Impact of Cardiac Progenitor Cells on Heart Failure and Survival in Single Ventricle Congenital Heart Disease.

RATIONALE: Intracoronary administration of cardiosphere-derived cells (CDCs) in patients with single ventricles resulted in a short-term improvement in cardiac function. OBJECTIVE: To test the hypothesis that CDC infusion is associated with improved cardiac function and reduced mortality in patients with heart failure. METHODS AND RESULTS: We evaluated the effectiveness of CDCs using an integrated cohort study in 101 patients with single ventricles, including 41 patients who received CDC infusion and 60 controls treated with staged palliation alone. Heart failure with preserved ejection fraction (EF) or reduced EF was stratified by the cardiac function after surgical reconstruction. The main outcome measure was to evaluate the magnitude of improvement in cardiac function and all-cause mortality at 2 years. Animal studies were conducted to clarify the underlying mechanisms of heart failure with preserved EF and heart failure with reduced EF phenotypes. At 2 years, CDC infusion increased ventricular function (stage 2: +8.4±10.0% versus +1.6±6.4%, P=0.03; stage 3: +7.9±7.5% versus -1.1±5.5%, P<0.001) compared with controls. In all available follow-up data, survival did not differ between the 2 groups (log-rank P=0.225), whereas overall patients treated by CDCs had lower incidences of late failure (P=0.022), adverse events (P=0.013), and catheter intervention (P=0.005) compared with controls. CDC infusion was associated with a lower risk of adverse events (hazard ratio, 0.411; 95% CI, 0.179-0.942; P=0.036). Notably, CDC infusion reduced mortality (P=0.038) and late complications (P<0.05) in patients with heart failure with reduced EF but not with heart failure with preserved EF. CDC-treated rats significantly reversed myocardial fibrosis with differential collagen deposition and inflammatory responses between the heart failure phenotypes. CONCLUSIONS: CDC administration in patients with single ventricles showed favorable effects on ventricular function and was associated with reduced late complications except for all-cause mortality after staged procedures. Patients with heart failure with reduced EF but not heart failure with preserved EF treated by CDCs resulted in significant improvement in clinical outcome. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01273857 and NCT01829750.

Cell Therapy Trials in Congenital Heart Disease.

Dramatic evolution in medical and catheter interventions and complex surgeries to treat children with congenital heart disease (CHD) has led to a growing number of patients with a multitude of long-term complications associated with morbidity and mortality. Heart failure in patients with hypoplastic left heart syndrome predicated by functional single ventricle lesions is associated with an increase in CHD prevalence and remains a significant challenge. Pathophysiological mechanisms contributing to the progression of CHD, including single ventricle lesions and dilated cardiomyopathy, and adult heart disease may inevitably differ. Although therapeutic options for advanced cardiac failure are restricted to heart transplantation or mechanical circulatory support, there is a strong impetus to develop novel therapeutic strategies. As lower vertebrates, such as the newt and zebrafish, have a remarkable ability to replace lost cardiac tissue, this intrinsic self-repair machinery at the early postnatal stage in mice was confirmed by partial ventricular resection. Although the underlying mechanistic insights might differ among the species, mammalian heart regeneration occurs even in humans, with the highest degree occurring in early childhood and gradually declining with age in adulthood, suggesting the advantage of stem cell therapy to ameliorate ventricular dysfunction in patients with CHD. Although effective clinical translation by a variety of stem cells in adult heart disease remains inconclusive with respect to the improvement of cardiac function, case reports and clinical trials based on stem cell therapies in patients with CHD may be invaluable for the next stage of therapeutic development. Dissecting the differential mechanisms underlying progressive ventricular dysfunction in children and adults may lead us to identify a novel regenerative therapy. Future regenerative technologies to treat patients with CHD are exciting prospects for heart regeneration in general practice.

Intracoronary Cardiac Progenitor Cells in Single Ventricle Physiology: The PERSEUS (Cardiac Progenitor Cell Infusion to Treat Univentricular Heart Disease) Randomized Phase 2 Trial.

