Absence of Gravin's Scaffolding Enhances Myofilament Sensitivity to Calcium by Modulating Actomyosin ATPase Activity
Authors: Suryavanshi Santosh , Singh Sonal , Gao Wei Dong , McConnell Bradley 
Published: November 2023

Background

Gravin is an A‐kinase anchoring protein (AKAP) that scaffolds β2‐adrenergic receptors, protein kinase A (PKA), protein kinase C (PKC), protein phosphatase 2B (PP2B) etc. and thus regulates β‐adrenergic (β‐AR) signaling. In our lab, we are inclined to find out gravin‐mediated effects on β‐AR pathway in the heart. While studying the β‐AR pathway in mice, we noticed that absence of gravin has significant effects on calcium cycling in cardiac cells and muscles.

Objective

The purpose of the current work is to study calcium cycling in the heart in the absence of gravin's scaffolding in mice.

Methods

Left ventricular cardiac cells were freshly isolated from 8–10 weeks old wild‐type (WT) and gravin knock‐out (t/t) mice. Heart failure was induced in these mice by chronic β‐AR stimulation with isoproterenol (ISO) at 60mg/kg/day for 14 days. Intracellular calcium transients and sarcomere shortening was measured and analyzed using IonOptix® system. Trabecular muscles were isolated from right ventricles of WT and gravin t/t mice. Intracellular calcium transients and contractile force generated were measured in the presence or absence of extracellular calcium. Actomyosin ATPase activity was measured in freshly isolated myofilaments according to the published protocol with few modifications. Hill coefficient and logEC50 values were analyzed and compared.

Summary of Results

The cardiac myocytes isolated from the gravin t/t mice had significantly lower calcium transients and higher corresponding sarcomere shortening as compared to the WT mice. This difference between the two parameters remained unaltered even when these cardiac myocytes were stimulated in vitro with ISO. Furthermore, we found similar results with chronic ISO‐induced heart failure in these mice. Also, the amount of force generated in right ventricular trabecular muscles of WT and gravin t/t mice was unchanged at any external calcium concentration. However, intracellular calcium release was significantly lower in gravin t/t muscles as compared to the WT. Lower intracellular calcium release and equal magnitude of contraction indicated myofilament sensitivity to calcium. To further elucidate mechanisms involved, we performed actomyosin ATPase activity assay in these mice as this enzyme is crucial in regulating actin‐myosin cross‐bridge cycle. There was no change in the enzyme activity parameters in the isolated myofilaments of baseline hearts. Interestingly, gravin t/t mice hearts showed higher hill coefficient (1.871 ± 0.2173) than WT (0.8218 ± 0.1201) hearts after chronic ISO‐induced heart failure. This suggests that disruption of gravin scaffolding might improve actomyosin ATPase activity under stressed conditions.

Conclusion

Ablation of gravin's scaffolding increases myofilament sensitivity to the calcium by augmenting actomyosin ATPase enzyme activity in mice.

Support or Funding Information

Research reported in this abstract was supported by the NHLBI Institute of NIH under Award Number R01HL085487 (to B.M.) and R15HL124458 (to B.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.