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Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression

ABSTRACT: Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labeled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CMUPRT mice) and tested our hypothesis that CM-derived miRNAs (CMmiRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PBsEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PBsEV of CMUPRT mice six hours after 4TUc injection. In PBsEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labeled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a (CMmiR-208a) levels peaked 12 hours after experimentally induced MI in PBsEV and 24 hours after MI in the lung. Notably, miR-208a is expressed from intron 29 of alpha myosin heavy chain (a-MHC), but a-MHC transcripts were nearly undetectable in the lung. When PBsEV from mice that underwent MI (MI-PBsEV) or sham surgery (Sham-PBsEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-kappa B pathway, was lower in the lungs of MI-PBsEV-treated animals than the lungs of animals treated with Sham-PBsEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, CMUPRT mice enables us to track sEV-mediated transport of CMmiRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

ORGANISM(S): Mus musculus

PROVIDER: GSE205405 | GEO | 2022/10/26

REPOSITORIES: GEO

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Project description:OBJECTIVE: To explore the cardioprotective effects of exercise-derived β-aminoisobutyric (BAIBA) on cardiomyocyte apoptosis and energy metabolism in a rat model of heart failure (HF).METHODS: In male Sprague-Dawley rats (8-week-old), myocardial infarction (MI) was used to induce HF by ligating the left anterior descending branch of the coronary artery. In the Sham group, the coronary artery was threaded but not ligated. After HF development, Sham and HF rats were exercised 60 min daily, 5 days per week on a treadmill for 8 weeks (50-60% maximal intensity) and exercise-induced cardiac remodeling after MI were assessed using echocardiography, hematoxylin and eosin (H&E), Masson’s Trichrome and TUNEL staining for the detection of apoptosis-associated factors in cardiac tissue. High-throughput sequencing and mass spectrometry were used to measure BAIBA production and to explore its cardioprotective effects and molecular actions. To further characterize the cardioprotective effects of BAIBA, an in vitro model of apoptosis was generated by applying H2O2 to H9C2 cells to induce mitochondrial dysfunction. In addition, cells were transfected with either a miR-208b analogue or a miR-208b inhibitor. Apoptosis-related proteins were detected by Western Blotting (WB). ATP production was also assessed by luminometry. After administration of BAIBA and Compound C, the expression of proteins related to apoptosis, mitochondrial function, lipid uptake and β-oxidative were determined. Changes in the levels of reactive oxygen species (ROS) were assessed by fluorescence microscopy. In addition, alterations in membrane potential (δψm) were obtained by confocal microscopy.RESULTS: Rats with HF after MI are accompanied by mitochondrial dysfunction, metabolic stress and apoptosis. Reduced expression of apoptosis-related proteins was observed, together with increased ATP production and reduced mitochondrial dysfunction in the exercised compared with the Sham (non-exercised) HF group. Importantly, exercise increased the production of BAIBA, irrespective of the presence of HF. To assess whether BAIBA had similar effects to exercise in ameliorating HF-induced adverse cardiac remodeling, rats were treated with 75 mg/kg/day of BAIBA and we found BAIBA had a similar cardioprotective effect. Transcriptomic analyses found that the expression of miR-208b was increased after BAIBA administration, and subsequent transfection with an miR-208b analogue ameliorated both the expression of apoptosis-related proteins and energy metabolism in H2O2-treated H9C2 cells. In combining transcriptomic with metabolomic analyses, we identified AMPK as a downstream target for BAIBA in attenuating metabolic stress in HF. Further cell experiments confirmed that BAIBA increased AMPK phosphorylation and had a cardioprotective effect on downstream fatty acid uptake, oxidative efficiency and mitochondrial function, which was prevented by the AMPK inhibitor Compound C.CONCLUSION: Exercise-generated BAIBA can reduce cardiomyocyte metabolic stress and apoptosis induced by mitochondrial dysfunction through the miR-208b/AMPK pathway.

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Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression

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