Mature Cardiomyocytes Research Articles

Epigenetic Regulation of Cardiomyocyte Maturation by Arginine Methyltransferase CARM1.

During the neonatal stage, the cardiomyocyte undergoes a constellation of molecular, cytoarchitectural, and functional changes known collectively as cardiomyocyte maturation to increase myocardial contractility and cardiac output. Despite the importance of cardiomyocyte maturation, the molecular mechanisms governing this critical process remain largely unexplored. We leveraged an in vivo mosaic knockout system to characterize the role of Carm1, the founding member of protein arginine methyltransferase, in cardiomyocyte maturation. Using a battery of assays, including immunohistochemistry, immuno-electron microscopy imaging, and action potential recording, we assessed the effect of loss of Carm1 function on cardiomyocyte cell growth, myofibril expansion, T-tubule formation, and electrophysiological maturation. Genome-wide transcriptome profiling, H3R17me2a chromatin immunoprecipitation followed by sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing were used to investigate the mechanisms by which CARM1 (coactivator-associated arginine methyltransferase 1) regulates cardiomyocyte maturation. Finally, we interrogated the human syntenic region to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks for single-nucleotide polymorphisms associated with human heart diseases. We report that mosaic ablation of Carm1 disrupts multiple aspects of cardiomyocyte maturation cell autonomously, leading to reduced cardiomyocyte size and sarcomere thickness, severe loss and disorganization of T tubules, and compromised electrophysiological maturation. Genomics study demonstrates that CARM1 directly activates genes that underlie cardiomyocyte cytoarchitectural and electrophysiological maturation. Moreover, our study reveals significant enrichment of human heart disease-associated single-nucleotide polymorphisms in the human genomic region syntenic to the H3R17me2a chromatin immunoprecipitation followed by sequencing peaks. This study establishes a critical and multifaceted role for CARM1 in regulating cardiomyocyte maturation and demonstrates that deregulation of CARM1-dependent cardiomyocyte maturation gene expression may contribute to human heart diseases.

Cfp1 Controls Cardiomyocyte Maturation by Modifying Histone H3K4me3 of Structural, Metabolic, and Contractile Related Genes.

Cardiomyocyte maturation is the final stage of heart development, and abnormal cardiomyocyte maturation will lead to serious heart diseases. CXXC zinc finger protein 1 (Cfp1), a key epigenetic factor in multi-lineage cell development, remains underexplored in its influence on cardiomyocyte maturation. This study investigates the role and mechanisms of Cfp1 in this context. Cardiomyocyte-specific Cfp1 knockout (Cfp1-cKO) mice died within 4 weeks of birth. Cardiomyocytes derived from Cfp1-cKO mice showed an inhibited maturation phenotype, characterized by structural, metabolic, contractile, and cell cycle abnormalities. In contrast, cardiomyocyte-specific Cfp1 transgenic (Cfp1-TG) mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing Cfp1 displayed a more mature phenotype. Mechanistically, deficiency of Cfp1 led to a reduction in trimethylation on lysine 4 of histone H3 (H3K4me3) modification, accompanied by the formation of ectopic H3K4me3. Furthermore, Cfp1 deletion decreased the level of H3K4me3 modification in adult genes and increased the level of H3K4me3 modification in fetal genes. Collectively, Cfp1 modulates the expression of genes crucial to cardiomyocyte maturation by regulating histone H3K4me3 modification, thereby intricately influencing the maturation process. This study implicates Cfp1 as an important molecule regulating cardiomyocyte maturation, with its dysfunction strongly linked to cardiac disease.

A Strongly Robust Chitosan‐Based Programmed Control Functional Hydrogel Improved Mitochondrial Function and Pro‐Vascularization for Adaptive Repair of Myocardial Infarction

AbstractHydrogel‐based engineered cardiac patches (ECP) hold great promise as potential treatment options for myocardial infarction (MI). However, optimizing the preparation of ECP with better biocompatibility, mechanical stability, and adaptation to MI repair remains a challenge. In this study, a chitosan (CS) hydrogel with good mechanical robustness through programmed control of hydrogen bonding is constructed to adapt to the continuous beating of myocardial tissue. With the synergistic effects of lipoic acid (TA), proanthocyanidins (PAs), and Eu3+, a functional platform capable of improving mitochondrial function, antioxidation, and pro‐vascularization is further constructed for the adaptive repair of the MI microenvironment. The fabricated functionalized chitosan hydrogel (CS/TA@PAs‐Eu) possessed good mechanical stability and ionic conductivity, showing the potential for long‐term adaptation to myocardial tissue pulsation. Also, the CS/TA@PAs‐Eu hydrogel promoted cardiomyocytes (CMs) maturation and functionalization, and effectively improved mitochondrial function, scavenged reactive oxygen species (ROS) as well as promoted angiogenesis. Animal studies indicated that the CS/TA@PAs‐Eu hydrogel can perform adaptive repair of MI to prevent left ventricular (LV) remodeling and restore cardiac function. This study highlights a functionalized hydrogel ECP with good biocompatibility and mechanical robustness for the adaptive repair of MI.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility withimmunostaining.

