Reduced Immunity Regulator MAVS Contributes to Non-Hypertrophic Cardiac Dysfunction by Disturbing Energy Metabolism and Mitochondrial Homeostasis

Abstract

Cardiac dysfunction is manifested as decline of cardiac systolic function, and multiple cardiovascular diseases (CVDs) can develop cardiac insufficiency. Mitochondrial antiviral signaling (MAVS) is known as an innate immune regulator involved in viral infectious diseases and autoimmune diseases, whereas its role in the heart remains obscure. The alteration of MAVS was analyzed in animal models with non-hypertrophic and hypertrophic cardiac dysfunction. Then, MAVS-deficient mice were generated to examine the heart function, mitochondrial status and energy metabolism. In vitro, CRISPR/Cas9-based gene editing was used to delete MAVS in H9C2 cell lines and the phenotypes of mitochondria and energy metabolism were evaluated. Here we observed reduced MAVS expression in cardiac tissue from several non-hypertrophic cardiac dysfunction models, contrasting to the enhanced MAVS in hypertrophic heart. Furthermore, we examined the heart function in mice with partial or total MAVS deficiency and found spontaneously developed cardiac pump dysfunction and cardiac dilation as assessed by echocardiography parameters. Metabonomic results suggested MAVS deletion probably promoted cardiac dysfunction by disturbing energy metabolism, especially lipid metabolism. Disordered and mitochondrial homeostasis induced by mitochondrial oxidative stress and mitophagy impairment also advanced the progression of cardiac dysfunction of mice without MAVS. Knockout of MAVS using CRISPR/Cas9 in cardiomyocytes damaged mitochondrial structure and function, as well as increased mitochondrial ROS production. Therefore, reduced MAVS contributed to the pathogenesis of non-hypertrophic cardiac dysfunction, which reveals a link between a key regulator of immunity (MAVS) and heart function.

Keywords: MAVS; cardiac dysfunction; energy metabolism; mitochondrial dysfunction; oxidative stress.

Figure 1

Differential regulation of MAVS in animal models with non-hypertrophic and hypertrophic cardiac dysfunction. (A). The data from GSE4105 illustrated that the MAVS expression (gene ID: 311430) was lower in rats challenged with cardiac ischemia-reperfusion injury (P=0.0019). (B). The mRNA levels of MAVS in heart tissues of mice treated with lipopolysaccharide (LPS, 10mg/kg, 12 h) were significantly downregulated. (C). Representative western blots of MAVS in LPS-treated hearts. Densitometry analysis of the western blots of MAVS. (D). Representative western blots of MAVS in the hearts of mice subjected to 5/6Nx. Densitometry analysis of the western blots of MAVS. (E). Representative western blots of MAVS in Angiotensin II (Ang II)-challenged mice hearts (Ang II, 1.4 mg/kg/day, 4 weeks). Densitometry analysis of the western blots of MAVS. The quantitative results are shown as means ± standard error of the mean (SEM) (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

Figure 2

MAVS depletion leads to left ventricular dilation and decreased systolic function in male mice. (A). Representative western blotting analysis of MAVS in the hearts of wild-type (WT) and MAVS^-/- mice. Densitometry analysis of the western blots of MAVS. (B). Representative short-axis M-mode echocardiographic images of WT and MAVS^-/- mice aged 12-16 weeks and 48 weeks. (C). Ejection fraction and fractional shortening of mice from WT and MAVS^-/- groups. (D). Cross-sectional view of the hearts from WT and MAVS^-/- mice at the papillary level, (scale bar=0.25mm). Cross-sectional areas of hearts from WT and MAVS^-/- mice. The quantitative results are shown as means ± SEM (NS, not significant; *P < 0.05, **P < 0.01).

Figure 3

MAVS deficiency resulted in energy metabolism disorder by disturbing lipid metabolism. (A). The ATP content in the hearts of MAVS^-/- mice was lower than that in WT mice (n: WT=7, knockout [KO]=9). (B). Serum CK-MB and LDH levels in WT and MAVS^-/- mice (n: WT=7, KO=7). (C). OPLS-DA analysis based on non-targeted metabolomics was conducted in the hearts of WT and MAVS^-/- mice (n: WT=5, KO=5). The levels of some medium- and long-chain fatty acids were decreased in MAVS^-/- mice (n: WT=5, KO=5). (D). RT-qPCR analysis showed downregulation of fatty acid metabolism related genes (ACC1/2, CPT1α, MCAD) in the hearts of MAVS^-/- mice (n: WT=9, KO=8), reduced expression of CD36 and PPARα (n: WT=5, KO=3). (E). Representative TEM images of hearts from WT and MAVS^-/- mice (Upper panel, scale bar: 2 µm; Lower panel, scale bar: 500 nm, red arrow: lipid droplets). (F). Representative Oil Red O-stained images of hearts from WT and MAVS^-/- mice (Upper panel, scale bar: 50 µm; Lower panel, scale bar: 20 µm, yellow arrow: lipid droplets). The quantitative results are shown as the means ± SEM (*P < 0.05 and **P < 0.01).

