Protein Kinase C Phosphomimetics Alter Thin Filament Ca2+ Binding Properties

Bin Liu,Joseph J Lopez,Jonathan P Davis,Brandon J Biesiadecki,Sakthivel Sadayappan

doi:10.1371/journal.pone.0086279

Abstract

Adrenergic stimulation modulates cardiac function by altering the phosphorylation status of several cardiac proteins. The Troponin complex, which is the Ca2+ sensor for cardiac contraction, is a hot spot for adrenergic phosphorylation. While the effect of β-adrenergic related PKA phosphorylation of troponin I at Ser23/24 is well established, the effects of α-adrenergic induced PKC phosphorylation on multiple sites of TnI (Ser43/45, Thr144) and TnT (Thr194, Ser198, Thr203 and Thr284) are much less clear. By utilizing an IAANS labeled fluorescent troponin C, , we systematically examined the site specific effects of PKC phosphomimetic mutants of TnI and TnT on TnC’s Ca2+ binding properties in the Tn complex and reconstituted thin filament. The majority of the phosphomemetics had little effect on the Ca2+ binding properties of the isolated Tn complex. However, when incorporated into the thin filament, the phosphomimetics typically altered thin filament Ca2+ sensitivity in a way consistent with their respective effects on Ca2+ sensitivity of skinned muscle preparations. The altered Ca2+ sensitivity could be generally explained by a change in Ca2+ dissociation rates. Within TnI, phosphomimetic Asp and Glu did not always behave similar, nor were Ala mutations (used to mimic non-phosphorylatable states) benign to Ca2+ binding. Our results suggest that Troponin may act as a hub on the thin filament, sensing physiological stimuli to modulate the contractile performance of the heart.

Highlights

The heart is a highly dynamic organ that can regulate both its contractile strength and speed to accommodate the demands of the body [1]
We studied the effects of these phosphomimetic mutants on Tn’s Ca2+ sensitivity, and their effects on Tn’s Ca2+ exchange kinetics, which may be even more significant to how the heart performs since the heart is dynamic and does not function in a static steady-state
Our results show that all the phosphorylation related protein modifications of troponin I (TnI) or troponin T (TnT) alter Tn’s Ca2+ binding in a way that can be related to previous physiological studies

Summary

Introduction

The heart is a highly dynamic organ that can regulate both its contractile strength and speed to accommodate the demands of the body [1]. A critical way to regulate cardiac function is through adrenergic pathways. It is well established that altered intracellular Ca2+ signaling contributes to the altered cardiac performance upon adrenergic stimulation [2,3]. Adrenergic stimulation and its subsequent phosphorylation of contractile proteins alters how the heart responds to the Ca2+ signal [4]. Numerous studies have demonstrated that the PKA-dependent TnI phosphorylation at Ser23/24 desensitizes the myofilament to Ca2+, accelerates thin filament deactivation and contributes to faster relaxation of the heart [5]. Adrenergic stimulation regulates cardiac function through altering both intracellular Ca2+ signaling and myofilament responsiveness to the Ca2+ signal

Methods

Results

Conclusion