This grant is focused on cardiac troponin I (cTnl), an essential protein of the cardiac sarcomere. Transgenic mouse studies have given conflicting results as to whether Tnl phosphorylation plays any role in the increased rate of myocardial relaxation during adrenergic stimulation. In addition, a major theme of this proposal is that heart function differs markedly between rodents and higher mammals due to key differences in contractile isoforms and calcium handling. Consequently, results obtained from transgenic experiments may not necessarily translate to cardiac myocytes that have more """"""""human-like"""""""" functional properties. The working hypothesis of this proposal is that cTnl phosphorylation status has an important role in the relaxation rate of canine myocytes that more closely resemble the functional properties of human cardiac muscle. Additionally, the acidosis accrued in myocardial infarction depresses cardiac function in part by altering Tnl function. The working hypothesis is that a charged amino-acid motif in the C-terminus of cTnl defines the critical switch domain responsible for acidic pH-induced alterations in Tnl function in myocytes from both small and large mammals. We have developed and optimized a gene transfer/primary culture system that takes advantage of the broad j species tropism of adenoviral vectors to accomplish rapid and efficient replacement of Tnl in living adult cardiac myocytes isolated from functionally divergent mammals. This technology is unique in the field as it allows rapid genetic engineering of TnI in living cardiac myocytes with """"""""human-like"""""""" contractile function.
The Specific Aims are:
Aim 1. To compare in adult cardiac myocytes from small and large mammals the direct effects of b-adrenergic-mediated Tnl phosphorylation on contractile performance.
Aim 2. To compare transgenesis versus gene transfer, focusing on the functional effects of phospho-mimetic Tnls in adult cardiac myocytes. Hypothesis: In comparison to wild-type rodent cardiac myocytes, myocytes expressing a phosphorylation mimetic cTnI (Ser23/24Asp) obtained by transgenesis or gene transfer will exhibit desensitization of calcium-activated isometric tension and enhanced relaxation kinetics.
Aim 3. To establish in a range of mammalian myocytes, the molecular basis for Tnl-isoform dependent altered calcium sensitivity of tension under normal and acidic pH conditions. Hypothesis: A highly charged and isoform variable motif in the C-terminus of Tnl defines the acidic pH-mediated decrease in tension in all mammalian species. Collectively, these studies will provide new information about Tnl function in cardiac myocytes from large versus small mammals, and will help identify new potential therapies for enhanced myofilament function in diseased myocardium.
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