Right ventricular contractile dysfunction after pressure overload

描述(由申请人提供)： 急性肺动脉高压所致的右室收缩衰竭是大面积肺栓塞、缺氧性肺血管收缩、体外循环和心脏移植后的发病率和死亡率的重要原因，但目前尚无有效的治疗策略。申请人实验室在一个大型动物模型中进行的研究表明，急性肺动脉高压引起的右室收缩衰竭与右室半胱氨酸蛋白酶Calain的激活有关，这种情况下的右心衰竭可能会被Calain抑制剂缓解。已证明在骨骼肌和心肌功能障碍的许多模型中都存在Calain的激活，但对于Calain激活的上游机制和激活的calain的下游靶点都存在争议：根据实验模型的不同，收缩功能障碍归因于结构蛋白如结蛋白、血影蛋白、1-肌动蛋白或肌动蛋白的破坏；调节蛋白的修饰，如肌钙蛋白-I、肌钙蛋白-T；或启动细胞凋亡。本项目的总体目标是确定急性右室压力超负荷在急性右室压力超负荷期间和之后引起钙蛋白激活和右室收缩功能障碍的机制。我们已经证明，已知的Calain底物--焦点黏附复合体蛋白talin的降解与RV功能障碍的严重程度高度相关，并获得了初步数据，表明在急性肺动脉高压期间，Calain重新分布到心肌细胞膜上。其他研究人员提出，Calain可能受非电压门控钙通道TRPM7、细胞外反应激酶ERK的直接磷酸化或酪氨酸激酶src对Calain靶标的磷酸化调控。因此，我们提出了以下两个假设：假设1：急性肺动脉高压引起的钙蛋白酶激活依赖于钙通道TRPM7、ERK和/或src。假设2：急性肺动脉高压中局部的钙蛋白酶激活通过降解粘着斑复合体(腔粒)蛋白，如Talin，导致右室功能障碍。为了验证这些假说，我们提出了以下具体目标：具体目标1：利用啮齿动物RV乳头肌建立新的急性RV压力超负荷的体外模型，以证实我们建立的活体猪急性RV压力超负荷模型的结果。具体目标2：确定导致压力超负荷所致RV功能障碍的信号通路和蛋白质修饰。具体目标3：确定压力超负荷引起的RV功能障碍是否依赖于TRPM7、ERK或src。具体目标4：确定目前正在进行临床试验的蛋白水解酶或蛋白激酶抑制剂是否可以减轻急性右室压力超负荷时的右心衰竭。我们将使用已建立的大动物(猪)体内急性肺动脉高压模型，以及利用分离的大鼠右室乳头肌建立的新的急性右室压力超负荷的体外模型。通过模拟急性RV压力超负荷的收缩应激状态下的大鼠RV乳头肌，将评估蛋白质重新分布和修饰的生化和组织学证据，并将确定Calain和蛋白激酶抑制剂对这些改变的影响。在猪模型中进行的平行实验将被用来建立体外啮齿动物乳头肌模型的发现的生理学意义。具有潜在临床实用价值的药物将使用分离的乳头肌模型进行鉴定，并在猪模型中进行测试。

DESCRIPTION (provided by applicant): Right ventricular (RV) contractile failure from acute pulmonary hypertension is an important cause of morbidity and mortality in conditions such as massive pulmonary embolism, hypoxic pulmonary vasoconstriction, and following cardiopulmonary bypass and cardiac transplantation, but effective therapeutic strategies are not currently available. Studies in the applicant's laboratory performed in a large animal model have demonstrated that RV contractile failure from acute pulmonary hypertension is associated with activation of the cysteine protease calpain in the RV, and that right heart failure in this setting may be attenuated by a calpain inhibitor. Calpain activation has been shown to occur in numerous models of both skeletal and cardiac muscle dysfunction, but there is controversy over both the upstream mechanism of calpain activation and the downstream targets of activated calpain: depending on the experimental model, contractile dysfunction has been attributed to disruption of structural proteins such as desmin, spectrin, 1-actinin or titin; modification of regulatory proteins such as troponin-I, troponin-T; or initiation of apoptosis. The overall goal of this project is to identify the mechanism by which acute RV pressure overload causes calpain activation and right ventricular contractile dysfunction during and following acute RV pressure overload. We have shown that degradation of the focal adhesion complex protein talin, a known calpain substrate, is highly correlated with the severity of RV dysfunction, and have obtained preliminary data showing that calpain redistributes to the myocyte membrane during acute pulmonary hypertension. Other investigators have proposed that calpain may be regulated by the non-voltage gated calcium channel TRPM7, by direct phosphorylation by the extracellular response kinase ERK, or by phosphorylation of calpain targets by the tyrosine kinase src. Therefore, we propose the following two hypotheses: Hypothesis #1: Acute pulmonary hypertension induced activation of calpain is dependent on the calcium channel TRPM7, ERK, and/or src. Hypothesis #2: Localized calpain activation in acute pulmonary hypertension causes RV dysfunction through degradation of focal adhesion complex (costameric) proteins such as talin. To test these hypotheses, we propose the following Specific Aims: Specific Aim 1: Employ a new ex vivo model of acute RV pressure overload using rodent RV papillary muscle to confirm findings from our established in vivo porcine model of acute RV pressure overload. Specific Aim 2: Identify signaling pathways and protein modifications that contribute to pressure overload induced RV dysfunction. Specific Aim 3: Determine if pressure overload induced RV dysfunction depends on TRPM7, ERK or src. Specific Aim 4: Determine whether protease or protein kinase inhibitors currently in clinical trials can attenuate right heart failure in acute RV pressure overload. We will use an established large animal (pig) in vivo model of acute pulmonary hypertension in conjunction with a new ex vivo model of acute RV pressure overload employing isolated rat RV papillary muscles. Rat RV papillary muscles subjected to contractile stress, modeling acute RV pressure overload, will be assessed for biochemical and histological evidence of protein redistribution and modification, and the effects of calpain and protein kinase inhibitors on these alterations will be determined. Parallel experiments performed in vivo in the pig model will be used to establish the physiological significance of findings from the ex vivo rodent papillary muscle model. Agents with potential clinical utility will be identified using the isolated papillary muscle model and tested in the pig model.