Nuclear Receptor Subfamily 4 Group A Member 1 Exacerbates Cardiac Remodeling by Inhibiting Mitochondrial Function Through the Peroxisome Proliferator‐Activated Receptor γ Coactivator‐1α/Nuclear Respiratory Factor 1/Transcription Factor A Mitochondrial Axis

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  Figure 1. NR4a1 is upregulated under heart failure. A, The transcriptional expression of NR4a1 was assessed in RNA‐sequencing data (GSE262105) including mice with sham (n=3) and mice with heart failure (n=3); B, The qRT‐PCR analysis comparing NR4a1 mRNA expression in mouse at sham and 8 wks after TAC (n=6); C, The qRT‐PCR analysis comparing NR4a1 mRNA expression in the NRVMs were stimulated in PBS/PE (n=6); D, Protein immunoblotting results for NR4a1 in mice hearts after 8 weeks of sham and TAC and quantitative results normalized to β‐actin (n=6); E, Protein immunoblotting results for NR4a1 in NRVMs were stimulated in PBS/PE, and quantitative results normalized to β‐actin (n=6); F, Immunofluorescence staining of NR4a1 in mice heart tissue at sham and TAC for 8 weeks (scale bar: 100 μm). All data are expressed as the mean±SEM. Statistics analyzed (A–E) were performed using an unpaired 2‐tailed Student’s t test. P values are indicated. FPKM indicates fragments per kilobase million; NR4a1, nuclear receptor subfamily 4 group A member 1; PE, phenylephrine; qRT‐PCR, quantitative real‐time polymerase chain reaction; and TAC, transverse aortic constriction.

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  Figure 2. NR4a1 overexpression exacerbates TAC‐induced myocardial remodeling. A, Protein immunoblotting results of NR4a1 overexpression efficiency and its quantification (n=6); B, Mortality statistics in mice (n=6); C, Representative mice B‐ and M‐mode ultrasound images; D, LVFS and LVEF (n=6); E, Measurements of diastolic and systolic LVIDd and LVIDs (n=6); F, Measurements of left ventricular end‐diastolic volume and end‐systolic volume (n=6); G, HW/BW, LW/BW and HW/TL ratios in mice (n=6); H and I, The mRNA levels of ANP, BNP, COL I and COL III detected by PCR were normalized to β‐actin (n=6); J and K, Representative images of WGA and PSR staining and their quantification (scale bar: 50 μm; n=6). All data are expressed as the mean±SEM. Statistics analyzed (A) were performed using an unpaired 2‐tailed Student’s t test and analyzed by 1‐way ANOVA followed by Bonferroni post hoc test (D–I and K). P values are indicated. AAV9 indicates adeno‐associated virus 9; ANP, atrial natriuretic peptide; BNP, B‐type natriuretic peptide; BW, body weight; CSA, cross‐sectional area; HW, heart weight; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left…

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  Figure 3. NR4a1 deletion attenuates TAC‐induced heart failure. A, Representative mice B‐mode and M‐mode ultrasound images; B, Representative segmental synchronicity ultrasound pictures of mouse; C, Measurements of LVEF and LVFS (n=6); D, Measurements of LVIDd and LVIDs (n=6); E, Measurements of left ventricular end‐diastolic volume, left ventricular end‐systolic volume (n=6); F, Representative hematoxylin–eosin staining images of mice heart (scale bar: 1 mm; n=6); G, HW/BW, LW/BW and HW/TL ratios in mice (n=6); H–J, Representative images of WGA and PSR staining and their quantification (scale bar: 50 μm; n=6). All data are expressed as the mean±SEM. Statistics analyzed (C–E; I–J) were performed using an unpaired 2‐tailed Student’s t test. P values are indicated. AAV9 indicates adeno‐associated virus 9; BW, body weight; CSA, cross‐sectional area; HW, heart weight; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left ventricular internal diastolic diameter; LVIDs, left ventricular internal systolic diameter; LW, lung weight; NR4a1, nuclear receptor subfamily 4 group A member 1; PSR, picrosirius red; TAC, transverse aortic constriction; TL, tibia…

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  Figure 4. NR4a1 drives cardiac dysfunction by impairing mitochondrial bioenergetics. A and B, The mRNA levels of DRP1, OPA1, Mfni, Mfn2, TOM20, NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A1 in mice hearts were normalized to statistics using VDAC1 (n=6); C, Representative mitochondrial transmission electron microscopy images (scale bar: 1 μm). D and E, The quantification of fragmented fused mitochondria and mitochondrial abundance by mitochondrial number and mtDNA/nDNA (n=6); F, Representative protein immunoblotting images of mitochondrial respiratory chain complex proteins and their quantification with β‐actin as an internal reference (n=6); G, Representative confocal image of mitochondrial morphology stained by MitoTracker and the quantification of mean length (scale bar: 50/10 μm; n=3). All data are expressed as the mean±SEM and analyzed by 1‐way ANOVA followed by Bonferroni post hoc test (A–G). P values are indicated. AAV9 indicates adeno‐associated virus 9; NR4a1, nuclear receptor subfamily 4 group A member 1; PE, phenylephrine; TAC, transverse aortic constriction; and VDAC1, voltage‐dependent anion channel 1.

