Wang Jinxi, Yang Bennett Daniela Sarahi, Echard Emma J, Chen Biyi, Ciampa Grace, Zhao Weiyang, Shi Qian, Yoon Jin-Young, Weiss Robert M, Grueter Chad E, Hall Duane D, London Barry, Song Long-Sheng
Abboud Cardiovascular Research Center, Division of Cardiovascular Medicine, Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA. (J.W., D.S.Y.B., E.J.E., B.C., W.Z., Q.S., J.-Y.Y., R.M.W., C.E.G., D.D.H., B.L., L.-S.S.).
Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA. (G.C., L.-S.S.).
Circulation. 2025 Jul 25. doi: 10.1161/CIRCULATIONAHA.125.073937.
Calcium (Ca) homeostasis in cardiac fibroblasts (CFs) plays a critical role in myocardial repair and remodeling after injury. JPH (junctophilin-2; human JPH2 or mouse Jph2) is a structural protein known to regulate intracellular Ca signaling and excitation-contraction coupling in cardiomyocytes. However, the role of JPH2 in CF biology remains unexplored.
Junctophilin expression was assayed in human and mouse CFs using reverse transcription quantitative polymerase chain reaction, Western blotting, and immunofluorescence. To investigate the functional role of Jph2 in CFs, we assessed Ca handling with live-cell confocal imaging, conducted RNA sequencing analysis, and assayed TGFβ (transforming growth factor β) responses after adenovirus-mediated gene silencing of Jph2 and fibroblast-specific Jph2 knockout. Jph2 interactions in CFs were identified using immunoprecipitation and proximity biotin ligation assays and confirmed by coimmunoprecipitation and rescue experiments. The in vivo role of Jph2 in CFs was investigated in fibroblast-specific Jph2 knockout mice (Col1a2/Jph2, Jph2) at baseline and after myocardial infarction and evaluated for changes in cardiac function, fibrosis, angiogenesis, CF activation, differentiation, and proliferation.
Jph2 was identified as the only junctophilin expressed in CFs and acutely upregulated in response to myocardial infarction. Cellular and RNA sequencing analyses revealed that isolated Jph2-deficient CFs exhibited impaired fibroblast activation, reduced extracellular matrix production, and diminished expression of VEGFA (vascular endothelial growth factor A), VEGFB, and VEGFC after TGFβ treatment. In vivo, Jph2 mice displayed exacerbated adverse cardiac remodeling after myocardial infarction, characterized by decreased CF activation/extracellular matrix production, enhanced CF proliferation, worsened systolic dysfunction, enlarged left ventricular dilation, impaired scar maturation, and decreased angiogenesis. Mechanistically, biochemical assays demonstrated that Jph2 interacts directly with the coiled-coil 2 domain of Stim1 (stromal interaction molecule 1) via its joining region. Jph2 knockdown led to Stim1 protein destabilization, defective store-operated Caentry, and a reduction in both canonical and noncanonical TGFβ signaling pathways. Stim1 overexpression partially rescued the sensitivity of Jph2-deficient CFs to TGFβ, as evidenced by increased expression of periostin and fibronectin-1, as well as enhanced Smad3 phosphorylation.
Our results demonstrate that Jph2 is required in CFs to orchestrate Ca homeostasis, CF activation, and extracellular matrix production and promotes angiogenesis in the infarcted heart, positioning it as a central regulator of cardiac repair after injury.
心脏成纤维细胞（CFs）中的钙（Ca）稳态在损伤后的心肌修复和重塑中起关键作用。连接蛋白（junctophilin-2；人类JPH2或小鼠Jph2）是一种结构蛋白，已知其可调节心肌细胞中的细胞内Ca信号传导和兴奋-收缩偶联。然而，JPH2在CF生物学中的作用仍未得到探索。
使用逆转录定量聚合酶链反应、蛋白质印迹法和免疫荧光法检测人及小鼠CFs中的连接蛋白表达。为了研究Jph2在CFs中的功能作用，我们通过活细胞共聚焦成像评估Ca处理情况，进行RNA测序分析，并在腺病毒介导的Jph2基因沉默和成纤维细胞特异性Jph2基因敲除后检测转化生长因子β（TGFβ）反应。使用免疫沉淀和邻近生物素连接测定法鉴定CFs中的Jph2相互作用，并通过共免疫沉淀和拯救实验进行确认。在基线期及心肌梗死后，在成纤维细胞特异性Jph2基因敲除小鼠（Col1a2/Jph2，Jph2）中研究Jph2在CFs中的体内作用，并评估心脏功能、纤维化、血管生成、CF激活、分化和增殖的变化。
Jph2被确定为CFs中唯一表达的连接蛋白，并在心肌梗死后急性上调。细胞和RNA测序分析显示，分离出的Jph2缺陷型CFs表现出成纤维细胞激活受损、细胞外基质产生减少，以及在TGFβ处理后血管内皮生长因子A（VEGFA）、血管内皮生长因子B（VEGFB）和血管内皮生长因子C（VEGFC）的表达降低。在体内，Jph2基因敲除小鼠在心肌梗死后表现出更严重的不良心脏重塑，其特征为CF激活/细胞外基质产生减少、CF增殖增强、收缩功能障碍加重、左心室扩张增大、瘢痕成熟受损和血管生成减少。从机制上讲，生化分析表明Jph2通过其连接区域直接与基质相互作用分子1（Stim1）的卷曲螺旋2结构域相互作用。Jph2敲低导致Stim1蛋白不稳定、储存性钙内流缺陷，以及经典和非经典TGFβ信号通路均减少。Stim1过表达部分挽救了Jph2缺陷型CFs对TGFβ的敏感性，这通过骨膜蛋白和纤连蛋白-1表达增加以及Smad3磷酸化增强得以证明。
我们的结果表明，CFs中需要Jph2来协调Ca稳态、CF激活和细胞外基质产生，并促进梗死心脏中的血管生成，使其成为损伤后心脏修复的核心调节因子。
