Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

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.

Phosphatidylserine Supplementation as a Novel Strategy for Reducing Myocardial Infarct Size and Preventing Adverse Left Ventricular Remodeling.

Phosphatidylserines are known to sustain skeletal muscle activity during intense activity or hypoxic conditions, as well as preserve neurocognitive function in older patients. Our previous studies pointed out a potential cardioprotective role of phosphatidylserine in heart ischemia. Therefore, we investigated the effects of phosphatidylserine oral supplementation in a mouse model of acute myocardial infarction (AMI). We found out that phosphatidylserine increases, significantly, the cardiomyocyte survival by 50% in an acute model of myocardial ischemia-reperfusion. Similar, phosphatidylserine reduced significantly the infarcted size by 30% and improved heart function by 25% in a chronic model of AMI. The main responsible mechanism seems to be up-regulation of protein kinase C epsilon (PKC-ε), the main player of cardio-protection during pre-conditioning. Interestingly, if the phosphatidylserine supplementation is started before induction of AMI, but not after, it selectively inhibits neutrophil's activation, such as Interleukin 1 beta (IL-1ß) expression, without affecting the healing and fibrosis. Thus, phosphatidylserine supplementation may represent a simple way to activate a pre-conditioning mechanism and may be a promising novel strategy to reduce infarct size following AMI and to prevent myocardial injury during myocardial infarction or cardiac surgery. Due to the minimal adverse effects, further investigation in large animals or in human are soon possible to establish the exact role of phosphatidylserine in cardiac diseases.

Oxidative stress in cardiac hypertrophy: From molecular mechanisms to novel therapeutic targets.

When faced with increased workload the heart undergoes remodelling, where it increases its muscle mass in an attempt to preserve normal function. This is referred to as cardiac hypertrophy and if sustained, can lead to impaired contractile function. Experimental evidence supports oxidative stress as a critical inducer of both genetic and acquired forms of cardiac hypertrophy, a finding which is reinforced by elevated levels of circulating oxidative stress markers in patients with cardiac hypertrophy. These observations formed the basis for using antioxidants as a therapeutic means to attenuate cardiac hypertrophy and improve clinical outcomes. However, the use of antioxidant therapies in the clinical setting has been associated with inconsistent results, despite antioxidants having been shown to exert protection in several animal models of cardiac hypertrophy. This has forced us to revaluate the mechanisms, both upstream and downstream of oxidative stress, where recent studies demonstrate that apart from conventional mediators of oxidative stress, metabolic disturbances, mitochondrial dysfunction and inflammation as well as dysregulated autophagy and protein homeostasis contribute to disease pathophysiology through mechanisms involving oxidative stress. Importantly, novel therapeutic targets have been identified to counteract oxidative stress and attenuate cardiac hypertrophy but more interestingly, the repurposing of drugs commonly used to treat metabolic disorders, hypertension, peripheral vascular disease, sleep disorders and arthritis have also been shown to improve cardiac function through suppression of oxidative stress. Here, we review the latest literature on these novel mechanisms and intervention strategies with the aim of better understanding the complexities of oxidative stress for more precise targeted therapeutic approaches to prevent cardiac hypertrophy.

Human-induced pluripotent stem cells for modelling metabolic perturbations and impaired bioenergetics underlying cardiomyopathies.

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.

Mitochondrial shaping proteins as novel treatment targets for cardiomyopathies.

Heart failure (HF) is one of the leading causes of death and disability worldwide. The prevalence of HF continues to rise, and its outcomes are worsened by risk factors such as age, diabetes, obesity, hypertension, and ischemic heart disease. Hence, there is an unmet need to identify novel treatment targets that can prevent the development and progression of HF in order to improve patient outcomes. In this regard, cardiac mitochondria play an essential role in generating the ATP required to maintain normal cardiac contractile function. Mitochondrial dysfunction is known to contribute to the pathogenesis of a number of cardiomyopathies including those secondary to diabetes, pressure-overload left ventricular hypertrophy (LVH), and doxorubicin cardiotoxicity. Mitochondria continually change their shape by undergoing fusion and fission, and an imbalance in mitochondrial fusion and fission have been shown to impact on mitochondrial function, and contribute to the pathogenesis of these cardiomyopathies. In this review article, we focus on the role of mitochondrial shaping proteins as contributors to the development of three cardiomyopathies, and highlight their therapeutic potential as novel treatment targets for preventing the onset and progression of HF.

Mitochondria in acute myocardial infarction and cardioprotection.

Acute myocardial infarction (AMI) and the heart failure (HF) that often follows are among the leading causes of death and disability worldwide. As such, new treatments are needed to protect the myocardium against the damaging effects of the acute ischaemia and reperfusion injury (IRI) that occurs in AMI, in order to reduce myocardial infarct (MI) size, preserve cardiac function, and improve patient outcomes. In this regard, cardiac mitochondria play a dual role as arbiters of cell survival and death following AMI. Therefore, preventing mitochondrial dysfunction induced by acute myocardial IRI is an important therapeutic strategy for cardioprotection. In this article, we review the role of mitochondria as key determinants of acute myocardial IRI, and we highlight their roles as therapeutic targets for reducing MI size and preventing HF following AMI. In addition, we discuss the challenges in translating mitoprotective strategies into the clinical setting for improving outcomes in AMI patients.

Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes.

BACKGROUND: Silica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2. RESULTS: The cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 µg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 µg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP. CONCLUSION: The aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure.

Mechanisms underlying diabetic cardiomyopathy: From pathophysiology to novel therapeutic targets.

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.

Myeloperoxidase As a Multifaceted Target for Cardiovascular Protection.

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.

Sheathless Acoustic Fluorescence Activated Cell Sorting (aFACS) with High Cell Viability.

In this work, we demonstrate a sheathless acoustic fluorescence activated cell sorting (aFACS) system by combining elasto-inertial cell focusing and highly focused traveling surface acoustic wave (FTSAW) to sort cells with high recovery rate, purity, and cell viability. The microfluidic sorting device utilizes elasto-inertial particle focusing to align cells in a single file for improving sorting accuracy and efficiency without sample dilution. Our sorting device can effectively focus 1 µm particles which represents the general minimum size for a majority of cell sorting applications. Upon the fluorescence interrogation at the single cell level, individual cells are deflected to the target outlet by a ∼50 µm wide highly focused acoustic field. We have applied our aFACS to sort three different cell lines (i.e., MCF-7, MDA-231, and human-induced pluripotent stem-cell-derived cardiomyocytes; hiPSC-CMs) at ∼kHz with a sorting purity and recovery rate both of about 90%. A further comparison demonstrates that the cell viability drops by 35-45% using a commercial FACS machine, while the cell viability only drops by 3-4% using our aFACS system. The developed aFACS system provides a benchtop solution for rapid, highly accurate single cell level sorting with high cell viability, in particular for sensitive cell types.

Manipulating energy migration within single lanthanide activator for switchable upconversion emissions towards bidirectional photoactivation.

Reliance on low tissue penetrating UV or visible light limits clinical applicability of phototherapy, necessitating use of deep tissue penetrating near-infrared (NIR) to visible light transducers like upconversion nanoparticles (UCNPs). While typical UCNPs produce multiple simultaneous emissions for unidirectional control of biological processes, programmable control requires orthogonal non-overlapping light emissions. These can be obtained through doping nanocrystals with multiple activator ions. However, this requires tedious synthesis and produces complicated multi-shell nanoparticles with a lack of control over emission profiles due to activator crosstalk. Herein, we explore cross-relaxation (CR), a non-radiative recombination pathway typically perceived as deleterious, to manipulate energy migration within the same lanthanide activator ion (Er3+) towards orthogonal red and green emissions, simply by adjusting excitation wavelength from 980 to 808 nm. These UCNPs allow programmable activation of two synergistic light-gated ion channels VChR1 and Jaws in the same cell to manipulate membrane polarization, demonstrated here for cardiac pacing.

Targeting Mitochondrial Fission Using Mdivi-1 in A Clinically Relevant Large Animal Model of Acute Myocardial Infarction: A Pilot Study.

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.

New insights provided by myofibril mechanics in inherited cardiomyopathies.

Cardiomyopathies represent a heterogeneous group of cardiac disorders that perturb cardiac contraction and/or relaxation, and can result in arrhythmias, heart failure, and sudden cardiac death. Based on morphological and functional differences, cardiomyopathies have been classified into hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). It has been well documented that mutations in genes encoding sarcomeric proteins are associated with the onset of inherited cardiomyopathies. However, correlating patient genotype to the clinical phenotype has been challenging because of the complex genetic backgrounds, environmental influences, and lifestyles of individuals. Thus, "scaling down" the focus to the basic contractile unit of heart muscle using isolated single myofibril function techniques is of great importance and may be used to understand the molecular basis of disease-causing sarcomeric mutations. Single myofibril bundles harvested from diseased human or experimental animal hearts, as well as cultured adult cardiomyocytes or human cardiomyocytes derived from induced pluripotent stem cells, can be used, thereby providing an ideal multi-level, cross-species platform to dissect sarcomeric function in cardiomyopathies. Here, we will review the myofibril function technique, and discuss alterations in myofibril mechanics, which are known to occur in sarcomeric genetic mutations linked to inherited HCM, DCM, and RCM, and describe the therapeutic potential for future target identification.

INDUCED PLURIPOTENT STEM CELLS FOR MODELLING ENERGETIC ALTERATIONS IN HYPERTROPHIC CARDIOMYOPATHY.

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.

Fatty acid metabolism driven mitochondrial bioenergetics promotes advanced developmental phenotypes in human induced pluripotent stem cell derived cardiomyocytes.

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 ß-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 ß-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.

Association of Cardiomyopathy With MYBPC3 D389V and MYBPC3Δ25bpIntronic Deletion in South Asian Descendants.

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.

Identification of a targeted and testable antiarrhythmic therapy for long-QT syndrome type 2 using a patient-specific cellular model.

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 ß-blockers is feasible and may represent a novel therapeutic option.

Acetylated Signal Transducer and Activator of Transcription 3 Functions as Molecular Adaptor Independent of Transcriptional Activity During Human Cardiogenesis.

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.

ErbB Receptor Tyrosine Kinase: A Molecular Switch Between Cardiac and Neuroectoderm Specification in Human Pluripotent Stem Cells.

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.

ErbB4 Activated p38γ MAPK Isoform Mediates Early Cardiogenesis Through NKx2.5 in Human Pluripotent Stem Cells.

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.