Nitroxyl: A positive inotrope enhancing SR CA(2+) cycling in isolated myocytes

Objectives. Nitroxyl (HNO) is a positive inotrope in vivo. Here we aim to elucidate the mechanisms for HNO-induced inotropy. We demonstrate that HNO enhances sarcoplasmic reticular (SR) Ca2+ cycling, in a thiol-sensitive manner. Methods and results. In murine isolated myocytes, the HNO donor Angeli's Salt (AS) increased sarcomere shortening in a dose-dependent manner (for instance, the increase with 1 mM AS was 117 ± 25%; p < .01 vs. base). This effect was not mimicked by the NO donor DEA/NO. Preincubation with reduced glutathione (GSH, 4 mM, 3 h) incremented intracellular thiol content by 6% (p < .05), and AS/HNO response was markedly blunted in these cells: 58 ± 19% (p = .05 vs. AS 1 mM alone). AS/HNO activated cardiac ryanodine receptor (RyR2). In reconstituted RyR2, HNO increased RyR2 open probability (Po) without altering the unitary conductance: Po increased from 0.16 ± 0.03 to 0.25 ± 0.05, 0.46 ± 0.07 and 0.69 ± 0.11 after 0.1, 0.3, and 1 mM AS, respectively. These changes were fully reverted by addition of the reducing agent DTT (2 mM) to the cis side (0.11 ± 0.04). In addition, HNO dose-dependently increased Ca2+ sparks frequency (CSF): with 0.5 mM AS/HNO CSF was 26 ± 3 vs. 4 ± 1 sparks/100 μm/s (p < .01). Again, pretreatment with cell-permeable GSH abrogated HNO-induced increase in CSF. Finally, to assess whether AS also affects SERCA2a function, AS (250 μM) was added to isolated cardiac mouse SR vesicles. HNO markedly increased the rate of SR Ca2+ uptake. Conclusions. Thus, HNO increases and sustain inotropy by enhancing SR Ca2+ cycling, without altering diastolic Ca2+ levels and/or SR Ca2+ load.