Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that ‘dispersion’ of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.


Sample-size estimation
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Replicates
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Information regarding computation of animal sample size and underlying assumptions is provided under the section 'Material and Methods', subsection 'Rat model of post-myocardial infarction congestive HF' and subsection 'Statistical Analyses'.
The performed live-cell Ca 2+ imaging and dSTORM super-resolution imaging do not allow for acquisition of technical replicates, since the research specimens are permanently altered by the experimental procedures. N values corresponding to the number of biological replicates for these experiments are presented in the legends of Figures 1, 2, 3, 4, and 6.
No data were excluded from the analyses, and consistent observations were made during analyses performed on different cardiomyocytes from different hearts. The performed mathematical simulations of Ca2+ release are true technical replicates, with repeated measures performed and analyzed. The n of these simulation is indicated in the legend for figure 5.
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