Troponin I isoforms play a key role in determining myofilament Ca2+ sensitivity in cardiac muscle. The goal here was to identify domain clusters and residues that confer troponin I isoform-specific myofilament Ca2+ and pH sensitivities of contraction. Key domains/residues that contribute to troponin I isoform-specific Ca2+ and pH sensitivity were studied using gene transfer of a slow skeletal troponin I (ssTnI) template, with targeted cardiac troponin I (cTnI) residue substitutions. Substitutions in ssTnI with cognate cTnI residues R125Q, H132A, and V134E, studied both independently and together (ssTnIQAE), resulted in efficient stoichiometric replacement of endogenous myofilament cTnI in adult cardiac myocytes. In permeabilized myocytes, the pCa50 of tension ([Ca2+] required for half maximal force), and the acidosis-induced rightward shift of pCa50 were converted to the cTnI phenotype in myocytes expressing ssTnIQAE or ssTnIH132A, and there was no functionally additive effect of ssTnIQAE versus ssTnIH132A. Interestingly, only the acidosis-induced shift in Ca2+ sensitivity was comparable to cTnI in myocytes expressing ssTnIV134E, while ssTnIR125Q fully retained the ssTnI phenotype. An additional ssTnIN141H substitution, which lies within the same structural region of TnI as V134, produced a shift in myofilament Ca2+ sensitivity comparable to cTnI at physiological pH, while the acidic pH response was similar to the effect of wild-type ssTnI. Analysis of sarcomere shortening in intact adult cardiac myocytes was consistent with the force measurements. Targeted substitutions in the carboxyl portion of TnI produced residue-specific influences on myofilament Ca2+ and pH sensitivity of force and give new molecular insights into the TnI isoform dependence of myofilament function.
Bibliographical noteFunding Information:
We gratefully acknowledge technical assistance provided by Andrea Borton, Stan Forfa, and Dustin Robinson, and helpful comments by Todd Herron on earlier versions of the manuscript. This study was supported by the National Institutes of Health grants HL67254 (MVW) and HL59301 (JMM). Immunohistochemical analysis of myofilament incorporation utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIH5P60 DK20572 from the National Institute of Diabetes and Digestive and Kidney Diseases.
- Ca sensitivity
- Troponin I