in myocardial retention of transplanted stem cells as compared to the LD hydrogel group. Collectively, myocardial transplantation of compliant scaffolding materials alone may physically improve wall mechanics, largely independent of stem cells. However, epicardially grafted cell patch conferred added systolic contractility by improving stem cell retention and cellular alignment leading to improved LV remodeling and geometric preservation postinfarction.