Key result
Mechanical stress loosens CapZ-actin binding via PIP2 interaction and PKC phosphorylation.
Why the study?
It was hypothesized that actin-capping protein (CapZ) integrates signaling by pathways including phosphorylation and PIP2 binding to regulate muscle fiber growth in response to mechanical load.
This study reveals that CapZ acts as a mechanotransduction integration node in cardiac myocytes, where its binding to actin is regulated by mechanical stiffness, PIP2, and phosphorylation, which may be relevant to fibrotic heart disease.
CapZ mechanosensitivity in rat myocytes leaves open its role as an integration node in human cardiac fibrosis; prospective studies needed before any therapeutic targeting.
Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.
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Solı́s et al. (2019) studied Mechanotransduction in cardiac myocytes. Substrate stiffness, neomycin, PMA, and CapZ mutations vs. Untreated cells or soft substrate (10 kPa) was evaluated on CapZ binding kinetics to sarcomeric thin filaments (FRAP) and molecular interactions with PIP2 (FRET). CapZ binding to actin is loosened by mechanical stimuli such as substrate stiffness, and this dynamic state is regulated by PIP2 binding and PKC phosphorylation at the β-tentacle.
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