Journal of molecular and cellular cardiology最新文献_第10页

Molecular mechanism for the interaction of MOG1 with the intracellular loop II of cardiac sodium channel Nav1.5 and its role in arrhythmias MOG1与心脏钠通道Nav1.5胞内环II相互作用的分子机制及其在心律失常中的作用。

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-19 DOI: 10.1016/j.yjmcc.2025.06.005

Xuemei Bai , Zhijie Wang , Hongbo Xiong , Chipeng Yan , Yufeng Yao , Chengqi Xu , Hui Li , Qing K. Wang

SCN5A encodes the cardiac sodium channel α-subunit Na_v1.5, and its variants cause long QT syndrome (LQTS), Brugada syndrome (BrS) and other arrhythmias. MOG1 interacts with Na_v1.5 to increase cardiac sodium current densities, however, molecular mechanisms remain poorly defined. The objectives of this study were to identify the crucial structural elements responsible for the interaction between MOG1 and Na_v1.5 intracellular Loop II, and determine the significance of this interaction to cardiac arrhythmias. Whole-cell patch-clamping was used to record sodium current I_Na in tsA201 and neonatal rat primary cardiomyocytes. Glutathione S-transferase (GST) pull-down assays were used to characterize protein-protein interactions. Mutagenesis was used to create deletions and point mutations. Characterization of large deletions and small deletions of Na_v1.5 Loop II 940–1200 defined the MOG1-interacting domain to V₁₁₉₀-H₁₂₀₀. Point mutation analysis revealed that amino acids R₁₁₉₅, Y₁₁₉₉ and H₁₂₀₀ were involved in MOG1-Na_v1.5 Loop II interaction. Two variants of MOG1-interacting domain from human patients showed important functional effects. Variant p.R1195C was identified in two individuals with cardiac arrhythmias in ClinVar, weakened the interaction between Na_v1.5 and MOG1, and reduced MOG1-enhanced cardiac sodium current densities. Variant p.Y1199S was identified in one individual with LQTS and one with cardiac arrhythmias, generated late I_Na, weakened the interaction between Na_v1.5 and MOG1, and reduced MOG1-enhanced cardiac sodium current densities. This study identifies three critical amino acids R₁₁₉₅, Y₁₁₉₉ and H₁₂₀₀ of Na_v1.5 Loop II for interaction with MOG1, and reveals the molecular mechanisms by which variants p.R1195C and p.Y1199S in MOG1-interacting domain cause LQTS and cardiac arrythmias.

SCN5A编码心脏钠通道α-亚基Nav1.5，其变异可引起长QT综合征（LQTS）、Brugada综合征（BrS）等心律失常。然而，MOG1与Nav1.5相互作用增加心脏钠电流密度的分子机制仍不明确。本研究的目的是确定MOG1和Nav1.5细胞内环路II之间相互作用的关键结构元件，并确定这种相互作用对心律失常的意义。采用全细胞膜片钳法记录tsA201和新生大鼠原代心肌细胞中的钠电流INa。谷胱甘肽s -转移酶（GST）下拉试验用于表征蛋白质-蛋白质相互作用。诱变用于产生缺失和点突变。Nav1.5 Loop II 940-1200的大缺失和小缺失将mog1相互作用域定义为V1190-H1200。点突变分析显示，氨基酸R1195、Y1199和H1200参与了MOG1-Nav1.5 Loop II互作。来自人类患者的mog1相互作用域的两个变体显示出重要的功能作用。变异p.R1195C在两例ClinVar心律失常患者中被发现，削弱了Nav1.5和MOG1之间的相互作用，降低了MOG1增强的心脏钠电流密度。变异p.Y1199S在1例LQTS患者和1例心律失常患者中被鉴定出，产生晚期INa，削弱Nav1.5和MOG1之间的相互作用，降低MOG1增强的心脏钠电流密度。本研究鉴定了Nav1.5 Loop II中与MOG1相互作用的三个关键氨基酸R1195、Y1199和H1200，揭示了MOG1相互作用域p.R1195C和p.Y1199S变异体导致LQTS和心律失常的分子机制。

