Selective phosphorylation of PKA targets after β-adrenergic receptor stimulation impairs myofilament function in Mybpc3-targeted HCM mouse model

Aims : Hypertrophic cardiomyopathy (HCM) has been associated with reduced b-adrenergic receptor (b-AR) signalling, leading downstream to a low protein kinase A (PKA)-mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished b-AR signalling in HCM. We aimed to investigate the role of b-AR signalling on regulating myofilament and calcium handling in an HCM mouse model harbouring a gene mutation (G >A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C. Methods and results : Cardiomyocyte contractile properties and phosphorylation state were measured in left ventricular permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. Significantly higher myofilament Ca2+ sensitivity and passive tension were detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force–sarcomere length relation was impaired in both HET and KI mice, suggesting a reduced length-dependent activation. Unloaded cardiomyocyte function revealed an impaired myofilament contractile response to isoprenaline (ISO) in KI, whereas the calcium-handling response to ISO was maintained. This disparity was explained by an attenuated increase in cardiac troponin I (cTnI) phosphorylation in KI, whereas the increase in phospholamban (PLN) phosphorylation was maintained to wild-type values. Conclusion : These data provide evidence that in the KI HCM mouse model, b-AR stimulation leads to preferential PKA phosphorylation of PLN over cTnI, resulting in an impaired inotropic and lusitropic response.