Highly Durable, Stretchable Multielectrode Array for Electro-mechanical Co-stimulation of Cells.

Electro-mechanical co-stimulation of cells can be a useful cue for tissue engineering. However, reliable co-stimulation platforms still have limitations due to low durability of the components and difficulty in optimizing the stimulation parameters. Although various electro-mechanical co-simulation systems have been explored, integrating materials and components with high durability is still limited. To tackle this problem, we designed an electro-mechanical co-stimulation system that facilitates uniaxial cyclic stretching, electrical stimulation, and optical monitoring. This system utilizes a robust and autoclavable stretchable multielectrode array housed within a compact mini-incubator. To illustrate its effectiveness, we conducted experiments that highlighted how electro-mechanical co-stimulation using this system can enhance the maturation of cardiomyocytes derived from human induced pluripotent stem cells. The results showed great potential of our co-stimulation platform as an effective tool for tissue engineering.

Cypher/ZASP drives cardiomyocyte maturation via actin-mediated MRTFA-SRF signalling.

Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.

Compact lens-free imager using a thin-film transistor for long-term quantitative monitoring of stem cell culture and cardiomyocyte production.

With advancements in human induced pluripotent stem cell (hiPSC) technology, there is an increasing demand for quality control techniques to manage the long-term process of target cell production effectively. While monitoring systems designed for use within incubators are promising for assessing culture quality, existing systems still face challenges in terms of compactness, throughput, and available metrics. To address these limitations, we have developed a compact and high-throughput lens-free imaging device named INSPCTOR. The device is as small as a standard culture plate, which allows for the installation of multiple units within an incubator. INSPCTOR utilises a large thin-film transistor image sensor, enabling simultaneous observation of six independent culture environments, each approximately 1 cm2. With this device, we successfully monitored the confluency of hiPSC cultures and identified the onset timing of epithelial-to-mesenchymal transition during mesodermal induction. Additionally, we quantified the beating frequency and conduction of hiPSC-derived cardiomyocytes by using high-speed imaging modes. This enabled us to identify the onset of spontaneous beating during differentiation and assess chronotropic responses in drug evaluations. Moreover, by tracking beating frequency over 10 days of cardiomyocyte maturation, we identified week-scale and daily-scale fluctuations, the latter of which correlated with cellular metabolic activity. The metrics derived from this device would enhance the reproducibility and quality of target cell production.

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Striated preferentially expressed gene deficiency leads to mitochondrial dysfunction in developing cardiomyocytes.

A deficiency of striatedpreferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg-/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg-/- hearts. Imaging of E18.5 Speg-/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg-/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg-/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α.

Carbon nanotube nanocomposite scaffolds: advances in fabrication and applications for tissue regeneration and cancer therapy.

Carbon nanotube (CNT) nanocomposite scaffolds have emerged as highly promising frameworks for tissue engineering research. By leveraging their intrinsic electrical conductivity and valuable mechanical properties, CNTs are commonly dispersed into polymers to create robust, electrically conductive scaffolds that facilitate tissue regeneration and remodeling. This article explores the latest progress and challenges related to CNT dispersion, functionalization, and scaffold printing techniques, including electrospinning and 3D printing. Notably, these CNT scaffolds have demonstrated remarkable positive effects across various cell culture systems, stimulating neuronal growth, promoting cardiomyocyte maturation, and facilitating osteocyte differentiation. These encouraging results have sparked significant interest within the regenerative medicine field, including neural, cardiac, muscle, and bone regenerations. However, addressing the concern of CNT cytotoxicity in these scaffolds remains critical. Consequently, substantial efforts are focused on exploring strategies to minimize cytotoxicity associated with CNT-based scaffolds. Moreover, researchers have also explored the intriguing possibility of utilizing the natural cytotoxic properties of CNTs to selectively target cancer cells, opening up promising avenues for cancer therapy. More research should be conducted on cutting-edge applications of CNT-based scaffolds through phototherapy and electrothermal ablation. Unlike drug delivery systems, these novel methodologies can combine 3D additive manufacturing with the innate physical properties of CNT in response to electromagnetic stimuli to efficiently target localized tumors. Taken together, the unique properties of CNT-based nanocomposite scaffolds position them as promising candidates for revolutionary breakthroughs in both regenerative medicine and cancer treatment. Continued research and innovation in this area hold significant promise for improving healthcare outcomes.

