Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes

Abstract

Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions

“…Accordingly, previous studies showed that following high frequency electrical stimulation in the presence of isoproterenol, the JKO cardiomyocytes developed spontaneous Ca 2+ aftertransients and aftercontractions at significantly increased percentage, compared to WT cardiomyocytes. 12, 13 Using similar stress conditions, we demonstrated that 52% of the JKO cardiomyocytes present with spontaneous Ca 2+ aftertransients compared to only 10% of WT cells (p=0.0002). Preincubation of the JKO cardiomyocytes with KN-93 reduced in part and significantly (by 35%), the percentage of cells with Ca 2+ aftertransients (p=0.022) (Fig.…”

“…Based on our biochemical data on RyR2 hyperphosphorylation and previous findings on increased SR Ca 2+ -leak in JKO cardiomyocytes 12, 13 , we proceeded to evaluate the effects of CaMKII inhibition on arrhythmogenesis, using isolated adult ventricular cardiomyocytes. One of the manifestations of arrhythmias associated with SR Ca 2+ leak at the cellular level is the appearance of spontaneous Ca 2+ aftertransients or arrhythmogenic Ca 2+ waves and aftercontractions.…”

“…Specifically, the junctin knock-out mouse presents with ventricular arrhythmias under stress conditions and an overall phenotype reminiscent of the classical CPVT symptoms, including aftercontractions, DADs and an increased propensity for spontaneous SR Ca 2+ release. 12, 13 Consistent with the human CPVT pathophysiology, approximately 25% of junctin-null mice die suddenly by 3 months of age with structurally normal hearts, suggesting acutely occurring fatal arrhythmias. 12 The trigger mechanism for arrhythmias in the junctin knock-out model is speculated to be the aberrant activity of RyR2, which leads to diastolic Ca 2+ leak from the SR, exacerbated by beta adrenergic activation.…”

“…Table 1). 12, 13 Since junctin is a key component of the RyR2 Ca 2+ release channel complex, and directly associated with CSQ2, JCN ablation may alter RyR2 Ca 2+ sensitivity through different mechanisms: i) by impairing the physical interaction among the proteins forming this complex and interfering with CSQ2 binding to Ca 2+ , which might induce excessive and aberrant RyR2 Ca 2+ release 12, 13 ; ii) by affecting RyR2 refractoriness in a similar way as CSQ2 mutants 35 ; or iii) by modifying RyR2 phosphorylation through a stress and/or Ca 2+ dependent protein kinase, which might in turn alter the RyR2 channel opening properties and promote aberrant SR Ca 2+ release. Our data do not support altered RyR2 refractoriness in the JKO mice, since the incidence of premature or late Ca 2+ release events is similar between WT and JKO cardiomyocytes.…”

Store-dependent deactivation: Cooling the chain-reaction of myocardial calcium signaling

“…Refilling the SR Ca 2+ stores by SERCA during the recovery (restitution) phase once again increases luminal Ca 2+ , causing CASQ2 to dissociate from the RyR2s rendering these SR Ca 2+ release channels once more sensitized for yet another activation by cytosolic Ca 2+ . There is also evidence that has implicated other proteins such as TRD [56,58], JNT [59] and histidine-rich Ca 2+ binding protein [60] in regulation of RyR2. The potential role of these proteins in mediating the effects of luminal Ca 2+ on RyR2 function, as well as that of Ca 2+ -dependent CASQ2 polymerization-depolymerization as part of the luminal signaling cascade [35] requires further investigation.…”