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引用次数: 0

Activation of FoxO1 prevents and reverses cardiac hypertrophy from diverse stimuli FoxO1的激活可以防止和逆转各种刺激引起的心脏肥厚

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-18 DOI: 10.1016/j.yjmcc.2025.06.008

Thomas G. Martin , Stephen J. Langer , Claudia Crocini , Eunhee Chung , Leslie A. Leinwand

The heart is a dynamic organ capable of structural and functional remodeling in the wake of changing mechanical and/or circulating cues. While the molecular underpinnings of cardiac hypertrophy are well-defined, the mechanisms of hypertrophy regression following stimulus removal are relatively less understood. Here, we demonstrate that activation of forkhead box proteins (FoxOs), and increased expression of their autophagy gene targets, are common features of hypertrophy regression after both exercise and pregnancy in mice. Additionally, we show FoxO1 activation is sufficient to prevent and reverse adrenergic agonist-dependent pathological hypertrophy. Our findings highlight the central role of FoxO1 in regulating cardiac mass.

心脏是一个动态的器官，能够在改变机械和/或循环信号后进行结构和功能重塑。虽然心肌肥厚的分子基础是明确的，但刺激去除后肥厚消退的机制却相对不太清楚。在这里，我们证明叉头盒蛋白（FoxOs）的激活及其自噬基因靶点的表达增加是小鼠运动和妊娠后肥厚消退的共同特征。此外，我们发现FoxO1激活足以预防和逆转肾上腺素能激动剂依赖性病理性肥大。我们的发现强调了fox01在调节心脏质量中的核心作用。

引用次数: 0

Endothelial CD36 mediates diet-induced increases in aortic stiffness 内皮细胞CD36介导饮食引起的主动脉僵硬增加

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-16 DOI: 10.1016/j.yjmcc.2025.06.009

Javad Habibi , Vincent G. DeMarco , Dongqing Chen , Adam Whaley-Connell , Michael A. Hill , Guanghong Jia

A Western diet (WD) contributes to the rising prevalence of obesity and insulin resistance, both of which are key risk factors for arterial stiffening and related cardiovascular diseases. We recently found that elevated CD36 is associated with increased ectopic lipid accumulation, systemic and tissue insulin resistance, and arterial stiffening. Here, we further examined whether endothelial cell (EC) specific CD36 (ECCD36) participates in WD-induced aortic insulin resistance, lipid accumulation, inflammation, fibrosis, remodeling, and associated aortic stiffening. Female ECCD36 knockout (ECCD36^−/−) and wild-type (ECCD36^+/+) mice, at six weeks of age, were fed either a Western diet (WD) or a standard chow diet (CD) for 16 weeks. Aortic stiffness and activity were investigated by ultrasound (pulse wave velocity) and wire myography, respectively. Gene expression was monitored by western blot and quantitative PCR. Lipid content and aortic remodeling were explored by Oil red O staining and immunostaining, respectively. 16 weeks of WD increased aortic stiffening that was associated with vascular insulin resistance and reduced insulin metabolic signaling via phosphoinositide 3-kinases/protein kinase B. The pathophysiological changes in vascular insulin resistance and stiffening were associated with activation of mammalian target of rapamycin/S6 kinase signaling, increased lipid disorders, decreased tight junction-associated protein occludin, and increased proinflammatory response, and aortic remodeling. These abnormalities were blunted in ECCD36^−/− mice fed a WD. These findings suggest that under an obesogenic Western diet (WD), heightened ECCD36 signaling contributes to aortic insulin resistance, increased lipid accumulation, increased endothelial permeability and proinflammatory responses, fibrosis, vascular remodeling, and consequent aortic stiffening.

