Background:
Diabetes mellitus type II (T2DM) is associated with an elevated risk of heart failure. Treatment options in particular for heart failure with preserved ejection fraction (HFpEF) are insufficient. Nitro-fatty acids (NO2-FAs) like nitro-oleic acid (NO2-OA) are electrophilic molecules with anti-oxidative effects, for which beneficial actions in several mouse models of cardiovascular diseases have been shown. Given that NO2-OA reduced blood glucose in a genetic mouse model of the metabolic syndrome in a previous study, we sought to investigate whether NO2-OA treatment can protect from HFpEF development and to unravel the underlying mechanisms.

Methods and Results:
We employed an established mouse model of diabetic cardiomyopathy induced by high fat diet (HFD) combined with the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Four weeks old C57bl/6N mice were fed with HFD and L-NAME provided via the drinking water (0.5 g/L) for 11 weeks. Thereafter, mice received either NO2-OA, oleic acid (OA) or vehicle via mini-osmotic pumps for 4 weeks. Echocardiography revealed, that LV diastolic function reflected by E/E´ and E´/A´ was significantly impaired after 11 weeks of HFD+L-NAME administration compared to mice receiving normal chow (ctrl) (ctrl. vs HFD+L-NAME, E´/A´: 1.14 ± 0.06, N=15 vs. 0.71 ± 0.04, N=45, p<0.0001; E/E´: 28.96 ± 1.74, N=15 vs. 48.00 ± 3.13, N=42, p=0.0008), whereas systolic LV function remained unchanged. Diastolic function was significantly improved after 4 weeks of treatment with NO2-OA compared to vehicle (NO2-OA vs. vehicle, E/E´: 34.49 ± 3.95, N=13 vs. 68.42 ± 9.90, N=10, p=0.002). Treatment with OA had no effect.
Given the well-known anti-oxidative properties of NO2-OA, we investigated the oxidative stress marker 8-hydroxydeoxy-guanosine (8-OHdG) and superoxide dismutase (SOD). 8-OHdG immunoreactivity in LV nuclei was significantly reduced in NO2-OA treated mice in comparison to vehicle (% area, NO2-OA vs. vehicle, 0.68 ± 0.17, N=9 vs. 1.97 ± 0.31, N=7, p=0.003). Likewise, SOD1 mRNA upregulation was significantly less pronounced in NO2-OA treated mice compared to vehicle (ctrl vs. vehicle vs. NO2-OA, 1.01 ± 0,07, N=8 vs. 1.33 ± 0.07, N=10, p=0.008 vs. 1.06 ± 0.06, N=11, p=0.015). Interestingly, no differences were found between the groups for LV hypertrophy and fibrosis.
Apart from these findings in the myocardium, systemic effects were investigated. Treatment with NO2-OA significantly improved glucose tolerance compared to vehicle (p<0.0001, N=11). Insulin content and secretion of isolated islets were studied by radioimmunassay. Both parameters were modified by application of HFD+L-NAME. Interestingly the insulin content was significantly less increased in NO2-OA- vs. vehicle-treated mice (p<0,05, N=10). Analysis of isolated pancreatic beta-cells disclosed, that HFD+L-NAME-mediated alterations in the profile of glucose-dependent ROS accumulation (DCF fluorescence) seem to be sensitive to NO2-OA treatment (N=7).

Conclusion:
Application of NO2-OA ameliorates HFpEF in diabetic mice. Anti-oxidative mechanisms in the LV myocardium as well as in pancreatic beta-cells may contribute to this effect. Further experiments are ongoing to expand these findings and to clarify mechanistic relations between regulation of oxidative stress and LV function, which can advance the identification of new pharmacological approaches for diabetic cardiomyopathy.