Amelioration of X-Linked Related Autophagy Failure in Danon Disease With DNA Methylation Inhibitor.

BACKGROUND: Danon disease is an X-linked disorder that leads to fatal cardiomyopathy caused by a deficiency in lysosome-associated membrane protein-2 (LAMP2). In female patients, a later onset and less severe clinical phenotype have been attributed to the random inactivation of the X chromosome carrying the mutant diseased allele. We generated a patient-specific induced pluripotent stem cell (iPSCs)-based model of Danon disease to evaluate the therapeutic potential of Xi-chromosome reactivation using a DNA methylation inhibitor. METHODS: Using whole-exome sequencing, we identified a nonsense mutation (c.520C>T, exon 4) of the LAMP2 gene in a family with Danon disease. We generated iPSC lines from somatic cells derived from the affected mother and her 2 sons, and we then differentiated them into cardiomyocytes (iPSC-CMs) for modeling the histological and functional signatures, including autophagy failure of Danon disease. RESULTS: Our iPSC-CM platform provides evidence that random inactivation of the wild-type and mutant LAMP2 alleles on the X chromosome is responsible for the unusual phenotype in female patients with Danon disease. In vitro, iPSC-CMs from these patients reproduced the histological features and autophagy failure of Danon disease. Administration of the DNA demethylating agent 5-aza-2'-deoxycytidine reactivated the silent LAMP2 allele in iPSCs and iPSC-CMs in female patients with Danon disease and ameliorated their autophagy failure, supporting the application of a patient-specific iPSC platform for disease modeling and drug screening. CONCLUSIONS: Our iPSC-CM platform provides novel mechanistic and therapeutic insights into the contribution of random X chromosome inactivation to disease phenotype in X-linked Danon disease.

An abnormal TRPV4-related cytosolic Ca2+ rise in response to uniaxial stretch in induced pluripotent stem cells-derived cardiomyocytes from dilated cardiomyopathy patients.

Dilated cardiomyopathy (DCM) is cardiac disease characterized by increased left ventricular chamber volume and decreased systolic function. DCM patient-specific human induced-pluripotent stem cells-derived cardiomyocytes (DCM-hiPSC-CMs) were generated. We found that uniaxial stretch elicited a cytosolic [Ca2+]i rise in hiPSC-CMs. Compared to control-hiPSC-CMs, DCM-hiPSC-CMs displayed a greater magnitude of [Ca2+]i responses to the cell stretch of 10-15% elongation in length. This stretch-induced [Ca2+]i rise was abolished by removal of extracellular Ca2+ and markedly attenuated by TRPV4 inhibitors HC-067047 and RN-1734. Application of nifedipine and tranilast also reduced the [Ca2+]i response but to a lesser degree. Moreover, the augmented [Ca2+]i was decreased by cytochalasin D treatment. Taken together, our study for the first time demonstrated an abnormal TRPV4-related mechanosensitive Ca2+ signaling in DCM-hiPSC-CMs.

Generation of Induced Cardiospheres via Reprogramming of Skin Fibroblasts for Myocardial Regeneration.

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3ß inhibitor-(2'Z,3'E)- 6-Bromoindirubin-3'-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3ß inhibitor-6-Bromoindirubin-3'-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016;34:2693-2706.

Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing.

In this paper, we report a novel heterozygous mutation of A285V codon conversion on exon 4 of the desmin (DES), using whole exome sequencing (WES) in an isolated proband with documented dilated cardiomyopathy (DCM). This mutation is predicted to cause three-dimensional structure changes of DES. Immunohistological and electron microscopy studies demonstrated diffuse abnormal DES aggregations in DCM-induced-pluripotent stem cell (iPSC)-derived cardiomyocytes, and control-iPSC-derived cardiomyocytes transduced with A285V-DES. DCM-iPSC-derived cardiomyocytes also exhibited functional abnormalities in vitro. This is the first demonstration that patient-specific iPSC-derived cardiomyocytes can be used to provide histological and functional confirmation of a suspected genetic basis for DCM identified by WES.

Modeling of Friedreich ataxia-related iron overloading cardiomyopathy using patient-specific-induced pluripotent stem cells.

