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Mathematical modeling of Ca2+dynamics in the heart has the potential to provide an integrated understanding of Ca2+-handling mechanisms. However, many previous published models used heterogeneous experimental data sources from a variety of animals and temperatures to characterize model parameters and motivate model equations. This methodology limits the direct comparison of these models with any particular experimental data set. To directly address this issue, in this study, we present a biophysically based model of Ca2+dynamics directly fitted to experimental data collected in left ventricular myocytes isolated from the C57BL/6 mouse, the most commonly used genetic background for genetically modified mice in studies of heart diseases. This Ca2+dynamics model was then integrated into an existing mouse cardiac electrophysiology model, which was reparameterized using experimental data recorded at consistent and physiological temperatures. The model was validated against the experimentally observed frequency response of Ca2+dynamics, action potential shape, dependence of action potential duration on cycle length, and electrical restitution. Using this framework, the implications of cardiac Na+/Ca2+exchanger (NCX) overexpression in transgenic mice were investigated. These simulations showed that heterozygous overexpression of the canine cardiac NCX increases intracellular Ca2+concentration transient magnitude and sarcoplasmic reticulum Ca2+loading, in agreement with experimental observations, whereas acute overexpression of the murine cardiac NCX results in a significant loss of Ca2+from the cell and, hence, depressed sarcoplasmic reticulum Ca2+load and intracellular Ca2+concentration transient magnitude. From this analysis, we conclude that these differences are primarily due to the presence of allosteric regulation in the canine cardiac NCX, which has not been observed experimentally in the wild-type mouse heart.

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O(2)()) production was increased in nNOS(-/-) mice and reduced by l-N(ω)-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)() production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Superoxide production from NADPH oxidases has increasingly been shown to play an important role in myocardial signalling. The activity of myocardial NADPH oxidases is known to be increased in the failing myocardium; however, whether this is a compensatory or maladaptive mechanism remains to be established. Gene deletion of the neuronal nitric oxide synthase (nNOS) is associated with an increase in myocardial superoxide production and with enhanced inotropy. Here we tested whether nNOS gene deletion leads to an increase in myocardial NADPH oxidase activity, which - in turn - causes a superoxide (O2-)-dependent increase in contraction.As expected, O2- production (measured by lucigenin 5μmol/L -enhanced chemiluminescence) was greater in nNOS−/− LV myocytes than in their wild type littermates (nNOS+/+). Pre-incubation of LV myocytes with the NADPH oxidase inhibitor apocynin (100 μmol/L, 30 min) reduced the level of O2- in nNOS−/− myocytes only, thereby abolishing the difference between genotypes. In agreement with these findings, apocynin significantly reduced cell shortening (%, field stimulation at 3Hz, 35°C) only in nNOS−/− myocytes. Inhibition of protein kinase A (amide 14-22, PKI, 2 μmol/L) reduced contraction to a larger extent in nNOS−/−. The effects of PKA inhibition were abolished after pre-incubation with apocynin. NADPH oxidase stimulation by endothelin-1 (ET-1, 10 nM, 5-10 min) caused an increase in cell shortening in both nNOS−/− and nNOS+/+ myocytes, which was abolished by apocynin. PKI significantly reduced the effect of ET-1 in both genotypes.Taken together, these findings suggest that nNOS-derived NO may tonically inhibit the activity of NADPH oxidase in murine LV myocytes and indicate that production of O2- by this oxidase system may account for the PKA-dependent increase in cell shortening in nNOS−/− mice.