Cardiac neuronal nitric oxide synthases (NOS1 or nNOS) has been well documented to regulate myocardial contraction and relaxation by targeting specific Ca2+ handling proteins, protein kinase- or phosphatase-dependent phosphorylation/dephosphorylation and redox homeostasis. SpecifICally, in left ventricular (LV) myocytes from healthy heart, NOS1 modulates myocardial inotropy and the amplitude of Ca2+ transient by limiting Ca2+ influx via attenuating L-type Ca2+ channel activity (ICa) through protein phosphatase 2A (PP2A)-dependent dephosphorylation of ICa and by increasing Ca2+ leak following s-nitrosylation of RyR (increase subconductance of RyR). On the other hand, NOS1 facilitates myocardial lusitropy by promoting sarcoplasmic Ca2+-ATPase activity and Ca2+ reuptake through increasing PKA-dependent (and cGMP/PKG-independent) phospholamban phosphorylation at serine 16 (PLN-Ser16) secondary to PP2A inhibition. Furthermore, NOS1 inhibits superoxide production from both XOR and NADPH oxidase. These compelling evidences show that NOS1 interacts with molecules in different subcellular compartments and exerts different effects in the heart. Importantly, the protein expression and the activity of NOS1 are increased in hypertrophic and failing myocardium and NOS1 up-regulation is associated with cardiac protection from oxidative stress, injury and arrhythmia. Under these conditions, NOS1 has been shown to traffic away from RyR to caveolin 3 in sarcolemma, suggesting that nNOS may target distinctive Ca2+ handling molecules from healthy conditions under stress. So far, our understandings of the upstream or downstream mechanisms mediating NOS1 transcription and NOS1 regulation of cardiac function in the heart under stress are sparse. Recently, we have demonstrated that the pathogenic stimulator, angiotension II (Ang II) is the upstream stimulator of NOS1 protein expression and activity. Our results demonstrated that type 1, type 2 Ang II receptors and Mas receptor (in the absence of Ang 1-7) mediated Ang II stimulation of NOS1 in LV myocytes, which in turn, inhibited NADPH oxidase activity and facilitated LV myocyte relaxation. Unexpectedly, NOS1 up-regulation was associated with unchanged ICa and myocyte contraction. In addition, NOS1 increased PLN-Ser16 through cGMP/PKG-dependent mechanism (independent of PKA, CaMKII or peroxynitrite signaling). Similarly, NOS1 was up-regulated in cardiac myocytes from Ang II-induced hypertensive hearts, facilitated LV myocyte relaxation and decay kinetics of Ca2+ transients (tau) and increased PLN-Ser16. Again, myocyte contraction and ICa were remained unaltered. Surprisingly, increased PLN-Ser16 and tau were ineffective to NOS1 inhibition; in contrast, NOS1 were shown to modulate myofilament Ca2+ sensitivity by regulating the phosphorylation of key proteins in sarcomere. Collectively, these results suggest that NOS1 shifts its target proteins for effective regulation of cardiac function in the myocardium under stress. This novel mechanism is important in better understanding of the endogenous cardiac protective mechanisms of NOS1 during disease progression.