Abstract
MicroRNAs (miRNAs/miRs) such as miR-1, miR-133a, miR-133b, miR-135a, and miR-29b play a key role in many cardiac pathological remodeling processes, including apoptosis, fibrosis, and arrhythmias, after a myocardial infarction (MI). Dietary flaxseed has demonstrated a protective effect against an MI. The present study was carried out to test the hypothesis that dietary flaxseed supplementation before and after an MI regulates the expression of above-mentioned miRNAs to produce its cardioprotective effect. Animals were randomized after inducing MI by coronary artery ligation into: (a) sham MI with normal chow, (b) MI with normal chow, and (c–e) MI supplemented with either 10% milled flaxseed, or 4.4% flax oil enriched in alpha-linolenic acid (ALA), or 0.44% flax lignan secoisolariciresinol diglucoside. The feeding protocol consisted of 2 weeks before and 8 weeks after the surgery. Dietary flax oil supplementation selectively upregulated the cardiac expression of miR-133a, miR-135a, and miR-29b. The levels of collagen I expression were reduced in the flax oil group. We conclude that miR-133a, miR-135a, and miR-29b are sensitive to dietary flax oil, likely due to its rich ALA content. The cardioprotective effect of flaxseed in an MI could be due to modulation of these miRNAs.
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Key Points
miRNAs are endogenous, non-coding, single-stranded RNAs of 22–26 nucleotides that regulate messenger RNA (mRNA) expression through posttranslational inhibition or degradation. Several miRNAs have been shown to be involved in cardiac pathological processes such as acute MI, cardiac arrhythmias, and hypertrophy. miRNAs can either promote or inhibit apoptosis of cardiomyocytes, they can modulate angiogenesis, alter cardiac regeneration and reprogram cardiac fibroblasts into cardiomyocytes. the mechanism responsible for this cardioprotective effect of flaxseed was unclear. It is possible that dietary flaxseed may alter miRNA expression and, ultimately, affect myocardial adaptive processes. In support of this hypothesis, diet is known to induce significant cardioprotective effects via a direct action on the epigenome. The current investigation was thus performed to test the hypothesis that flaxseed supplementation exerts its cardioprotective effect by modulating the expression of miR-1, miR-133a, miR-133b, miR-135a, and miR-29b, which are involved in the adverse cardiac remodeling process after an MI.
The data showed that the expression of miR-133a but not miR-133b was upregulated by dietary flaxseed oil supplementation in rat hearts 8 weeks after coronary artery ligation. The location of miR-133a and miR-133b on separate chromosomes may explain why only one of the isoforms was upregulated. Overexpression of miR-133 reduces the apoptosis of cardiomyocytes. Pro-apoptotic genes such as DAPK2, APAF1, caspase-9, Bcl-2, and BMF have been identified as the target of miR-133. miR 133a also improved cardiac reprogramming to produce beating cardiomyocytes from murine and human fibroblasts by repressing Snai1, which regulates epithelial to mesenchymal transition. This is important because cardiomyocytes are unable to replicate and regenerate any lost contractile tissue after an MI. By upregulating the expression of cardiogenic miR-133, dietary flaxseed may be a viable strategy to promote cardiac repair.
The results also demonstrated that flaxseed oil treatment upregulated the expression of miR-135a and miR-29b after an MI. The upregulation of miR-135a has previously reduced MI size and exerted an anti-apoptotic and anti-fibrotic effect in a model of ischemia-reperfusion injury. An increased expression of collagen I was detected in the present study after the MI which was normalized when dietary flaxseed oil was included in the diet of the rats. These effects on myocardial fibrosis by miR-29b and miR-135a are complemented by the actions of miR-133. miR-133 inhibits the expression of collagen 1A1 and connective tissue growth factor (CTGF) to exert its anti-fibrotic effect. During overexpression of miR-133, inflammatory cell infiltration in the myocardium is reduced after an MI. Upregulation of miR-29b, miR-135b and miR-133a and the subsequent decrease in collagen I levels by flaxseed oil may represent the primary mechanisms through which the ALA content of flaxseed exhibits its anti-fibrotic effect.
The anti-arrhythmic action of dietary flaxseed oil may also be associated with its capacity to alter miRNA expression. Previous work from this lab has shown dietary flaxseed and flaxseed oil is anti-arrhythmic in models of ischemia-reperfusion injury to the heart and coronary artery ligation-induced MI. Flaxseed was particularly effective against ventricular fibrillation (VF) and decreased expression of miR-133 increases the incidence of VF. The capacity of dietary flaxseed oil to induce the expression of miR-133 and miR-135a may, therefore, represent the anti-arrhythmic mechanism of dietary flaxseed oil. Overall, these studies suggest that the reduced MI size, fibrosis, inflammation and incidence of arrhythmias shown previously may have been associated with the ALA content of dietary flaxseed.