Sarah E Lang1, Tamara K Stevenson2, Dongyang Xu3, Ryan O'Connell4, Margaret V Westfall5. 1. Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA. 2. Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. 3. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. 4. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. 5. Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: wfall@med.umich.edu.
Abstract
A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.
A phospho-null Ala substitution at protein kinase C (n>an class="Gene">PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.
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