Abstract
AimsCardiomyocyte β2-adrenergic receptor (β2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors’ subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown.Methods and resultsRat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β2AR-mediated signal compartmentation or reduce cAMP diffusion.ConclusionAlthough changes in T-tubule structure and β2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β2AR-mediated cAMP is temporarily reversed. Complete disorganization of β2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.
Highlights
Heart failure (HF) is a complex clinical syndrome arising from myocardial injury or dysfunction
Single cell hypertrophy subsequently developed as early as 4-week post-myocardial infarction (MI) to compensate for reduced muscle function and an overall increase in cardiomyocyte volume was detectable at 8- and 16-week post-MI
As we reported previously in,[13] we observed that in control cardiomyocytes, the b2AR-dependent cyclic adenosine monophosphate (cAMP) response is five-fold greater in the T-tubule openings than at the crests (Figure 5A,B,C), whereas in cardiomyocytes isolated from rat hearts at end stage HF (16-week post-MI), the b2AR-cAMP response was found to be present in T-tubules and in the crests (Figure 5A,B,C)
Summary
Heart failure (HF) is a complex clinical syndrome arising from myocardial injury or dysfunction. Together with other cardiovascular complications, HF accounts up to 40% of all deaths worldwide.[1] HF does not occur instantaneously but develops over time due to mechanical and hormonal stresses and adverse cardiac remodelling, as shown in multiple human studies and recapitulated in animal models.[2] The stages of disease progression may manifest themselves, at first, as compensatory hypertrophy as decompensated hypertrophy and as HF. When HF develops, ventricular myocytes undergo a plethora of functional and structural changes.[3,4] During the initial phase of HF progression, the sympathetic nervous system compensates for the diminished cardiac output through an increase in catecholaminergic stimuli[3] and cardiomyocytes experience hypertrophic growth.[5] This results in transiently advantageous changes in the b-adrenergic receptor (b-AR)- and cyclic adenosine monophosphate (cAMP)-dependent inotropic response of cardiomyocytes[4] but transforms into a maladaptive decrease of myocardial responsiveness at more advanced stages of HF.[6,7]
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