Validating a Model of Nitric Oxide-Ca2+ Crosstalk in Cardiac Myocytes

Recently, we have developed a mathematical model of activation of constitutive nitric oxide synthase (NOS)-cGMP signaling pathway and its crosstalk with the excitation-contraction coupling in rat cardiac myocytes. This model describes a dynamic regulation of intracellular nitric oxide ([NO]i) formation by changes in intracellular calcium [Ca2+]i signals. In particular, a key prediction of the model is that the formation of NO is sensitive to the frequency and magnitude of [Ca2+]i signaling. To validate these predictions, we tested experimentally correlations between increases in pacing frequency and the level of intracellular [Ca2+]i and formation of [NO]i in neonatal rat cardiac myocytes which were loaded with membrane permeable fluorescent indicators DAF-FM and Fluo-4, respectively. As predicted by the model, increasing pacing frequency from 0.5 Hz to 2.0 Hz enhanced diastolic [Ca2+]i and [NO]i formation. NO production was markedly blocked by eliminating the extracellular Ca2+ with 2 mM EGTA or by increasing Ca2+ buffering with BAPTA-AM or by treatment with non-specific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), confirming that pacing-frequency-dependent NO synthesis was Ca2+ and NOS dependent. Several of our model simulations compared the effects of cytosolic vs. dyadic Ca2+ signals in NOS signaling, predicting that cytosolic Ca2+ signals may be sufficient to initiate NOS activation. Using the computational model, we found that reduction of extracellular sodium [Na+]0 from 137 mM to 70 mM under condition of inhibited SR would induce a Ca2+signal with magnitude similar to pacing-induced cytosolic Ca2+ signals. This occurs due to influx of Ca2+ through the Na+/Ca2+ exchanger. Experimental testing of this new protocol confirmed a broad, slow Na+-depletion-induced Ca2+ signal. The Na+ depletion protocol was sufficient to enhance NO synthesis similar to 0.5 Hz pacing. These results confirm the model prediction that cytosolic Ca2+ signals regulate NOS activity in cardiac myocytes.