The H2Bub1-deposition complex is required for human and mouse cardiogenesis.

De novo variants affecting monoubiquitylation of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, how H2Bub1 affects cardiogenesis is unknown. We show that the H2Bub1-deposition complex (RNF20-RNF40-UBE2B) is required for mouse cardiogenesis and for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific Rnf20 deletion are embryonic lethal and have abnormal myocardium. We then analyzed H2Bub1 marks during differentiation of human iPSCs into cardiomyocytes. H2Bub1 is erased from most genes at the transition from cardiac mesoderm to cardiac progenitor cells but is preserved on a subset of long cardiac-specific genes. When H2Bub1 is reduced in iPSC-derived cardiomyocytes, long cardiac-specific genes have fewer full-length transcripts. This correlates with H2Bub1 accumulation near the center of these genes. H2Bub1 accumulation near the center of tissue-specific genes was also observed in embryonic fibroblasts and fetal osteoblasts. In summary, we show that normal H2Bub1 distribution is required for cardiogenesis and cardiomyocyte differentiation, and suggest that H2Bub1 regulates tissue-specific gene expression by increasing the amount of full-length transcripts.

Biophysical properties of NaV1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes expressed higher atrial- and sinoatrial-specific markers (MYL7, CACNA1D) and lower ventricular-specific markers (MYL2, CACNA1C, GJA1) than ventricular cardiomyocytes. The amplitude, duration, and steady-state phase of APs in atrial cardiomyocytes decreased, and had a shape similar to that of mature atrial cardiomyocytes. Interestingly, NaV1.5 channels in atrial cardiomyocytes exhibited lower mRNA transcripts and protein expression, which could explain the lower current densities recorded by patch-clamp. Moreover, Na+ currents exhibited differences in activation and inactivation parameters. These differences could be explained by an increase in SCN2B regulatory subunit expression and a decrease in SCN1B and SCN4B regulatory subunit expressions. Our results show that a RA treatment made it possible to obtain atrial cardiomyocytes and investigate differences in NaV1.5 channel properties between ventricular- and atrial-like cells.

Direct contractility measurement in cardiomyocytes via thin PDMS diaphragm integration with a moving coil actuator: Synergistic maturation through combined topographical and mechanical stimulation

Mechanical stimulation within diaphragm-based cell culture platforms presents a promising strategy to enhance cardiomyocyte maturation. However, most of the proposed techniques have met with limited success due to their inability to directly measure the contractile force of cardiomyocytes. This issue primarily stems from the typically thicker diaphragm structure which is necessary to withstand high strain ranges induced by mechanical stimulation. Herein, we propose a moving coil actuator integrated with a thin polydimethylsiloxane (PDMS) diaphragm for precise mechanical stimulation, cell culture, and drug screening applications. This mechanical stimulation system generates desired mechanical deformation to cultured cardiomyocytes without compromising the integrity of the diaphragm structure. The 10 µm thin diaphragm produces significant displacement from the contractility of cultured cardiomyocytes, which can be directly measured using a laser vibrometer system. Cardiomyocytes cultured on nanogroove PDMS diaphragms with and without stimulation and flat diaphragms with stimulation exhibited 4.25-, 2.81-, 2.39-fold enhancements in contraction force and 1.09-, 1.06-, 1.04-, and 1.32-, 1.17-, 1.16-fold enhancements in sarcomere lengths and Connexin 43 (Cx43) compared to counterparts cultured on flat and nanogroove PDMS diaphragms without stimulation. The practicality of the device as a drug screening platform is demonstrated by examining the drug-induced effects on cardiomyocytes using cardiovascular drugs.

Bridging the Translational Gap in Heart Failure Research: Using Human iPSC-derived Cardiomyocytes to Accelerate Therapeutic Insights.

