Cardiac Myocyte Differentiation Research Articles

Abstract P3181: A Cyclophilin D-mitochondria-redox Differentiation Pathway Regulates Neonatal Heart Bioenergetics And Function

Background: During birth, demands on the heart increase when exposed to high oxidative stress. Cardiac myocyte differentiation is dependent on changes in mitochondrial function that regulate cell stress pathways to enhance differentiation. We have shown these changes are dependent on closure of the mitochondrial permeability transition pore (mPTP) controlled by the protein cyclophilin D (CypD). These studies aim to determine the effects of independently manipulating CypD, mitochondrial membrane potential, and reactive oxygen species (ROS) production on mitochondrial function, myocyte differentiation, and cardiac function in the neonate. Methods: Neonatal C57Bl6 mice were intraperitoneally injected on postnatal day (P) 1-6 of with mitoparaquat (0.025-0.05 mg/kg), Tempol (0.7 mg/kg), mitoTempo (0.7 mg/kg), 2,4-dinitrophenol (2 mg/kg), or vehicle. Hearts were harvested at P7 and mitochondrial respiration, enzymatic activity of the electron transport chain (ETC) complex (Cx) I and 2, and assembly of mitochondrial supercomplexes were measured by native gel electrophoresis. Results: Treatment with mitoparaquat to increase mitochondrial ROS caused a dose-dependent decrease of Cx I activity measured by oxygen consumption and enzyme assays. This corresponded to decrease in assembly of Cx-I containing supercomplexes. Decreasing mitochondrial ROS using mitoTempo had no effect on Cx I activity but increased assembly of Cx-III containing supercomplexes. Decreasing ROS in the cell with Tempol decreased Cx-I activity but had no effect on supercomplex assembly. Uncoupling at the mitochondrial membrane using 2,4-dinitrophenol increased electron flux from Cx I to Cx III but may decrease supercomplex assembly. Treatment of isolated mitochondria with cyclosporin A to inhibit CypD had no effects on these measures. Conclusions: Changes in ROS and mitochondrial membrane potential have selective effects on ETC assembly, activity in the neonatal period, and are not inhibited by closure of the mPTP. This data suggests a pathway where CypD regulates the mPTP to regulate the ETC, membrane potential, and ROS production to regulate myocyte differentiation; such a pathway could be targeted to increase cardiac maturation in infants with poor cardiac function.

Cyclophilin D inhibition rescues cardiac function in neonatal hypoxia.

The heart develops in utero under hypoxic conditions, and exposure to higher oxygen levels at birth activates changes in the heart to allow survival ex-utero. Mitochondria are critical in terminal cardiac myocyte differentiation after birth, and inhibition of the mitochondrial chaperone cyclophilin D (CypD) enhances this process by regulating the electron transport chain (ETC) to meet increased metabolic demands. Disruption of this process may play a role in neonatal cardiomyopathy and congenital heart disease. We tested the hypothesis that inhibition of CypD with cyclosporin A (CsA) or N-methyl-4-isoleucine cyclosporin (NIM811) will rescue the effects of hypoxia on cardiac and mitochondrial function. Mice were exposed to hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (NoTx) or intraperitoneal injections 10mg/kg of vehicle, CsA, or NIM811 from P1 to P6. Room air neonates served as controls. On P7, mice underwent echocardiography and hearts were harvested for mitochondrial isolation for various assays of mitochondrial structure and function. Hypoxia decreased survival and body weight, caused cardiomegaly, decreased cellular organization, and decreased cardiac function in the NoTx and vehicle groups when compared to room air controls, while the enzymatic function of ETC complexes I and III (P<0.01) and complex I-mediated respiration (P<0.0001) was decreased. Treatment with CsA and NIM811 normalized cardiomegaly, cardiac function, cellular structure, ETC activity of complexes I and III and the mitochondrial respiratory capacity for complex I. Thus, pharmacologic inhibition of CypD reversed the effects of hypoxia on ETC activity and overall cardiac function and structurein the neonatal heart. Our study may help develop therapies to treat cardiomyopathies and the effects of hypoxia on the neonatal heart.

