Experiments proposed here test the hypothesis that modulation of the structure, activity, and isoform population of troponin (TnI) are important determinants of the relative susceptibility of hearts to injury resulting from ischemia/reperfusion. We have focused on TnI, a key thin filament regulatory protein, in view of evidence that the extent of ischemia/reperfusion injury has been correlated: 1) with cardiac development which involves isoform switching from the embryonic slow skeletal (ssTnI) to the adult cardiac (cTnI) isoform, 2) with specific proteolysis of TnI, and 3) with protein kinase (PKC) dependent phosphorylation of TnI. Our approach involves the use of hearts of two transgenic (TG) mouse models with specific and functionally significant alterations in TnI. One transgenic model (ssTnI- TG) expresses the embryonic/neonatal isoform, slow skeletal TnI (ssTnI), in place of adult cardiac TnI (cTnI). The other transgenic model (cTnIS43,45A- TG) express a mutant, in which functionally significant serine PKC sites have been rendered non-phosphorylatable by mutation to alanines.
The specific aims are:
Aim #1) to compare the effects of ischemia/reperfusion of pressure developed by NTG control hearts with both ssTnI-TG and cTnIS43,45A-TG hearts, Aim #2) to determine whether proteolytic breakdown of cTnI is altered in hearts containing myofilaments in which cTnI is replaced with either ssTnI or cTnIS43,45A, Aim #3) to determine whether the functional properties (pH, length dependence of Ca2+-activation and tension cost) of myofilaments isolated from hearts stressed by ischemia/reperfusion are altered differently in NTG and the two TG models, and Aim #4) to determine whether treatment of hearts with calcium-sensitizers (CGP 48506 and EMD 57033), alters the functional consequences of ischemia/reperfusion NTG and TG models. Experiments proposed here provide new evidence on the molecular basis of ischemia/reperfusion injury in both neonatal and adult myocardium. The results are also of significance with regard to the design of future therapies.
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