Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca²⁺) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca²⁺ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca²⁺ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the Stemdiff^TM kit. Confocal Ca²⁺ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca²⁺ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC₅₀ = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca²⁺ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca²⁺ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.     

The role of mitochondria in sepsis-induced cardiomyopathy

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Myocardial dysfunction, often termed sepsis-induced cardiomyopathy, is a frequent complication and is associated with worse outcomes. Numerous mechanisms contribute to sepsis-induced cardiomyopathy and a growing body of evidence suggests that bioenergetic and metabolic derangements play a central role in its development; however, there are significant discrepancies in the literature, perhaps reflecting variability in the experimental models employed or in the host response to sepsis. The condition is characterised by lack of significant cell death, normal tissue oxygen levels and, in survivors, reversibility of organ dysfunction. The functional changes observed in cardiac tissue may represent an adaptive response to prolonged stress that limits cell death, improving the potential for recovery. In this review, we describe our current understanding of the pathophysiology underlying myocardial dysfunction in sepsis, with a focus on disrupted mitochondrial processes.     

不同频率慢性电刺激膈神经对兔膈肌骨骼肌型二氢吡啶受体α1亚单位、ryanodine受体mRNA和蛋白表达的影响

测定不同频率慢性电刺激(chronic electrical stimulation,CES)膈神经5周后对兔膈肌钙释放单位中骨骼肌型二氢吡啶受体(DHPR)α1亚单位和ryanodine受体(RyRs)的mRNA和蛋白表达水平的影响,探讨CES后兔膈肌钙释放单位结构组成的变化和可能的临床应用价值.封闭群日本大耳白兔30只,随机分为正常对照组、10、20、50和100Hz,每组6只;以10和20 Hz为慢性低频电刺激组,50和100Hz为慢性高频电刺激组.CES参数为波宽0.2 ms 3～6个波/次,45次/min,电压10～20 V.刺激时间2×2 h/日,每周刺激6 d,连续刺激5周.分别采用RT-PCR和免疫组织化学法测定兔膈肌骨骼肌型DHPRα1-亚单位和RyR1、RyR2和RyR3的mRNA和蛋白表达.结果显示与对照组比较,慢性低频电刺激10和20 Hz组骨骼肌型DHPRα1、RyR的mRNA和蛋白表达明显降低(P＜0.01),有低度的RyR,mRNA的表达出现;慢性高频电刺激50和100Hz组骨骼肌型DHPRα1、RyR1的mRNA和蛋白表达明显升高(P＜0.01),未检测到RyR2mRNA的阳性表达.本实验提示慢性低频电刺激膈神经5周后,膈肌质膜上DHPR与RyRs之间的信号转导方式已从变构耦联为主转变为以Ca2+诱导Ca2+释放耦联为主.     

Lipid modulation of ion channels through specific binding sites

Ion channel conformational changes within the lipid membrane are a key requirement to control ion passage. Thus, it seems reasonable to assume that lipid composition should modulate ion channel function. There is increasing evidence that this implicates not just an indirect consequence of the lipid influence on the physical properties of the membrane, but also specific binding of selected lipids to certain protein domains. The result is that channel function and its consequences on excitability, contractility, intracellular signaling or any other process mediated by such channel proteins, could be subjected to modulation by membrane lipids. From this it follows that development, age, diet or diseases that alter lipid composition should also have an influence on those cellular properties. The wealth of data on the non-annular lipid binding sites in potassium channel from Streptomyces lividans (KcsA) makes this protein a good model to study the modulation of ion channel structure and function by lipids. The fact that this protein is able to assemble into clusters through the same non-annular sites, resulting in large changes in channel activity, makes these sites even more interesting as a potential target to develop lead compounds able to disrupt such interactions and hopefully, to modulate ion channel function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

Transport and transporters in the endoplasmic reticulum

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.     

滨蒿内酯对哮喘豚鼠气管平滑肌细胞RyR2 mRNA表达影响

研究哮喘豚鼠气管平滑肌细胞Ryanodine受体亚型的变化及滨蒿内酯对其的影响。将动物分为3组:正常组、哮喘组和滨蒿内酯组,采用卵蛋白致敏复制豚鼠哮喘模型,滨蒿内酯组雾化吸入滨蒿内酯。应用RT-PCR鉴定豚鼠气管平滑肌细胞(TSMC)中RyR亚型分布并观察各组豚鼠气管平滑肌细胞RyR2 mRNA表达的变化。豚鼠TSMC表达RyR2和RyR3两个亚型;哮喘时豚鼠TSMC中RyR2 mRNA表达量(以RyR2区带与-βactin区带的密度百分比表示)为47.15%±15.30%,明显低于对照组(77.12%±6.16%)(P<0.01);滨蒿内酯组豚鼠TSMC中RyR2 mRNA量为63.77%±9.09%,较哮喘组增强(P<0.05)。豚鼠TSMC表达RyR2和RyR3两个亚型;哮喘时TSMC RyR2 mRNA表达下调;滨蒿内酯可使哮喘豚鼠TSMC RyR2表达水平得以恢复。     

The cardiac ryanodine receptor: Looking for anomalies in permeation properties

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba²⁺ with other divalents (Ca²⁺, Sr²⁺), of Ca²⁺ with monovalents (Li⁺, Cs⁺), and of Na⁺ with other monovalents (Cs⁺, Li⁺) were used. We revealed a clear anomaly only for the ion conductance measured in the Na⁺-Cs⁺ and Ca²⁺-Li⁺ mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li⁺/Ca²⁺ bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.