RATIONALE: Patients with single ventricle physiology are at high risk of mortality resulting from ventricular dysfunction. The preliminary results of the phase 1 trial showed that cardiosphere-derived cells (CDCs) may be effective against congenital heart failure. OBJECTIVE: To determine whether intracoronary delivery of autologous CDCs improves cardiac function in patients with single ventricle physiology. METHODS AND RESULTS: We conducted a phase 2 randomized controlled study to assign in a 1:1 ratio 41 patients who had single ventricle physiology undergoing stage 2 or 3 palliation to receive intracoronary infusion of CDCs 4 to 9 weeks after surgery or staged reconstruction alone (study A). The primary outcome measure was to assess improvement in cardiac function at 3-month follow-up. Four months after palliation, controls had an alternative option to receive late CDC infusion on request (study B). Secondary outcomes included ventricular function, heart failure status, somatic growth, and health-related quality of life after a 12-month observation. At 3 months, the absolute changes in ventricular function were significantly greater in the CDC-treated group than in the controls (+6.4% [SD, 5.5] versus +1.3% [SD, 3.7]; P=0.003). In study B, a late CDC infusion in 17 controls increased the ventricular function at 3 months compared with that at baseline (38.8% [SD, 7.7] versus 34.8% [SD, 7.4]; P<0.0001). At 1 year, overall CDC infusion was associated with improved ventricular function (41.4% [SD, 6.6] versus 35.0% [SD, 8.2]; P<0.0001) and volumes (P<0.001), somatic growth (P<0.0001) with increased trophic factors production, such as insulin-like growth factor-1 and hepatocyte growth factor, and quality of life, along with a reduced heart failure status (P<0.0001) and cardiac fibrosis (P=0.014) relative to baseline. CONCLUSIONS: Intracoronary infusion of CDCs after staged palliation favorably affected cardiac function by reverse remodeling in patients with single ventricle physiology. This impact may improve heart failure status, somatic growth, and quality of life in patients and reduce parenting stress for their families. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01829750.

Transcoronary cell infusion with the stop-flow technique in children with single-ventricle physiology.

BACKGROUND: Almost all reports on cardiac regeneration therapy have referred to adults, and only a few have focused on transcoronary infusion of cardiac progenitor cells using the stop-flow technique in children. METHODS: Intracoronary autologous cardiosphere-derived cell (CDC) transfer was conducted at Okayama University as a phase 1 clinical trial for seven patients with hypoplastic left heart syndrome between January 2011 and December 2012, and as a phase 2 clinical trial for 34 patients with single-ventricle physiology between July 2013 and March 2015. RESULTS: A total of 41 patients with single-ventricle physiology underwent transcoronary infusion of CDC with the stop-flow technique. The median age was 33 months (range, 5-70 months) and the median weight was 10.1 kg (range, 4.1-16.0 kg). Transient adverse events occurred during the procedure, including ST-segment elevation or depression, hypotension, bradycardia, and coronary artery vasospasm. All patients completely recovered. There were no major procedure-related adverse events. In this study, transcoronary infusion of CDC using the stop-flow technique was successfully completed in all patients. CONCLUSION: Transcoronary infusion of CDC using the stop-flow technique in children is a feasible and safe procedure.

Intracoronary autologous cardiac progenitor cell transfer in patients with hypoplastic left heart syndrome: the TICAP prospective phase 1 controlled trial.

RATIONALE: Hypoplastic left heart syndrome (HLHS) remains a lethal congenital cardiac defect. Recent studies have suggested that intracoronary administration of autologous cardiosphere-derived cells (CDCs) may improve ventricular function. OBJECTIVE: The aim of this study was to test whether intracoronary delivery of CDCs is feasible and safe in patients with hypoplastic left heart syndrome. METHODS AND RESULTS: Between January 5, 2011, and January 16, 2012, 14 patients (1.8±1.5 years) were prospectively assigned to receive intracoronary infusion of autologous CDCs 33.4±8.1 days after staged procedures (n=7), followed by 7 controls with standard palliation alone. The primary end point was to assess the safety, and the secondary end point included the preliminary efficacy to verify the right ventricular ejection fraction improvements between baseline and 3 months. Manufacturing and intracoronary delivery of CDCs were feasible, and no serious adverse events were reported within the 18-month follow-up. Patients treated with CDCs showed right ventricular ejection fraction improvement from baseline to 3-month follow-up (46.9%±4.6% to 52.1%±2.4%; P=0.008). Compared with controls at 18 months, cardiac MRI analysis of CDC-treated patients showed a higher right ventricular ejection fraction (31.5%±6.8% versus 40.4%±7.6%; P=0.049), improved somatic growth (P=0.0005), reduced heart failure status (P=0.003), and lower incidence of coil occlusion for collaterals (P=0.007). CONCLUSIONS: Intracoronary infusion of autologous CDCs seems to be feasible and safe in children with hypoplastic left heart syndrome after staged surgery. Large phase 2 trials are warranted to examine the potential effects of cardiac function improvements and the long-term benefits of clinical outcomes. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01273857.

