The Right Ventricular Fibroblast Secretome Drives Cardiomyocyte Dedifferentiation. The Right Ventricular Fibroblast Secretome Drives Cardiomyocyte Dedifferentiation

Rationale: In virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We’ve previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells. Objective: To determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture. Methods and Results: Brief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation. Conclusions: These data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction. Overall design: Two experiments were performed. The first was to identify transcriptome differences between bovine cardiac fibroblasts from high altitude animals to low altitude animals. The second experiment was to determine the effects of the fibroblast conditioned media from the high altitude animals on adult rat ventricular myocytes vs control, untreated cells.

研究背景：在几乎所有心力衰竭（heart failure）模型中，预后均由右心室（Right Ventricular, RV）功能决定；因此，明确导致右心室功能障碍的细胞机制至关重要。器官水平的整体重构与细胞特异性改变相关，包括心肌细胞去分化以及心脏成纤维细胞（cardiac fibroblasts, Cfib）的激活，而后者又与细胞骨架蛋白紊乱及肌节结构丧失相关。然而，这些细胞改变如何影响右心室功能仍不明晰。我们此前在大型肺动脉高压（pulmonary hypertension, PH）动物模型中观察到显著的器官水平右心室功能障碍，但分离得到的心肌细胞功能并未出现对应降低。我们提出假说：内源性心脏成纤维细胞分泌的因子通过构建由去分化细胞构成的异质性细胞环境，进而导致整体右心室功能障碍。
研究目的：明确肺动脉高压犊牛心脏成纤维细胞条件培养基（pulmonary hypertension conditioned media, PH-CM）对体外培养的成年大鼠心室肌细胞（adult rat ventricular myocytes, ARVM）的作用。
方法与结果：将成年大鼠心室肌细胞短时间（<2天）暴露于PH-CM，可使其快速、显著地去分化为具备自发收缩行为的新生样表型。去分化细胞可维持存活超过30天，持续表达心肌肌钙蛋白I（troponin I, TnI）与α-辅肌动蛋白（α-actinin）等心肌细胞蛋白，但同时表现出成肌纤维细胞特征，包括α-平滑肌肌动蛋白（α-smooth muscle actin）的表达。通过生物信息学（bioinformatics）方法筛选介导去分化的调控因子，我们发现肺动脉高压来源的心脏成纤维细胞激活后呈现独特的转录组（transcriptome）特征，其与分泌组（secretome）中的因子以及去分化肌细胞中的激活通路均存在显著关联。进一步研究证实，心脏成纤维细胞及条件培养基中骨桥蛋白（periostin）的表达上调，且骨桥蛋白足以诱导心肌细胞去分化。
结论：上述数据表明，肺动脉高压犊牛的心脏成纤维细胞释放的旁分泌因子（paracrine factor）可诱导部分心肌细胞发生表型转化，这一过程可能参与右心室功能障碍的发生。针对该过程的干预手段（如骨桥蛋白抑制）或可预防右心室功能障碍。
整体实验设计：本研究共开展两项实验。第一项实验旨在对比高海拔与低海拔牛源心脏成纤维细胞的转录组差异。第二项实验则探究高海拔动物来源的成纤维细胞条件培养基对成年大鼠心室肌细胞的影响，并以未经处理的细胞作为对照。