Background: Synthesized endogenously, nitrated fatty acids are nitric oxide (NO) derived highly electrophilic molecules that have been identified as anti-inflammatory signaling mediators in different cardiovascular diseases. Nitro-oleic acid (NO₂-OA) reduces infarct size and improves left ventricular function after myocardial ischemia/reperfusion damage in mice and is currently being tested in a phase-2-clinical trial for treatment of pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp^-/-) are characterized by impaired left ventricular function (LVEF) and increased ventricular fibrosis at the age of 8 weeks and serve as a well-established animal model for dilated cardiomyopathy (DCM). This study investigates the therapeutical effects of NO₂-OA in a murine DCM model in vivo and in vitro.

Methods and Results: 12-week-old Mlp^-/- mice were treated with NO₂-OA (1.04 nmol/g/h) or vehicle only (PEG) continuously for 4 weeks via subcutaneous osmotic minipumps. WT littermates treated with PEG served as controls. Echocardiography was performed at baseline, and at 16 weeks of age. Vehicle-treated Mlp^-/- mice showed a decreased left ventricular ejection fraction (LVEF) and had a higher heart weight to body weight ratio (HWBW) compared to WT animals. Treatment of Mlp^-/- mice with NO₂-OA significantly reduced HWBW and improved LVEF, cardiac output (CO) and global longitudinal strain. The level of ventricular fibrotic remodeling was histologically assessed by Picrosirius red staining. Untreated Mlp^-/- ventricles showed significantly higher levels of fibrosis than WT littermates and treatment with NO₂-OA attenuated ventricular fibrosis in Mlp^-/- hearts. This finding was confirmed by determination of total collagen levels by measuring free hydroxyproline residues. Interestingly, increased protein and mRNA levels of TGFβ, the hallmark inducer of fibrosis, were found in LV homogenates of Mlp^-/- mice. To investigate effects of NO₂-OA on TGFβ signaling, primary cardiac fibroblasts were stimulated with TGFβ (10 ng/ml) and concomitantly treated with NO₂-OA (1 µM). Analysis of downstream targets of TGFβ revealed that NO₂-OA significantly attenuates phosphorylation of Smad2/3, STAT3 and Erk1/2. These findings were supported by a reduction of alpha smooth muscle actin (aSMA) expression in primary cardiac fibroblasts.

Conclusions: NO₂-OA treatment improves left ventricular function and attenuates myocardial fibrosis in a murine model of DCM. In vitro, NO₂-OA inhibits TGFβ-induced fibroblast to myofibroblast transdifferentiation. Taken together these data indicate a therapeutic anti-fibrotic effect of NO₂-OA in DCM.