Protein Kinase A Inhibitor Peptide (PKI) and Cardiac Protection in Heart Failure

描述(由申请人提供)：心肌梗死(MI)是心力衰竭的主要原因。MI需要交感肾上腺素能系统(SAS)的持续激活，以维持心脏的泵功能。SAS的激活导致蛋白激酶A(PKA)和钙/钙调蛋白依赖的激酶II(CaMK II)的过度激活，从而导致不利的心脏重构，促进心力衰竭的发展。因此，限制过度的PKA活性可能对心肌梗死后的心脏有有益的影响。心脏中存在可能调节PKA活性的内源性PKA抑制蛋白(PKI)。然而，PKI在正常和疾病心脏中的作用仍不清楚。我们发现心肌梗死后小鼠心脏内源性PKIa表达上调，心肌梗死后PKIa缺乏增强了心脏的肾上腺素能反应，但加速了心衰的发生。β-AR还激活了非依赖于PKA的心肌保护信号通路，因为：(1)抑制PKA使cAMP信号转导到EPAC/Rap1/Raf/ERK通路，从而保护培养的心肌细胞免于凋亡。(2)PKI-GFP转基因小鼠心肌梗死后心功能改善，肥大减轻。(3)β-受体阻滞剂美托洛尔可减少PKI对心肌梗死后心脏的部分有益作用。在这项研究中，我们将确定KI是否以及如何调节正常和梗死心脏的肾上腺素能信号。我们假设，PKI介导的抑制应激心脏中过度的PKA激活将减少PKA和CaMK II信号的潜在有害影响，并将通过cAMP/EPAC和B2AR/GI/Akt通路保留SAS信号的有益效果。我们的假设是，对于心脏应激状态下过度的SAS活动的有害影响，PKA是一个重要的结点控制点。我们预测，临床上有效的BAR拮抗剂用于治疗心力衰竭患者，可能会减少BAR信号的有害和心脏保护功能。因此，通过PKI的选择性PKA抑制方法将模仿一种“优化的”偏向的β-受体阻滞剂，这可能比通常使用的β-受体阻滞剂疗法提供更多的益处。为了验证这些想法，我们建立了一个PKIa基因敲除小鼠系，以及高表达不同水平(高、中、低)PKI-GFP融合基因的转基因小鼠系。为了探索EPAC激活在心肌梗死后通过抑制PKA而免于心脏保护的作用，我们将使用EPAC1或Epac2基因缺陷的小鼠。我们的具体目标是：1.确定PKI对内源性PKA抑制在心梗后心力衰竭发生中的作用。PKI-A基因敲除小鼠和对照小鼠将受到MI的应激。2.确定选择性抑制PKA和过表达PKI微型基因(无论是通过基因操作还是通过病毒基因传递)是否以及如何减少MI诱导的引起心衰的结构和功能变化。我们还将比较PKI和β-受体阻滞剂抑制PKA的保护作用。我们的长期目标是揭示PKA/PKI在HF中的作用，并探索利用PKI治疗HF的可能性。

DESCRIPTION (provided by applicant): Myocardial infarction (MI) is a major cause of HF. MI requires persistent activation of the sympathetic adrenergic system (SAS) in order to maintain the pump function of the heart. SAS activation causes excessive activation of protein Kinase A (PKA) and Ca2+/calmodulin-dependent kinase II (CaMK II), which causes adverse cardiac remodeling and promotes HF development. Thus, limiting excessive PKA activity could have beneficial effects in hearts after MI. There are endogenous PKA inhibitor proteins (PKI) in the heart that may regulate PKA activity. However, the role of PKI in normal and diseased hearts remains unclear. We have found that the endogenous PKIa is upregulated in mouse hearts after MI and PKIa deficiency enhances cardiac adrenergic responses but precipitates HF development after MI. beta-AR stimulation also activates PKA- independent cardioprotective signaling pathways because: (1) PKA inhibition spares cAMP signaling to EPAC/Rap1/Raf/ERK pathway to protect cultured myocytes from apoptosis~ (2) PKI-GFP transgenic mice had improved cardiac function and reduced hypertrophy than control mice after MI. (3) Metoprolol, a beta-blocker may reduce some of beneficial effects of PKI in post-MI hearts. In this study we will determine if and how KI regulates adrenergic signaling in the normal and infarcted heart. We hypothesize that PKI-mediated inhibition of excessive PKA activation in stressed hearts will reduce the potentially detrimental effects of PKA and CaMK II signaling and will preserve beneficial effects f SAS signaling through cAMP/EPAC and b2AR/Gi/Akt pathways. Our hypothesis is that PKA is an essential nodal control point for the detrimental effects of excessive SAS activity i cardiac stress states. We predict that clinically effective bAR antagonists used to tret HF patients will probably reduce both detrimental and cardioprotective features of bAR signaling. Therefore, a selective PKA inhibitory approach through PKI will mimics an "optimized" biased beta-blocker, which may provide more benefit than commonly used beta-blocker therapies. To test these ideas, we have established a PKIa knockout mouse line, and transgenic mouse lines overexpressing different levels (high, medium and low) of a PKI-GFP fusion gene. To explore the role of EPAC activation in cardiac protection spared by PKA inhibition after MI, we will use mice deficient in EPAC1 or EPAC2. Our SPECIFIC AIMS are: 1. To determine the role of endogenous PKA inhibition by PKI in HF development after MI. PKI-a knockout and control mice will be stressed with MI. 2. To determine if and how selective inhibition of PKA, with overexpression of a PKI minigene (either by genetic manipulation or alternatively by viral gene delivery), can reduce MI-induced structural and functional changes that cause HF. We will also compare the protective effects of PKA inhibition with PKI to those of beta-blockers. Our long-term goal is to reveal the roles of PKA/PKI in HF and explore the possibility of using PKI to treat HF.