MURC/Cavin-4 facilitates recruitment of ERK to caveolae and concentric cardiac hypertrophy induced by α1-adrenergic receptors.

The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, α1-ARs localize to flask-shaped membrane microdomains known as "caveolae." Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated α1-AR-induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by α1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to α1A- and α1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to α1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient α1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy.

Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy.

Hypertrophic growth is a risk factor for mortality in heart diseases. Mechanisms are lacking for this global increase in RNA and protein per cell, which underlies hypertrophy. Hypertrophic signals cause phosphorylation of the RNA polymerase II C-terminal domain, required for transcript elongation. RNA polymerase II kinases include cyclin-dependent kinases-7 (Cdk7) and Cdk9, components of two basal transcription factors. We report activation of Cdk7 and -9 in hypertrophy triggered by signaling proteins (Galphaq, calcineurin) or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by endothelin. A preferential role for Cdk9 was shown in RNA polymerase II phosphorylation and growth induced by endothelin, using pharmacological and dominant-negative inhibitors. All four hypertrophic signals dissociated 7SK small nuclear RNA, an endogenous inhibitor, from cyclin T-Cdk9. Cdk9 was limiting for cardiac growth, shown by suppressing its inhibitor (7SK) in culture and preventing downregulation of its activator (cyclin T1) in mouse myocardium.Note: In the AOP version of this article, the numbering of the author affiliations was incorrect. This has now been fixed, and the affiliations appear correctly online and in print.

Cardiosphere-derived exosomal microRNAs for myocardial repair in pediatric dilated cardiomyopathy.

Although cardiosphere-derived cells (CDCs) improve cardiac function and outcomes in patients with single ventricle physiology, little is known about their safety and therapeutic benefit in children with dilated cardiomyopathy (DCM). We aimed to determine the safety and efficacy of CDCs in a porcine model of DCM and translate the preclinical results into this patient population. A swine model of DCM using intracoronary injection of microspheres created cardiac dysfunction. Forty pigs were randomized as preclinical validation of the delivery method and CDC doses, and CDC-secreted exosome (CDCex)-mediated cardiac repair was analyzed. A phase 1 safety cohort enrolled five pediatric patients with DCM and reduced ejection fraction to receive CDC infusion. The primary endpoint was to assess safety, and the secondary outcome measure was change in cardiac function. Improved cardiac function and reduced myocardial fibrosis were noted in animals treated with CDCs compared with placebo. These functional benefits were mediated via CDCex that were highly enriched with proangiogenic and cardioprotective microRNAs (miRNAs), whereas isolated CDCex did not recapitulate these reparative effects. One-year follow-up of safety lead-in stage was completed with favorable profile and preliminary efficacy outcomes. Increased CDCex-derived miR-146a-5p expression was associated with the reduction in myocardial fibrosis via suppression of proinflammatory cytokines and transcripts. Collectively, intracoronary CDC administration is safe and improves cardiac function through CDCex in a porcine model of DCM. The safety lead-in results in patients provide a translational framework for further studies of randomized trials and CDCex-derived miRNAs as potential paracrine mediators underlying this therapeutic strategy.

Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells.

The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-beta and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB-431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-beta, and inhibition of transforming growth factor-beta-mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-beta negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes.

Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling.

The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-beta family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.

Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases.

