Cardiac Troponin I N-Terminal Pseudo-Phosphorylation Regulates Power Output and Stretch Activation

Abstract

Beat-to-beat heart function is modulated by β-adrenergic stimulation, which elicits PKA phosphorylation of several cardiac myocyte proteins including cardiac troponin I (cTnI). PKA phosphorylation of the cTnI N-terminal region is known to regulate myofilament Ca2+ sensitivity of force. Our lab has also observed PKA phosphorylation increases cardiac myofilament power and augments transient force overshoot. However, it is unknown if cTnI N-terminal phosphorylation per se affects myofilament power and stretch activation. We tested the hypothesis that cTnI N-terminal pseudo-phosphorylation is sufficient to increase power and stretch activation. Molecular sufficiency was investigated by measuring power and stretch activation after cTn exchange into rat permeabilized slow-twitch skeletal muscle fibers, since slow-skeletal (ss)TnI lacks N-terminal phosphorylation sites. Four different human cTnI N-terminal pseudo-phosphorylation states were tested: (i) non-phosphorylated WT, (ii) the canonical S22/23D PKA sites, (iii) the tyrosine kinase Y26E site, also known to modulate myofilament Ca2+ sensitivity, and (iv) the combinatorial S22/23D + Y26E cTnI. Following S22/23D cTnI exchange, fiber power increased ∼18% compared to WT cTn exchange where power decreased ∼3% (p < 0.05). Following Y26E cTnI exchange, power also increased (∼6%), albeit to a less degree than S22/23D. Exchange of combined S22/23D + Y26E cTnI resulted in a similar power increase (∼17%) as S22/23D alone, indicating S22/23D cTnI pseudo-phosphorylation is sufficient to regulate myofilament power. Exchange with S22/23D, Y26E, and S22/23D + Y26E cTnI all augmented stretch activation to a similar amount, implicating redundant site-specific stretch regulation. Finally, only the combinatorial S22/23D + Y26E cTnI increased stretch-activated rates of force development, implicating synergistic regulation of stretch-activated force kinetics. Overall, cTnI N-terminal pseudo-phosphorylation regulates myofilament power and stretch activation by a combination of dominant, redundant and synergistic biophysical mechanisms, which may complement each other to tune beat-to-beat myocardial output demand.