Abstract P446: Cyclophilin D Inhibition Rescues Cardiac Function And Electron Transport Chain Assembly And Activity In Hypoxia Exposed Neonatal Mice

Introduction: Mitochondria play a critical role in cardiac myocyte physiology and differentiation. Hypoxia decreases cardiac function. Changes in embryonic heart metabolism at the level of the electron transport chain (ETC) are regulated by a chaperone protein known as cyclophilin D (CypD). Inhibition of CypD with chemicals such as cyclosporin A (CsA) and N-methyl-4-isoleucine cyclosporin (NIM811) leads to more complex mitochondrial structure and effective oxidative phosphorylation. Hypothesis: Inhibition of CypD with CsA or NIM811 will rescue the detrimental effects of hypoxia on cardiac function and on ETC assembly and activity. Methods: Mice were exposed to continuous hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (No Tx) or intraperitoneal injections 10mg/kg of vehicle (VEH), CsA, or NIM811 from P1 to P6. Litters of mice born into room air served as controls. On P7, mice were anesthetized and underwent echocardiography and/or hearts were harvested for mitochondrial isolation. Enzymatic activity of ETC complexes was quantified using spectrophotometry and normalized to total protein. To measure physical assembly of complex I &amp; V of the ETC, high resolution clear native polyacrylamide gel electrophoresis (HCRN PAGE) followed by in-gel assays were utilized using appropriate protein loading controls. Results: Cardiac ejection fraction was decreased in hypoxic No Tx (P&lt;0.0001) and VEH (P&lt;0.0001), but was rescued by CsA or NIM811 when compared to room air controls (P&gt;0.05). Heart weight to body weight ratio was increased in No Tx and VEH groups (P&lt; 0.0001) and rescued in the CsA and NIM811 groups when compared to room air controls (P&gt;0.05). Complex I enzymatic activity was rescued with treatment with CsA and NIM811. HCRN PAGE followed by in-gel ETC complex assay demonstrated assembly of complexes I and V into dimers and tetramer in the room air, CsA, and NIM811 groups that was not seen in the No Tx and VEH groups. Conclusion: Pharmacologic inhibition of CypD reversed the effects of hypoxia on cardiac function and ETC activity and assembly in the neonatal heart. Our studies may help develop therapies to treat neonatal cardiomyopathies and the effects of hypoxia on the neonatal heart.

Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity invitro.

Identifying the particular transcription factors that maintain cell type invitro is important for manipulating cell type. Identifying such transcription factors by their cell-type-specific expression or their involvement in developmental regulation has had limited success. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would reveal identity-maintaining transcription factors. We developed perturbation panel profiling (P3) as a framework for perturbing cells across many conditions and measuring gene expression responsiveness transcriptome-wide. In human iPSC-derived cardiac myocytes, P3 showed that transcription factors important for cardiac myocyte differentiation and maintenance were among the most frequently upregulated (most responsive). We reasoned that one function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming. We propose that responsiveness to perturbations is a property of transcription factors that help maintain cellular identity invitro. A record of this paper's transparent peer review process is included in the supplemental information.

Abstract 534: Chemically Defined Cell Culture Media for High Yield Differentiation of Functional Human Cardiac Myocytes

Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising technology for regenerative medicine and pharmacology. However, existing protocols are limited due to the high costs when generating cells at scale, to low yield, and to a lack of protocol replicability. Additionally, the use of animal-derived or recombinant proteins in cell differentiation creates concerns in human compatibility and presents technical challenges due to batch-to-batch variability. Aim: Develop a protocol for functional hiPSC-CM differentiation with high yield without the use of proteins. Methods: We expanded the CDM3 protocol by testing approximately 200 combinations of cell culture media during hiPSC differentiation. The differentiation was induced with a 24h incubation in 6 μM CHIR (d0), followed by 24 h in basal media (d1) and 48h in media supplemented with AA2P and Wnt-C59 (d2-d4). From day 4 onwards, cells were kept in media supplemented with insulin and AA2P, and in some conditions with polyvinyl alcohol. On day 8, hiPSC-CMs were antibiotically selected using geneticin. Tissues were imaged on day 13 to assess myocyte morphology and contraction. Videos were scored from 0 (dead/delaminated cells) to 9 (confluent tissues beating at least 4 times in a 5 sec interval). Results: We identified 6 basal media combinations that yield confluent monolayers on day 13. Out of these, 3 conditions consistently resulted in confluent contractile tissues. Conclusions: Our data demonstrate that a protein-free high yield hiPSC-CM differentiation is possible with the appropriate selection of basal media based on cellular nutritional requirements.

