Prolonged β-Adrenergic Stimulation Disperses Ryanodine Receptor Clusters in Cardiomyocytes: Implications for Heart Failure

Abstract

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 hour triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of 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.

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks

“…For the no phosphorylation condition, see red dots in Fig 3 , the K + value was set to 55 μm. This value was chosen by optimizing K + values to achieve physiologically realistic spark property values [ 21 ]. A particular focus was set on obtaining non-phosphorylated sparks with time to peak (TTP) values of around 7 ms [ 21 ].…”

“…This value was chosen by optimizing K + values to achieve physiologically realistic spark property values [ 21 ]. A particular focus was set on obtaining non-phosphorylated sparks with time to peak (TTP) values of around 7 ms [ 21 ]. For phosphorylated RyRs, we differentiated between two specific cases.…”

“…In this case, for phosphorylated RyRs (yellow dots in Fig 3 ) the K + value was set to 25 μm. This K + value was estimated based on obtaining sparks with spark properties within the same range as previous experimental results [ 6 , 21 ]. The selection of K + values for blanket phosphorylation was set such that the Ca 2+ dynamics from a blanket phosphorylation pattern would match the Ca 2+ kinetics obtained from using a uniform pattern.…”

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks

“…The first case is one where a single geometry contains both phosphorylated and non phosphorylated RyRs, see the outer, inner and uniform phoshorylation patterns in Fig 3 . In this case, for phosphorylated RyRs (yellow dots in Fig 3) the K + value was set to 25 μm. This K + value was estimated based on obtaining sparks with spark properties within the same range as previous experimental results [6,21]. The selection of K + values for blanket phosphorylation was set such that the Ca 2+ dynamics from a blanket phosphorylation pattern would match the Ca 2+ kinetics obtained from using a uniform pattern.…”