Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere

Abstract

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca2+ sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.

The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

“…Both residues are located on the H1 alphahelix of cTnI [106]. A similar regulatory mechanism to S23/24 phosphorylation was predicted upon phosphorylation of S43/45, yielding an accelerated dissociation of Ca 2+ from cTnC [103]. However, S43/45 phosphorylation induces additional conformational changes that have not been observed with S23/24 pseudophosphorylation.…”

“…PKC mediated phosphorylation on cTnI S43 or S45 contributes to cardiac dysfunction in heart disease [19,103–105]. Both residues are located on the H1 alphahelix of cTnI [106].…”

“…Phosphorylation at S43/45 extends the interaction of cTnI and cTnC while keeping thin filaments in an inactive state [103,108]. In isolation, this S43/45 effect could significantly alter contractility, but it is tempered by cellular adaptions via cardiac myosin binding protein-C S282 and cTnI S23/24 phosphorylation that normalize cell shortening or re-lengthening [103].…”

“…In isolation, this S43/45 effect could significantly alter contractility, but it is tempered by cellular adaptions via cardiac myosin binding protein-C S282 and cTnI S23/24 phosphorylation that normalize cell shortening or re-lengthening [103]. Complete replacement with cTnI phosphomimetics of PKC targets S23/24, S43/45 and T144 resulted in a modest decrease in several components of cardiac function including pressure development, rate ejection fraction, and relaxation rate.…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Pseudo-phosphorylated Tm was shown to increase maximal myosin ATPase activity without altering the steadystate Ca 2+ sensitivity of the myofilament, thereby regulating muscle relaxation dynamics (Nixon et al 2013). Similarly, viral-based gene transfer studies show that replacing myofilament cardiac TnI with phosphomimetic cTnI S43/S45D may serve as modulatory brakes by slowing/reducing contraction as well as leading to adaptive changes to modulate crossbridges cycling and fine-tune contractile performance (Lang et al 2015). Finally, phosphorylation in myosin essential light chain has also been studied.…”