Abstract
Diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR2) contributes to arrhythmias. Protein kinase A (PKA) and Ca2+/Calmodulin- dependent protein kinase II (CaMKII) have been involved in SR Ca2+ leak by altering RyR2 gating. Under these conditions, JTV519 (K201), a benzothiazepine derivate, has been shown to stabilize the modified RyR2 and reduce diastolic SR Ca2+ leak. We tested, whether JTV519 reduces SR Ca2+ leak induced by Na+- and Ca2+ overload (induced by 100 µM ouabain), i.e. independent of CaMKII signaling. Methods: [Ca2+]i was measured (Fluo4-AM, confocal) in paced murine cardiomyocytes ±JTV519 (1 µM, >1h preincubation). [Ca2+]-transients, diastolic SR Ca2+ leak (Ca2+ spark (SparkF, in s−1∗pL−1) and Ca2+ wave frequency) and SR [Ca2+] (caffein) were measured ±ouabain (OUAB) and KN93 (CaMKII-inhibitor, 1 µM). Phosphorylation of RyR2 (pSer2814, CaMKII site) was quantified by Western blot.Results: With OUAB, total cellular [Ca2+] increased from 3.3±0.3 to 4.3±0.3 (F/F0, mean±SE), SparkF increased from 31±20 to 85±30 (both pl0.05). Ouabain did not increase pSer2814, and KN93 had no effect on elevated SparkF with ouabain (89±24), indicating no contribution of CaMKII to increased SR Ca2+ leak. JTV519 decreased SparkF (25±4), and Ca2+ waves, but also attenuated the increase in SR [Ca2+] in OUAB (F/F0: 5.2±0.3 with JTV+Ouab vs. 7.7±0.6 OUAB, pl0.05). However, matching cells for equal SR [Ca2+] revealed an SR [Ca2+] - independent effect of JTV519 on SparkF. In contrast, propagation speed of Ca2+ waves and the ratio between [Ca2+] transient amplitude and SR [Ca2+] were unchanged.Conclusion: In conditions not related to CaMKII-mediated alterations of RyR2 gating, JTV519 reduces spontaneous SR Ca2+ release, but does not influence propagated Ca2+ release or the fraction of SR Ca2+ released with each beat.
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