Wenjuan Liu

Polydatin protects cardiac function against burn injury by inhibiting sarcoplasmic reticulum Ca2+ leak by reducing oxidative modification of ryanodine receptors.

Our recent studies demonstrate that burn trauma induces leaky sarcoplasmic reticulum (SR) in heart due to excessively active ryanodine receptor (RyR) function. SR Ca(2+) leak causes partial depletion of SR Ca(2+) content and disturbances in intracellular Ca(2+) homeostasis, resulting in the pathogenesis of burn-generated cardiac dysfunction. This study investigated the role of polydatin, a resveratrol glucoside, in preventing SR leak and its therapeutic effect against burn-generated cardiac dysfunction. We found that polydatin treatment improved cardiac function impaired by burn injury of 30% of total body surface area. Parallel to the alterations in cardiac function, polydatin significantly increased the defective systolic Ca(2+) transient and contractility in burn-traumatized cardiomyocytes. Burn injury increased the occurrence of Ca(2+) sparks. The enhancement of Ca(2+) spark-mediated SR leak caused partial depletion of SR Ca(2+) content in burn-traumatized cardiomyocytes. Furthermore, we found that the content of free thiols (the number of reduced cysteines) in RyR2 in cardiomyocytes determined by the monobromobimane fluorescence of RyR2 was decreased markedly in burn-traumatized hearts. Polydatin treatment decreased intracellular reactive oxygen species levels and restored the amount of free thiols in RyR2 in burns. Concomitantly, polydatin corrected Ca(2+) spark-mediated SR leak and restored SR Ca(2+) load. The systolic Ca(2+) transient and cellular contractility were significantly increased by polydatin treatment. Taken together, the present findings provide the first evidence demonstrating that polydatin prevents enhanced Ca(2+) spark-mediated SR leak by reducing oxidative stress in RyR2 in burn-traumatized heart, leading to protection of cardiac function against burn injury.

Polydatin modulates Ca2 + handling, excitation–contraction coupling and β-adrenergic signaling in rat ventricular myocytes

Polydatin (PD), a resveratrol glucoside, has recently been suggested to have cardioprotective effects against heart diseases, including ischemia-reperfusion injury and pressure-overload induced ventricular remodeling. However, the mechanisms are poorly understood. This study aims to investigate the direct effects of PD on cardiac Ca(2+) handling and excitation-contraction (EC) coupling to explore the potential role of which in PD-mediated cardioprotection. We found that micromolar PD decreased action potential-elicited Ca(2+) transient, but slightly increased cell shortening. The contradictory response could be attributed to PD increasing myofilament Ca(2+) sensitivity. Exploring the activities of the two types of Ca(2+) channels, L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs), reveals that PD dose-dependently decreased LCC current (I(Ca)), but increased frequency of spontaneous Ca(2+) sparks, the elementary Ca(2+) releasing events reflecting RyR activity in intact cells. PD dose-dependently increased the gain of EC coupling. In contrast, PD dose-dependently decreased SR Ca(2+) content. Furthermore, PD remarkably negated β-adrenergic receptor (AR) stimulation-induced enhancement of I(Ca) and Ca(2+) transients, but did not inhibit β-AR-mediated inotropic effect. Inhibition of nitric oxide synthase (NOS) with L-NAME abolished PD regulation of I(Ca) and Ca(2+) spark rate, and significantly inhibited the alteration of Ca(2+) transient and myocyte contractility stimulated by PD. These results collectively indicate that PD modulated cardiac EC coupling mainly by inversely regulating LCC and RyR activity and increasing myofilament Ca(2+) sensitivity through increasing intracrine NO, resulting in suppression of Ca(2+) transient without compromising cardiac contractility. The unique regulation of PD on cardiac EC coupling and responsiveness to β-AR signaling implicates that PD has potential cardioprotective effects against Ca(2+) mishandling related heart diseases.

Effects of advanced glycation end products on calcium handling in cardiomyocytes.

Background and Aims: Advanced glycation end products (AGEs) accumulate in diabetes and the engagement of receptor for AGE (RAGE) by AGEs contributes to the pathogenesis of diabetic cardiomyopathy. This study aims to investigate the effects of AGE/RAGE on ryanodine receptor (RyR) activity and Ca²⁺ handling in cardiomyocytes to elucidate the possible mechanism underlying cardiac dysfunction in diabetic cardiomypathy. Methods and Results: Confocal imaging Ca²⁺ spark, the elementary Ca²⁺ release event reflecting RyR activity in intact cell, as well as SR Ca²⁺ content and systolic Ca²⁺ transient were performed in cultured neonatal rat ventricular myocytes. The results show that 50 mg/ml AGE increased the frequency of Ca²⁺ sparks by 160%, while 150 mg/ml AGE increased it by 53%. AGE decreased the amplitude, width and duration of Ca²⁺ sparks. Blocking RAGE with anti-RAGE IgG completely abolished the alteration of Ca²⁺ sparks. The SR Ca²⁺ content indicated by the amplitude (ΔF/F0) of 20 m<smlcap>M</smlcap> caffeine-elicited Ca²⁺ transient was significantly decreased by 150 mg/ml AGE. In parallel, the amplitude of systolic Ca²⁺ transient evoked by 1 Hz-field stimulation was remarkably decreased by 150 mg/ml AGE. The anti-RAGE antibody completely restored the impaired SR load and systolic Ca²⁺ transient. Conclusion: AGE/RAGE signal enhanced Ca²⁺ spark-mediated SR Ca²⁺ leak, causing partial depletion of SR Ca²⁺ content and consequently decreasing systolic Ca²⁺ transient, which may contribute to contractile dysfunction in diabetic cardiomyopathy.

