Voltage-induced Ca2+ release is supported by junctophilins 1, 2 and 3, and not by junctophilin 4

In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1, which then activate RYR1 to release calcium from the sarcoplasmic reticulum (SR). Because it does not require extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPH) 1 and 2 are responsible for forming the SR–PM junctions at which voltage-induced calcium release occurs; structurally similar junctions with different molecular constituents are formed in neurons by JPH3 and JPH4. Studies on mice models demonstrated that JPH1 knockout mice can still perform voltage-induced calcium release, although the complementary approach to verify whether JPH1 alone also supports this release is not easily practicable due to the embryonic lethality of JPH2 knockout mice. In a previous work, we showed that voltage-induced calcium release could be recapitulated in HEK293-derived cells transfected with cDNAs for JPH2 and CaV1.1, β1a, Stac3, and RYR1. Here, we used this reconstitutional approach to test whether JPH1 and the more distantly related JPH3 and JPH4 can also support voltage-induced calcium release in HEK293-derived cells. Our data show that all the four isoforms colocalize with CaV1.1 at ER–PM junctions and that JPH1, JPH2, and JPH3, but not JPH4, cause colocalization of RYR1 with CaV1.1 at the junctions. To test for function, potassium depolarization was applied to cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D) to eliminate extracellular calcium entry. Calcium transients were observed in cells expressing JPH1, JPH2, and JPH3, indicating that these isoforms support voltage-induced calcium release, but not in cells expressing JPH4. Thus, the JPHs seem to act primarily to (1) form ER–PM junctions and (2) recruit the required set of signaling proteins to these junctions; voltage-induced calcium release can be supported by any JPH isoform fulfilling both of these functions.

Possible mechanisms underlying the dynamic assembly of calcium entry units: The role of temperature and pH

Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e., the sarcoplasmic reticulum; SR) and from extracellular space: (1) excitation–contraction (EC) coupling and (2) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of calcium entry units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR stacks and transverse tubules (TTs). Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in the presence of extracellular Ca2+. The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated. Here, we first verified whether CEUs can assemble ex vivo (in the absence of blood supply and innervation), subjecting excised EDL muscles from mice to an ex vivo incremental fatigue protocol (80 Hz tetanus stimulation lasting 45 min): the data collected demonstrate that CEUs can assemble ex vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C versus 25°C) and lower pH (7.2 versus 7.4) promotes formation of CEUs increasing the percentage of fibers containing SR stacks, the number of SR stacks/area, and the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36°C) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+, suggesting that CEUs assembled ex vivo are functional.

Apoptotic Cells Trigger Calcium Entry in Phagocytes by Inducing the Orai1-STIM1 Association

Swift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP3R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level.

In silico Identification of Disrupted Myocardial Calcium Homeostasis as Proarrhythmic Trigger in Arrhythmogenic Cardiomyopathy

Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear.Aim: To unravel the mechanisms by which calcium-handling abnormalities in plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy.Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model.Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings.Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to plakophilin-2 loss-of-function.