西方饮食（WD）导致肥胖和胰岛素抵抗的患病率上升，这两者都是动脉硬化和相关心血管疾病的关键危险因素。我们最近发现CD36升高与异位脂质积累增加、全身和组织胰岛素抵抗以及动脉硬化有关。在这里，我们进一步研究了内皮细胞（EC）特异性CD36 （ECCD36）是否参与了wd诱导的主动脉胰岛素抵抗、脂质积累、炎症、纤维化、重塑和相关的主动脉硬化。雌性ECCD36敲除（ECCD36−/−）和野生型（ECCD36+/+）小鼠在6周龄时，分别饲喂西方饮食（WD）或标准食物（CD） 16周。分别用超声（脉冲波速度）和钢丝肌图检查主动脉僵硬度和活动。western blot和定量PCR检测基因表达。分别采用油红O染色和免疫染色检测主动脉脂质含量和主动脉重构。16周的WD增加了主动脉硬化，这与血管胰岛素抵抗和通过磷酸肌肽3-激酶/蛋白激酶b减少胰岛素代谢信号有关。血管胰岛素抵抗和硬化的病理生理变化与哺乳动物雷帕霉素/S6激酶信号靶的激活、脂质紊乱增加、紧密连接相关蛋白occludin减少、促炎反应增加和主动脉重塑有关。在饲喂WD的ECCD36 - / -小鼠中，这些异常被减弱。这些研究结果表明，在致肥胖的西方饮食（WD）下，ECCD36信号的升高有助于主动脉胰岛素抵抗、脂质积累增加、内皮通透性增加和促炎反应、纤维化、血管重塑以及随之而来的主动脉硬化。

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引用次数: 0

Mechanisms underlying atrial fibrillation in chronic kidney disease 慢性肾脏疾病心房颤动的潜在机制

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-09 DOI: 10.1016/j.yjmcc.2025.06.002

Jose Alberto Navarro-Garcia , Joshua A. Keefe , Jia Song , Na Li , Xander H.T. Wehrens

Chronic kidney disease (CKD) is a serious and progressive worldwide health problem affecting 15 % of the global population. CKD is associated with higher mortality rates due to secondary complications such as cardiovascular disease. Common cardiovascular complications found in CKD patients include left ventricular hypertrophy, heart failure, and cardiac arrhythmias. The most common type of cardiac arrhythmia in CKD patients is atrial fibrillation (AF). Proper management of AF is important due to its high risk of cardiovascular complications and stroke. The incidence of AF remains higher in CKD patients than in the healthy population, highlighting the need to improve our understanding of the mechanisms underlying CKD-induced AF. In this review, we discuss well-known systemic factors linking CKD to AF pathogenesis. We highlighted the involvement of several inflammatory mediators in the CKD-induced atrial arrhythmogenesis. We also address special considerations for experimental models of CKD and AF management in CKD patients. Finally, we emphasize the need for a deeper understanding of the molecular underpinning, and for high-quality clinical investigations into the CKD-AF connection.

慢性肾脏疾病（CKD）是一种严重的进行性全球健康问题，影响全球15%的人口。由于继发性并发症，如心血管疾病，CKD与较高的死亡率相关。慢性肾病患者常见的心血管并发症包括左心室肥厚、心力衰竭和心律失常。CKD患者最常见的心律失常类型是心房颤动（AF）。由于房颤具有较高的心血管并发症和卒中风险，因此适当的治疗非常重要。与健康人群相比，慢性肾脏病患者的房颤发病率仍然更高，这凸显了我们需要提高对慢性肾脏病诱发的房颤机制的理解。在这篇综述中，我们讨论了众所周知的将慢性肾脏病与房颤发病机制联系起来的系统性因素。我们强调了几种炎症介质在ckd诱导的心房心律失常发生中的作用。我们还讨论了CKD实验模型和CKD患者AF管理的特殊考虑。最后，我们强调需要更深入地了解分子基础，并对CKD-AF联系进行高质量的临床研究。

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引用次数: 0

Optogenetic mitochondrial preconditioning enhances cardiomyocyte survival under stress 光遗传线粒体预处理提高心肌细胞在应激下的存活率