High-mobility group box 1 (HMGB1) impaired cardiac excitation–contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca2 + leak through TLR4–ROS signaling in cardiomyocytes

High-mobility group box 1 (HMGB1) is a proinflammatory mediator playing an important role in the pathogenesis of cardiac dysfunction in many diseases. In this study, we explored the effects of HMGB1 on Ca(2+) handling and cellular contractility in cardiomyocytes to seek for the mechanisms underlying HMGB1-induced cardiac dysfunction. Our results show that HMGB1 increased the frequency of Ca(2+) sparks, reduced the sarcoplasmic reticulum (SR) Ca(2+) content, and decreased the amplitude of systolic Ca(2+) transient and myocyte contractility in dose-dependent manners in adult rat ventricular myocytes. Inhibiting high-frequent Ca(2+) sparks with tetracaine largely inhibited the alterations of SR load and Ca(2+) transient. Blocking Toll-like receptor 4 (TLR4) with TAK-242 or knockdown of TLR4 by RNA interference remarkably inhibited HMGB1 induced high-frequent Ca(2+) sparks and restored the SR Ca(2+) content. Concomitantly, the amplitude of systolic Ca(2+) transient and myocyte contractility had significantly increased. Furthermore, HMGB1 increased the level of intracellular reactive oxygen species (ROS) and consequently enhanced oxidative stress and CaMKII-activated phosphorylation (pSer2814) in ryanodine receptor 2 (RyR2). TAK-242 pretreatment significantly decreased intracellular ROS levels and oxidative stress and hyperphosphorylation in RyR2, similar to the effects of antioxidant MnTBAP. Consistently, MnTBAP normalized HMGB1-impaired Ca(2+) handling and myocyte contractility. Taken together, our findings suggest that HMGB1 enhances Ca(2+) spark-mediated SR Ca(2+) leak through TLR4-ROS signaling pathway, which causes partial depletion of SR Ca(2+) content and hence decreases systolic Ca(2+) transient and myocyte contractility. Prevention of SR Ca(2+) leak may be an effective therapeutic strategy for the treatment of cardiac dysfunction related to HMGB1 overproduction.

KCNE2 modulates cardiac L-type Ca2+ channel

KCNE2 plays an important role in maintaining cardiac electrical stability. Mutations in KCNE2 have been linked to long-QT syndrome (LQT6) and atrial fibrillation/short QT syndrome. It has been suggested that KCNE2 has the most promiscuity of function which can interact with multiple-subunits of voltage-dependent cation channels and modulate their functions. However, whether KCNE2 regulates voltage-dependent L-type Ca(2)(+) channel (LCC) remains unknown. This study investigated the possible role of KCNE2 in regulating cardiac LCCs and the pathophysiological relevance of this regulation. We found that overexpression of KCNE2 in Sprague-Dawley rat cardiomyocytes decreased L-type Ca(2+)current (ICa,L), whereas KCNE2 knockdown by RNA interference increased ICa,L. Upregulation of KCNE2 caused a slight positive shift of the voltage-dependent activation and a negative shift of the steady-state voltage-dependent inactivation, and slowed the recovery from inactivation of ICa,L, while knockdown of KCNE2 had the contrary effects. Similar regulation of ICa,L magnitude had been observed in transfected HEK 293 cells. Coimmunoprecipitation and colocalization assays in both cardiomyocytes and the transfected cell line suggest that Cav1.2 physically interacted with KCNE2. Deletion of the N-terminal inhibitory module (NTI) of Cav1.2 results in the large loss of KCNE2 regulation of ICa,L and interaction with Cav1.2. Furthermore, we found that the familial atrial fibrillation related KCNE2 mutation R27C enhanced the effect of KCNE2 on suppressing ICa,L. Taken together, our findings indicate that KCNE2 modulates ICa,L by regulating NTI function of Cav1.2. The KCNE2 mutation R27C may induce familial atrial fibrillation partially through enhancing the suppression of ICa,L.

