Yingxin Li

GSK-3α directly regulates β-adrenergic signaling and the response of the heart to hemodynamic stress in mice

The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, alpha and beta. Although GSK-3beta has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3alpha in the mouse heart using gene targeting. Gsk3a(-/-) mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired beta-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3alpha appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of beta-adrenergic responsiveness. In the absence of GSK-3alpha, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of beta-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3.

Finding the Rhythm of Sudden Cardiac Death New Opportunities Using Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death.Sudden Cardiac Death (SCD) is a common cause of death in patients with structural heart disease, genetic mutations or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with SCD. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell derived Cardiomyocytes (iPSC-CMs) resemble, but are not identical, to adult human cardiomyocytes, and provide a new platform for studying arrhythmic disorders leading to SCD. A variety of platforms exist to phenotype cellular models including conventional and automated patch clamp, multi-electrode array, and computational modeling. iPSC-CMs have been used to study Long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy and other hereditary cardiac disorders. Although iPSC-CMs are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of SCD.

Increasing Cardiac Contractility After Myocardial Infarction Exacerbates Cardiac Injury and Pump Dysfunction

Rationale: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. Objectives: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca2+-handling defects could avert post-MI cardiac pump dysfunction. Methods and Results: MI was produced in mice with inducible, cardiac-specific expression of the &bgr;2a subunit of the L-type Ca2+ channel. Myocyte and cardiac function were compared in control and &bgr;2a animals before and after MI. &bgr;2a myocytes had increased Ca2+ current; sarcoplasmic reticulum Ca2+ load, contraction and Ca2+ transients (versus controls), and &bgr;2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in &bgr;2a versus control hearts after MI. &bgr;2a animals also had poorer survival after MI. Myocytes isolated from &bgr;2a hearts after MI did not develop depressed Ca2+ handling, and Ca2+ current, contractions, and Ca2+ transients were still above control levels (before MI). Conclusions: Maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

Cardiac G-Protein–Coupled Receptor Kinase 2 Ablation Induces a Novel Ca2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of &bgr;-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca2+ content because of increased sodium-Ca2+ exchanger activity and inhibited SR Ca2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca2+ handling phenotype is explained by a higher fractional SR Ca2+ release induced by increased L-type Ca2+ channel currents. After &bgr;-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca2+ transients, which were not mediated through cardiac L-type Ca2+ channels but through an increased SR Ca2+. Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca2+ handling phenotype. The SR Ca2+ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca2+ channel current density and no increase in sodium-Ca2+ exchanger in GRK2KO myocytes. An L-type Ca2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca2+ handling phenotype resisting adverse remodeling after MI.

Enhanced basal contractility but reduced excitation-contraction coupling efficiency and β-adrenergic reserve of hearts with increased Cav1.2 activity

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca(2+) channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the beta-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavbeta2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon beta-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca(2+) current (I(Ca,L)) density, myocyte fractional shortening (FS), peak Ca(2+) transients, and sarcoplasmic reticulum (SR) Ca(2+) content (caffeine-induced Ca(2+) transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the beta-adrenergic agonist isoproterenol (2 microg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of beta-adrenergic stimulation on contraction, Ca(2+) transients, I(Ca,L), and SR Ca(2+) content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.

β-Adrenergic stimulation increases Cav3.1 activity in cardiac myocytes through protein kinase A.

The T-type Ca2+ channel (TTCC) plays important roles in cellular excitability and Ca2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca2+ current (ICa-T(3.1)). ICa-T(3.1) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased ICa-T(3.1) as well as ICa-L in TG-VMs at both physiological and room temperatures. The ISO effect on ICa-L and ICa-T in TG myocytes was blocked by H89, a PKA inhibitor. ICa-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of ICa-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. ICa-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases ICa-T(3.1) in cardiomyocytes, which is mediated by the cAMP/PKA pathway. The upregulation of ICa-T(3.1) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.

Cross-Site Reliability of Human Induced Pluripotent stem cell-derived Cardiomyocyte Based Safety Assays Using Microelectrode Arrays: Results from a Blinded CiPA Pilot Study

Abstract Recent in vitro cardiac safety studies demonstrate the ability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to detect electrophysiologic effects of drugs. However, variability contributed by unique approaches, procedures, cell lines, and reagents across laboratories makes comparisons of results difficult, leading to uncertainty about the role of hiPSC-CMs in defining proarrhythmic risk in drug discovery and regulatory submissions. A blinded pilot study was conducted to evaluate the electrophysiologic effects of 8 well-characterized drugs on 4 cardiomyocyte lines using a standardized protocol across 3 microelectrode array platforms (18 individual studies). Drugs were selected to define assay sensitivity of prominent repolarizing currents (E-4031 for IKr, JNJ303 for IKs) and depolarizing currents (nifedipine for ICaL, mexiletine for INa) as well as drugs affecting multichannel block (flecainide, moxifloxacin, quinidine, and ranolazine). Inclusion criteria for final analysis was based on demonstrated sensitivity to IKr block (20% prolongation with E-4031) and L-type calcium current block (20% shortening with nifedipine). Despite differences in baseline characteristics across cardiomyocyte lines, multiple sites, and instrument platforms, 10 of 18 studies demonstrated adequate sensitivity to IKr block with E-4031 and ICaL block with nifedipine for inclusion in the final analysis. Concentration-dependent effects on repolarization were observed with this qualified data set consistent with known ionic mechanisms of single and multichannel blocking drugs. hiPSC-CMs can detect repolarization effects elicited by single and multichannel blocking drugs after defining pharmacologic sensitivity to IKr and ICaL block, supporting further validation efforts using hiPSC-CMs for cardiac safety studies.

Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant

BACKGROUND The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity. OBJECTIVES The purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy. METHODS Peripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs). RESULTS Whole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (IKr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent IKr activator, enhanced IKr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder. CONCLUSIONS The results suggest that the KCNH2T983I VUS may be classified as potentially pathogenic.

Increasing T‐type calcium channel activity by β‐adrenergic stimulation contributes to β‐adrenergic regulation of heart rates

Cav3.1 T‐type Ca2+ channel current (ICa‐T) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β‐adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β‐adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β‐adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. ICa‐T in sinoatrial nodal cells can be upregulated by β‐adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T‐type Ca2+ channel can be a target for heart disease treatments that aim to slow down the heart rate

Finding the Rhythm of Sudden Cardiac Death

Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death.Sudden Cardiac Death (SCD) is a common cause of death in patients with structural heart disease, genetic mutations or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with SCD. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell derived Cardiomyocytes (iPSC-CMs) resemble, but are not identical, to adult human cardiomyocytes, and provide a new platform for studying arrhythmic disorders leading to SCD. A variety of platforms exist to phenotype cellular models including conventional and automated patch clamp, multi-electrode array, and computational modeling. iPSC-CMs have been used to study Long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy and other hereditary cardiac disorders. Although iPSC-CMs are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of SCD.