Human Cardiomyocyte Function Research Articles

Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.

Minimal contribution of IP3R2 in cardiac differentiation and derived ventricular-like myocytes from human embryonic stem cells.

Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Identification of IRX5 transcription factor regulatory mechanisms in human cardiomyocyte function

In the heart, several transcription factors (TF) are powerful regulators of cardiac cell fate and organogenesis. Less well studied are the roles of TF in physiological functions. Several studies on animal models have shown that the Iroquois homeobox (Irx) TFs are key patterning and differentiation regulators that confer cell type-specific properties to specialized cardiac lineages. Importantly, studies in mice have shown that IRX5 is expressed very early during cardiogenesis and maintained in adult heart where it regulates ventricular electrophysiological properties, the repolarizing gradient. It has also been shown that IRX5 target-genes regulation occurs through recruitment of histone modifiers. In order to study IRX5 in human, we used cardiomyocytes differentiated from induced pluripotent stem cells (hiPS-CMs) generated from patients carrying loss of function mutations in IRX5 (IRX5Mut) and control individuals. As we detected IRX5 protein as early as in the pluripotent state, we also analyzed cells at hiPS stage in our experiments. High-throughput transcriptomic and epigenetic analyses were performed to obtain a global understanding of IRX5 function. The transcriptomic experiments allowed us to determine the entire set of differentially expressed genes in hiPS and hiPS-CMs IRX5Mut cells as compared to control cells. The epigenetic experiments, performed by ChIPseq, suggested that IRX5 binds to already open chromatin, and preferentially, to enhancer rich regions rather than proximal promoters. Combining these high-throughput experiments, we found direct target genes of IRX5 at hiPS-CM stage, and some of them are relevant to cardiac function. Notably, we showed that this TF regulates the expression of several actors of electrical conduction, such as SCN5A, at hiPS-CM stage. IRX5 seems to be an important factor for the regulation of human cardiac function, and more specifically of its electrical function.

LAMP-2B regulates human cardiomyocyte function by mediating autophagosome–lysosome fusion

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.

Abstract 534: Modulatory Effect of Nkx2.5+ Cardiomyoblast Secreted Exosomes in Cardiometabolism

It was found cardiac precursors secreted exosomes (CPC-exo) may have anti-apoptotic and anti-fibrotic actions to facilitate cardiac repair in the damaged hearts. It remains unclear the effect of CPC-exo in cardiometabolism. The aim of the study was to assess the modulatory action of CPC-exo in cardiometabolism of cardiomyopathic cardiomyocytes. Nkx2.5+ cardiomyoblasts were enzymatically isolated and were purified by FACS sorter from mouse embryonic stem cells under cardiomyogenic differentiation or transgenic mice hearts (2 weeks old) carrying GFP reporter driven by Nkx2.5-enhancer for the purification of the exosomes (mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo) from the culture medium by ExoQuick-TC. The vesicle size was further measured by Nanosight. The exosomal miRNA profile was characterized by miRNA microarray. It was further compared the different effect of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo in the mitochondrial function of human cardiomyocytes derived from iPSC of left ventricle non compaction (LVNC) patients (LVNC-hiPSC-CM) by Agilent seahorse XF24 analyzer. Cell shortening was measured by Ionoptix system. It was found the defect of cardiac mitochondrial function associated with the weak cell shortening in LVNC-hiPSC-CM on day 70 after differentiation. With the treatment of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo during day 50-70, the mitochondrial function and cell shortening of LVNC-hiPSC-CM were significantly improved on day 70. The functional exosomal miRNAs were characterized in advance in this study. In conclusion, CPC-exo may exert the benefit in the improvement of cardiometabolism in cardiomyopathic cardiomyocytes.

Abstract 504: LAMP-2B Regulates Human Cardiomyocyte Function by Mediating Autophagosome-lysosome Fusion

