Cardiac Myosin Binding Protein-C in Development and Reversal of Heart Failure

2017年，心力衰竭(HF)困扰着650万美国人，5年死亡率约为50%，而且还可能增加 到2030年将达到800万英镑。过去46年来死亡率没有显著改善，现实需要 因心力衰竭射血分数降低(HFrEF)出现低血压而停止有效药物， 而有效治疗心力衰竭的保留射血分数(HFpEF)结合起来 表明需要新的治疗方法。心肌肌球蛋白结合蛋白-C(CMyBPC)驻留在厚壁 心肌的细丝。CMyBPC在其M结构域的磷酸化增加了跨桥循环的速度。 因为增加跨桥循环速度可以改善收缩性能和弹性，所以我们假设 CMyBPC的磷酸化提供了一种新的中枢机制，可以预防和治疗不同类型的心力衰竭 原因。使用模拟3个丝氨酸位点去磷酸化和磷酸化cMyBPC的小鼠模型(S) 在M域，我们最近发现cMyBPC的磷酸化减缓了与年龄相关的发育 HFpEF和经主动脉缩窄(TAC)手术诱导的HFrEF：存活率和更好的 保留舒张期功能。因此，cMyBPC的磷酸化具有治疗HFrEF和 HFpEF。然而，我们发现有证据表明，在我们研究的3个S位点之外，有2个新的S磷酸化位点增加了 显著的功能效应。因此，我们已经制作了2个新的敲入鼠标模型，以包括新的 阐明最大磷酸化cMyBPC(5SD)和去磷酸化cMyBPC(5SA)作用的位点 M-域。我们还发现了压力应激可以激活粘着斑激酶(FAK)磷酸化的证据 酪氨酸(Y)残基(S)。这一发现导致了一个新的支持假说，即压力应激 作为一种代偿反应，触发FAK磷酸化cMyBPC以增加收缩能力。有了这些 考虑到这些发现，我们打算确定最大M结构域磷酸化的有效性，并阐明 新的FAK-cMyBPC机制。目标1：确定磷酸化的疗效和伴随机制 CMyBPC可在心力衰竭诱导的衰老、压力应激和肥胖条件下保护心脏功能。我们 将挑战两年老化、TAC或高脂肪饮食的小鼠。我们将使用功能和生物化学的组合 确定潜在机制的技术。CMyBPC(5SD)小鼠的抗衰老和抗衰老能力 使用cMyBPC(5SD)基因治疗逆转WT衰竭心脏的有效性将量化cMyBPC的疗效 磷酸化分别用于预防和治疗。目标2：阐明信号机制和 压力应激诱导cMyBPC酪氨酸磷酸化的功能结果。我们将确定Y站点(S)， 确认FAK-cMyBPC相互作用，阐明FAK磷酸化cMyBPC对模型测距的影响 从完整的乳头肌到新的敲击小鼠。影响：疗效结果和机械洞察力 本研究为将cMyBPC的磷酸化转化为新的HF治疗方法提供了证据。

Heart failure (HF) afflicted 6.5 million Americans in 2017, carries 5-year mortality of ~50%, and will likely increase to > 8 million by 2030. The lack of significant improvement in mortality for the last 46 years, the reality of needing to discontinue effective medications due to hypotension in heart failure with reduced ejection fraction (HFrEF), and the paucity of effective treatment for heart failure with preserved ejection fraction (HFpEF) combine to suggest that new treatments are needed. Cardiac myosin binding protein-C (cMyBPC) resides on the thick filament of the heart muscle. Phosphorylation of cMyBPC at its M-domain increases cross-bridge cycling rate. Because increasing cross-bridge cycling rate can improve both contractility and lusitropy, we hypothesize that phosphorylation of cMyBPC provides a novel central mechanism that can prevent and treat HF due to different causes. Using mouse models that mimic de-phosphorylated and phosphorylated cMyBPC at 3 serine (S) sites in the M-domain, we recently discover that cMyBPC phosphorylation mitigates both aged-related development of HFpEF and trans-aortic constriction (TAC) surgery induced HFrEF, as seen by improved survival and better preservation of diastolic function. Thus, cMyBPC phosphorylation holds potential to treat both HFrEF and HFpEF. However, we find evidence that 2 new S phosphorylation sites outside our study of 3 S sites add significant functional effects. Consequently, we have made 2 new knock-in mouse models to include the new sites for elucidating the effects of maximally phosphorylated cMyBPC(5SD) and dephosphorylated cMyBPC(5SA) M-domain. We also find evidence that pressure stress can activate focal adhesion kinase (FAK) to phosphorylate tyrosine (Y) residue(s) in cMyBPC. This discovery leads to a new supporting hypothesis that pressure stress triggers FAK to phosphorylate cMyBPC to increase contractility as a compensatory response. With these discoveries in mind, we intend to determine the efficacy of maximal M-domain phosphorylation and elucidate the novel FAK-cMyBPC mechanism. Aim #1: Determine the efficacy and companion mechanisms of phosphorylated cMyBPC to preserve cardiac function under HF inducing conditions of aging, pressure stress, and obesity. We will challenge mice with 2-yr aging, TAC, or high fat diet. We will use a combination of functional and biochemistry techniques to determine the underlying mechanisms. Ability of cMyBPC(5SD) mouse to resist deterioration and effectiveness of using cMyBPC(5SD) gene therapy to reverse failing WT hearts will quantify the efficacy of cMyBPC phosphorylation for prevention and treatment respectively. Aim #2: Elucidate signaling mechanism and functional results of pressure stress induced tyrosine phosphorylation of cMyBPC. We will identify Y site(s), confirm FAK-cMyBPC interaction, and elucidate effects of FAK phosphorylating cMyBPC on models ranging from intact papillary muscle to new knock-in mouse. Impact: Efficacy results and mechanistic insights stemming from this study provide evidence for translating cMyBPC phosphorylation to new HF treatment.