Search

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.)

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.)

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.)

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.)

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.

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.)

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.)

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.

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.)

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.)

Aims: Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes., Methods and Results: The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization., Conclusion: Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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±17.89ms vs. 536.59±37.1ms; 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±0.27pA/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. [ABSTRACT FROM AUTHOR]