Autophagy plays a crucial role in cell homeostasis and function. Two types of autophagy have been well studied. Macroautophagy (referred to as autophagy hereafter) is mediated by double-membrane autophagosomes that enclose cytosolic cargoes, followed by fusion with late endosomes/lysosomes for degradation. Chaperone-mediated autophagy (CMA) is a process of chaperon-dependent selection of cytosolic proteins that are translocated into the lysosome for degradation. LAMP-2A, the A isoform of LAMP-2, is an essential receptor for CMA. However, alternative splicing of pre-LAMP-2 mRNA produces another two isoforms, LAMP-2B and LAMP-2C, with unclear functions. In non-cardiomyocytes, STX17 is required for autophagosome-lysosome fusion by interacting with SNAP29 and VAMP8. Whether cardiomyocytes (CMs) utilize the STX17-dependent mechanism to mediate autophagic fusion remains elusive. Using CMs derived from induced human pluripotent stem cells (hiPSC-CMs) and genome editing approaches, we identified previously undefined biological and molecular roles for LAMP-2B in the control of autophagosome-lysosome fusion. Remarkably, LAMP-2B functions independently of STX17. Instead, LAMP-2B interacts with ATG14 and VAMP8 through its C-terminal coiled-coil domain to promote autophagic fusion. Knockout of LAMP-2B in hiPSC-CMs decreased colocalization of ATG14 with VAMP8, and autophagosome-lysosome fusion. Forced expression of LAMP-2B in LAMP-2 deficient hiPSC-CMs restored autophagic signaling. In addition, LAMP-2B -deficient hiPSC-CMs recapitulated the metabolic and contractile defects observed in hiPSC-CMs derived from Danon patients. Danon disease represents a severe form of cardiomyopathy and has been associated with mutations in X-linked LAMP-2 gene. However, mechanisms by which LAMP-2 deficiency leads to cardiomyopathy are poorly understood. Given the similarity of phenotype between Danon and LAMP-2B knockout hiPSC-CMs, we conclude that LAMP-2B deficiency is sufficient and necessary to cause Danon cardiomyopathy. Therefore, our findings not only reveal a STX17-independent autophagic fusion mechanism mediated by LAMP-2B in human CMs, but also provide a molecular mechanism underlying Danon cardiomyopathy.

Iron deficiency impairs contractility of human cardiomyocytes through decreased mitochondrial function.

AimsIron deficiency is common in patients with heart failure and associated with a poor cardiac function and higher mortality. How iron deficiency impairs cardiac function on a cellular level in the human setting is unknown. This study aims to determine the direct effects of iron deficiency and iron repletion on human cardiomyocytes.Methods and resultsHuman embryonic stem cell‐derived cardiomyocytes were depleted of iron by incubation with the iron chelator deferoxamine (DFO). Mitochondrial respiration was determined by Seahorse Mito Stress test, and contractility was directly quantified using video analyses according to the BASiC method. The activity of the mitochondrial respiratory chain complexes was examined using spectrophotometric enzyme assays. Four days of iron depletion resulted in an 84% decrease in ferritin (P < 0.0001) and significantly increased gene expression of transferrin receptor 1 and divalent metal transporter 1 (both P < 0.001). Mitochondrial function was reduced in iron‐deficient cardiomyocytes, in particular ATP‐linked respiration and respiratory reserve were impaired (both P < 0.0001). Iron depletion affected mitochondrial function through reduced activity of the iron–sulfur cluster containing complexes I, II and III, but not complexes IV and V. Iron deficiency reduced cellular ATP levels by 74% (P < 0.0001) and reduced contractile force by 43% (P < 0.05). The maximum velocities during both systole and diastole were reduced by 64% and 85%, respectively (both P < 0.001). Supplementation of transferrin‐bound iron recovered functional and morphological abnormalities within 3 days.ConclusionIron deficiency directly affects human cardiomyocyte function, impairing mitochondrial respiration, and reducing contractility and relaxation. Restoration of intracellular iron levels can reverse these effects.

Abstract 240: Asb2-dependent Proteolysis of the Cytoskeleton Directs Cardiac Development and Disease

Congenital heart diseases (CHDs) account for 25% of birth defects and are major risk factors for adult cardiovascular problems. Partial disease penetrance is seen even in autosomal dominant disorders and genotype/phenotype correlations remain a clinical challenge; thus, the need to understand different regulators of cardiac formation. The Ubiquitin-Proteasome System (UPS) is important in controlling protein turnover during organ development but its role in the mammalian heart remains ambiguous. We have identified a specificity subunit of ubiquitin-mediated proteolysis (Asb2) as being specific for the cardiac myogenic lineage. Asb2 was previously shown to regulate hematopoietic and skeletal muscle cell differentiation through targeting filamin proteins (FlnA, B and C), actin-binding proteins important for cytoskeleton stabilization. In our present study, we show that Asb2 is markedly enriched in myocardial progenitor cells and cardiomyocytes. To investigate the role of Asb2 and UPS dependent proteolysis in heart development, we generated two cardiac-specific murine knockouts (KOs): Nkx Cre .Asb2 -/- and Mef2c Cre .Asb2 -/- (deleting Asb2 in early cardiomyocyte progenitors and anterior heart field progenitors, respectively). Both KOs are embryonic lethal with pericardial edema. We used tissue clarifying and confocal microscopy to define the morphological defects of the Asb2 null heart. Moreover, we found that FlnA is overexpressed in the hearts of these mice and its deletion therein partially rescues their lethality. In addition, using transcriptomic analysis on Asb2-null e9.5 hearts, we identified novel potential Asb2 targets in the heart. Finally, to understand the role of Asb2 in the differentiation and function of human cardiomyocytes, we used CRISPR/Cas9 genome editing technique to generate Asb2-null human induced pluripotent stem cells. Collectively, our study provides a novel mechanistic understanding of the role of the UPS proteasome in cardiac development, myocardial function, and disease pathogenesis. Given recent interests in both the UPS and the cytoskeleton as therapeutic targets, our study provides an innovative platform for the development of pharmacotherapy for cardiac disease.

