The overarching goal of this research is to design, test, and implement sarcomere-based therapies for contractile dysfunction in ischemic heart failure (IMF). Health relevance stems from IMF being a leading cause of combined mortality and morbidity in this country. In ischemic myocardium, the intracellular acidosis that accrues in ischemic muscle uncouples the sarcomere from Ca2+; hence, the contractile machinery does not respond effectively to the normal Ca2+ transient. Troponin I (Tnl) is a molecular switch of the sarcomere and has a central, isoform-dependent role in ischemic contractile failure. In fetal/neonatal heart, the slow skeletal Tnl (ssTnl) gene is expressed and confers protection from ischemia-mediated contractile failure relative to the cardiac Tnl (cTnl) isoform expressed exclusively in adult heart. We found that a single histidine residue, present in ssTnl and absent in cTnl, confers pH-dependent alterations in the molecular switch function of Tnl, reminiscent of archetypal histidine buttons in biology, e.g., the pH-dependent histidine switch in hemoglobin. The overarching hypothesis of this proposal is histidine-substituted cTnl serves as a titratable, """"""""myofilament sensor"""""""", that tunes the gain-of-contractile function in response to the changing biochemical milieu of the ischemic and failing myocardium in vivo.
The Specific Aims are:
(Aim 1) To determine the mechanism underlying histidine-substituted cTnl's titratable gain-of-contractile function by targeted modifications at cTnl codon 164 in rodent and human adult cardiac myocytes. Cardiac Tnl codon 164 resides in the molecular switch regulatory domain of cTnl that toggles between actin and troponin C (TnC) during the contractile cycle. Hypothesis. The protonated chemical switch of cTnl A164H, as in the case of ischemic myocardium, strengthens Tnl-TnC interactions.
(Aim 2) To determine in genetically engineered animals in vivo the short- and long-term cardiovascular effects of cTnl A164H in models of ischemia and failure. Hypothesis: Cardiac Tnl A164H expression by transgenesis and by adeno-associated vector systemic gene transfer in vivo will confer long-term improvements in systolic and diastolic performance, compared to wild-type mice, by virtue of improved synergy between the sarcomere and intracellular Ca2+ in experimental and genetic models of myocardial ischemia, stunning, and failure. ? ? ?
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