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-08 DOI: 10.1016/j.yjmcc.2025.06.004

Seulhee Kim , Hwayeon Lim , Patrick Ernst , Li Zhu , Jiashuai Zhang , Min Xie , Xiaoguang “Margaret” Liu , Lufang Zhou

Mitochondria play a central role in preconditioning-mediated cytoprotection, yet the specific role of mitochondrial membrane potential (ΔΨ_m) in this process remains incompletely understood. In this study, we employed a next-generation, mitochondrial-targeted optogenetic system (mOpto) to induce precisely controlled (partial and transient) ΔΨ_m depolarization and investigate its role in enhancing cardiomyocyte resilience to stress. Human AC16 cardiomyocytes expressing mOpto were subjected to low-intensity LED illumination for preconditioning, followed by exposure to stressors including FCCP, H₂O₂, or simulated ischemia-reperfusion. mOpto-preconditioned cells exhibited significantly improved viability, attenuated ΔΨ_m depolarization, and reduced reactive oxygen species (ROS) production compared to non-preconditioned controls. Notably, this cytoprotective effect occurred independently of canonical ROS signaling and mitochondrial ATP-sensitive potassium channel (mitoK_ATP) activation. Transcriptional analysis revealed coordinated mitochondrial and metabolic reprogramming, including upregulation of genes involved in lipid biosynthesis, mitochondrial quality control, energy homeostasis, and a shift toward mitochondrial fusion. Importantly, mOpto preconditioning conferred similar cytoprotective effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), underscoring the translational potential of this approach. These findings demonstrate that mOpto-mediated transient ΔΨ_m depolarization induces a preconditioning effect that enhances cardiomyocyte resilience through the establishment of a mitochondrial “memory” and dynamic remodeling of mitochondrial function.

线粒体在预调节介导的细胞保护中起着核心作用，但线粒体膜电位在这一过程中的具体作用（ΔΨm）仍未完全了解。在这项研究中，我们采用了下一代线粒体靶向光遗传系统（mOpto）来诱导精确控制的（部分和短暂的）ΔΨm去极化，并研究其在增强心肌细胞抗应激能力中的作用。将表达mOpto的人AC16心肌细胞置于低强度LED照明下进行预处理，然后暴露于包括FCCP、H2O2或模拟缺血再灌注在内的应激源。与未预处理的对照组相比，mopto预处理的细胞表现出显著提高的活力，减弱ΔΨm去极化，减少活性氧（ROS）的产生。值得注意的是，这种细胞保护作用独立于典型ROS信号和线粒体atp敏感钾通道（mitoKATP）激活而发生。转录分析揭示了线粒体和代谢重编程的协调，包括参与脂质生物合成、线粒体质量控制、能量稳态和向线粒体融合转变的基因的上调。重要的是，mOpto预处理在人诱导多能干细胞来源的心肌细胞（hiPSC-CMs）中具有类似的细胞保护作用，强调了该方法的转化潜力。这些发现表明，mopto介导的瞬时ΔΨm去极化诱导了一种预处理效应，通过建立线粒体“记忆”和线粒体功能的动态重塑来增强心肌细胞的弹性。

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引用次数: 0

The BAG3-HSP70-CHIP axis controls the degradation of TGFBR2 in cardiac fibroblasts BAG3-HSP70-CHIP轴控制TGFBR2在心脏成纤维细胞中的降解。

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-05 DOI: 10.1016/j.yjmcc.2025.06.003

Margaretha A.J. Morsink , Josephine M. Watkins , Katelyn Zhu , Xiaokan Zhang , Lori J. Luo , Barry M. Fine , Bryan Z. Wang , Gordana Vunjak-Novakovic

