Cardioprotection and uncoupling myofibroblast-myocyte communications

项目总结/摘要 作为对损伤的反应，病理性心脏重塑经常表现为心肌肥大， 纤维化、收缩功能障碍和/或增加的心律失常脆弱性。肌成纤维细胞（Mfbs）通常不 存在于工作心肌中，但在受伤的心脏中大量积聚。分泌过多 来自活化的Mfbs的胶原和旁分泌因子导致电绝缘隔膜的形成， 心肌细胞（CM）的离子重构，分别促进心律失常。除了他们的屏障和 旁分泌功能，使用活体电压成像和免疫组织化学的初步实验表明， Mfbs通过以下途径影响纤维化重塑心肌的功能特性的有趣可能性 通过由细胞间连接蛋白（Cx）43组成的间隙连接与周围CM直接电紧张性相互作用 亚单位。初步的小鼠模型数据还显示，慢性全身性输注β-肾上腺素能或 血管紧张素II受体激动剂以及急性心肌梗死（MI）导致Mfb限制性错误表达 Cx43、牵张诱导的促纤维化细胞因子Ccl 2（MCP-1）和促纤维化基质细胞骨膜蛋白（Postn） 与病理性重塑一致。同样，在急性心肌梗死或压力超负荷患者中， MCP-1被强烈上调，Cx43（GJA 1）分布受到干扰。Cx43，主要间隙连接 负责心室中动作电位传播的蛋白质，通常在肥大和 缺血性心脏病患者我们和其他人已经证明，Cx43也存在于病理性Mfb中， 另外的初步数据显示，Ccl 2在Mfbs中转录（而其受体Ccr 2在Mfbs中转录）。 CM）。内源性Postn在功能上促进心脏纤维化/心室僵硬，因为存活的Postn无效 心肌梗死后纤维化程度较低，功能较好。此外，我们还构建了一个3. 9 kb的Postn增强子 仅在受损心脏的Mfbs中驱动Cre重组酶表达的报告基因系（命名为Postn-Cre）。因此，在本发明中， 这是对心脏激活的损伤位点Mfbs进行遗传操作的最有用的工具。然而，实际作用 表达Postn的Mfb谱系中的电偶联作用和Mfb-CM的潜在机制 病理性重塑过程中的相互作用仍然未知。因此，提出了三个相互关联的目标， 检查这些初步数据。目的1将检验通过以下途径解偶联Mfb-CM电信号的假设： Cx43的Mfb限制性cKO可预防不良心脏重塑。目标2将测试Mfb限制的Cx43是否 cKO提供心律失常保护。目的3将确定Mfb限制的Ccl 2细胞因子损失是否改善 邻近CM收缩功能障碍。

PROJECT SUMMARY/ABSTRACT In response to damage, pathological cardiac remodeling frequently manifests as myocardial hypertrophy, fibrosis, contractile dysfunction and/or increased arrhythmia vulnerability. Myofibroblasts (Mfbs) are not normally present in the working myocardium but accumulate in large numbers in the injured heart. Hypersecretion of collagen and paracrine factors from activated Mfbs results in the formation of electrically insulating septa and ionic remodeling of cardiomyocytes (CMs), respectively, promoting arrhythmia. Besides their barrier and paracrine function, preliminary experiments using intravital voltage imaging and immunohistochemistry, suggest the intriguing possibility that Mfbs influence the functional properties of fibrotically remodeled myocardium via direct electrotonic interactions with surrounding CMs via gap junctions composed of cell-to-cell Connexin(Cx)43 subunits. Preliminary mouse modeling data also revealed that chronic systemic infusion of β-adrenergic or angiotensin-II receptor agonists, as well as acute myocardial infarction (MI) result in Mfb-restricted misexpression of Cx43, a stretch-induced profibrotic cytokine Ccl2 (MCP-1) and profibrotic matricellular Periostin (Postn) coincident with pathological remodeling. Likewise, in patients with acute MI or pressure overload, POSTN and MCP-1 are robustly upregulated and Cx43 (GJA1) distribution is perturbed. Cx43, the principal gap junction protein responsible for action potential propagation in ventricles, is often mis-expressed in hypertrophied and ischemic patient hearts. We and others have demonstrated that Cx43 is also present in the pathological Mfb population, and additional preliminary data revealed Ccl2 is transcribed in Mfbs (whereas its receptor Ccr2 is in CMs). Endogenous Postn functionally promotes cardiac fibrosis/ventricular stiffness, as surviving Postn nulls exhibit less fibrosis and better function after MI. Moreover, we generated a unique 3.9kb-driven Postn enhancer reporter line that drives Cre-recombinase expression only in Mfbs in injured hearts (designated Postn-Cre). Thus, this is a most useful tool for genetic manipulation of cardiac activated injury-site Mfbs. However, the actual role that electrical coupling in Postn-expressing Mfb-lineage plays and the mechanism underlying Mfb-CM interactions during pathological remodeling remain unknown. Thus, three interrelated aims are proposed to examine these preliminary data. Aim 1 will test the hypothesis that uncoupling Mfb-CM electrical signaling via Mfb-restricted cKO of Cx43 prevents adverse cardiac remodeling. Aim 2 will test whether Mfb-restricted Cx43 cKO offers arrhythmia protection. Aim 3 will determine if Mfb-restricted loss of the Ccl2 cytokine ameliorates adjacent CM contractile dysfunction.