Title

The role of cardiac troponin I in the regulation of cardiac muscle contraction

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

James D. Potter, Committee Chair

Abstract

Our cloned cDNA of mouse cardiac troponin I (CTnI) showed that compared to skeletal TnI (STnI), mouse CTnI contained additional 32 amino-terminal residues including two adjacent serines (Serine 22,23). The Northern blot data showed that slow skeletal TnI (ssTnI) was the major form in the early stage of cardiac development, and it was replaced by cardiac TnI in adult cardiac muscle. The phosphorylation of CTnI at the two adjacent serines has been implicated in the inotropic response of the heart to $\beta$-agonists. In this study we found that phosphorylation of a cardiac skinned muscle preparation (CSM) by PKA, mainly at CTnI, resulted in a decrease in the Ca$\sp{2+}$-sensitivity of muscle contraction. To determine the role of the two PKA phosphorylation sites in mouse CTnI, serine 22 or 23, or both were mutated to alanine. We found that serine 23 was phosphorylated more rapidly than serine 22, and both serines were required to be phosphorylated in order to observe the characteristic reduction in Ca$\sp{2+}$-sensitivity of force development seen in skinned cardiac muscle preparation. To study the role of CTnI phosphorylation in the regulation of cardiac muscle relaxation, we studied the effect of CTnI phosphorylation on the rate of cardiac muscle relaxation. We found that the rate (t$\sb{1/2}$) of cardiac muscle relaxation increased from 110 $\pm$ 10 ms to 70 $\pm$ 8 ms after CTnI phosphorylation. Model calculations showed that increased dissociation of Ca$\sp{2+}$ from CTnC, coupled with the faster uptake of Ca$\sp{2+}$ by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, could account for the faster relaxation seen in the inotropic response of the heart to catecholamines. To further study the possible role of TnI isoform switch, endogenous CTnI from CSM was extracted, and reconstituted with either rat ssTnI or CTnI. The CSM reconstituted with either ssTnI or CTnI was tested for its Ca$\sp{2+}$-sensitivity of force development at pH 6.5 and pH 7. It was surprising to find that there was no difference of changes in the Ca$\sp{2+}$-sensitivity of force development in acidic pH between CSMs reconstituted with either ssTnI or CTnI. These data suggested that other factors, in addition to TnI isoforms, might also play a role in the different responses of neonatal or adult cardiac muscle to acidic pH.

Keywords

Biology, Molecular; Health Sciences, Pharmacology; Biology, Animal Physiology; Biophysics, Medical

Link to Full Text

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