Heart failure (HF) remains a leading cause of death worldwide, with increasing prevalence and burden. Despite extensive research, a cure for HF remains elusive. Traditionally, the study of HF's pathogenesis and therapies has relied heavily on animal experimentation. However, these models have limitations in recapitulating the full spectrum of human HF, resulting in challenges for clinical translation. To address this translational gap, research employing human cells, especially cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs), offers a promising solution. These cells facilitate the study of human genetic and molecular mechanisms driving cardiomyocyte dysfunction and pave the way for research tailored to individual patients. Further, engineered heart tissues combine hiPSC-CMs, other cell types, and scaffold-based approaches to improve cardiomyocyte maturation. Their tridimensional architecture, complemented with mechanical, chemical, and electrical cues, offers a more physiologically relevant environment. This review explores the advantages and limitations of conventional and innovative methods used to study HF pathogenesis, with a primary focus on ischemic HF due to its relative ease of modeling and clinical relevance. We emphasize the importance of a collaborative approach that integrates insights obtained in animal and hiPSC-CMs-based models, along with rigorous clinical research, to dissect the mechanistic underpinnings of human HF. Such an approach could improve our understanding of this disease and lead to more effective treatments.

Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development

Cardiomyocytes are highly metabolic cells responsible for generating the contractile force in the heart. During fetal development and regeneration, these cells actively divide but lose their proliferative activity in adulthood. The mechanisms that coordinate their metabolism and proliferation are not fully understood. Here, we study the role of the transcription factor NFYa in developing mouse hearts. Loss of NFYa alters cardiomyocyte composition, causing a decrease in immature regenerative cells and an increase in trabecular and mature cardiomyocytes, as identified by spatial and single-cell transcriptome analyses. NFYa-deleted cardiomyocytes exhibited reduced proliferation and impaired mitochondrial metabolism, leading to cardiac growth defects and embryonic death. NFYa, interacting with cofactor SP2, activates genes linking metabolism and proliferation at the transcription level. Our study identifies a nodal role of NFYa in regulating prenatal cardiac growth and a previously unrecognized transcriptional control mechanism of heart metabolism, highlighting the importance of mitochondrial metabolism during heart development and regeneration.

Full cardiac regeneration by MicroRNA and G-actin monomer binding protein

Abstract Myocardial infarction causes massive loss of cardiomyocytes, leading to high mortality rate and heart failure. Cardiac regeneration is thought to have therapeutic potential for myocardial infarction and heart failure. We recently found that Nkx2.5+ cardiac myoblasts exist in declining number in the hearts of postnatal mice. The Nkx2.5+ cardiac myoblasts reactivate after myocardial infarction. They reside mostly in the subepicardium. Using inducible Nkx2.5 enhancer-Cre/ROSA26 reporter double transgenic mice to lineage trace the fate of activated Nkx2.5+ cardiac myoblasts, we documented that the activated Nkx2.5+ cardiac myoblasts proliferate and differentiate into mature cardiomyocyte in vivo. We also documented that Nkx2.5+ cardiac myoblasts originate from the embryonic epicardial cells. Thymosin ß4 is a G-actin monomer binding protein, involved in cell proliferation, migration, and differentiation. We hypothesized that Thymosin ß4 would promote Nkx2.5+ cardiac myoblasts, an embryonic epicardial-derived cells, mobilization and cardiomyogenesis, after myocardial injury. Various microRNAs (miR) have been reported to control cardiac development and promote cardiac regeneration. We have transfected neonatal Nkx2.5 cardiac myoblasts with various microRNAs and found miR-590 markedly promotes cardiomyogenesis capacity of cardiac progenitor cells. We have proved that miR-590 plus Thymosin ß4 (intraperitoneal injection) markedly improved murine cardiac function after myocardial infarction. In the treatment group with miR-590 plus Thymosin ß4 (n=9), LVEF improved from 45% (Day 0) to 77% (Day 7), to 83% (Day 28), to 85% (Day 56) with no LV regional wall motion abnormality and minimal aneurysm formation. In comparison, the control group with miR-mimic plus PBS (n=7), LVEF was 50% (Day 0) to 51% (Day 7), to 56% (Day 28), to 51% (Day 56) with persistent hypokinesia at apex and anterior wall with impaired LV contractility and larger aneurysm was revealed (p<0.001). Heart section of the post-MI mouse receiving miR-590 plus Thymosin ß4 showed minimal fibrosis in the outer layer of myocardium. In contrast, large fibrotic area was noted in the no treatment group. The mice receiving miR-590 plus Thymosin ß4 had a 2-fold increase in survival rate than the control group. Enhanced cardiomyocytes proliferation was documented by phosphorylated histone H3 stain at treated 1-week post-MI heart section. The study applies the results of our recent studies and successfully demonstrated systemic administration of miR-590 plus Thymosin ß4 markedly enhance cardiac repair/ regeneration. We anticipate the result would promote cardiac regeneration in clinical use.

