Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes.

Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; however, its function in cardiomyocytes is unknown. Under basal conditions, PP5 is autoinhibited, but enzymatic activity rises upon binding of specific factors, such as the chaperone Hsp90. Here we show that PP5 binds and dephosphorylates the elastic N2B-unique sequence (N2Bus) of titin in cardiomyocytes. Using various binding and phosphorylation tests, cell-culture manipulation, and transgenic mouse hearts, we demonstrate that PP5 associates with N2Bus in vitro and in sarcomeres and is antagonistic to several protein kinases, which phosphorylate N2Bus and lower titin-based passive tension. PP5 is pathologically elevated and likely contributes to hypo-phosphorylation of N2Bus in failing human hearts. Furthermore, Hsp90-activated PP5 interacts with components of a sarcomeric, N2Bus-associated, mechanosensor complex, and blocks mitogen-activated protein-kinase signaling in this complex. Our work establishes PP5 as a compartmentalized, well-controlled phosphatase in cardiomyocytes, which regulates titin properties and kinase signaling at the myofilaments

Multiple Potential Molecular Contributors to Atrial Hypocontractility Caused by Atrial Tachycardia Remodeling in Dogs

Background-Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility. Methods and Results-ATR (7-day tachypacing) decreased APD (perforated patch recording) by approximate to 50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca2+](i) transients. ATR AP waveforms suppressed [Ca2+](i) transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca2+](i) transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had approximate to 60% smaller [Ca2+](i) transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced I-CaL; I-CaL inhibition superimposed on ATR APs further suppressed [Ca2+](i) transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca2+ release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca2+ sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca2+-calmodulin-dependent protein kinase II delta and protein phosphatase 1 activity were enhanced. Recovery of [Ca2+](i) transients and cell shortening occurred in parallel after ATR cessation. Conclusions-Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include I-CaL current reduction, impaired subcellular Ca2+ signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements. (Circ Arrhythm Electrophysiol. 2010;3:530-541.