Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala.

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

PKCßII modulation of myocyte contractile performance.

Significant up-regulation of the protein kinase Cß(II) (PKCß(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCß(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCß(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCß(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCß(II) (PKCßDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCß(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCß(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCß inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCß(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCß(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCß(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.

Calcitriol modulation of cardiac contractile performance via protein kinase C.

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.

Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response.

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.