Stimulation Of P2x Purinergic Receptors Increases Calcium Spark Frequency, But Does Not Normalize Calcium Transient Synchronization, In Mouse Cardiomyocytes From The Calsequestrin Model Of Cardiomyopathy (Csq)

Sarcoplasmic reticulum (SR) Ca2+ release is impaired in cardiomyocytes from failing hearts. In studies of cardiomyocytes from CSQ hearts Ca2+ spark frequency and synchronization of Ca2+ release were reduced. Interestingly, binary overexpression of CSQ and the human P2X4 purinergic receptor prolongs CSQ survival. Our objective was to determine if amelioration of the CSQ phenotype through Binary (CSQ+P2X4) overexpression was due to purinergic effects on Ca2+ release function. Cardiomyocytes isolated from the hearts of wild-type (WT), P2X4, CSQ or Binary mice were loaded with Fluo-4AM, superfused with a modified Tyrode's solution (22°C) and paced at 0.5 Hz in the presence/absence of the P2-receptor agonist 2-MeSATP (3 μM). Line-scans were recorded with a Zeiss LSM510. Under basal conditions, 2-MeSATP responsive cardiomyocyte Ca2+ spark frequencies (sparks/μm/sec) did not differ (WT 1.04±0.23; P2X4 1.78±0.28; CSQ 1.60 ± 0.87; Binary 0.79±0.27; p=0.73). When 2-MeSATP was applied, Ca2+ spark frequency increased significantly compared to basal for each genotype (WT 2.89±0.32; P2X4 5.79±1.02; CSQ 5.13±1.53; Binary 3.45±0.89; p<0.01). These data suggest that a P2X4R-mediated mechanism can influence SR Ca2+ load and/or release. Effects of purinergic stimulation on coordination of SR Ca2+ release were investigated by determining the dyssynchrony index (DI) in paced cardiomyocytes. Under basal conditions the CSQ and Binary DIs were dramatically increased compared to WT (13.59±1.39 and 14.28±1.89 vs 4.34±0.93; p<0.01). Application of 2-MeSATP did not decrease the DI in myocytes from failing CSQ and Binary hearts (15.82±3.03 and 13.85±1.01). Conclusion: Cardiac P2X purinergic receptor stimulation increases Ca2+ spark frequency, suggesting a beneficial effect on SR Ca2+ loading or release. However, P2X receptor activation does not normalize DI, determined from confocal line-scans, in CSQ or Binary cardiomyocytes.