Tropomyosin pseudo-phosphorylation and muscle kinetics

Tropomyosin contains a phosphorylation site at Ser-283 located within the head-to-tail overlap region that regulates muscle contraction. Previously we demonstrated that recombinant wild-type (Tm-WT) and pseudo-phosphorylated (Tm-S283D) alpha tropomyosin, expressed and purified from insect cells, exhibit regulated ATPase activity and troponin T binding similar to that reported for non-phosphorylated and phosphorylated tropomyosin (pTm) purified from muscle. We further demonstrated transgenic mice expressing the pseudo-phosphorylated tropomyosin (Tm-S283D Tg) exhibit increased mortality and decreased rate of relaxation in the work performing heart preparation. These changes occur in the absence of altered steady state maximal force or calcium-sensitive force development in skinned papillary bundles. In light of these findings, we sought to investigate the effect of pTm on the kinetics of cardiac muscle function. Using an extraction/replacement protocol, we measured force kinetics in a myofibril preparation. Results demonstrate no significant difference between myofibrils replaced with Tm- S283D or Tm-WT in maximal force, the rate of force activation, or the rate of force redevelopment, implying pTm does not play a role in altering muscle activation or cycling kinetics. To investigate if pTm affects the kinetics of thin filament inactivation we measured the rate of calcium disassociation from troponin C. Results demonstrate thin filaments reconstituted with Tm-S283D decreased the rate of calcium disassociation from troponin C consistent with the previously observed relaxation impairment. Finally, to determine the effects of pTm on systemic alterations in cardiovascular performance we measured heart function in Tm-S283D Tg mice by echocardiography. Results demonstrate Tm-S283D Tg mice exhibit trends towards impaired cardiac contractility compared to non-transgenic mice including decreased peak systolic velocity and ejection fraction. Overall, these findings demonstrate tropomyosin phosphorylation contributes to the regulation of cardiac dynamics; however, the precise role of pTm in the development of cardiac dysfunction remains elusive.