Human Cardiac Myocytes Research Articles

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

Interactions of N-acetyl-D-glucosamine-conjugated silk fibroin with lectins, cytoskeletal proteins and cardiomyocytes

We have reported that cytoskeletal proteins such as desmin and vimentin are expressed on the surface of muscle, mesenchymal and cancer cells, and possess N-acetyl-β-D-glucosamine (β-GlcNAc) residue-binding properties. As cell-recognizable β-GlcNAc residue-bearing biopolymer, we prepared glycoconjugates (SF-GlcNAc) composed of silk fibroin (SF) and monosaccharide N-acetyl-D-glucosamine (GlcNAc) by chemical modification using cyanuric chloride. The covalent immobilization of GlcNAc into SF was assessed by 1H-NMR measurements. The 1H-NMR spectrum of SF-GlcNAc conjugates showed new peaks attributed to the methyl protons of the N-acetyl group in GlcNAc, and the integration of these peaks revealed that the GlcNAc content in the conjugates was 9 wt%. The existence of β-GlcNAc residues in SF-GlcNAc was examined by the criteria using lectins such as wheat germ agglutinin (WGA). Addition of WGA to SF-GlcNAc solution caused an increase in the turbidity of the solution due to lectin-mediated aggregation. Solid-phase lectin binding assay based on the biotin-avidin interaction showed that biotinylated succinylated WGA bound more strongly onto SF-GlcNAc conjugate-coated wells compared to SF-coated well. Following the establishment of the existence of β-GlcNAc residues in SF-GlcNAc, the interaction of SF-GlcNAc with desmin was examined by enzyme-linked immunosorbent assay using anti-desmin antibody. The stronger binding of desmin was observed for SF-GlcNAc conjugate-coated wells compared to SF-coated wells. The use of SF-GlcNAc conjugates as a substrate for culturing desmin-expressing human cardiac myocytes demonstrated an increase in the numbers of attached cells and proliferating cells on the conjugate-coated wells compared to SF-coated wells. These results suggest that the immobilization of monosaccharide GlcNAc is a useful method for the versatile functionalization of SF as an application in tissue engineering.

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.

Using cardiac ionic cell models to interpret clinical data.

For over 100 years cardiac electrophysiology has been measured in the clinic. The electrical signals that can be measured span from noninvasive ECG and body surface potentials measurements through to detailed invasive measurements of local tissue electrophysiology. These electrophysiological measurements form a crucial component of patient diagnosis and monitoring; however, it remains challenging to quantitatively link changes in clinical electrophysiology measurements to biophysical cellular function. Multi-scale biophysical computational models represent one solution to this problem. These models provide a formal framework for linking cellular function through to emergent whole organ function and routine clinical diagnostic signals. In this review, we describe recent work on the use of computational models to interpret clinical electrophysiology signals. We review the simulation of human cardiac myocyte electrophysiology in the atria and the ventricles and how these models are being used to link organ scale function to patient disease mechanisms and therapy response in patients receiving implanted defibrillators, \cardiac resynchronisation therapy or suffering from atrial fibrillation and ventricular tachycardia. There is a growing use of multi-scale biophysical models to interpret clinical data. This allows cardiologists to link clinical observations with cellular mechanisms to better understand cardiopathophysiology and identify novel treatment strategies. This article is categorized under: Cardiovascular Diseases > Computational Models Cardiovascular Diseases > Biomedical Engineering Cardiovascular Diseases > Molecular and Cellular Physiology.

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling.

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.

Dietary Glycine Prevents FOLFOX Chemotherapy-Induced Heart Injury: A Colorectal Cancer Liver Metastasis Treatment Model in Rats.

Introduction: FOLFOX chemotherapy (CTx) is used for the treatment of colorectal liver metastasis (CRLM). Side effects include rare cardiotoxicity, which may limit the application of FOLFOX. Currently, there is no effective strategy to prevent FOLFOX-induced cardiotoxicity. Glycine has been shown to protect livers from CTx-induced injury and oxidative stress, and it reduces platelet aggregation and improves microperfusion. This study tested the hypothesis of glycine being cardioprotective in a rat model of FOLFOX in combination with CRLM. Materials and Methods: The effect of glycine was tested in vitro on human cardiac myocytes (HCMs). To test glycine in vivo Wag/Rij rats with induced CRLM were treated with FOLFOX ±5% dietary glycine. Left ventricle ejection fraction (LVEF), myocardial fibrosis, and apoptosis, also heart fatty acid binding protein (h-FABP) and brain natriuretic peptide levels were monitored. PCR analysis for Collagen type I, II, and brain natriuretic peptide (BNP) in the heart muscle was performed. Results: In vitro glycine had no effect on HCM cell viability. Treatment with FOLFOX resulted in a significant increase of h-FABP levels, increased myocardial fibrosis, and apoptosis as well as increased expression of type I Collagen. Furthermore, FOLFOX caused a decrease of LVEF by 10% (p = 0.028). Dietary glycine prevented FOLFOX-induced myocardial injury by preserving the LVEF and reducing the levels of fibrosis (p = 0.012) and apoptosis (p = 0.015) in vivo. Conclusions: Data presented here demonstrate for the first time that dietary glycine protects the heart against FOLFOX-induced injury during treatment for CRLM.