The role of latrophilin-2 in maintaining cardiac cell-to-cell junctions and its implications for dilated cardiomyopathy and sudden death

Abstract Background Identifying genes that are important for maintaining heart function and characterizing their roles and pathways are essential for developing therapeutic approaches. Latrophilin-2 (LPHN2), a relatively unknown GPCR, critically contributes to differentiation from pluripotent stem cells (PSC) to the cardiac lineage, but it is not known whether LPHN2 is active in the adult heart. Purpose In this study, we explored the functions of LPHN2 in adult hearts. Methods and Results To determine whether there is an association between variation in LPHN2 and cardiac phenotypes in humans, we used gnomAD and UK Biobank data. The data suggested that LPHN2 variants could be fatal. Accordingly, we developed a mouse model for further analysis of Lphn2. Deletion of Lphn2 causes embryonic lethality, so we generated a cardiac-specific, tamoxifen-inducible Lphn2 conditional knockout mouse (Lphn2icKO mouse) by cross-breeding Lphn2flox/flox mice with the TMX-inducible Cre recombinase (MerCreMer) under control of the α-Myh6 (α-myosin heavy chain) promoter mice. After administering tamoxifen, we found that the left ventricle became dilated and the systolic function was decreased, and gaps were created between cardiomyocytes. We also observed ventricular tachycardia and atrioventricular block associated with the dilated cardiomyopathy and subsequent sudden cardiac death. Cell-to-cell junctions (adherence, gap, and desmosomes,) were found to be dysregulated in the Lphn2icKO adult mouse heart and the heart at embryonic day 13.5 of Lphn2−/− (homozygous Lphn2 deficiency). To investigate Lphn2 function in a disease model, we generated a myocardial infarction (MI) in Lphn2icKO mice. MI with tamoxifen group showed wide QRS tachycardia and various types of block, and a survival reduction of 40% compared with that of the MI group. Regarding underlying mechanisms, we found that PGC-1α, a factor downstream of p38-MAPK that contributes to arrhythmia and maturation of PSC-derived cardiomyocytes, was downregulated (Figure). Conclusions LPHN2 is a key molecule that maintain adult heart integrity via its effects on cell-to-cell junctions, and the deficiency of Lphn2 exacerbates heart failure. Our findings provide evidence in support of LPHN2 being a pivotal regulator of heart function through the p38-MAPK-PGC-1α pathway.Schematic illustration

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling

BackgroundCardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation.MethodshiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli.ResultsTreatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features—including enhanced Ca2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells.ConclusionAMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

Abstract 11679: The Crucial Role of Estrogen-Related Receptor in Human Cardiomyocyte Maturation

During the developmental transition from fetal to adult, the heart undergoes dramatic mitochondrial and sarcomeric maturation. This process is partially reversed during development of heart failure. We have identified the nuclear receptors, estrogen-related receptor (ERR) α and γ, as critical transcriptional regulators of energy metabolic and sarcomeric genes during postnatal maturation of mouse heart. Here, we aimed to define the transcriptional mechanisms involved in ERR-mediated maturation in human cardiomyocytes. Genomic interrogation with published datasets revealed that approximately 50% of super-enhancer regions (SEs) overlapped with ERRγ peaks in human-induced pluripotent stem cell-derived cardiac myocytes. Intersection of the ERRγ cistrome with a published H3K27ac Hi-ChIP dataset from human hearts identified over 3,000 ERR-containing enhancer-promoter loops, which are associated with genes related to cardiac contractile function. These results highlighted the essential role of ERR in cardiomyocyte enhancer function. Among the SEs defined by mediator complex deposition, we found that both ERRα and γ occupied the enhancer region flanking the MYH6/MYH7 cluster, a well-characterized locus for fetal to adult developmental switching. Targeting ERR recruitment on the enhancer using catalytically-dead Cas9-KRAB or proteolysis targeting chimeric (PROTAC) strategies resulted in the reversal of MYH6/7 switching. RNA-sequencing analysis following PROTAC treatment revealed that deactivation of ERRα resulted in downregulation of a wide array of genes encoding cardiac structural proteins, particularly adult-type ventricular sarcomeric proteins. Additional cistrome analyses demonstrated that PROTAC treatment resulted in decreased ERRα peak signals on cardiac structural genes, including enhancer regions directing MYH6/7 transcription consistent with the observed gene expression changes. In summary, ERR signaling is essential for the transcriptional program of structural maturation in human cardiomyocytes. This process likely involves establishing proper cardiac chromatin architecture. These results support a novel therapeutic strategy of utilizing ERR activators to reverse fetal shifts in failing hearts.