Sedentary ageing remodels the structure–function relationships that underlie calcium signalling at super‐resolution scales in the heart

Intracellular calcium handling underpins excitation-contraction coupling in the heart, and its dysfunction has been linked to both mechanical and electrical impairments. Age-related remodelling of the structure-function relationships that underpin calcium spark morphology and dynamics may therefore be causatively linked to associated cardiac dysfunction. We aim to uncover age-dependent features of calcium spark morphology and the distribution of type-2 ryanodine receptor (RyR) clusters, responsible for intracellular calcium release. Isolated ventricular cardiomyocytes were obtained from male Wistar rats at 6, 18 and 24 months of age. Subcellular structure was assessed using expansion microscopy to localize RyR, and calcium sparks were recorded in the absence and presence of 1 µM isoprenaline using spinning disk confocal microscopy. Super-resolution imaging revealed that both aged points were associated with a greater degree of cluster fragmentation, sparseness and structural disorder than the 6-month group, leading to an increased prevalence of remodelled phenotype cells in the aged groups. The 18-month group exhibited the most substantial remodelling of many properties, including the prevalence of remodelled cells, indicating either a genuine biphasic progression of remodelling with age or the divergence of structurally healthy and structurally remodelled groups with age. Moreover, ageing enhanced the impact of β-adrenergic stimulation on calcium spark morphology, increasing spark amplitude, full-width half maximum and spark mass. Our study reveals that RyR cluster properties are non-linearly remodelled with age, with RyR phosphorylation leading to remodelling of calcium spark morphology with age, which may contribute to reduced contractile performance and increased arrhythmia incidence. KEY POINTS: Calcium spark morphology and ryanodine receptor cluster properties were assessed in a rat model of sedentary ageing at three age points. Calcium spark morphology was much more strongly influenced by β-adrenergic stimulation in the aged cohorts compared to the young group, corresponding to an increase in the spatial extent and overall spark mass and indicating an increased responsiveness to sympathetic regulation in ageing. Ageing was associated with a non-linear remodelling of the ryanodine receptor cluster pattern. At the submicron scale ageing led to an increase in fragmented-like clusters; at the broader scale ageing led to an increase in axial spacing and sparseness, as well as a reduction in the regularity of cluster distribution. Across all metrics the younger of the two aged groups exhibited the most substantial and highest probability of remodelling, indicating a potential biphasic progression of structure-function relationships with age, corresponding to remodelling and partial recovery.