Abstract 142: Defining the Non-Myocyte Compartment is Key for Enhanced Maturation of Human Engineered Heart Muscle

Background: Tissue engineering of heart muscle from human pluripotent stem cells holds great potential for in vitro studies, disease modeling, and cardiac replacement therapy. A number of variables may however affect maturation and function of human cardiomyocytes (CM) in tissue engineered heart muscle (EHM). Here, we hypothesized that defined non-myocyte (NM) populations support structural and functional maturation of EHM. Methods and Results: To investigate the role of non-myocytes (NM) for heart muscle assembly in vitro we generated EHM from purified CM (93±1.5% actinin+) and a mixture of CM and NM (70/30%). Notably, only the NM-supplemented EHM generated measurable forces (0.8±0.1 mN, n=9) with anisotropically aligned cardiomyocytes. Depending on pluripotent stem cell line and differentiation protocol the NM compartment may vary considerably. To further define the influence of the NM compartment we generated EHM from HES2-derived CM with undefined NM, i.e the NM typically derived during cardiac differentiation, and defined NM (fibroblasts). Defined EHM were more mature with higher forces and lower variability between experimental series (defined: 9.8±0.9 nN/CM, undefined: 4.7±1.4 nN/CM, n=10/9), higher EC50 for calcium, and enhanced inotropic response to isoprenaline despite comparable CM:NM composition of 1:1. Increased actinin protein per CM, a reduction of MLC2V/2A double positive CM, and evidence of CM cycle withdrawal indicated enhanced ventricular maturation in defined EHM. Next, we tested whether defining cell composition and NM in iPS-derived EHM will yield a comparable functional phenotype to HES2-EHM. In agreement with the above data, defined iPS-EHM displayed advanced functional maturation with high specific forces, comparable calcium EC50, and inotropic response to isoprenaline. Summary and Conclusions: Here we demonstrate that defining the NM compartment is essential for optimized human heart muscle formation and maturation in vitro. Moreover, our data provide (1) evidence for the applicability of EHM in modelling of heart muscle development and (2) a strong rationale for the need to define CM and NM compartments in tissue engineered myocardium to reduce variability in applications such as disease modelling.

Acoustical sensing of cardiomyocyte cluster beating

Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.

The effects of jaspamide on human cardiomyocyte function and cardiac ion channel activity

Jaspamide (jasplakinolide; NSC-613009) is a cyclodepsipeptide that has antitumor activity. A narrow margin of safety was observed between doses required for efficacy in mouse tumor models and doses that caused severe acute toxicity in rats and dogs. We explored the hypothesis that the observed toxicity was due to cardiotoxicity. Jaspamide was tested in a patch clamp assay to determine its effect on selected cardiac ion channels. Jaspamide (10μM) inhibited Kv1.5 activity by 98.5%. Jaspamide also inhibited other channels including Cav1.2, Cav3.2, and HCN2; however, the Kv11.1 (hERG) channel was minimally affected. Using spontaneously contracting human cardiomyocytes derived from induced pluripotent stem cells, effects on cardiomyocyte contraction and viability were also examined. Jaspamide (30nM to 30μM) decreased cardiomyocyte cell indices and beat amplitude, putative measurements of cell viability and cardiac contractility, respectively. Concentration-dependent increases in rhythmic beating rate were noted at ⩽6h of treatment, followed by dose-dependent decreases after 6 and 72h exposure. The toxic effects of jaspamide were compared with that of the known cardiotoxicant mitoxantrone, and confirmed by multiparameter fluorescence imaging analysis. These results support the hypothesis that the toxicity observed in rats and dogs is due to toxic effects of jaspamide on cardiomyocytes.