Monoamine oxidase A deletion leads to autophagy inhibition and impairment in cardiomyocyte differentiation from hiPSCs

Reactive oxygen species (ROS) and autophagy play a major role in cardiac pathophysiology, including cardiomyocyte differentiation. Yet, the exact source of ROS controlling the process of differentiation remains unknown. Within mitochondria, monoamine oxidases A and B (MAO-A and -B) are one of the major sources of ROS and play an important role in cardiac tissue homeostasis. Nevertheless, their involvement in cardiomyocyte differentiation has not been investigated yet. Here, we assessed whether ablation of MAO-A, the isoform expressed in human induced pluripotent stem cells (hiPSCs), could affect cardiomyocyte differentiation. MAO-A deletion negatively affected contractile properties of hiPSC-derived cardiomyocytes, resulting in higher frequency and lower amplitude of calcium transients. These features were accompanied by sarcomere disorganization and a reduction in the α- to β-myosin heavy chain ratio in MAO-A−/− cells, similarly to what occurs in failing hearts. GATA4, one of the key genes involved in cardiac remodeling, was significantly upregulated in these cells. To understand the underlying mechanisms, we investigated whether MAO-A deletion affected macroautophagy, one of the key processes required for cardiomyocyte differentiation. Macroautophagy levels were reduced in MAO-A−/− hiPSCs both at baseline and upon starvation or rapamycin administration, as evidenced by reduced accumulation of LC3-II in the presence of inhibitors of lysosomal degradation. The inhibitory effect on macroautophagy is likely due to the reduction of AMPK phosphorylation levels observed in MAO-A−/− hiPSCs. Taken together, these results suggest that MAO-A is required for the regulation of macroautophagy in hiPSCs and, consequently, for the process of differentiation and maturation of cardiac myocytes.

Expression and effects of TSP50 in mouse embryo and cardiac myocyte development

TSP50, a testis-specific gene encoding a serine protease-like protein, was specifically expressed in the spermatocytes of testes but abnormally activated and expressed in many different kinds of cancers. Here, we aimed to analyze the expression of TSP50 in mouse embryo and its function in early embryonic development. Firstly, the distribution of TSP50 in oocytes and embryonic development was characterized by immunofluorescence, RT-PCR and western blotting, and the results showed that TSP50 was detected at all studied stages with a dynamic expression pattern. When overexpressed TSP50 in zygotes by microinjection, the zygotes development was highly accelerated. On the contrary, knocking down TSP50 expression by RNA interference greatly retarded the zygote development. Furthermore, TSP50 expression at embryonic day 6.5 (E6.5), day 8.5 (E8.5) and day 10.5 (E10.5) were increasingly enhanced, However, the expression of TSP50 decreased gradually in the development and differentiation of cardiac myocyte from E12.5 to postnatal (P0).Additionally, we found that TSP50 expression was decreased during cardiac myocyte differentiation of P19 cells. Overexpression of TSP50 could decrease the expression of GATA-4, and knockdown of TSP50 markedly increase the expression of GATA-4. Taken together, our data indicate that TSP50 may play an important role during the process of mouse embryonic development as well as myocardial cell differentiation.

Micro‐RNA‐205 Regulates Heart Size Through Direct Modulation of the Hippo Pathway