Pathogenesis of SARS-CoV-2 induced cardiac injury from the perspective of the virus

Pathogenesis of SARS-CoV-2 induced cardiac injury from the perspective of the virus

Abstract 222: The Estrogen-related Receptor (ERR) Drives Cardiac Myocyte Maturation in Cooperation With GATA4

During transition from fetal to adult developmental stages, the heart undergoes robust mitochondrial biogenesis in parallel with growth and structural maturation. Recently we identified a role for ERRα/γ in this broad program of postnatal cardiac maturation. We sought to delineate the mechanisms whereby ERRs coordinate transcriptional regulation of both metabolic and structural genes in the maturation of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). CRISPR-based gene deletion studies demonstrated that ERRα/γ is necessary for activation of genes involved in mitochondrial and structural maturation during hiPSC-CM differentiation including mitochondrial function, ion transport, Ca 2+ handling, and the adult sarcomere. Whole-genome RNA-sequencing and ChIP-sequencing (ChIP-seq) studies indicated that ERRγ directly regulates both metabolic and structural genes by remodeling H3K27ac depositions in related enhancer regions. Integration of the ERRγ ChIP-seq data with published hiPSC-CM datasets demonstrated that approximately 50% of super-enhancer regions overlapped ERRγ peaks, suggesting ERR is involved in the maintenance and function of cardiac super-enhancer regions. Corresponding motif analyses and intersection analyses with published GATA4 ChIP-seq datasets suggested that ERRγ cooperates with GATA4 on many structural, but not metabolic targets. Specifically, binding sites for both factors are often co-localized around cardiac contractile, ion channel, and Ca 2+ handling genes, including TNNI3, MYBPC3, MYH7, TTN, KCNQ1, and RYR2 . In contrast, ERRγ sites on target genes involved in mitochondrial maturation such as ATP5B, COX4I1, CPT1B , and FABP3 lacked GATA4 sites. Experiments using luciferase reporters demonstrated functional cooperation between ERRγ and GATA4 that depends on the known ERR coactivator, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α). We conclude that the ERR cooperates with GATA4 to drive cardiac structural gene transcription during cardiac differentiation, whereas ERR regulates genes involved in energy metabolism independent of GATA4. Activation of ERR signaling could prove to be an effective tool to drive hiPSC-CM maturation.

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.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1β (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1β or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

Remifentanil preconditioning protects against hypoxia-induced senescence and necroptosis in human cardiac myocytes in vitro.

Remifentanil and other opioids are suggested to be protective against ischemia-reperfusion injury in animal models and coronary artery bypass surgery patients, however the molecular basis of such protection is far from being understood. In the present study, we have used a model of human cardiomyocytes treated with the hypoxia-mimetic agent cobalt chloride to investigate remifentanil preconditioning-based adaptive responses and underlying mechanisms. Hypoxic conditions promoted oxidative and nitrosative stress, p21-mediated cellular senescence and the activation of necroptotic pathway that was accompanied by a 2.2-, 9.6- and 8.2-fold increase in phosphorylation status of mixed lineage kinase domain-like pseudokinase (MLKL) and release of pro-inflammatory cytokine IL-8 and cardiac troponin I, a marker of myocardial damage, respectively. Remifentanil preconditioning was able to lower hypoxia-mediated protein carbonylation and limit MLKL-based signaling and pro-inflammatory response to almost normoxic control levels, and decrease hypoxia-induced pro-senescent activity of about 21% compared to control hypoxic conditions. In summary, we have shown for the first time that remifentanil can protect human cardiomyocytes against hypoxia-induced cellular senescence and necroptosis that may have importance with respect to the use of remifentanil to diminish myocardial ischemia and reperfusion injury in patients undergoing cardiac surgery.

Regulation of Myofilament Contractile Function in Human Donor and Failing Hearts.

