Contractile Response to Endothelin in Myocytes Expressing Troponin I Ser43/45 Substitution Mutants

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Activation of protein kinase C (PKC) by the neurohormone, endothelin increases peak shortening and accelerates relaxation in adult rat cardiac myocytes. Endothelin-activated PKC also phosphorylates cardiac troponin I (cTnI), and our laboratory previously demonstrated a temporal role for the cTnIThr144 and cTnISer23/24 phosphorylation sites in the accelerated relaxation response to endothelin. The goal of the present study is to determine whether cTnISer43/45, a third potential target for PKC-mediated phosphorylation, plays a significant role in the contractile response to endothelin. Contractile function in adult rat cardiac myocytes expressing either cardiac troponin I with Ser43/45Ala (cTnIS43/45A) or Ser43/45Asp (cTnIS43/45D) substitutions was measured 4 days after gene transfer. Western analysis demonstrated 85-90% replacement of endogenous cTnI with cTnIFLAG, cTnIS43/45AFLAG, or cTnIS43/45DFLAG by 4 days post-gene transfer. The amplitude of sarcomere shortening increased 13.2+4.6% and the return velocity, a measure of relaxation rate increased 14.4+6.3% (n=14) in response to 10 nM endothelin over 15 min. In myocytes expressing cTnIS43/45A or cTnIS43/45D, the relaxation response to endothelin was similar to myocytes expressing cTnI or cTnIFLAG over the same time interval. The substitution mutant, cTnISer43/45Ala/Thr144Pro (cTnIA2P) was then studied to further investigate the role of individual cTnI phosphorylation sites in the endothelin response. Gene transfer of cTnIA2P produced significant cTnI replacement in preliminary studies of adult myocytes. However, in contrast to cTnIS43/45A or cTnIS43/45D, the accelerated relaxation response to 15 min of 10 nM endothelin was reduced in myocytes expressing cTnIA2P compared to cTnI-expressing myocytes (-2.3+ 4.7%, n=3). These preliminary results suggest Thr144, but not Ser43/45 plays a role in accelerating relaxation rate during the initial response to PKC activation by endothelin.