Figure 4

MAVS deletion resulted in mitochondrial dysfunction by inducing oxidative stress and impaired mitophagy. (A). Representative TEM images of hearts showed breaks in myocardial fibers, mitochondrial disorganization, and mitophagy (Upper panel, scale bar: 2 µm; lower panel, scale bar: 500 nm; red circle: myocardial fiber rupture; red arrow: damaged mitochondria; yellow arrow: autophagosomes containing mitochondrion). Quantification of damaged mitochondria in the hearts of MAVS-/- mice by TEM images (n: WT=3, KO=3). (B), RT-qPCR analysis of mitochondrial genes in the hearts of WT and MAVS^-/- mice (n: WT=9, KO=8). (C). Western blotting analysis showed reduced expression of mitochondrial electron transfer chain protein ND3 in MAVS-/- mice. Densitometry analysis of the western blots of ND3 (n: WT=6, KO=7). (D). Increased level of MDA was detected in MAVS^-/- mice hearts (n: WT=6, KO=10). (E). The level of 2-hydroxybutyric acid was markedly increased in the hearts of MAVS^-/- mice (n: WT=5, KO=5). 2-Hydroxybutyric acid is a byproduct in the synthesis of glutathione in response to oxidative stress. The quantitative results are shown as the means ± SEM (*P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 5

Loss of MAVS resulted in mitochondrial dysfunction in H9C2 cells. (A). Representative western blotting verified the deletion of MAVS by CRISPR/Cas9 in H9C2 cells. (B). ATP production was reduced in H9C2-/- cells. (C). RT-qPCR analysis of some genes (FASN, ACC1, ACC2, CPT1α) involved in lipid metabolism in H9C2-/- cells (n=6) (D). Representative TEM images of damaged mitochondria and autophagosomes in H9C2-/- cells, (Left panel, scale bar: 2 µm; Right panel, scale bar: 500 nm; red arrow: damaged mitochondria; blue arrow: autophagosomes). Quantification of damaged mitochondria in H9C2-/- cells by TEM images (n=3). (E). RT-qPCR analysis for mitochondrial genes in control and H9C2-/- cells (n=6). (F). Western blotting analysis of mitochondrial electron transfer chain proteins ND1 and Cytochrome C in H9C2-/- cells. Densitometry analysis of the western blots of ND1 and Cytochrome C (n=3). (G). Representative fluorescence images of tetramethylrhodamine, methyl ester (TMRM) in H9C2-/- cells (original magnification, 400×; scale bar: 20 µm). (H), Quantitative analysis of MFI of TMRM by flow cytometry (n=6). (I). Representative fluorescence images from confocal microscope confirmed the staining signals of MMP was from mitochondria. TMRM (red); Mito-tracker staining (green); Hoechst (blue); merge image (yellow) (original magnification, 400×; scale bar: 20 µm). (J). Quantitative analysis of MFI of MitoSOX by flow cytometry in H9C2 cells (n=6). (K). Western blotting analysis of mitophagy marker proteins Parkin, PINK1, LC3-II, and P62. Densitometry analysis of the western blots of above proteins (n=3). The quantitative results are shown as the means ± SEM (*P < 0.05, **P < 0.01, ***P<0.001 and ****P < 0.0001).

Figure 6

MAVS deletion leads to left ventricular dilation and systolic function decrease in female mice. (A), Representative short axis M-mode echocardiographic images of female WT and MAVS^-/- mice at the age of 6 months. (B), EF, FS, LVVs/d, LVIDs/d, and LV mass of mice in WT and MAVS^-/- mice. The quantitative results were shown as the means ± SEM (NS not significant, *p < 0.05 and **p < 0.01).

Figure 7

MAVS deficiency did not affect cardiac development. (A). Representative short-axis M-mode echocardiographic images of WT and MAVS^-/- mice at the age of 2-3 weeks. EF, FS, and LVVs/d did not differ between WT and MAVS^-/- mice. (B). The ATP content in the hearts of mice did not differ between WT and MAVS^-/- mice (n: WT=4, KO=6). (C). Serum CK-MB and LDH levels in mice (n: WT=4, KO=6). (D), RT-qPCR analysis of mitochondrial genes in the hearts of WT and MAVS^-/- mice (n: WT=4, KO=6). (E). Representative HE staining images of hearts from WT and MAVS^-/- mice revealed no morphologic changes of cardiomyocytes (scale bar: 20 µm). (F). Representative TEM images of hearts showed no intergroup difference in mitochondrial morphology (scale bar: 500 nm). The quantitative results are shown as the means ± SEM. (NS not significant).