Stretch regulation of β2-Adrenoceptor signalling in cardiomyocytes requires caveolae.

AIMS: Caveolin-3 is essential for the formation of caveolae in cardiomyocytes. Caveolar microdomains have been shown to regulate the distribution of signalling proteins such as beta-adrenoceptors (βAR) and may act as membrane reserves to protect the cell from damage during the mechanical stretch. Myocardial stretch occurs during haemodynamic overload and may be normal (e.g. exercise) or pathological (e.g. heart failure); therefore, it is important to understand the effect of stretch on signalling pathways associated with mechanosensitive structures, such as caveolae. In this study, we investigate the role of caveolae in regulating the effect of stretch on βAR-signalling. METHODS AND RESULTS: We used osmotic swelling of isolated rat ventricular cardiomyocytes as a method to stretch the cell membrane and investigate the effect of βAR stimulation on cyclic adenosine monophosphate (cAMP) activity and contractility. βAR response was measured using a Förster Resonance Energy Transfer reporter for the second messenger cAMP and using CytoCypher for the measurement of cell contractility. β1AR and β2AR blockers were used to selectively allow stimulation of β2AR and β1AR, respectively. We also investigated the effect of stretch on βAR response to isoprenaline stimulation in left ventricular trabeculae dissected from control and cardiac-specific caveolin-3 knock-out mice (Cav3KO). Stretching trabeculae produces increased baseline adenylyl cyclase activity and a higher level of cAMP and a greater β2AR-induced positive inotropy after stimulation of the β2AR but not β1AR, by isoprenaline. Similar findings were confirmed for isolated myocytes subjected to hypoosmotic conditions. In isolated cardiomyocytes, caveolae depletion using methyl-beta-cyclodextrin or Cav3KO abolished the increase in β2AR response induced by stretch. CONCLUSION: Our study reveals a stretch-regulation of the β2AR signalling pathway, which requires functional caveolae. This indicates caveolae are mechanosensitive membrane domains that undergo structural and functional changes in response to stretch, thus leading to mechanical regulation of caveolae-associated signalling pathways.