Heart failure (HF) often includes changes in myocardial contractile function. This study addressed the myofibrillar basis for contractile dysfunction in failing human myocardium. Regulation of contractile properties was measured in cardiac myocyte preparations isolated from frozen, left ventricular mid-wall biopsies of donor (n = 7) and failing human hearts (n = 8). Permeabilized cardiac myocyte preparations were attached between a force transducer and a position motor, and both the Ca2+ dependence and sarcomere length (SL) dependence of force, rate of force, loaded shortening, and power output were measured at 15 ± 1°C. The myocyte preparation size was similar between groups (donor: length 148 ± 10 μm, width 21 ± 2 μm, n = 13; HF: length 131 ± 9 μm, width 23 ± 1 μm, n = 16). The maximal Ca2+-activated isometric force was also similar between groups (donor: 47 ± 4 kN⋅m–2; HF: 44 ± 5 kN⋅m–2), which implicates that previously reported force declines in multi-cellular preparations reflect, at least in part, tissue remodeling. Maximal force development rates were also similar between groups (donor: ktr = 0.60 ± 0.05 s–1; HF: ktr = 0.55 ± 0.04 s–1), and both groups exhibited similar Ca2+ activation dependence of ktr values. Human cardiac myocyte preparations exhibited a Ca2+ activation dependence of loaded shortening and power output. The peak power output normalized to isometric force (PNPO) decreased by ∼12% from maximal Ca2+ to half-maximal Ca2+ activations in both groups. Interestingly, the SL dependence of PNPO was diminished in failing myocyte preparations. During sub-maximal Ca2+ activation, a reduction in SL from ∼2.25 to ∼1.95 μm caused a ∼26% decline in PNPO in donor myocytes but only an ∼11% change in failing myocytes. These results suggest that altered length-dependent regulation of myofilament function impairs ventricular performance in failing human hearts.

Oxidative stress in bisphenol AF-induced cardiotoxicity in zebrafish and the protective role of N-acetyl N-cysteine

Research has shown that there is a relationship between bisphenol A (BPA) exposure and the incidence of cardiovascular diseases. However, the effect of bisphenol AF (BPAF), a main substitute for BPA, on heart development remains unclear. In this study, the cardiotoxicity of BPAF was evaluated in zebrafish in vivo and in human cardiac myocytes (HCMs) in vitro. Our results showed that BPAF at a concentration of 200 μg/L results in cardiotoxicity, including a reduced number of cardiomyocytes and endocardial cells in the heart, and reduced heart size in two transgenic zebrafish models (myl7:: dsred2-nuc and fli1a::nGFP). An increase in apoptosis was observed along with antioxidant enzyme inhibition and lipid peroxidation. In addition, the mRNA expression levels of several key genes involved in cardiac development were suppressed by BPAF treatment. In the HCM cell model, BPAF at 2 mg/L induced reactive oxygen species generation, antioxidant enzyme inhibition, mitochondrial dysfunction and oxidative DNA damage. These adverse outcomes can be attenuated by the antioxidant N-acetyl-L-cysteine (NAC), suggesting that oxidative stress is involved in BPAF-induced cardiotoxicity. These data indicated that BPAF exposure increased oxidative stress and apoptosis and that it suppressed the expression of genes involved in cardiac development, which may play crucial roles in the mechanisms of BPAF-induced cardiotoxicity.

Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection.

In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2-5years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.

Calreticulin expression in human cardiac myocytes induces ER stress-associated apoptosis.

The global burden of heart failure following myocardial ischemia‐reperfusion (IR) injury is a growing problem. One pathway that is key to understanding the progression of myocardial infarction and IR injury is the endoplasmic reticulum (ER) stress pathway, which contributes to apoptosis signaling and tissue death. The role of calreticulin in the progression of ER stress remains controversial. We hypothesized that calreticulin induction drives proapoptotic signaling in response to ER stress. We find here that calreticulin is upregulated in human ischemic heart failure cardiac tissue, as well as simulated hypoxia and reoxygenation (H/R) and thapsigargin‐mediated ER stress. To test the impact of direct modulation of calreticulin expression on ER stress‐induced apoptosis, human cardiac‐derived AC16 cells with stable overexpression or silencing of calreticulin were subjected to thapsigargin treatment, and markers of apoptosis were evaluated. It was found that overexpression of calreticulin promotes apoptosis, while a partial knockdown protects against the expression of caspase 12, CHOP, and reduces thapsigargin‐driven TUNEL staining. These data shed light on the role that calreticulin plays in apoptosis signaling during ER stress in cardiac cells.

A Critical Role for Estrogen-Related Receptor Signaling in Cardiac Maturation.