Friedreich ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is due to GAA repeat expansions within the first intron of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. The triplet codon repeats lead to heterochromatin-mediated gene silencing and loss of frataxin. Nevertheless, inadequacy of existing FRDA-cardiac cellular models limited cardiomyopathy studies. We tested the hypothesis that iron homeostasis deregulation accelerates reduction in energy synthesis dynamics which contributes to impaired cardiac calcium homeostasis and contractile force. Silencing of FXN expressions occurred both in somatic FRDA-skin fibroblasts and two of the induced pluripotent stem cells (iPSC) clones; a sign of stress condition was shown in FRDA-iPSC cardiomyocytes with disorganized mitochondrial network and mitochondrial DNA (mtDNA) depletion; hypertrophic cardiac stress responses were observed by an increase in α-actinin-positive cell sizes revealed by FACS analysis as well as elevation in brain natriuretic peptide (BNP) gene expression; the intracellular iron accumulated in FRDA cardiomyocytes might be due to attenuated negative feedback response of transferring receptor (TSFR) expression and positive feedback response of ferritin (FTH1); energy synthesis dynamics, in terms of ATP production rate, was impaired in FRDA-iPSC cardiomyocytes, which were prone to iron overload condition. Energetic insufficiency determined slower Ca(2+) transients by retarding calcium reuptake to sarcoplasmic reticulum (SR) and impaired the positive inotropic and chronotropic responses to adrenergic stimulation. Our data showed for the first time that FRDA-iPSCs cardiac derivatives represent promising models to study cardiac stress response due to impaired iron homeostasis condition and mitochondrial damages. The cardiomyopathy phenotype was accelerated in an iron-overloaded condition early in calcium homeostasis aspect.

A review of the anticancer and immunomodulatory effects of Lycium barbarum fruit.

The anticancer effects of traditional Chinese medicine (TCM) have attracted the attention of the public vis-à-vis existing cancer therapies with various side effects. Lycium barbarum fruit, commonly known as Gou Qi Zi in China, is a potential anticancer agent/adjuvant. Its major active ingredients, L. barbarum polysaccharides (LBP), scopoletin and 2-O-ß-D-glucopyranosyl-L-ascorbic acid (AA-2ßG), are found to have apoptotic and antiproliferative effects on cancer cell lines. Moreover, LBP also contributes to body's immunomodulatory effects and enhances effects of other cancer therapies. It is not known whether there are any undesirable effects. Further studies on its pharmacological mechanisms and toxicology could facilitate a safe usage of this TCM herb.

Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway.

AIM: To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs). METHODS: Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 µmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging. RESULTS: Compared with control, mESCs treated with ouabain (20 µmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and ß-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 µmol/L) inhibited cardiac differentiation while ouabain (20 µmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR). CONCLUSION: Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.

MTMR4 SNVs modulate ion channel degradation and clinical severity in congenital long QT syndrome: insights in the mechanism of action of protective modifier genes.

AIMS: In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. METHODS AND RESULTS: We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. CONCLUSION: Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.

Exogenous expression of HIF-1 alpha promotes cardiac differentiation of embryonic stem cells.

Hypoxia plays an important role in the proliferation, differentiation and maintenance of the cardiovascular system during development. While low oxygen tension appears to direct the cultured embryonic stem cells (ESCs) to differentiate into cardiomyocytes, the underlying molecular mechanism remains unclear. At a molecular level, hypoxia inducible factor-1 (HIF-1) plays an important role in handling the hypoxia signal. In the present study, we demonstrated that expression of exogenous HIF-1 alpha cDNA into murine ESCs significantly promoted cardiogenesis as indicated by a higher percentage of beating embryoid body and troponin-T positive cell counts as well as increased expression of early and late cardiac markers, such as GATA-binding protein 4 and 6, NK2 transcription factor related locus 5, alpha-myosin heavy chain, beta-myosin heavy chain and myosin light chain 2 ventricular transcripts. In addition, the transduced cells exhibited increased mRNA levels of cardiotrophin-1 and vascular endothelial growth factor, along with phosphorylation of eNOS [p-eNOS (ser1171)]. Application of NOS inhibitors, diphenyleneiodonium chloride (DPI), N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) or N(omega)-Nitro-L-arginine (L-NNA) abolished the HIF-1 alpha stimulated cardiac differentiation. With the clues of upregulated mRNA expression of calcium handling proteins, ryanodine receptor 2, sodium calcium exchanger and sarcoplasmic/endoplasmic reticulum calcium ATPase, in the transduced HIF-1 alpha ESCs, further study indicated that the maximum upstroke and decay velocity was significantly increased in both non-caffeine and caffeine-induced calcium transient in ESCs-derived cardiomyocytes. This suggests a well developed function of the sarcoplasmic reticulum in ESC-derived cardiomyocytes. Electrophysiological study also indicated that a portion of the HIF-1 alpha-transduced cells exhibited prominent phase-4 depolarization. These findings suggest that keen activation of the HIF-1 pathway enhances differentiation and maturation of cardiomyocytes derived from ESCs.

