Cardioprotective effects of omega‐3 fatty acids and ascorbic acid improve regenerative capacity of embryonic stem cell‐derived cardiac lineage cells

Abstract

One of the major issues in cell therapy of myocardial infarction (MI) is early death of engrafted cells in a harsh oxidative stress environment, which limits the potential therapeutic utility of this strategy in the clinical setting. Increasing evidence implicates beneficial effects of omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) Abbreviations: AA, ascorbic acid; ANOVA, analysis of variance; ARE, antioxidant response element; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; Col1, Collagen type 1; CX43, connexin 43; DAPI, 4 0 ,6-diamidino-2-phenylindole; DCF, 2 0 ,7 0 -dichlorofluorescein diacetate; DHA, docosahexaenoic acid; ECL, enhanced chemiluminescence; EF, ejection fraction; EPA, eicosapentaenoic acid; ESC, embryonic stem cell; FS, fractional shortening; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCl, hydrochloric acid; HO-1, heme oxygenase-1; HRP, horseradish peroxidase; IgG, immunoglobulin G; LAD, left anterior descending artery; LDL, low-density lipoprotein; LV, left ventricle; LVD d , LV end-diastolic dimension; LVD s , LV end-systolic dimension; MI, myocardial infarction; mtDNA, mitochondrial DNA; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuminner salt; MYH6, myosin heavy chain, α isoform; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; TBST, tris buffered saline with tween; TNNT2, cardiac troponin † Nasser Aghdami and Hossein Baharvand contributed equally to this work. BioFactors 427Research Communication and ascorbic acid (AA) in cardiovascular diseases, in particular their role in ameliorating fibrosis. In the current study, we aim to assess the cytoprotective role of EPA + DHA and AA in protecting embryonic stem cell (ESC)-derived cardiac lineage cells and amelioration of fibrosis. Herein, we have shown that preincubation of the cells with EPA + DHA + AA prior to H 2 O 2 treatment attenuated generation of reactive oxygen species (ROS) and enhanced cell viability. Gene expression analysis revealed that preincubation with EPA + DHA + AA followed by H 2 O 2 treatment, upregulated heme oxygenase-1 (HO-1) along with cardiac markers (GATA4, myosin heavy chain, α isoform [MYH6]), connexin 43 [CX43]) and attenuated oxidative stressinduced upregulation of fibroblast markers (vimentin and collagen type 1 [Col1]). Alterations in gene expression patterns were followed by marked elevation of cardiac troponin (TNNT2) positive cells and reduced numbers of vimentin positive cells. An injection of EPA + DHA + AA-pretreated ESC-derived cardiac lineage cells into the ischemic myocardium of a rat model of MI significantly reduced fibrosis compared to the vehicle group. This study provided evidence that EPA + DHA + AA may be an appropriate preincubation regimen for regenerative purposes.

Roles of Reactive Oxygen Species in Cardiac Differentiation, Reprogramming, and Regenerative Therapies

“…The development of scaffold-based cell delivery techniques is in the early stages for cardiac tissue engineering, and there are still opportunities to incorporate additional treatments to modulate the antioxidative and anti-inflammatory process. Pharmacological pretreatments (such as omega-3 fatty acids and cobalt protoporphyrin) have beneficent effects on survival of ESC-CMs as evidenced by upregulation of HO-1 and decreased ROS levels under oxidative or hypoxic conditions [ 151 , 152 ]. Future study will reveal new targets and pharmacological compounds to enhance cell engraftment of EHTs after delineating the mechanisms by which the fate of transplanted cells is mediated by increased ROS or downregulated endogenous antioxidant system.…”

Bioactive Lipid Signaling in Cardiovascular Disease, Development, and Regeneration

“…Incubation with this bioactive lipid cocktail before hydrogen peroxide treatment increases the expression of cardiomyocyte markers, decreases the expression of fibroblast markers, and reduces the production of harmful reactive oxygen species. It is also notable that these oxylipins reduce the accumulation of fibrosis after myocardial infarction in ischemic rat hearts [ 189 ]. One particular area of study that is generating much interest is the beneficial pro-resolution of inflammation mediated by docosanoids.…”

Therapeutic Effects of Specialized Pro-Resolving Lipids Mediators on Cardiac Fibrosis via NRF2 Activation

“…In clinical trials, high-dose EPA (4 g/day) reduced the incidences of clinical events by 25% in statin-treated patients with established CVD or diabetes and other cardiovascular risk factors [193]. The cardioprotective effect of omega-3 fatty acids and ascorbic acid improves the regenerative capacity of embryonic stem cell-derived cardiac lineage cells [194]. Pre-incubation of cells with EPA + DHA + AA before H 2 O 2 treatment weakened the production of reactive oxygen species (ROS), improved cell viability and increased HO-1 and CX43 (connexin43) expression, leading to a marked rise in cardiac troponin (TNNT2) positive cells and a decrease in vimentin-positive cells [194].…”

“…The cardioprotective effect of omega-3 fatty acids and ascorbic acid improves the regenerative capacity of embryonic stem cell-derived cardiac lineage cells [194]. Pre-incubation of cells with EPA + DHA + AA before H 2 O 2 treatment weakened the production of reactive oxygen species (ROS), improved cell viability and increased HO-1 and CX43 (connexin43) expression, leading to a marked rise in cardiac troponin (TNNT2) positive cells and a decrease in vimentin-positive cells [194]. In particular, the injection of EPA + DHA + AA pretreated ESC (embryonic stem cell)-derived cardiac lineage cells into the ischemic myocardium of the MI rat model significantly reduced fibrosis compared to the vehicle group [194].…”