Beta-adrenergic Response in Cardiac Hypertrophy/Failure

描述（由申请人提供）：信号转导的改变

DESCRIPTION (provided by applicant): Alterations in the signal transduction pathways which regulate Ca2+ dependent force in the heart contribute to the impaired contractile function in heart failure. These functional changes are likely to be mediated by altered phosphorylation of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) substrates. One of the major PKA/PKC substrates in the cardiac muscle cell is the thin filament regulatory protein, troponin I (TnI). As a result of conformational changes in the TnI molecular upon phosphorylation of the different PKA and PKC sites TnI, interactions between TnI with other proteins of the thin filament - and thus contractile function - are altered. In other words, TnI and its phosphorylation fingerprint represent a critical control point in the pathway regulating contractile state as a function of the incominb Ca2+ signal. We have shown that PKA phosphorylation of TnI is decreased by 25% in human heart failure. This results in increased Ca2+ affinity of troponin C (TnC), and may contribute to enhanced myofilament Ca2+ sensitivity, and prolonged relaxation of failing hearts. In contrast, PKC is reportedly increased in failing hearts; increased PKC phosphorylation of one or more sites on TnI decreases maximal actomyosin (AM) ATPase activity and thus could also contribute to impaired contraction in heart failure. However, reports on the effect of elevated PKC activity on TnI phosphorylation and cardiac function are conflicting. Finally, activity of protein phosphatases - protein phosphatase 1 (PP1) and/or PP2A - will also determine the phosphorylation state of TnI. In Specific Aim 1, we will identify the complete phosphorylation profile of TnI in failing human hearts with dilated cardiomopathy (DCM) and compare this with non-failing hearts. Electrospray ionization mass spectrometry (ESI/MS) will be used to quantify stoichiometry of the phosphorylated residues in tryptic digests of TnI obtained from failing and non-failing hearts, by a rapid one-step isolation to trop the in vivo phosphorylation state. In Specific Aim 2, we will (a) examine conformational changes that result from the combined changes of PKC and PKA phosphorylation of TnI in failing vs non-failing hearts. This will be achieved by measurement of fluorescence quenching tryptophan residues in cTnI, with selected serines and threonine mutated to aspartates or alanines, then reconstituted with human cardiac TnT and TnC. (b) The functional consequences of altered TnI phosphorylation will be assessed by measurement of Ca2+ dependent force in skinned cardiac trabeculae from failing and non-failing hearts. Specific Aim 3 will test the hypothesis that activity of TnI targeted phosphatases is altered in failing hearts. These studies should provide new information on the complete complement of changes in PKA and PKC-dependent TnI phosphorylation in human heart failure. Structural and functional outcomes of these changes plus identification of the altered phosphatase activity will shed light on mechanisms responsible for the functional decline in heart failure.