The heart undergoes dramatic developmental changes during the prenatal to postnatal transition, including maturation of cardiac myocyte energy metabolic and contractile machinery. Delineation of the mechanisms involved in cardiac postnatal development could provide new insight into the fetal shifts that occur in the diseased heart and unveil strategies for driving maturation of stem cell-derived cardiac myocytes. To delineate transcriptional drivers of cardiac maturation. We hypothesized that ERR (estrogen-related receptor) α and γ, known transcriptional regulators of postnatal mitochondrial biogenesis and function, serve a role in the broader cardiac maturation program. We devised a strategy to knockdown the expression of ERRα and γ in heart after birth (pn-csERRα/γ [postnatal cardiac-specific ERRα/γ]) in mice. With high levels of knockdown, pn-csERRα/γ knockdown mice exhibited cardiomyopathy with an arrest in mitochondrial maturation. RNA sequence analysis of pn-csERRα/γ knockdown hearts at 5 weeks of age combined with chromatin immunoprecipitation with deep sequencing and functional characterization conducted in human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) demonstrated that ERRγ activates transcription of genes involved in virtually all aspects of postnatal developmental maturation, including mitochondrial energy transduction, contractile function, and ion transport. In addition, ERRγ was found to suppress genes involved in fibroblast activation in hearts of pn-csERRα/γ knockdown mice. Disruption of Esrra and Esrrg in mice during fetal development resulted in perinatal lethality associated with structural and genomic evidence of an arrest in cardiac maturation, including persistent expression of early developmental and noncardiac lineage gene markers including cardiac fibroblast signatures. Lastly, targeted deletion of ESRRA and ESRRG in hiPSC-CM derepressed expression of early (transcription factor 21 or TCF21) and mature (periostin, collagen type III) fibroblast gene signatures. ERRα and γ are critical regulators of cardiac myocyte maturation, serving as transcriptional activators of adult cardiac metabolic and structural genes, an.d suppressors of noncardiac lineages including fibroblast determination.

TWIK-1 channels contribute to hypokalemia induced paradoxical depolarization in cardiac myocytes

TWIK-1 potassium channels contributes to background K+ conductance in diverse cell types including human cardiac myocytes. TWIK-1 potassium channels changes ion selectivity and conduct inward Na+ currents at sub-physiological [K+]e. However, the pathological implications of the functional changes of TWIK-1 channels remains largely unknown. In hypokalemia, human cardiomyocytes show paradoxical depolarization which cause cardiac arrhythmia. However, the underlying mechanism is unknown. In the present study, we show that TWIK-1 channels conduct inward leak Na+ currents and contribute to the paradoxical membrane potential depolarization in cardiac myocytes in hypokalemia. First, under hypokalemia conditions, Chinese hamsterovary cells that heterologously express TWIK-1-WT channels show depolarization; in contrast, the cells with expression of TWIK-1-T118I mutant channels show hyperpolarization. Second, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL-1 cardiomyocytes with ectopic expression of TWIK-1 channels show paradoxical depolarization phenomenon. These cardiomyocytes display a nearly linear current-voltage relationship with a reversal potential at about -30 mV at 1 mM [K+]e, indicating a cation conductance. Substitution extra-cellular Na+ with NMDG+ shift the reversal potential to -110 mV and abolish the paradoxical depolarization phenomenon. Besides, inhibition of K2P1 expression eliminates the phenomenon, suggesting contributions of TWIK-1 channels to paradoxical depolarization. These findings reveal the mechanisms underlying hypokalemia induced paradoxical depolarization in human cardiac myocytes, and support that the inward leak Na+ currents carried by TWIK-1 channels induce paradoxical depolarization in cardiac myocytes under hypokalemia conditions.

Hypoxia Produces Pro-arrhythmic Late Sodium Current in Cardiac Myocytes by SUMOylation of NaV1.5 Channels

SUMMARYAcute cardiac hypoxia produces life-threatening elevations in late sodium current (ILATE) in the human heart. Here, we show the underlying mechanism: hypoxia induces rapid SUMOylation of NaV1.5 channels so they reopen when normally inactive, late in the action potential. NaV1.5 is SUMOylated only on lysine 442, and the mutation of that residue, or application of a deSUMOylating enzyme, prevents hypoxic reopenings. The time course of SUMOylation of single channels in response to hypoxia coincides with the increase in ILATE, a reaction that is complete in under 100 s. In human cardiac myocytes derived from pluripotent stem cells, hypoxia-induced ILATE is confirmed to be SUMO-dependent and to produce action potential prolongation, the pro-arrhythmic change observed in patients.

Prestin Amplifies Cardiac Motor Functions

Cardiac cells generate and amplify force in the context of cardiac load, yet the membranous sheath enclosing the fibers, the sarcolemma, does not experience displacement. The fact that the sarcolemma sustains beat-to-beat pressure changes without experiencing significant distortion is the muscle contraction paradox the authors sought to resolve. Here, we report that an elastic element, the motor protein prestin (Slc26a5), serves to amplify actin-myosin force generation in mouse and human cardiac myocytes, accounting for the nonlinear properties of contraction. The functional significance of prestin is underpinned by alterations of cardiac contractility in Prestin knockout mice. Prestin was previously considered exclusive to outer hair cells of the inner ear; however, our results show that prestin serves a broader cellular motor function.