Transforming Growth Factor Beta (TGF-β) is a master regulator of cardiac fibrosis, in part through the type II TGF-β receptor (TGFBR2) which initiates signaling after ligand binding. We previously identified the co-chaperone protein Bcl2-associated athanogene (BAG3) as a modulator of TGFBR2 through ubiquitination and proteasomal degradation. However, the E3 ligase of TGFBR2 was not known. Using induced pluripotent stem cell-derived cardiac fibroblasts, we identified C-terminal interacting protein of HSP70 (CHIP) as an E3 ubiquitin ligase utilized by BAG3 for TGFBR2 degradation in cardiac fibroblasts. Overexpression of CHIP significantly decreased TGFBR2 stability, while inhibition of CHIP led to increased sensitivity to TGF-β and subsequent promotion of a fibrogenic program. Further, the BAG3-HSP70 interaction was crucial to this process, as disruption of the axis increased TGFBR2 stability and sensitivity to TGF-β signaling. Together, these findings demonstrate that the BAG3-HSP70-CHIP axis controls TGF-β signaling in cardiac fibroblasts and could serve as a new therapeutic target for cardiac fibrosis.

转化生长因子β (TGF-β)是心脏纤维化的主要调节因子，通过II型TGF-β受体（TGFBR2）在配体结合后启动信号传导。我们之前发现了bcl2相关的协同伴侣蛋白（BAG3）通过泛素化和蛋白酶体降解作为TGFBR2的调节剂。然而，TGFBR2的E3连接酶是未知的。利用诱导多能干细胞衍生的心脏成纤维细胞，我们发现HSP70的c端相互作用蛋白（CHIP）是一种E3泛素连接酶，被BAG3利用来降解心脏成纤维细胞中的TGFBR2。CHIP的过表达显著降低了TGFBR2的稳定性，而CHIP的抑制导致对TGF-β的敏感性增加，随后促进了纤维化程序。此外，BAG3-HSP70相互作用对这一过程至关重要，因为轴的破坏增加了TGFBR2的稳定性和对TGF-β信号的敏感性。综上所述，这些发现表明BAG3-HSP70-CHIP轴控制心肌成纤维细胞中TGF-β信号，可以作为心脏纤维化的新治疗靶点。

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引用次数: 0

Cytoplasmic mutant RBM20 causes arrhythmogenicity in murine atria. 细胞质突变体RBM20引起小鼠心房心律失常。

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-06-04 DOI: 10.1016/j.yjmcc.2025.06.001

Kensuke Ihara , Satoshi Iwamiya , Masaki Ikuta , Yurie Soejima , Yuichi Hiraoka , Atsushi Nakano , Susumu Minamisawa , Tetsushi Furukawa , Hidehito Kuroyanagi , Tetsuo Sasano

RNA binding motif protein 20 (RBM20) is a critical splicing regulator in cardiomyocytes, and mutations in its RSRSP domain are associated with severe dilated cardiomyopathy (DCM) and a high prevalence of atrial fibrillation (AF). RBM20 mutation has long been thought to cause DCM through the disturbed splicing of the target genes by its loss of function. However, recent studies have highlighted that the gain of function of mutant RBM20, independent of splicing defects, may also play a critical role in the pathogenesis of DCM. Despite these findings, the contribution of the gain of function of mutant RBM20 to the development of AF remains poorly understood. In this study, we aimed to elucidate the contribution of mutant RBM20 in atrial arrhythmogenicity by generating a novel atrial-specific mutant RBM20-expressing mouse model (Sln^Cre/+; LSL-Rbm20^S637A mice). These mice specifically expressed mutant RBM20 in the atria while maintaining RBM20-dependent alternative splicing. Analyses revealed the spontaneous development of atrial tachycardia and increased inducibility of AF, despite the absence of atrial structural remodeling or heart failure in Sln^Cre/+; LSL-Rbm20^S637A mice. Reduced atrial conduction velocity was observed, along with decreased and mislocalized expression of connexin 43, as well as abnormal Ca²⁺ handling and altered phosphorylation of Ca²⁺-handling proteins. These findings suggest that mutant RBM20 contributes to the arrhythmogenicity through mechanisms independent of splicing regulation, involving alterations in Ca²⁺ handling and electrical conduction property in murine atria.

