In cardiomyocytes, as in other muscle cells, an increased level of cytosolic Ca²⁺ concentration initiates the production of force and cellular signaling. Unregulated Ca²⁺ release and an uncontrolled increase of Ca²⁺ concentration can induce severe contractile dysfunction, arrhythmias, high blood pressure, protein degradation and cell death. Therefore, the stabilization of intracellular Ca²⁺ is one of the major goals in order to counteract the progression of the disease. During the last decades, nitric oxide (NO) has been shown to have modulating effects on molecules involved in Ca²⁺ handling, as well as regulation of contractility, mitochondrial oxygen metabolism and glucose. The absence of NO induces abnormal electrophysiological parameters seen in the electrocardiogram compared to normal mice. For instance, restoration of nNOS via transgenic overexpression reduces the macrophage infiltration, cardiac fibrosis and normalizes the ECG parameters, such as Q-wave amplitude, heart rate variability and reduces the polyphasic R-waves and arrhythmias. The use of NO donors also showed an improvement of the dystrophic phenotype. However, lacunae exist regarding the effect of NO on biophysical parameters of temporal properties of Ca²⁺ handling within isolated murine cardiomyocytes.

Material & Methods:

Cardiomyocytes isolation from adult wild-type mouse was performed using Langendorff perfusion system. Fluo-4 (5 µM) stained isolated cardiomyocytes were electrically stimulated (1Hz, 18V, 10 ms) and Ca²⁺ waves were recorded using a new type fluorescence microscopy of simplified system based on LED excitation. Effects of NO were investigated using spontaneous NO donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP, 100 µM) and sarco-(endo-)plasmic reticulum adenosine triphosphatase isoform 2 (SERCA₂) blocker cyclopiazonic acid (CPA, 5 µM).

Results:

SNAP was able to reduce the time to fall, as well the inhibitory effect of CPA on SERCA₂ thereby increasing Ca²⁺ sequestration into sarcoplasmic reticulum (SR). Moreover, SNAP modified the Ca²⁺ release from SR; NO increased the time to peak, but reduced the increased effect of CPA.

Conclusion:

These results support previous reports, which showed that NO is able to act on SERCA₂ regulation via cyclic adenosine monophosphate (cAMP) dependent pathway. Moreover, gained results suggest that NO is able to act on cardiac isoform of ryanodine receptor (RyR₂) as well, either via cAMP dependent pathway, direct interaction or intra-sarcoplasmic Ca²⁺ concentration.