Rationale:Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca²⁺-sensitivity, and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model.Objective:Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed.Methods and Results:Dahl salt–sensitive rats fed a high-salt diet, with echocardiographically verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt–sensitive rats receiving low salt diet served as controls. Protein and phosphorylated Ser, Thr, and Tyr residues from left ventricular tissue were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared with control. Of those, 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, β, and δ concentration, whereas CDC treatment led to a reversion of PKCβ. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests that PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment.Conclusions:Increased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCβ upregulation, PKCβ merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention.

中文翻译：

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干细胞治疗舒张性心力衰竭中的肌丝磷酸化

基本原理：肌节蛋白的磷酸化与射血分数保留的心力衰竭 (HFpEF) 有关；^{这些变化可能通过改变收缩力、心脏硬度、Ca 2+}而导致舒张功能障碍-灵敏度和机械感应。在大鼠 HFpEF 模型中，心球衍生细胞 (CDC) 治疗可恢复正常的舒张功能，减轻纤维化和炎症，并提高存活率。分析方法和结果：Dahl 盐敏感大鼠喂食高盐饮食，超声心动图证实存在舒张功能障碍，随机分配至冠状动脉内 CDC 或安慰剂组。接受低盐饮食的 Dahl 盐敏感大鼠作为对照。来自左心室组织的蛋白质和磷酸化 Ser、Thr 和 Tyr 残基通过质谱法进行量化。HFpEF 心脏表现出广泛的过度磷酸化，与对照相比，529 个显着改变的磷酸位点中有 98% 增加。其中，39% 位于肌节亚蛋白质组内，一大群蛋白质位于或与 Z 盘相关。CDC 治疗部分恢复了过度磷酸化，其中 85% 的显着改变的 76 个残基被低磷酸化。差异磷酸化蛋白残基的生物信息学上游分析表明 PKC 是主要的假定调节激酶。PKC 异构体​​分析表明 PKC α、β 和 δ 浓度增加，而 CDC 处理导致 PKCβ 逆转。使用 PKC 异构体​​特异性抑制和各种 PKC 异构体​​的过表达强烈表明 PKCβ 是参与 HFpEF 过度磷酸化的主要激酶，并且随着 CDC 治疗而改变。结论：Z 盘上增加的蛋白质磷酸化与舒张功能障碍相关，PKC亚型驱动最量化的磷酸化变化。由于 CDC 逆转 HFpEF 的关键异常并选择性逆转 PKCβ 上调，因此 PKCβ 值得被归类为 HFpEF 的潜在治疗靶点，HFpEF 是一种众所周知的医学干预难治性疾病。