RNA结合基序蛋白20 （RBM20）是心肌细胞中关键的剪接调节因子，其RSRSP结构域的突变与严重扩张型心肌病（DCM）和房颤（AF）的高患病率相关。长期以来，RBM20突变被认为是通过其功能丧失对靶基因剪接的干扰而导致DCM。然而，最近的研究强调，突变体RBM20的功能获得，独立于剪接缺陷，也可能在DCM的发病机制中发挥关键作用。尽管有这些发现，突变体RBM20的功能获得对房颤发展的贡献仍然知之甚少。在这项研究中，我们旨在通过建立一种新的心房特异性RBM20突变表达小鼠模型(SlnCre/+；LSL-Rbm20S637A老鼠)。这些小鼠在心房中特异性表达突变的RBM20，同时维持RBM20依赖性的选择性剪接。分析显示，尽管在SlnCre/+中没有心房结构重构或心力衰竭，但心房心动过速的自发发展和AF的诱导性增加；LSL-Rbm20S637A老鼠。观察到心房传导速度降低，同时连接蛋白43的表达减少和错位，以及Ca2+处理异常和Ca2+处理蛋白磷酸化改变。这些发现表明，突变体RBM20通过独立于剪接调节的机制参与心律失常，涉及小鼠心房Ca2+处理和电传导特性的改变。

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引用次数: 0

The role of connexin-43 in modeling arrhythmogenic diseases with induced pluripotent stem cell-derived cardiomyocytes 连接蛋白43在诱导多能干细胞源性心肌细胞模拟心律失常疾病中的作用

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-05-26 DOI: 10.1016/j.yjmcc.2025.05.008

Xijian Ke , Jonathan S. Baillie , Enrico D. Lemma , Martin Bastmeyer , Markus Hecker , Nina D. Ullrich

A common pathophysiological characteristic of arrhythmic diseases is the disruption of electrical signal transmission across the heart causing life-threatening rhythm disorders. These conditions are associated with decreased expression of connexin-43 (Cx43) at intercalated discs and its translocation to the lateral membranes, however, the underlying mechanisms remain unclear. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) offer a model for studying these pathophysiological processes. Here, we tested the hypothesis that chronic stress, usually preceding arrhythmic developments, modulates Cx43 expression. iPSC-CM were electrically stimulated at a normal rate and by tachypacing, and their electrical and Ca²⁺ signaling properties were analyzed. Our data revealed that tachypacing significantly reduced Cx43 expression by a micro-RNA miR-1-dependent mechanism. Anti-miR-1 treatment restored Cx43 expression in conditions of stress, enhanced Na⁺ currents, improved Ca²⁺ propagation and synchronized electrical activity. These findings suggest miR-1 as a potential pharmacological target for mitigating arrhythmogenic remodeling and restoring robust electrical signal transmission in cardiomyocytes.

心律失常疾病的一个共同病理生理特征是通过心脏的电信号传输中断，导致危及生命的节律障碍。这些疾病与嵌入椎间盘的连接蛋白43 （Cx43）表达减少及其向外侧膜的易位有关，然而，其潜在机制尚不清楚。诱导多能干细胞衍生的心肌细胞（iPSC-CM）为研究这些病理生理过程提供了一个模型。在这里，我们验证了慢性应激（通常在心律失常发展之前）调节Cx43表达的假设。对iPSC-CM进行正常速率和速搏电刺激，分析其电学和Ca2+信号传导特性。我们的数据显示，通过微rna mir -1依赖机制，速配显著降低了Cx43的表达。抗mir -1处理恢复了应激条件下Cx43的表达，增强了Na+电流，改善了Ca2+传播和同步电活动。这些发现表明miR-1可能是减轻心律失常重塑和恢复心肌细胞强大电信号传递的潜在药理学靶点。

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引用次数: 0

A Langendorff-heart discovery pipeline demonstrates cardiomyocyte targeting by extracellular vesicles functionalized with beta-blockers using click-chemistry Langendorff-heart发现管道证明了细胞外囊泡与β受体阻滞剂使用点击化学功能化的心肌细胞靶向。