During fetal development, the mammalian myocardium undergoes a period of hyperplastic growth, which establishes the number of cardiomyocytes in the adult heart. After birth, cardiomyocytes proceed through a final round of cell division in the absence cytokinesis that results in binucleation of &gt;95% of adult cardiomyocytes. Nearly all subsequent increase in myocardial mass is due to myocardial hypertrophy, with extremely low number of new cardiomyocytes being produced throughout post‐natal life. Despite the importance of this phenomenon, little is known about the molecular/genetic basis, especially with regard to the role of micro‐RNAs, for the transition from hyperplastic to hypertrophic‐based myocardial growth.We hypothesize a specific perinatal heart micro‐RNA‐mediated gene program is necessary for the normal transition from a fetal heart to an adult heart gene program.To identify the molecular mechanisms and genetic pathways involved in cardiac myocyte differentiation, RNA was isolated from E19, and 1, 3, 5, 7, 10 and 35‐day old mouse hearts (n=9 hearts/time point pooled). Cardiomyocyte micro‐RNA profiles (n=3 arrays/time point) were measured and bioinformatic analysis was used to identify genes that are transiently and significantly changing (p&lt;0.05, fold change &gt;1.5) during the perinatal period.Our analysis identified microRNA‐205 as a candidate for playing a role in the cardiac transitional program. Previous studies have shown a global knockout of miR‐205 to be neonatally lethal. We observe a transient 20‐fold increase in miR‐205 expression between day 1 and day 5 of post‐natal life, with levels returning to baseline by day 10. In‐situ hybridization revealed miR‐205 expression to be restricted to the epicardium of the heart.Mice harbouring a cardiomyocyte‐specific deletion of miR‐205 using αMHC‐Cre are born healthy with expected Mendelian ratios, and develop through the neonatal period normally. Hearts collected from adult mice show signs of abnormal growth and hypertrophy, up to 50% larger than controls. Previous studies demonstrated that miR‐205 directly targets YAP within the evolutionarily conserved hippo pathway that controls organ size. Hearts lacking miR‐205 exhibit a substantial increase in YAP protein expression. Increased cardiac size has been demonstrated in a constitutively active YAP transgenic mouse model. We conclude that miR‐205 plays a direct role in regulating post‐natal heart size through direct modulation of the Hippo pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vangl2 is essential for myocardial remodeling activated by Wnt/JNK signaling

The Wnt/JNK pathway, responsible for tissue polarity in cardiogenesis in vertebrates, has been shown to play numerous roles during differentiation and development of cardiac myocytes. Van Gogh-like-2 (Vangl2) is a core component that regulates the induction of polarized cellular and tissue morphology during animal development. However, little is known about Wnt/JNK signaling pathway in the process of myocardial remodeling. In present study, we found that activation of Wnt/JNK signaling by Wnt5a stimulates enlargement of cardiomyocyte surface area. The hypertrophic features were inhibited in Vangl2 depleted cells. Meanwhile, Wnt/JNK activation induced cytoskeleton rearrangement but failed to activate these effects in cells lacking Vangl2. Moreover, Wnt/JNK activation significantly increased the cell apoptosis by mediating the mitochondrial permeability transition pore (mPTP) dysfunction, whereas knockdown of Vangl2 partly reversed these effects. These results suggest that activation of Wnt/JNK signaling stimulates myocardial remodeling (cell morphological changes, apoptosis and mitochondrial dysfunction), in which Vangl2 may play an essential role.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.

Regulation of cardiac myocyte cell death and differentiation by myocardin.

Myocardin is a cardiac- and smooth muscle-enriched transcriptional co-activator that was originally described as an interacting partner of the serum response factor. Shortly after myocardin's discovery, a wealth of published literature described the role of myocardin as a regulator of smooth muscle differentiation and phenotype modulation, while gene-targeting studies confirmed the essential role of myocardin in vascular development. More recently, myocardin has been implicated as an important regulator of cardiac myocyte differentiation in studies demonstrating direct programming of fibroblasts towards the cardiac lineage. This function of myocardin has been attributed to its physical interaction with cardiac-enriched transcription factors such as MEF2C, GATA4, and TBX5. Moreover, conditional knockout models have revealed a critical role for myocardin during cardiac chamber maturation, and a surprising function for myocardin in the regulation of cardiomyocyte proliferation, cell death, and possibly mitochondrial function. This review summarizes the literature surrounding the cardiac-specific roles of myocardin during development and post-natal cardiac remodeling. In addition, we take a bioinformatics and computational approach to discuss known and predicted interactions and biological functions of myocardin, which suggests areas for future research.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols.

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.

Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation

INTRODUCTION: Protein acetylation is an important regulator of cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases its ability to open the mitochondria permeability transition pore (PTP), which is an important regulator of mitochondrial metabolism and myocyte differentiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mitochondrial structure and function, and myocyte differentiation in the developing heart. METHODS: We use immunoblotting and immunoprecipitation to determine the global protein acetylation state and that of CyPD during embryonic cardiac development in wild type (WT) and CyPD-null mice. We also re-expressed WT and mutant CyPD in CyPD-null embryonic and neonatal cardiac myocyte cultures to determine effects on mitochondrial structure and function and myocyte differentiation. RESULTS: Immunoblotting revealed that global protein acetylation was high in the embryo but fell during later development. In contrast, CyP-D was highly acetylated in the early embryonic heart, but this decreased dramatically during subsequent development. When re-expressed in cultures of CyPD-null myocytes, WT CyPD recapitulated the WT cell phenotype of fragmented mitochondrial structure, low mitochondrial membrane potential (Δψm), and decreased differentiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had similar or greater effects on these parameters than WT CyPD, while an inactivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. CONCLUSION: These data suggest that myocyte differentiation during cardiac development is associated with a decrease in overall protein acetylation, perhaps because of the changes that occur in metabolism as the heart develops. However, specific acetylation of CyPD appears to be particularly important for maturation of mitochondrial function and regulation of cardiac myocyte differentiation.

Preventing permeability transition pore opening increases mitochondrial maturation, myocyte differentiation and cardiac function in the neonatal mouse heart

In embryonic myocytes, closure of the mitochondrial permeability transition pore (PTP) drives mitochondrial maturation and cardiac myocyte differentiation. Since neonatal cardiac myocytes remain relatively immature, we hypothesized that inducing PTP closure at this age, by inhibiting the PTP regulator, cyclophilin D (CyPD), genetically or with Cyclosporin A (CsA) and NIM811, would increase cardiac function by increasing mitochondrial maturation and myocyte differentiation. Cultured neonatal myocytes or neonatal mice were treated for 5 d with vehicle, CsA or NIM811. Mitochondrial function and structure were measured in vitro. Myocyte differentiation was assessed by immunolabeling for contractile proteins. Cardiac function was determined using echocardiography. The probability of PTP opening was high in WT neonatal myocytes. Treatment with CsA or NIM811 in vitro increased mitochondrial structural complexity and membrane potential, decreased reactive oxygen species levels, and increased myocyte differentiation. WT mice treated with either CsA or NIM811 in vivo for the first 5 d of life had higher ejection fractions. Deleting CyPD had similar effects as CsA and NIM811 on all parameters. It may be feasible to inhibit the PTP using available drugs to increase mitochondrial maturation, myocyte differentiation, and cardiac function in neonates.

Epigenetic Mechanisms: New Targets for Heart Failure Pharmacopuncture.

Pharmacopuncture treatments are proposed for cardiovascular disease and heart failure [1]. Heart failure results from complex environment-gene interactions. β-adrenergic receptors and protein kinase A (PKA) interact with histones and related segments of deoxyribonucleic acid (DNA) at promoter regions of genes for messenger ribo nucleic acid (mRNA) transcription. This increases cardiac muscle mass and contractility in normal hearts. In heart failure, activation of β-adrenergic receptors and PKA promote pathological hypertrophy and decreased contractility. Experimental models show that prenatal exposure to hypoxia, cocaine, or nicotine increases susceptibility to heart failure when animals reach adulthood. Hypermethylation of DNA is an epigenetic mechanism associated with downregulation of the protein kinase C (PKC) gene in such models of heart failure. Downregulation of PKC is also produced by the stress-related hormone norepinephrine with upregulation of the hypoxia-inducible differentiation regulator Nix in norepinephrine-induced cardiac fibrosis [2]. Norepinephrine is also the main mediator of sympathetic neural activity. Sympathetic neural overactivity, a significant cofactor in human heart failure, is, therefore, implicated as a cofactor in this epigenetic mechanism for heart failure. Other epigenetic mechanism for cardiac hypertrophy and heart failure involve endothelin- 1 induced downregulation of the cardiac myocyte differentiation factor RE1-silencing transcription signaler (REST) and GATA zinc-finger domain-containing protein-1 (GATAD1) induced inhibition of histone deacetylase (HDAC-2) in cardiac myocytes harvested from autosomal-recessive dilated cardiac myopathy patients with heart failure [3]. In contrast, both the HDAC-2 in cardiac myocytes and the HDAC-1 in cardiac fibroblasts are upregulated in experimental animal models of congestive heart failure [4]. A prominent role for inflammation in heart failure is suggested by tumor necrosis factor (TNF-α), a proinflammatory cytokine, inducing hypermethylation of the sarcoplasmic reticulum calcium ATPase (SERCA-2A) gene in cardiac myocytes, associated with diastolic dysfunction and heart failure. An important role for epigenetic mechanisms in heart failure is also suggested by HDAC-dependent stimulation of the stress-apoptosis intracellular signaling pathway, which induces hypertrophy of both cardiac and vascular smooth muscle [5].

Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes

AimsMutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells. Methods and resultsWe generated a human embryonic stem cell (hESC) line carrying a naturally occurring mutation of MYPBC3 (c.2905 +1 G >A) to study HCM pathogenesis during cardiac differentiation. HCM-specific hESC-derived cardiomyocytes (hESC-CMs) displayed hallmark aspects of HCM including sarcomere disarray, hypertrophy and impaired calcium impulse propagation. HCM hESC-CMs presented a transient haploinsufficiency of cMyBP-C during cardiomyocyte differentiation, but by day 30 post-differentiation cMyBP-C levels were similar to control hESC-CMs. Gene transfer of full-length MYBPC3 during differentiation prevented hypertrophy, sarcomere disarray and improved calcium impulse propagation in HCM hESC-CMs. Conclusion(s)These findings point to the critical role of MYBPC3 during sarcomere assembly in cardiac myocyte differentiation and suggest developmental influences of MYBPC3 truncating mutations on the mature hypertrophic phenotype.

Abstract 110: The Estrogen-related Receptor Gamma Promotes Maturation of iPSC-derived Cardiac Myocytes

The mammalian heart has a tremendous energy requirement to support normal contractile function. Recently, our group discovered that the estrogen-related receptor γ (ERRγ) coordinately controls oxidative energy metabolism and the type I fiber program in skeletal muscle. We hypothesized that this circuit is also operative in heart, serving to match capacity to produce ATP with energy utilizing processes such as contraction and ion transport. To identify downstream targets and pathways of ERRγ in cardiac myocytes, RNA-sequencing (RNA-seq) was conducted in neonatal rat ventricular myocytes (NRVMs) overexpressing ERRγ. ERRγ induced the expression of genes involved in mitochondrial oxidative phosphorylation, fatty acid oxidation, and the TCA cycle. Interestingly, ERRγ also induced expression of a broad program of adult cardiac contractile and structural genes including Myh6 (αMHC) and Ca 2+ handling genes such as Atp2a2 (SERCA2A). ERRγ-mediated activation of adult genes occurred concomitant with the suppression of fetal cardiac and embryonic/perinatal fast skeletal isoform contractile protein gene expression including Myh7 (βMHC), Myl1 and Myh3/8 . These results prompted us to explore the role of ERRγ in differentiation and maturation of human iPS-derived cardiac myocytes (iPSC-CM). The expression level of ERRγ was markedly upregulated during the transition to cardiac lineage commitment in parallel with induction of cardiogenic transcription factors (e.g. NKX2.5 and GATA4). Forced expression of ERRγ in iPSC-CM activated the broad adult cardiac myocyte program including oxidative metabolism and adult contractile gene expression. Of note, ERRγ increased MYH6 expression while decreasing expression of MYH7 and the fetal gene markers NPPA (ANF) and NPPB (BNP). The expression of TNNI3 , thought to be a marker of mature adult cardiomyocytes, was also induced by ERRγ. Conversely, CRISPR-Cas9n deletion of ERRγ in iPSC-CM resulted in diminished mitochondrial energy metabolic and adult cardiac contractile gene expression. These data provide evidence that ERRγ is a key nodal regulator of cardiac myocyte determination and terminal maturation. ERR signaling is a candidate target for driving iPSC-CM to the “adult” cardiac myocyte phenotype.

Identifying circulating microRNAs as biomarkers of cardiovascular disease: a systematic review.

