Abstract
Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (β2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in β-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute β-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca2+ in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased β2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent β2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin’s scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute β-AR stimulation by decreasing β2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.
Highlights
Activation of the beta-adrenergic receptors (β-ARs) via the sympathetic nervous system is the primary mechanism through which cardiac contractility and output is modulated [1]
In agreement with Fink et al, we suggest that the lack of change in the Ca2+ transients may involve altered Ca2+ influx through the L-type Ca2+ channel and/or altered Ca2+ release from SR through the ryanodine receptor as protein kinase A (PKA) phosphorylation modulates the activity of these two channels [42]
We have observed that cMyPBC Ser-273 phosphorylation was significantly increased in the gravin-t/t mice compared to WT mice in the absence of ISO. We propose that this baseline increase in Ser-273 phosphorylation in the gravin-t/t mice may help to explain the increased cardiac function seen in the gravin-t/t mice as studies have shown that increased cardiac myosin binding protein C (cMyBPC) phosphorylation results in enhancement of the actin-myosin interaction resulting in the augmentation of the cross-bridge cycling rate and increased cardiac contractility [38,44,45]
Summary
Activation of the beta-adrenergic receptors (β-ARs) via the sympathetic nervous system is the primary mechanism through which cardiac contractility and output is modulated [1]. Norepinephrine or epinephrine binds to β-ARs to activate GTPbinding proteins, which regulate the activity of adenylyl cyclase. Stimulatory GTP-binding proteins (Gs) prompt the formation of cAMP, which activates protein kinase A (PKA), the main effector of β-AR signaling. PKA phosphorylates an assortment of proteins involved in the regulation of calcium movement and contractility. Inhibitory G-proteins (Gi) attenuate adenylyl cyclase activity resulting in the reduction of PKA-. Mediated regulation of cardiac function [2,3].
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