Transcriptomic analyses of cardiomyopathy development in mice carrying the phospholamban p.(Arg14del) pathogenic variant, and comparison with ischemic heart disease

Background: The p.(Arg14del) pathogenic variant (R14del) of the phospholamban (PLN) gene is a prevalent cause of cardiomyopathy with heart failure. The exact underlying pathophysiology is unknown, and a suitable therapy is unavailable. We aim to identify molecular perturbations underlying this cardiomyopathy in a clinically relevant PLN-R14del mouse model. Methods: We investigated progression of cardiomyopathy in PLN-R14?/? mice using echocardiography, electrocardiography and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 weeks of age (before onset of disease), 5 weeks (mild cardiomyopathy), and 8 weeks (end-stage). Data were compared with cardiac expression levels of mice that underwent myocardial ischemia-reperfusion or myocardial infarction surgery, in an effort to identify alterations that are specific to PLN-R14del-related cardiomyopathy. Results: At 3 weeks of age, PLN-R14?/? mice had normal cardiac function, but from the age of 4 weeks, we observed increased myocardial fibrosis and impaired global longitudinal strain. From 5 weeks onwards, ventricular dilatation, decreased contractility and diminished electrocardiogram voltages were observed. Strikingly, PLN protein aggregation was present prior to onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodeling, inflammation and metabolic dysfunction, in part similar to ischemic cardiomyopathy. Altered protein homeostasis pathways were identified exclusively in PLN-R14?/? mice, even before disease onset, in concert with aggregate formation. Conclusions: We mapped the development of PLN-R14del-related cardiomyopathy, and identified alterations in proteostasis and PLN protein aggregation amongst the first manifestations of this disease, which could possibly be a novel target for therapy. Overall design: RNA-Seq on mouse hearts from WT and phospholamban (Pln) R14del mutant mice (3, 5, or 8 weeks old), and ischemic heart diease induced by ischemia-reperfusion (I/R) injury, myocardial infarction (MI) or sham surgery (n=4/group).

背景：受磷蛋白（phospholamban, PLN）基因的p.(Arg14del)（精氨酸14缺失）致病变异体（R14del）是引发伴心力衰竭的心肌病的常见病因。其确切潜在病理生理学机制尚未明确，且尚无针对性的有效治疗方案。本研究旨在于临床相关的PLN-R14del小鼠模型中，阐明该心肌病的分子扰动机制。

方法：本研究通过超声心动图、心电图及组织病理学分析，探究PLN-R14Δ/Δ小鼠的心肌病进展情况。分别于小鼠3周龄（疾病发作前）、5周龄（轻度心肌病阶段）及8周龄（终末期）采集心脏组织，进行RNA测序与质谱分析法分析。同时将检测数据与接受心肌缺血再灌注手术、心肌梗死手术的小鼠的心脏基因表达水平进行对比，以期筛选出PLN-R14del相关心肌病所特有的分子改变。

结果：3周龄时，PLN-R14Δ/Δ小鼠的心脏功能正常，但自4周龄起，可见心肌纤维化程度升高及整体纵向应变受损。自5周龄起，可观察到心室扩张、收缩力下降及心电图电压降低。值得注意的是，在功能缺陷发作前，即可检测到PLN蛋白质聚集现象。转录组学与蛋白质组学分析显示，心肌重塑、炎症及代谢功能障碍相关过程存在差异调控，部分特征与缺血性心肌病相似。仅在PLN-R14Δ/Δ小鼠中可检测到蛋白质稳态（protein homeostasis）通路发生改变，且该改变早在疾病发作前即已出现，与聚集体形成过程同步发生。

结论：本研究明确了PLN-R14del相关心肌病的进展历程，并鉴定出蛋白质稳态与PLN蛋白质聚集改变是该疾病的早期表现之一，或可成为新型治疗靶点。

总体实验设计：对野生型（Wild Type, WT）及受磷蛋白（Pln）R14del突变型小鼠（3、5或8周龄）的心脏组织进行RNA测序，同时纳入由缺血再灌注（I/R）损伤、心肌梗死（MI）或假手术诱导的缺血性心脏病模型小鼠（每组n=4）。