Cardiac troponin I tyrosine 26 phosphorylation decreases myofilament Ca2+ sensitivity and accelerates deactivation

Abstract

Troponin I (TnI), the inhibitory subunit of the troponin complex, can be phosphorylated as a key regulatory mechanism to alter the calcium regulation of contraction. Recent work has identified phosphorylation of TnI Tyr-26 in the human heart with unknown functional effects. We hypothesized that TnI Tyr-26 N-terminal phosphorylation decreases calcium sensitivity of the thin filament, similar to the desensitizing effects of TnI Ser-23/24 phosphorylation. Our results demonstrate Tyr-26 phosphorylation and pseudo-phosphorylation decrease calcium binding to Troponin C (TnC) on the thin filament and calcium sensitivity of force development to a similar magnitude as TnI Ser-23/24 pseudo-phosphorylation. To investigate the effects of TnI Tyr-26 phosphorylation on myofilament deactivation, we measured the rate of calcium dissociation from TnC. Results demonstrate filaments containing Tyr-26 pseudo-phosphorylated TnI accelerate the rate of calcium dissociation from TnC similar to that of TnI Ser-23/24. Finally, to assess functional integration of TnI Tyr-26 with Ser-23/24 phosphorylation, we generated recombinant TnI phospho-mimetic substitutions at all three residues. Our biochemical analyses demonstrated no additive effect on calcium sensitivity or calcium-sensitive force development imposed by Tyr-26 and Ser-23/24 phosphorylation integration. However, integration of Tyr-26 phosphorylation with pseudo-phosphorylated Ser-23/24 further accelerated thin filament deactivation. Our findings suggest that TnI Tyr-26 phosphorylation functions similarly to Ser-23/24 N-terminal phosphorylation to decrease myofilament calcium sensitivity and accelerate myofilament relaxation. Furthermore, Tyr-26 phosphorylation can buffer the desensitization of Ser-23/24 phosphorylation while further accelerating thin filament deactivation. Therefore, the functional integration of TnI phosphorylation may be a common mechanism to modulate Ser-23/24 phosphorylation function.

Myofilament Calcium Sensitivity: Mechanistic Insight into TnI Ser-23/24 and Ser-150 Phosphorylation Integration

“…However, the combination of Ser-23/24 and Ser-150 phosphorylation exhibit functional integration by differentially regulating cardiac function from that of either phosphorylation alone, retaining contractile force (normal Ca 2+ sensitivity) but accelerating relaxation (accelerated Ca 2+ dissociation) (Nixon et al, 2012, 2014). This and others' findings demonstrate the significance of TnI phosphorylation integration to cardiac function during disease (Kooij et al, 2010; Boontje et al, 2011; Taglieri et al, 2011; Salhi et al, 2014). Ultimately, elucidating the different molecular mechanisms utilized during simultaneous TnI phosphorylation will provide insight into the regulatory protein interactions that can be therapeutically targeted, such as by altered phosphorylation, to modulate contraction and relaxation in cardiac disease.…”

“…Therefore, it is not the isolated effect of a single TnI phosphorylation that is responsible for overall cardiac regulation, but rather the simultaneous presence of multiple TnI phosphorylations that combine to exhibit expected or unexpected functional integration. The significance of these multiple phosphorylations results in a functional integration as demonstrated by the finding that the contractile effects of TnI Ser-23/24 phosphorylation are dependent on the myofilament phosphorylation background (Biesiadecki et al, 2007; Kooij et al, 2010; Nixon et al, 2012; Salhi et al, 2014; Lang et al, 2015). The mechanisms underlying how combined TnI phosphorylations integrate to regulate the myofilament response to Ca 2+ remain poorly understood.…”

TNNI1, TNNI2 and TNNI3: Evolution, regulation, and protein structure–function relationships

“…Thr 31 is a substrate of mammalian sterile 20-like kinase 1 (Mst1) (You et al, 2009). Tyr 26 phosphorylation was shown to function similarly to that of Ser 23/24 in decreasing myofilament Ca 2+ sensitivity and increasing cardiac muscle relaxation (Salhi et al, 2014). Physiological significance and regulatory mechanisms of these phosphorylation sites remain to be further investigated.…”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…Salhi et al . demonstrated that Tyr-26 phosphorylation, glutamic acid substitution (Y26E) or aspartic acid substitution (Y26D) decreased the Ca 2+ binding to thin filament, decreased the Ca 2+ sensitivity of force development (by exchanging the recombinant human cTn into skinned rat myocyte), and accelerated the Ca 2+ dissociation rate from cTnC [110]. Wijnker et al .…”