Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

Abstract

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as ‘junctions’, are fast communication hubs within excitable cells such as neurones and muscle. Here we have examined two transgenic murine models probing the role of junctophilin-2, a membrane tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR). Although this change was associated with much enlarged RyR clusters that due to their size should be more excitable, functionally it caused a mild inhibition in the calcium signalling output of the junctions (calcium sparks). Analysis of the single molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the calcium signalling properties of the junction and thus the contractile function of the cell.

Junctophilin-2 physically interacts with ryanodine receptor type 2 for peripheral coupling of mouse cardiomyocytes

“…In the present study, we further confirmed that a siRNA based knockdown of JPH2 resulted in reduced amplitudes of Ca 2+ transient in cardiomyocytes, but the respective levels of expression of RyR2 mRNA and proteins showed no significant changes in our reports [15]. This result is consistent with previous findings from similarly oriented analyses by authors of this study and other researchers [14,15,23,28], and was a result likely due to decreasing levels of SR Ca 2+ stores which resulted in an SR Ca 2+ leak by directly increasing the probability that RyR2 receptors would be open.…”

“…JPH2 has been previously suggested to interact with RyR2 and Cav1.2 such as to regulate their gating channel functions [23,24]. Previous observations suggested that both JPH2 and RyR2 labeling puncta strongly overlapped, and that the amount of JPH2 associated with RyR2 clusters was greatly reduced after JPH2 was manually knocked down [14,25]. In the present study, the interaction between JPH2…”

“…Recent studies have also demonstrated that cardiac-specific knockdown of JPH2 triggers an SR Ca 2+ leak by directly increasing the probability of RyR2 receptors being open in cardiomyocytes [14,23,28]. Moreover, downregulation of JPH2 has been observed in patients with hypertrophic cardiomyopathy, for some rodent based model analyses of hypertrophic cardiomyopathy, overexpression of JPH2 which could correct cardiac function with early stage heart failure, for approaches to inhibition of SR Ca 2+ leak induced by RyR2 [12,13].…”

“…Quantitative single-molecule localization microscopy also indicated that colocalization between RyR2 and JPH2 in JPH2 knocked down cardiomyocytes was reduced. In contrast, for cases of induced overexpression of JPH2 in cardiomyocytes, there was colocalization of RyR2 with JPH2 at levels that were significantly increased [14].However, the binding domain between JPH2 and RyR2 channel has not been fully elucidated.…”

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

“…While there is abundant evidence that junctophilins associate with the plasma membrane, and that the MORN repeat-containing region is likely to mediate this, there is to the authors’ knowledge no paper demonstrating that the junctophilin MORN repeats directly interact with lipids (Takeshima et al, 2000, Munro, Jayasinghe et al, 2016, Woo, Srikanth et al, 2016, Perni, Lavorato et al, 2017, Jayasinghe, Clowsley et al, 2018, Nakada et al, 2018, Rossi et al, 2019). Despite this, the evidence that the MORN repeat-containing region mediates plasma membrane targeting has been repeatedly cited as evidence that MORN repeats directly bind lipids.…”