Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium.

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca2+ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca2+ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines43/45 and threonine144 (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca2+ sensitivity required the presence of phosphorylatable cTnI. For comparison, the beta-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca2+ sensitivity. These beta-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca2+ and ATP, thereby promoting efficient ATP utilisation during contraction.

Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice.

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during beta-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine(43/45) and threonine(144) mutated to alanine) and for PKA (serine(23/24) mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by beta-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the beta-agonist isoproterenol decreased twitch duration and relaxation time constant (tau) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after beta-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased tau by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that beta-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.