Heart failure remains the leading cause of death worldwide, and is a burgeoning problem in public health due to the limited capacity of postnatal hearts to self-renew. The pathophysiological changes in injured hearts can sometimes be manifested as scar formation or myocardial degradation, rather than supplemental muscle regeneration to replenish lost tissue during the healing processes. Stem cell therapies have been investigated as a possible treatment approach for children and adults with potentially fatal cardiovascular disease that does not respond to current medical therapies. Although the heart is one of the least regenerative organs in mammals, discoveries made during the past few decades have improved our understanding of cardiac development and resident stem/progenitor pools, which may be lineage-restricted subpopulations during the post-neonatal stage of cardiac morphogenesis. Recently, investigation has specifically focused on factors that activate either endogenous progenitor cells or preexisting cardiomyocytes, to regenerate cardiovascular cells and replace the damaged heart tissues. The discovery of induced pluripotent stem cells has advanced our technological capability to direct cardiac reprogramming by essential factors that are crucial for heart field completion in each stage. Cardiac tissue engineering technology has recently shown progress in generating myocardial tissue on human native cardiac extracellular matrix scaffolds. This review summarizes recent advances in the field of cardiac cell therapies with an emphasis on cellular mechanisms, such as bone marrow stem cells and cardiac progenitor cells, which show the high potential for success in preclinical and clinical meta-analysis studies. Expanding our current understanding of mechanisms of self-renewal in the neonatal mammalian heart may lead to the development of novel cardiovascular regenerative medicines for pediatric heart diseases.

Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells.

BACKGROUND: Cultivation of endothelial progenitor cells (EPCs) leads to premature replicative senescence, limiting ex vivo expansion for potential clinical cell therapy. Recent studies have linked senescence to the dysfunction of telomeres, the "ends" of chromosomes, via the so-called mitotic clock or culture-induced stress. The purpose of this study was to elucidate a possible role of telomere biology in the functional augmentation of EPCs by statins. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood. Using flow cytometry after fluorescence in situ hybridization with a telomere-specific (C3TA2)3 peptide nucleic acid probe (Flow-FISH), we found mean telomere length in untreated EPCs from healthy subjects to range between 8.5+/-0.2 and 11.1+/-0.5 kb with no change over 6 days of culture, excluding telomere erosion as one cause for premature senescence. Although mean telomere length did not differ between statin-treated and untreated EPCs, atorvastatin (0.1 micromol/L) and mevastatin (1.0 micromol/L) both led to a more than 3-fold increase in the expression of the telomere capping protein TRF2 (telomere repeat-binding factor), as shown by immunoblotting, whereas quantitative reverse transcription-polymerase chain reaction demonstrated no increase in TRF2 mRNA. Telomere dysfunction of EPCs was also paralleled by a 4-fold increase in the DNA damage checkpoint-kinase 2 (Chk2). Conversely, statin cotreatment or overexpression of TRF2 completely suppressed Chk2 induction. Finally, overexpression of a dominant negative mutant of the TRF2 protein abrogated statin-induced enhancement of migratory activity down to baseline values. CONCLUSIONS: Ex vivo culturing of EPCs leads to "uncapping" of telomeres, indicated by the loss of TRF2. Statin cotreatment of EPCs prevents impairment of their functional capacity by a TRF2-dependent, posttranscriptional mechanism. This is the first time a beneficial effect of statins on telomere biology has been described.

[New era of cardiac stem cell therapy in heart failure].

Despite of plethora of reports on stem cell transplantation leading to neovascularization in infarct models, whether sustaining clinical benefit in post-myocardial infarction patients is manifested by myocyte repair remains unclear. Cardiac muscle regeneration in adult heart is thought to occur through the mobilization and differentiation of mesenchymal stem cells in bone marrow origin, however, recent studies have suggested that substantial cardiac stem cells may exist in the heart itself, repopulating the damaged cardiac muscle during injury or aging processes. The implications of cardiac stem cells-based myocyte plasticity have recently begun to define in human heart, neither arisen from bone marrow nor circulating precursors. Introduction of cardiac stem cells may improve myocardial function, but several hurdles exist and should be coaxed far beyond the clinical application of cardiac regenerative therapies. On-going investigations may lead to the discovery of mediators of cardiac stem cells migration, proliferation and differentiation that, in turn, might result in the mending of the broken heart after injury.

Transcoronary infusion of cardiac progenitor cells in hypoplastic left heart syndrome: Three-year follow-up of the Transcoronary Infusion of Cardiac Progenitor Cells in Patients With Single-Ventricle Physiology (TICAP) trial.

