Co-treatment with conjugated linoleic acid and nitrite protects against myocardial infarction

Abstract

According to the CDC, the most common type of heart disease is coronary artery disease, which commonly leads to myocardial infarction (MI). Therapeutic approaches to lessen the resulting cardiovascular injury associated with MI are limited. Recently, MicroRNAs (miRNAs) have been shown to act as negative regulators of gene expression by inhibiting mRNA translation and/or stimulating mRNA degradation. A single miRNA can modulate physiological or disease phenotypes by regulating whole functional systems. Importantly, miRNAs can regulate cardiac function, thereby modulating heart muscle contraction, heart growth and morphogenesis. MicroRNA-499 (miRNA-499) is a cardiac-specific miRNA that when elevated causes cardiomyocyte hypertrophy, in turn preventing cardiac dysfunction during MI. Previous studies revealed that combination treatment with conjugated linoleic acid (cLA) and nitrite preserved cardiovascular function in mice. Therefore, it was hypothesized that cLA and nitrite may regulate miRNA-499, thus providing cardiac protection during MI. To test this hypothesis, 12-week old mice were treated with cLA (10 mg/kg/d-via osmotic mini-pump) or cLA and nitrite (50 ppm-drinking water) 3 days prior to MI (ligation of the left anterior descending artery). Echocardiography and pressure-volume (PV)-loop analysis revealed that cLA and nitrite-treated MI mice had improved heart function (10 days following MI) compared to untreated MI mice. Treatment with cLA and nitrite significantly induced levels of miRNA-499 compared to untreated MI mice. In addition, treatment with cLA and nitrite abolished MI-induced protein expression of p53 and dynamin-related protein-1 (DRP-1). Moreover, the antioxidant enzyme expression of heme oxygenase-1 (HO-1) was elevated in MI mice treated with cLA and nitrite compared to untreated MI mice. Confocal imaging on heart tissue confirmed expression the levels of HO-1 and p53. Taken together, these results suggest that therapeutic treatment with cLA and nitrite may provide significant protection during MI through regulation of both cardiac specific miRNA-499 and upregulation of phase 2 antioxidant enzyme expression.

Keywords: Apoptosis; Conjugated linoleic acid; Drp-1; Myocardial infarction; Nitrite.

Graphical abstract

Fig. 1

Co-treatment with cLA and nitrite increases heart weight following MI. Heart weight is significantly increased following cLA or nitrite with MI, or in MI alone (A). cLA and nitrite co-treatment results in a further increase in heart weight, which is significant compared to MI alone (A) (^⁎p<0.05 to control), ^#p<0.05 to MI, n=8), where overall body weight is unchanged (B).

Fig. 2

Co-treatment with cLA and nitrite significantly improves cardiac function after MI. Treatment with cLA exacerbates cardiac injury after MI, where combination treatment (supplementation with nitrite) rescues heart function following MI (^⁎p<0.05 to control, ^#p<0.05 to MI, n=8).

Fig. 3

Mitochondrial fission factor DRP-1 is attenuated in mice treated with cLA and nitrite following MI. Nitrite- or cLA-treated MI mice have decreased levels of DRP-1 compared to MI alone. Combination treatment with cLA and nitrite significantly lowers the level of Drp-1 in MI mice, comparable to control values (^⁎p<0.05 to control, ^#p<0.05 to MI).

Fig. 4

Co-treatment with cLA and nitrite increases pre-miRNA-499 and miRNA-499 during MI. Pre-miRNA-499 (A) and miRNA-499 (B) is increased in MI mice after combination treatment with cLA and nitrite (^⁎p<0.05).

Fig. 5

Co-treatment with cLA and nitrite attenuates p53 and induces HO-1 expression following MI. Confocal microscopy (A): heart tissue was labeled with p53 (green), HO-1 (red), and cell nuclei was labeled with DAPI (blue). Western blot analysis reveals that p53 is significantly increased in cardiac tissue following MI (B). Quantitated protein expression reveals that co-administration of cLA and nitrite lowers p53 levels, while increasing expression of HO-1 (C) and (D) (^⁎p<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)