Angiotensin II (Ang II) impairs fatty acid (FA) oxidation (Ref 1) which may account for contractile dysfunction in diseased heart. Since nitric oxide is involved in the regulation of FA uptake (Ref 2) and modulates cardiac contractility (Ref 3), we tested whether neuronal nitric oxide synthase (nNOS) contributes to reduced FA-dependent cardiac contractility after Ang II stimulation in rat LV myocytes. Our results showed that palmitic acid (PA, 1-100 microM) increased the amplitude of sarcomere shortening in LV myocytes from normal rats (at 100 microM, P<0.001, n=20) without affecting the amplitude of Ca2+ transient (P=0.9, n=11) or the relationship between Ca2+ and myocyte shortening. Similar responses of PA were observed in sham-operated rats (P<0.001, n=18). In contrast, Ang II treatment (via osmotic minipump for 4 weeks) abolished the positive inotropic effect of PA (P=0.2, n=18). 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron) pretreatment did not affect the response to PA in either group (P=0.005, PA vs. control in the presence of tiron in shams, n= 7; P=0.2, PA vs. control in the presence of tiron in Ang II-rats, n=6). Immunoblotting results revealed that nNOS protein expression was greater in LV myocyte homogenates from Ang II-rats (nNOS/GAPDH: P=0.03, n=8); inhibition of nNOS (S-methyl-L-thiocitrulline, SMTC or vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine, L-VNIO) restored the positive inotropic effect of PA (P=0.004, between PA and control in the presence of SMTC, n=14). Furthermore, nNOS up-regulation was associated with enhanced s-nitrosylation of transglutaminase 2 (TG2, P<0.05, n=3) and reduced activity in LV myocytes from Ang II-rats (63%) . Inhibition of TG2 (cystamine or gene deletion, TG2-/-) abolished PA-enhancement of LV myocyte contraction in basal (P=0.02 between PA and PA+cystamine in normal rats, n=6 and P=0.91 between PA and control in TG2-/-, n=7; in TG+/+, P=0.007 between PA and control, n=19) and in Ang II-rats after nNOS inhibition (P=0.67 between cystamine and cystamine+PA in the presence of L-VNIO, n=3). These results indicate that nNOS is up-regulated by Ang II and is responsible for impaired PA-dependent myocyte contraction, at least in part, via modulating TG2 activity. Our findings suggest a novel mechanism through which nNOS modulates myocardial contractility in diseased heart. Rats (of 8 weeks old) were anesthetized with isoflurane (2.5 %). An osmotic minipump (Alzet model 2004) containing Ang II (200μl, 6 mM, infusion rate 125 ng/min/kg) was implanted in the midscapular region under sterile condition. Sham-operated animals underwent the same surgical procedure, except for no pump insertion.