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-05-23 DOI: 10.1016/j.yjmcc.2025.05.007

Kyung Chan Park , Amir Mashia Jaafari , Christopher Anthony Smith , Althea Rennisa Lobo , Lorenzo Errichelli , Gül Şimşek , Mala Gunadasa-Rohling , Alexander Marchant , Maria O. Levitin , Virginia Castilla-Llorente , Patrick Vilela , Pawel Swietach

Extracellular vesicles (EVs) are widely explored as vehicles for delivering therapeutic or experimental cargo to cardiomyocytes. Efforts to improve EV bioavailability in the heart, and reduce their off-target actions, require screening methods that can replicate the physiological and anatomical barriers present in the myocardium. Additionally, discovery pipelines must exercise control over EV dosage and timing, and provide a means of assessing cargo incorporation into cardiomyocytes specifically. These criteria are not generally met by experiments on cultured cells or animals. Here, we present a Langendorff-heart discovery pipeline that combines the strengths of in vivo and in vitro approaches. Langendorff-mode perfusion enables controlled exposure of beating hearts to re-circulated EVs. Following perfusion, cardiomyocytes can be isolated enzymatically for analysis, such as imaging. We tested this discovery pipeline by functionalizing EVs with beta-blockers (atenolol, metoprolol) using click-chemistry and incorporating the fluorescent protein NeonGreen2 to track the fate of EV cargo. Fluorescence in cardiomyocytes, including their nuclear regions, increased after Langendorff-treatment with beta-blocker decorated EVs, but only if these contained NeonGreen2, implicating the fluorescent cargo as the source of signal. Superior binding efficacy of beta-blockers was confirmed by referencing to the substantially lower signals obtained using wild-type EVs or EVs presenting myomaker or myomixer proteins, motifs that modestly enrich cardiac EV uptake in mice. Our findings demonstrate successful cardiomyocyte targeting using EVs decorated with beta-receptor binders. We propose the Langendorff-perfused heart as an intermediate step - nested between in vitro characterisation and animal testing - in discovery pipelines for seeking improved cardiac-specific EV designs.

细胞外囊泡（EVs）作为向心肌细胞输送治疗或实验货物的载体被广泛探索。努力提高EV在心脏中的生物利用度，减少它们的脱靶作用，需要能够复制心肌中存在的生理和解剖屏障的筛选方法。此外，发现管道必须控制EV的剂量和时间，并提供一种评估货物特异性进入心肌细胞的方法。在培养细胞或动物上进行的实验通常不符合这些标准。在这里，我们提出了一个朗根多夫心脏发现管道，结合了体内和体外方法的优势。langendorff模式灌注使跳动的心脏受控地暴露于再循环的ev中。灌注后，心肌细胞可以被酶分离用于分析，如成像。我们测试了这一发现管道，使用点击化学方法将β受体阻滞剂（阿替洛尔、美托洛尔）功能化电动汽车，并结合荧光蛋白NeonGreen2来跟踪电动汽车货物的命运。使用β受体阻滞剂修饰的ev进行langendorff处理后，心肌细胞（包括其核区域）的荧光增加，但仅当这些ev含有NeonGreen2时，这表明荧光货物是信号来源。通过参考野生型EV或呈现myomaker或myomixer蛋白的EV获得的显著较低的信号，证实了β受体阻滞剂的优越结合效率，这些基序适度地增加了小鼠心脏EV摄取。我们的研究结果表明，利用β受体结合物修饰的ev成功靶向心肌细胞。我们建议将langendorff灌注心脏作为中间步骤，嵌套在体外表征和动物试验之间，以寻求改进的心脏EV设计。

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引用次数: 0

The role of the troponin T interactions with actin in regulation of cardiac thin filament revealed by the troponin T pathogenic variant Ile79Asn 肌钙蛋白T致病变异Ile79Asn揭示的肌钙蛋白T与肌动蛋白相互作用在心脏细丝调控中的作用

