Characterisation of the cardiac ryanodine receptor in obesity and the role of caveolin-3 as a putative regulator of receptor function

Abstract

Obesity is linked to the development of cardiovascular diseases including type 2 diabetes mellitus (T2DM). Impaired excitation-contraction (EC) coupling, due to aberrant calcium homeostasis, is a hallmark of obesity and T2DM. EC coupling is maintained by the finely tuned process termed calcium induced calcium release. The ryanodine receptor (RyR2) is a crucial component of the EC coupling apparatus, regulating the release of Ca2+ from the sarcoplasmic reticulum, SR, into the cytosol initiating contraction. Previously published data from our group has shown that caveolin-3 (Cav3), a component of caveolae small cave-like folds within sarcolemma, interacts with skeletal muscle isoform RyR1. The expression of Cav3, a negative regulator of nitric oxide (NO) synthase (NOS) and thus NO bioavailability, has been reported to be decreased in several animal models of diabetes. Increased NO bioavailability has been linked to RyR2 S-nitrosylation, through the addition of a nitroso group, resulting in leaky RyRs. However, the role of Cav3 in modulating this process is yet to be described. Therefore, the aims of this research work were to; i) investigate the hypothesis that obesity leads to down regulation of Cav3 and increased bioavailability of NO which in turn causes S-nitrosylation of RyR2 resulting in impaired calcium homeostasis and ii) determine if Cav3 interacts with RyR2 and the structural basis for a Cav3-RyR2 interaction. A model of obesity was developed using male Sprague Dawley by feeding a 45% fat diet (HFD) ad libitum for 16 weeks. It was determined that a HFD led to the development of hyperinsulinemia but with no evidence of cardiac dysfunction. However, at the molecular level Western blotting showed a reduction in Cav1, nNOS and eNOS but with no change to Cav3, SERCA2a (a Ca2+ ATPase that pumps Ca2+ back into the SR leading to relaxation) or RyR2 in the obese left ventricle (LV). There was no change to NOS activity and using an HPLC method it was determined NO bioavailability was unaltered. In agreement with these data a biotinylation assay revealed that the S-nitrosylation state of RyR2 was unchanged in the obese LV. However, a key finding was that there was an increase in SR Ca2+ release after high fat feeding. Morphological analysis of the cardiomyocytes applying serial block face scanning electron microscopy revealed that the caveolae density was reduced after high fat feeding, 2.07  0.04 (n = 3) compared to 3.14  0.43 (n =3) control group, p

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ashraf Kitmitto (Supervisor) & Elizabeth Cartwright (Supervisor)