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  Figure 5. NR4a1 affects mitochondrial function by inhibiting PGC1α transcription in vivo. A, Immunofluorescence staining of PGC1α in mice heart tissue at sham and TAC for 8 wk (scale bar: 100 μm; n=3); B, The mRNA of PGC1α in mice hearts were normalized to statistics using β‐actin (n=6); C, Representative protein immunoblotting images of PGC1α and the quantification with β‐actin as an internal reference(n=6); D and E, Representative protein immunoblotting images of of PGC1α, NRF1, TFAM, DRP1, OPA1, Mfn1, Mfn2, and TOM20 and the quantification with β‐actin or VDAC1 as an internal reference (n=6); F, The mRNA of PGC1α, NRF1, TFAM, DRP1, OPA1, Mfn1, Mfn2, and TOM20 in mice heart (n=6). All data are expressed as the mean±SEM, and analyzed by one‐way ANOVA followed by Bonferroni post hoc test (B, C; E, F). P values are indicated. AAV9 indicates adeno‐associated virus 9; NR4a1, nuclear receptor subfamily 4 group A member 1; PGC1α, peroxisome proliferator‐activated receptor γ coactivator‐1α; TAC, transverse aortic constriction; and VDAC1, voltage‐dependent anion channel 1.

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  Figure 6. NR4a1 affects mitochondrial function by inhibiting PGC1α transcription in vitro. A and B, Representative protein immunoblotting images of PGC1α, NRF1, and TFAM and the quantification with β‐actin as an internal reference in NRVMs transfected with siRNA and siNR4a1(n=6); C, Representative images for immunofluorescence staining of NR4a1 (green) and PGC1α (red) in NRVMs stimulated with PE (50 μmol/L) or PBS for 24 h (Scale bar, 50 μm); D, OCR assay was performed on NRVMs transfected with siRNA and siNR4a1. OCR was normalized to the total number of cardiomyocytes in each group (n=6); E, The basal respiration, ATP production and maximal respiration of OCR. All data are expressed as the mean±SEM and analyzed by 1‐way ANOVA followed by Bonferroni post hoc test (B, E). P values are indicated. NR4a1 indicates nuclear receptor subfamily 4 group A member 1; NRVM, neonatal rat ventricular myocyte; OCR, oxygen consumption rate; PE, phenylephrine; and PGC1α, peroxisome proliferator‐activated receptor γ coactivator‐1α.

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  Figure 7. Activation of PGC1α transcription counteracts impaired mitochondrial function caused by NR4a1 overexpression in vitro. A and B, Representative protein immunoblotting images of NRF1, TFAM, and DRP1 and the quantification with VDAC1 as an internal reference in NRVMs (n=6); C, The mRNA levels of NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A1 in NRVMs were normalized to statistics using VDAC1 (n=6); D, OCR assay was performed on NRVMs transfected with AdNC and AdNR4a1. OCR was normalized to the total number of cardiomyocytes in each group (n=6); E and F, The basal respiration, ATP production, and maximal respiration of OCR. All data are expressed as the mean±SEM and analyzed by 1‐way ANOVA followed by Bonferroni post hoc test (B, C; E, F). P values are indicated. NR4a1 indicates nuclear receptor subfamily 4 group A member 1; NRVM, neonatal rat ventricular myocyte; OCR, oxygen consumption rate; PE, phenylephrine; and VDAC1, voltage‐dependent anion channel 1.

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  Figure 8. PGC1α knockdown reverses heart failure improved by NR4a1 knockdown. A, Schematic diagram depicting the experimental strategy; B, Measurements of LVEF and LVFS (n=6); C, Representative mice B‐mode and M‐mode ultrasound images; D, Measurements of LVIDd and LVIDs (n=6); E, Measurements of left ventricular end‐diastolic volume and end‐systolic volume (n=6); F, Representative hematoxylin–eosin staining images of mice hearts (scale bar: 1 mm; n=6); G–I, Representative images of wheat germ agglutinin and picrosirius red staining and their quantification (scale bar: 50 μm; n=6). All data are expressed as the mean±SEM. Statistics analyzed (B, D, E, H, I) were performed using an unpaired 2‐tailed Student’s t test. P values are indicated. cKO indicates cardiac knockout; CSA, cross‐sectional area; i.v., intravenous; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left ventricular internal diastolic diameter; LVIDs, left ventricular internal systolic diameter; NR4a1 nuclear receptor subfamily 4 group A member 1; PGC1α, peroxisome proliferator‐activated receptor γ coactivator‐1α; TAC, transverse aortic constriction; and WT, wild type.