OBJECTIVES: Our aim was to assess midterm safety and clinical outcomes of intracoronary infusion of cardiosphere-derived cells (CDCs) after staged palliation in patients with hypoplastic left heart syndrome (HLHS). METHODS: In this prospective, controlled study, 14 consecutive patients with HLHS who were undergoing 2- or 3-stage surgical palliations were assigned to receive intracoronary CDC infusion 1 month after cardiac surgery (n = 7), followed by 7 patients allocated to a control group with standard care alone. The primary end point was to assess procedural feasibility and safety; the secondary end point was to evaluate cardiac function and heart failure status through 36-month follow-up. RESULTS: No complications, including tumor formation, were reported within 36 months after CDC infusion. Echocardiography showed significantly greater improvement in right ventricular ejection fraction (RVEF) in infants receiving CDCs than in controls at 36 months (+8.0% ± 4.7% vs +2.2% ± 4.3%; P = .03). These cardiac function improvements resulted in reduced brain natriuretic peptide levels (P = .04), lower incidence of unplanned catheter interventions (P = .04), and higher weight-for-age z score (P = .02) at 36 months relative to controls. As independent predictors of treatment responsiveness, absolute changes in RVEF at 36 months were negatively correlated with age, weight-for-age z score, and RVEF at CDC infusion. CONCLUSIONS: Intracoronary CDC infusion after staged procedure in patients with HLHS is safe and improves RVEF, which persists during 36-month follow-up. This therapeutic strategy may enhance somatic growth and reduce incidence of heart failure.

Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells.

The evidence of cardiomyocyte proliferation in damaged heart implied cardiac regeneration might occur by resident or extra cardiac stem cells. However, the specification and origin of these cells remain unknown. Here, we report using fluorescence-activated cell sorting that cardiac progenitor cells resided in adult heart and colocalized with small capillary vessels, within the stem cell antigen (Sca-1) population expressing high telomerase activity. Notably, hematopoietic stem cells capable of efflux Hoechst 33342, termed side population cells, also were identified within the heart-derived cells. The cardiac progenitor cells (CD45(-)/CD34(-)) express neither cardiac muscle nor endothelial cell markers at an undifferentiated stage. The exposure of 5-azacytidine induced cardiac differentiation, which depends, in part, on Bmpr1a, a type IA receptor for bone morphogenetic protein (BMP). The capability of adult Sca1(+) cells to adopt a cardiac muscle in embryogenesis was substantiated by blastocyst injection, using progenitors from the adult hearts of transgenic mice that harbor a bacterial artificial chromosome expressing GFP via the Nkx-2.5 locus. Intravenously injected progenitors, shortly after ischemic/reperfusion, homed and functionally differentiated 3.5% of total left ventricle in the host myocardium. Differentiation included both fusion-independent and fusion-associated components, proved by the Cre/loxP donor/recipient system. Our studies suggest that endogenous cardiac progenitors reside in the adult heart, regenerate cardiomyocytes functionally, and integrate into the existing heart circuitry.

Stem cell therapies in patients with single ventricle physiology.

Single ventricle physiology, especially hypoplastic left heart syndrome, is one of the most high-risk lesions in children with congenital heart disease, and the ensuing heart failure remains as a major problem related to adverse outcomes in these patients. The field of stem cell therapy for heart failure has shown striking advances during the past 10 years, and many clinical trials using stem cell technologies have been conducted in adults, which suggest that stem cell therapy is associated with long-term improvement in cardiac function. Cardiac progenitor cells have recently been discovered, and their strong regenerative ability has been demonstrated in several studies. Although no large clinical trials have been performed in the field of congenital heart disease, recent investigations indicate that stem cell therapy may hold great potential to treat children with cardiac defects.

Directed differentiation of patient-specific induced pluripotent stem cells identifies the transcriptional repression and epigenetic modification of NKX2-5, HAND1, and NOTCH1 in hypoplastic left heart syndrome.

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

Molecular genetic and functional characterization implicate muscle-restricted coiled-coil gene (MURC) as a causal gene for familial dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies. METHODS AND RESULTS: We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ(2)=8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC. CONCLUSIONS: MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.

PARM-1 is an endoplasmic reticulum molecule involved in endoplasmic reticulum stress-induced apoptosis in rat cardiac myocytes.

To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.