Functional LTCC-₂AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure

Background:Beta-2 adrenergic receptors (& beta;(2)ARs) but not beta-2 adrenergic receptors (& beta;(1)ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and & beta; adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Methods:Global signaling between LTCCs and & beta; adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and & beta;(1)AR or & beta;(2)AR in different membrane microdomains in control and failing cardiomyocytes. Results:LTCC open probability (Po) showed an increase from 0.054 & PLUSMN;0.003 to 0.092 & PLUSMN;0.008 when & beta;(2)AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and & beta;(2)AR was lost. Interestingly, local stimulation of & beta;(1)AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via & beta;(1)AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the & beta;(2)AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream & beta;(1)AR and results in an increase in LTCC current. Conclusions:Regulation of the LTCC activity by proximity coupling mechanisms occurs only via & beta;(2)AR, but not & beta;(1)AR. This may explain how & beta;(2)ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.