Troponin I Ser-150 Phosphorylation Sustains Troponin Ca2+ Sensitivity in an Acidic Environment

Abstract

A hallmark of cardiac ischemia is decreased intracellular pH which can affect a number of cellular processes. Such an acidic environment alters cardiac troponin (Tn) myofilament regulation to decrease Ca2+ sensitive force production. Tn also undergoes cardiac ischemia-induced AMPK troponin I (TnI) Ser-150 phosphorylation. We recently characterized the effects of TnI Ser-150 phosphorylation demonstrating that it blunted the functional effects of canonical TnI Ser-23/24 phosphorylation; however, the role of Ser-150 phosphorylation in ischemia remains unknown. As an initial step, we sought to investigate the effect of acidic pH on myofilament regulation in the presence of TnI Ser-150 phosphorylation alone and in combination with Ser-23/24 phosphorylation. We first investigated the effect of in vivo cardiac ischemia on levels of TnI Ser-150 and Ser-23/24 phosphorylation. Exposure to 30 minutes of regional ischemia resulted in elevation of both TnI Ser-150 and Ser-23/24 phosphorylation. Next we determined the effects of TnI Ser-150 pseudo-phosphorylation (S150D) on the myofilament by measuring troponin C (TnC) Ca2+ binding properties at normal and acidic pH. Results demonstrate acidic pH decreases steady-state Ca2+ binding to TnC in reconstituted thin filaments across all Tn (WT, S150D, S23/24D, and S23/24/150D) such that TnI S150D Ca2+ sensitivity at pH 6.5 is similar to WT at pH 7. Decreasing the pH had no effect on Ca2+ dissociation such that compared to WT, S23/24/150D remained fast while S150D was slowed. We conclude that TnI Ser-150 phosphorylation imparts resistance to acidic pH-induced myofilament Ca2+ desensitization while retaining increased Tn Ca2+ dissociation when in combination with Ser-23/24 phosphorylation suggesting the potential for an increase in force while maintaining accelerated Ca2+ dissociation. Future investigations are aimed at examining the effect of TnI Ser-150 and Ser-23/24 phosphorylation on protease cleavage of TnI.