IF 4.9 2区医学 Q1 CARDIAC & CARDIOVASCULAR SYSTEMS

Journal of molecular and cellular cardiology

Pub Date : 2025-05-22 DOI: 10.1016/j.yjmcc.2025.05.005

Cristina M. Risi , Maicon Landim-Vieira , Betty Belknap , P. Bryant Chase , Jose R. Pinto , Vitold E. Galkin

Cardiac muscle contraction/relaxation cycle depends on the rising and falling Ca²⁺ levels in sarcomeres that control the extent of interactions between myosin-based thick and actin-based thin filaments. Cardiac thin filament (cTF) consists of actin, tropomyosin (Tm) that regulates myosin binding to actin, and troponin complex that governs Tm position upon Ca²⁺-binding. Troponin has three subunits – Ca²⁺-binding troponin C (TnC), Tm stabilizing troponin T (TnT), and inhibitory troponin I (TnI). TnT N-terminus (TnT1) interactions with actin stabilize the inhibited state of cTF. TnC, TnI, and Tm work in concert to control actomyosin interactions. Cryo-electron microscopy (cryo-EM) provided factual structures of healthy cTF, but structures of cTF carrying missense mutations linked to human cardiomyopathy are unknown. Variant Ile79Asn in human cardiac TnT (TnT-I79N) increases myofilament Ca²⁺ sensitivity and slows cross-bridge kinetics, leading to severe hypertrophic/restrictive cardiomyopathy. Here, we used TnT-I79N mutation as a tool to examine the role of TnT1 in the complex mechanism of cTF regulation. Comparison of the cryo-EM structures of murine wild type and TnT-I79N native cTFs at systolic Ca²⁺ levels (pCa = 5.8) demonstrates that TnT-I79N causes 1) dissociation of the TnT1 loop from its actin interface that results in Tm release to a more activated position, 2) reduced interaction of TnI C-terminus with actin-Tm, and 3) increased frequency of Ca²⁺-bound regulatory units. Our data indicate that the TnT1 loop is a crucial element of the allosteric regulatory network that couples Tn subunits and Tm to maintain adequate cTF response to physiological Ca²⁺ levels during a heartbeat.

心肌收缩/松弛周期依赖于肌节中Ca2+水平的上升和下降，控制肌球蛋白基粗丝和肌动蛋白基细丝之间相互作用的程度。心肌细丝（cTF）由肌动蛋白、调节肌动蛋白与肌动蛋白结合的原肌球蛋白（Tm）和控制Ca2+结合时Tm位置的肌钙蛋白复合物组成。肌钙蛋白有三个亚基——钙结合肌钙蛋白C （TnC）、稳定肌钙蛋白T （TnT）和抑制肌钙蛋白I （TnI）。TnT n端（TnT1）与肌动蛋白的相互作用稳定了cTF的抑制状态。TnC、TnI和Tm协同作用，控制肌动球蛋白的相互作用。冷冻电子显微镜（cryo-EM）提供了健康cTF的真实结构，但携带与人类心肌病相关的错义突变的cTF结构尚不清楚。人类心脏TnT （TnT- i79n）变异Ile79Asn增加肌丝Ca2+敏感性，减缓过桥动力学，导致严重的肥厚/限制性心肌病。在这里，我们使用TnT-I79N突变作为工具来研究TnT1在cTF调节的复杂机制中的作用。在收缩期Ca2+水平（pCa = 5.8）下，比较野生型和天然cTFs的冷冻电镜结构表明，TnT-I79N导致1)TnT1环从其肌动蛋白界面分离，导致Tm释放到更活化的位置，2)减少TnI c端与actin-Tm的相互作用，3)增加Ca2+结合的调节单元的频率。我们的数据表明，TnT1环是变构调节网络的一个关键元素，该网络将Tn亚基和Tm偶联，以维持心跳期间cTF对生理Ca2+水平的足够反应。

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