Relationship of circulating endothelial progenitor cells to the recurrence of atrial fibrillation after successful conversion and maintenance of sinus rhythm.

AIMS: To determine whether the number of circulating endothelial progenitor cells (EPCs) in patients with persistent atrial fibrillation (AF) predicts arrhythmia recurrence after direct current cardioversion (DCCV). METHODS AND RESULTS: The numbers of circulating CD34+/KDR+ EPCs were determined with flow cytometry in 51 consecutive patients with persistent AF [the mean age: 67 +/- 1.3 years, male (65%)] prior to DCCV and were compared with that of age- and sex-matched controls, and cohorts of patients with coronary artery disease and ischaemic stroke. The AF recurrence rate at 1 year was also determined. The EPCs in patients with persistent AF, patients with coronary artery disease, and patients with ischaemic stroke were significantly lower than that of the age- and sex-matched controls (P < 0.01). One year after successful DCCV, patients with high EPC count (50th to 100th percentile) had a higher recurrence rate of AF when compared with those with low EPC count (less than 50th percentile) (73 vs. 40%, P = 0.02). Cox regression analysis revealed the high EPC count was the only independent predictors for the AF recurrence (HR: 2.29, P = 0.047). CONCLUSION: The number of EPCs is reduced in patients with persistent AF and predicts the recurrence of AF after successful DCCV.

Expression of Lmna-R225X nonsense mutation results in dilated cardiomyopathy and conduction disorders (DCM-CD) in mice: Impact of exercise training.

AIMS: To recapitulate progressive human dilated cardiomyopathy (DCM) and heart block in the Lmna R225X mutant mice model and investigate the molecular basis of LMNA mutation induced cardiac conduction disorders (CD); To investigate the potential interventional impact of exercise endurance. METHODS AND RESULTS: A Lmna R225X knock-in mice model in either heterozygous or homozygous genotype was generated. Electrical remodeling was observed with higher occurrence of AV block from neonatal and aged mutant mice as measured by surface electrocardiogram and atrio-ventricular Wenckebach point detection. Histological and molecular profiles revealed an increase in apoptotic cells and activation of caspase-3 activities in heart tissue. Upon aging, extracellular cellular matrix (ECM) remodeling appeared with accumulation of collagen in Lmna R225X mutant hearts as visualized by Masson's trichrome stain. This could be explained by the upregulated ECM gene expression, such as Fibronectin: Fn1, collagen: Col12a1, intergrin: Itgb2 and 3, as detected by microarray gene chip. Also, endurance exercise for 3 month improved the ventricular ejection fraction, attenuated fibrosis and cardiomyocytes apoptosis in the aged mutant mice. CONCLUSIONS: The mechanism of LMNA nonsense mutation induced cardiac conduction defects through AV node fibrosis is due to upregulated ECM gene expression upon activation of cardiac apoptosis. Lmna R225X mutant mice hold the potential for serving as in vivo models to explore the mechanism and therapeutic methods for AV block or myopathy associated with the aging process.

Effects of iron oxide nanoparticles on cardiac differentiation of embryonic stem cells.

The therapeutic potential of transplantation of embryonic stem cells (ESCs) in animal model of myocardial infarction has been consistently demonstrated. The development of superparamagnetic iron oxide (SPIO) nanoparticles labeling and cardiac magnetic resonance imaging (MRI) have been increasingly used to track the migration of transplanted cells in vivo allowing cell fate determination. However, the impact of SPIO- labeling on cell phenotype and cardiac differentiation capacity of ESCs remains unclear. In this study, we demonstrated that ESCs labeled with SPIO compared to their unlabeled counterparts had similar cardiogenic capacity, and SPIO-labeling did not affect calcium-handling property of ESC-derived cardiomyocytes. Moreover, transplantation of SPIO-labeled ESCs via direct intra-myocardial injection to infarct myocardium resulted in significant improvement in heart function. These findings demonstrated the feasibility of in vivo ESC tracking using SPIO-labeling and cardiac MRI without affecting the cardiac differentiation potential and functional properties of ESCs.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi004-A from a carrier of the KCNQ1-R594Q mutation.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a male carrier of the heterozygous mutation c.1781 G > A p.R594Q on the KCNQ1 gene. hiPSCs, generated using four retroviruses each encoding for OCT4, SOX2, KLF4 and cMYC, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi005-A from a patient carrying the KCNQ1-R190W mutation.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a woman carrier of the heterozygous mutation c.568C > T p.R190W on the KCNQ1 gene. hiPSCs, obtained using four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

Empagliflozin Ammeliorates High Glucose Induced-Cardiac Dysfuntion in Human iPSC-Derived Cardiomyocytes.