Resveratrol and polydatin as modulators of Ca2+ mobilization in the cardiovascular system

In the cardiovascular system, Ca2+ controls cardiac excitation–contraction coupling and vascular contraction and dilation. Disturbances in intracellular Ca2+ homeostasis induce malfunctions of the cardiovascular system, including cardiac pump dysfunction, arrhythmia, remodeling, and apoptosis, as well as hypertension and impairment of vascular reactivity. Therefore, developing drugs and strategies manipulating Ca2+ handling are highly valued in the treatment of cardiovascular disease. Resveratrol (Res) and polydatin (PD), a Res glucoside, have been well established to have beneficial effects on improving cardiovascular function. Studies from our laboratory and others have demonstrated that they exhibit inotropic effects on normal heart and therapeutic effects on hypertension, cardiac ischemia/reperfusion injury, hypertrophy, and heart failure by manipulating Ca2+ mobilization. The actions of Res and PD on Ca2+ signals delicately manipulated by multiple Ca2+‐handling proteins are pleiotropic and somewhat controversial, depending on cellular species and intracellular oxidative status. Here, we focus on the effects of Res and PD on controlling Ca2+ homeostasis in the heart and vasculature under normal and diseased conditions and highlight the key direct and indirect molecules mediating these effects.

Manipulation of KCNE2 expression modulates action potential duration, and Ito and IK in rat and mouse ventricular myocytes

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K(+) channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K(+) current (Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K(+) current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.

Toll-like receptor 4–induced ryanodine receptor 2 oxidation and sarcoplasmic reticulum Ca2+ leakage promote cardiac contractile dysfunction in sepsis

Studies suggest the potential role of a sarcoplasmic reticulum (SR) Ca2+ leak in cardiac contractile dysfunction in sepsis. However, direct supporting evidence is lacking, and the mechanisms underlying this SR leak are poorly understood. Here, we investigated the changes in cardiac Ca2+ handling and contraction in LPS-treated rat cardiomyocytes and a mouse model of polymicrobial sepsis produced by cecal ligation and puncture (CLP). LPS decreased the systolic Ca2+ transient and myocyte contraction as well as SR Ca2+ content. Meanwhile, LPS increased Ca2+ spark–mediated SR Ca2+ leak. Preventing the SR leak with ryanodine receptor (RyR) blocker tetracaine restored SR load and increased myocyte contraction. Similar alterations in Ca2+ handling were observed in cardiomyocytes from CLP mice. Treatment with JTV-519, an anti-SR leak drug, restored Ca2+ handling and improved cardiac function. In the LPS-treated cardiomyocytes, mitochondrial reactive oxygen species and oxidative stress in RyR2 were increased, whereas the levels of the RyR2-associated FK506-binding protein 1B (FKBP12.6) were decreased. The Toll-like receptor 4 (TLR4)–specific inhibitor TAK-242 reduced the oxidative stress in LPS-treated cells, decreased the SR leak, and normalized Ca2+ handling and myocyte contraction. Consistently, TLR4 deletion significantly improved cardiac function and corrected abnormal Ca2+ handling in the CLP mice. This study provides evidence for the critical role of the SR Ca2+ leak in the development of septic cardiomyopathy and highlights the therapeutic potential of JTV-519 by preventing SR leak. Furthermore, it reveals that TLR4 activation-induced mitochondrial reactive oxygen species production and the resulting oxidative stress in RyR2 contribute to the SR Ca2+ leak.

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like &agr;) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene (himf) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction–induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and &bgr;-MHC [myosin heavy chain-&bgr;]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction–induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca2+ concentration and activated the CaN–NFAT (calcineurin–nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca2+ concentration with L-type Ca2+ channel blocker nifedipine or inhibiting the CaSR (Ca2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1&agr; (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1&agr; upregulation in transverse aortic constriction mice. Knockdown of HIF-1&agr; attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy.

Sarcoplasmic reticulum calcium leak and heart failure

Calcium ions play a key role in maintaining the equilibrium of excitation-contraction coupling process in the heart. The increased calcium concentration is primarily responsible for the whole cell activation, relevant signaling pathways involved in regulation of sarcomeric gene expression, and have broader effects on cardiac functioning. Sarcoplasmic reticulum, playing an important role in maintaining the intracellular calcium homeostasis, is the critical factor involved in excitation-contraction coupling in cardiomyocytes. The increased activity of ryanodine receptor 2 (RyR2) channel in diastolic phase leads to its abnormal opening and/or incomplete closing, inducing significant calcium leak from sarcoplasmic reticulum. More and more evidences indicate that, abrupt increase in local calcium concentration, especially in end-stage heart failure, leads to sarcoplasmic reticulum abnormalities as well as reorganization of cardiac structure and its function. Here, we primarily emphasized on the mechanism responsible for calcium leak and explored its therapeutic option, if any, to prevent the development and progression of heart failure by regulating the increased calcium concentration locally.