miR‐21‐5p Regulation of Cardiac Fatty Acid Oxidation and Mitochondrial Respiration
Authors: Nasci Victoria , Chuppa Sandra , Griswold Lindsey , Kriegel Alison 
Published: June 2021

Cardiovascular related pathologies are the leading single cause of death in chronic kidney disease (CKD) patients. Previously, we found that 5/6 nephrectomy model of CKD leads to an upregulation of miR‐21‐5p in the left ventricle, targeting peroxisome proliferator‐activated receptor alpha (PPARα) and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. We also observed that overexpression of miR‐21‐5p attenuates both lipid content and lipid peroxidation in H9C2 (cardiomyoblast, ATCC) cells. These findings lead us to hypothesize that miR‐21‐5p regulates cardiac mitochondrial respiration and fatty acid oxidation. In this study we utilized the Seahorse XF Fatty Acid Oxidation Assay (Agilent) to evaluate the potential for knockdown or overexpression of miR‐21‐5p to impact oxygen consumption rate in the H9C2 cells. Cells were plated into 96 well Seahorse assay plates and transfected with anti‐miR‐21‐5p (40nM), pre‐miR‐21‐5p (20nM) or appropriate scrambled oligonucleotide controls. After a 24‐hour incubation medium was changed from 4.5g/L glucose to 1.0g/L glucose and incubated for an additional 24 hours. In preparation for the Seahorse Assay, medium was changed and half of the cells from each transfection group were treated with etomoxir, a compound that prevents fatty acid oxidation, for 30 minutes. This was followed by addition of palmitic acid (PA), a saturated fatty acid, to half of the cells of each transfection/treatment group. When we compare mitochondrial respiration rate (oxygen consumption rate normalized by total cellular protein in that sample) we find that both anti‐miR‐21‐5p and pre‐miR‐21‐5p increase basal and maximal respiration rates. However, it should be noted that maximal respiration is significantly higher with anti‐miR‐21‐5p than with pre‐21‐5p (172% vs. 139% of control, respectively). Similarly, we find that non‐mitochondrial respiration, basal respiration, maximal respiration, ATP production and proton leak are all significantly increased in response to either pre‐miR‐21‐5p or anti‐miR‐21‐5p when compared to their respective control treatment. Neither etomoxir, nor PA had any impact on oxygen consumption rate in pre‐miR‐21‐5p treated cells, suggesting that fatty acid was not contributing significantly to the oxygen consumption when miR‐21‐5p is elevated. Conversely, anti‐miR‐21‐5p cell transfected cells exhibited reduced oxygen consumption rate with both etomoxir and PA, with PA treated cells exhibiting signs of lipotoxicity. To determine if miR‐21‐5p manipulation altered mitochondria oxidative capacity, we transfected and treated cells as described above, and then measured the amount of lactate produced over a one‐hour using the Lactate Glo Assay (Promega). Cellular lactate production was not increased, and tended to decrease in both pre‐miR‐21‐5p and anti‐miR‐21‐5p treatment groups, when compared to controls, suggesting oxidative capacity is not impaired in either condition. In sum these results suggest that overexpression and suppression of miR‐21‐5p augment mitochondrial respiration in H9C2 cells through different mechanisms. Together with previous findings, our results indicate that the overexpression of miR‐21‐5p reduces cellular fatty acid utilization, potentially shifting cellular metabolism toward reliance on the glycolytic pathway.

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R01HL128332

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.