Empagliflozin, a sodium-glucose co-transporter (SGLT) inhibitor, reduces heart failure and sudden cardiac death but the underlying mechanisms remain elusive. In cardiomyocytes, SGLT1 and SGLT2 expression is upregulated in diabetes mellitus, heart failure, and myocardial infarction. We hypothesise that empagliflozin exerts direct effects on cardiomyocytes that attenuate diabetic cardiomyopathy. To test this hypothesis, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were used to test the potential effects of empagliflozin on neutralization of cardiac dysfunction induced by diabetic-like cultures. Our results indicated that insulin-free high glucose culture significantly increased the size of and NPPB, SGLT1 and SGLT2 expression of hiPSC-derived cardiomyocytes. In addition, high glucose-treated hiPSC-derived cardiomyocytes exhibited reduced contractility regardless of the increased calcium transient capacity. Interestingly, application of empagliflozin before or after high glucose treatment effectively reduced the high glucose-induced cardiac abnormalities. Since application of empagliflozin did not significantly alter viability or glycolytic capacity of the hiPSC-derived cardiomyocytes, it is plausible that empagliflozin exerts its effects via the down-regulation of SGLT1, SGLT2 and GLUT1 expression. These observations provide supportive evidence that may help explain its unexpected benefit observed in the EMPA-REG trial.

Coverage and diagnostic yield of Whole Exome Sequencing for the Evaluation of Cases with Dilated and Hypertrophic Cardiomyopathy.

Targeted next generation sequencing of gene panels has become a popular tool for the genetic diagnosis of hypertrophic (HCM) and dilated cardiomyopathy (DCM). However, it is uncertain whether the use of Whole Exome Sequencing (WES) represents a more effective approach for diagnosis of cases with HCM and DCM. In this study, we performed indirect comparisons of the coverage and diagnostic yield of WES on genes and variants related to HCM and DCM versus 4 different commercial gene panels using 40 HCM and DCM patients, assuming perfect coverage in those panels. We identified 6 pathogenic or likely pathogenic among 14 HCM patients (diagnostic yield 43%). 3 pathogenic or likely pathogenic were found among the 26 DCM patients (diagnostic yield 12%). The coverage was similar to that of four existing commercial gene panels due to the clustering of mutation within MYH7, MYBPC3, TPM1, TNT2, and TTN. Moreover, the coverage of WES appeared inadequate for TNNI3 and PLN. We conclude that most of the pathogenic variants for HCM and DCM can be found within a small number of genes which were covered by all the commercial gene panels, and the application of WES did not increase diagnostic yield.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi002-A from a patient affected by the Jervell and Lange-Nielsen syndrome and carrier of two compound heterozygous mutations on the KCNQ1 gene.

We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells.

BACKGROUND: Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the LMNA gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different LMNA mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in LMNA-related cardiomyopathy. METHODS AND RESULTS: We generated human induced pluripotent stem cells lines from 3 patients who carried distinctive mutations (R225X, Q354X, and T518fs) in the LMNA gene. The cardiomyocytes derived from these human induced pluripotent stem cells lines reproduced the pathophysiological hallmarks of LMNA-related cardiomyopathy. Interestingly, PTC124 treatment increased the production of full-length LMNA proteins in only the R225X mutant, not in other mutations. Functional evaluation experiments on the R225X mutant further demonstrated that PTC124 treatment not only reduced nuclear blebbing and electrical stress-induced apoptosis but also improved the excitation-contraction coupling of the affected cardiomyocytes. CONCLUSIONS: Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations, we demonstrated that the effect of PTC124 is codon selective. A premature stop codon UGA appeared to be most responsive to PTC124 treatment.

Efficient attenuation of Friedreich's ataxia (FRDA) cardiomyopathy by modulation of iron homeostasis-human induced pluripotent stem cell (hiPSC) as a drug screening platform for FRDA.

BACKGROUND: Friedreich's ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS: Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog, idebenone (IDE) or the iron chelator, deferiprone (DFP), which are both under clinical trial. RESULTS: DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 µM and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 µM. With regard to cardiac electrical-contraction (EC) coupling function, decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein, succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition, iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS: DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events, cardiac electrical-coupling during contraction.

Generation and Characterization of Patient-Specific iPSC Model for Cardiovascular Disease.

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening, biomarker discovery, and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here, we describe the (1) retroviral reprogramming strategies in the generation of human iPSC, (2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality, and (3) protocol of cardiac differentiation by modulation of Wnt signaling and ß-catenin pathway.