miR‐21‐5p Regulation of Fatty Acid Oxidation and Mitochondrial Respiration in Intact and Permeabilized H9C2 Cardiomyoblast Cells
Authors: Nasci Victoria , Paterson Mark , Hall Raven , Harris Cayla , Kriegel Alison 
Published: April 2020

Previous studies in our laboratory have shown that in the 5/6 nephrectomy model of CKD a pathological upregulation of miR‐21‐5p occurs in the left ventricle, which suppresses peroxisome proliferator‐activated receptor alpha and impacts regulation of fatty acid oxidation and glycolysis. Subsequent studies in H9C2 (cardiomyoblast, ATCC) cell showed that overexpression of miR‐21‐5p reduced lipid content and lipid peroxidation in response to fatty acid treatment in culture. Studies of oxygen consumption rate (OCR) showed that upregulation of miR‐21‐5p induced a shift in cellular metabolism toward reliance on the glycolytic pathway, even when lipid is present in abundance. The goal of this study was to determine whether the regulatory effects of miR‐21‐5p on metabolism are isolated to cytoplasmic pathway components, or if miR‐21‐5p could have a direct effect on mitochondrial function. To accomplish this, we performed additional studies evaluating OCR (Seahorse System, Agilent) in intact and permeabilized H9C2 cells, as well as miR‐21−/− H9C2 cells which we generated by CRISPR/Cas9 editing. We also evaluated miR‐21‐5p rescue potential through overexpression in miR‐21−/− H9C2 cells. Cells were plated into 96 well Seahorse assay plates (7000/well) and transfected with pre‐miR‐21‐5p (20nM) or a scrambled oligonucleotide control. After a 24‐hour incubation medium was changed from 4.5g/L to 1.0g/L glucose for an additional 24 hours. Half of the plates were treated with Agilent Seahorse XF Plasma Membrane Permeabilizer (PMP). This was followed by addition of Palmitate‐BSA (intact) or palmitoylcarnitine‐malate (PM; permeabilized), a saturated fatty acid, to half of the cells of each transfection/treatment group. Intact cells were evaluated by Agilent Seahorse XF fatty acid oxidation assay and permeabilized cells were as in Salabei et al. Nature Protocols, 2014. Higher basal respiration, proton leak and ATP production were observed in miR‐21−/− versus WT H9C2 cells. Maximal respiration was not different between genotypes. Evaluation of mitochondrial OCR in permeabilized cells revealed that state 3 and state 4 respiration was higher in miR‐21−/− than WT H9C2 cells. Overexpression of miR‐21‐5p prevented this difference. In intact cells, palmitate treatment had no further effect on basal respiration, however maximal respiration was augmented in all groups except miR‐21‐5p overexpressing miR‐21−/− cells where it was reduced. In permeabilized cells, PM treatment reduced state 3, state 4 and RCR similarly in WT and miR‐21−/− cells overexpressing miR‐21‐5p. In summary, these results indicate that miR‐21‐5p regulation of H9C2 cellular metabolism is not mediated solely through effects on non‐mitochondrial cytoplasmic proteins. Cellular miR‐21‐5p abundance has a direct impact on mitochondrial function. Our results also suggest that the loss of miR‐21‐3p in the miR‐21−/− may have independent effects on cell metabolism. These data establish a strong rationale for further investigation of the regulation of miR‐21‐5p and miR‐21‐3p and their effect on cardiomyocyte metabolism.Support or Funding InformationNIH: R01‐HL‐128332‐01A1 AHA: 18PRE34000045