A101. Uncovering the mechanisms of CapZ control of myofilament activation

Abstract

Cardiac Z-discs act as intracellular communications centres, anchoring and controlling molecular messengers. We have shown that reductions in the Z-disc protein CapZ alter myofilament activation and regulation by several intracellular signalling pathways. While these protein messengers are unable to exert control over myofilament function, they are still able to alter the phosphorylation status of several myofilament proteins. We hypothesized that reductions in CapZ protein render myofilaments insensitive to changes in protein phosphorylation, thereby disrupting contractile regulation. To test this hypothesis we treated cardiac myofilaments with type 1α protein phosphatase (PP1α) before or after reducing myofilament CapZ protein levels. By extracting CapZ after PP1α treatment we ensure that the proper amino acids are targeted and that any alterations in PP1α-dependent control are not due to the targeting of different amino acids or insufficient protein dephosphorylation. Treatment of cardiac myofilaments with exogenous PP1α significantly increased maximum Ca2+-dependent actomyosin MgATPase activity and Ca2+ sensitivity. Extraction of 20% CapZ prior to PP1α treatment significantly attenuated these changes, while CapZ extraction after PP1α treatment did not impact the effects of PP1α on myofilament activation. These results demonstrate that reduced CapZ protein levels inhibit myofilament regulation by the intracellular messenger PP1α. Moreover, our findings indicate that cardiac myofilaments are not insensitive to the covalent modification of their components when CapZ levels are decreased, which contradicts our hypothetical mechanism.