Background: Myofilament protein phosphorylation is central to cardiac muscle contractile regulation. Results: AMP-activated protein kinase (AMPK) phosphorylates troponin I (TnI) to increase cardiac contraction and blunt the effects of TnI protein kinase A phosphorylation. Conclusion: AMPK myofilament signaling represents a novel mechanism to regulate cardiac contraction. Significance: AMPK links metabolics to TnI regulation of cardiac muscle function and adrenergic regulation.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then ‘stitch’ together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

The binding of Ca2+ to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin-myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca2+ to TnC and impaired contraction. To determine the pathological relevance of simultaneous TnI phosphorylation we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca2+ binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca2+ from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylation to maintain Ca2+ binding while accelerating Ca2+ dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca2+ sensitive activation. These data suggest TnI Ser-150 phosphorylation attenuation of the pH-dependent decrease in Ca2+ sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.

Tropomyosin (Tm) is a central protein in the Ca2+ regulation of striated muscle. The Tm isoform undergoes phosphorylation at serine residue 283. While the biochemical and steady-state muscle function of muscle purified Tm phosphorylation have been explored, the effects of Tm phosphorylation on the dynamic properties of muscle contraction and relaxation are unknown. To investigate the kinetic regulatory role of Tm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants from insect cells. Purified Tm’s regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm’s, however disassociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to WT Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. These data demonstrate that Tm pseudo-phosphorylation slows deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle activation. This supports a role for Tm as a key protein in the regulation of muscle relaxation dynamics.

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