The aim of the present study is to identify microRNAs (miRs) with high potential to be used as biomarkers in plasma and/or serum to clinically diagnose, or provide accurate prognosis for survival in, patients with atherosclerosis, coronary artery disease, and acute coronary syndrome (ACS). A systematic search of published original research yielded a total of 72 studies. After review of the risk of bias of the published studies, according to Cochrane Collaboration and the QUADUAS Group standards, 19 studies were selected. Overall 52 different miRs were reported. In particular, miR-133a/b (5 studies), miR-208a/b (6 studies), and miR-499 (7 studies) were well studied and found to be significant diagnostic and/or prognostic markers across different cardiovascular disease progression stages. miR-1 and miR-145b are potential biomarkers of ACS; miR-1 with higher sensitivity for all acute myocardial infarction (AMI), and miR-145 for STEMI and worse outcome of AMI. But when miRs were studied across different ACS study populations, patients had varying degrees of coronary stenosis, which was identified as an important confounder that limited the ability to quantitatively pool the study results. The identified miRs were found to regulate endothelial function and angiogenesis (miR-1, miR-133), vascular smooth muscle cell differentiation (miR-133, miR-145), communication between vascular smooth muscle and endothelial cell to stabilize plaques (miR-145), apoptosis (miR-1, miR-133, miR-499), cardiac myocyte differentiation (miR-1, miR-133, miR-145, miR-208, miR-499), and to repress cardiac hypertrophy (miR-133). Their role in these processes may be explained by regulation of shared RNA targets such as cyclin-dependent kinase inhibitor 1A (or p21), ETS proto-oncogene 1, fascin actin-bundling protein 1, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4, insulin-like growth factor 1 receptor LIM and SH3 protein 1, purine nucleoside phosphorylase, and transgelin 2. These mechanistic data further support the clinical relevance of the identified miRs. miR-1, miR-133a/b, miR-145, miR-208a/b, and miR-499(a) in plasma and/or serum show some potential for diagnosis of cardiovascular disease. However, biased selection of miRs in most studies and unexplained contrasting results are major limitations of current miR research. Inconsistencies need to be addressed in order to definitively identify clinically useful miRs. Therefore, this paper presents important aspects to improve future miR research, including unbiased selection of miRs, standardization/normalization of reference miRs, adjustment for patient comorbidities and medication, and robust protocols of data-sharing plans that could prevent selective publication and selective reporting of miR research outcomes.

Permeability Transition Pore Closure Increases Mitochondrial Maturation and Myocyte Differentiation in the Neonatal Heart

Background: Recent studies in embryonic mice demonstrate that mitochondrial maturation and cardiac myocyte differentiation are linked. Closure of the mitochondrial permeability transition pore (mPTP), by pharmacologically or genetically manipulating Cyclophilin D (CyPD), drives this process of differentiation. Myocytes in the neonatal heart are relatively immature, but the effects of mPTP closure have not been determined at this developmental stage.Objective: We hypothesized that closure of the mPTP in the neonatal mouse heart will increase cardiac function by increasing mitochondrial maturation and enhancing cardiac myocyte differentiation.Methods: In vitro: Cardiac myocytes cultured from the ventricles of 1-day old wild type (WT) or CyPD null mice were treated with Vehicle (Veh), 500nM Cyclosporin A (CsA), or 500nM NIM811 for 5 days. Assays to determine mitochondrial maturation, both structure and function, and cardiac myocyte differentiation were performed. In vivo: One day old WT or CyPD null mice were treated with daily intraperitoneal injections of Veh, or 10mg/kg of CsA or NIM811 for 5 days. Hearts were processed then analyzed for changes in ventricular wall thickness.Results: In vitro: WT neonatal cardiac myocytes treated with either CsA or NIM811 had more complex mitochondrial structure (P < 0.05), higher mitochondrial membrane potential (NIM811, P <0.01), and more differentiated cardiac myocytes relative to Veh (p<0.05). Deleting CyPD to close the mPTP also increased mitochondrial maturation and myocyte differentiation. In vivo: WT mice hearts treated with NIM811 had thicker left ventricular walls relative to Veh (P < 0.01).Conclusion: Closure of the mPTP using pharmacologic or genetic approaches increased mitochondrial maturation and cardiac myocyte differentiation in cultured neonatal myocytes. Early in vivo experiments demonstrate that mPTP closure in the neonatal heart increased LV wall, suggesting altered myocyte proliferation or differentiation.