Nrf1 promotes heart regeneration and repair by regulating proteostasis and redox balance

Figures

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  Fig. 1. Nrf1 is expressed in regenerative CM4 cardiomyocytes. a Violin plot showing the expression of Acta2 in CM1–CM5 cardiomyocyte populations. Expression levels are shown as reads per 10k counts. b Acta2 and Tnnt2 RNA transcripts detected by RNA-scope probes in the left ventricular myocardium on a transverse section of P1 hearts. Scale bar, 20 μm. c Quantification of Acta2 transcript levels in cardiomyocytes from P1 and P8 heart; n = 29–34 cardiomyocytes were quantified. d Acta2 and Tnnt2 RNA transcripts detected by RNA-scope probes in the left ventricular myocardium on a transverse section of P8 hearts. Scale bar, 20 μm. e Schematic diagram showing the spatial transcriptome analysis of heart sections to identify anatomic locations of cardiac cell type populations. f Hematoxylin and eosin (H&E) image of a heart transverse section collected at 7-day post P1 MI. Left and right ventricles are indicated. Black arrowheads depict fibrotic tissue; Scale bar, 500 μm. g Left: cell type composition shown in pie-charts for individual spatial spots mapped onto the heart section with H&E staining shown in (f). Right: the boxed region is shown in high magnification overlayed with the H&E image to…

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  Fig. 2. Nrf1 deletion impairs neonatal heart regeneration. a Illustration showing timeline of MI and Sham surgeries performed at P3 and subsequent days of sample collection. b, c Fractional shortening (b) and ejection fraction (c) of Nrf1fl/fl and Nrf1 cKO mouse hearts at 25 days after MI or Sham surgery; n = 4 animals for Sham groups and n = 7–8 animals for MI groups; **p = 0.0022 (b), **p = 0.0009 (c) by Student’s t-test two-tailed. d Masson’s trichrome staining showing fibrotic scarring in Nrf1fl/fl and Nrf1 cKO mouse hearts at 25-day after MI; similar results were observed in six animals; Scale bar, 200 μm. e Immunohistochemistry showing pH3+ cardiomyocytes (cTnT+), marked by arrows, in heart sections collected from Nrf1 cKO and Nrf1fl/fl mice 3-day post-MI; Scale bar, 50 μm. f Quantification of pH3+ cardiomyocytes (cTnT+) in heart sections from (e); n = 12 animals for WT and n = 11 animals for Nrf1 cKO, **p = 0.0034 by Student’s t-test two-tailed. g Chymotrypsin-like activity of the proteasome in hearts from Nrf1 cKO and Nrf1fl/fl mice at P4. arb. units, arbitrary units; n = 6 animals for each group; *p = 0.0373 by Student’s t-test one-tailed. h UMAP visualization of cardiomyocyte…

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  Fig. 3. Nrf1 overexpression protects cardiomyocytes against I/R and cardiotoxin injury. a Western blot analysis showing Nrf1 expression in hearts from mice at 4-week after AAV9-TdTomato and AAV9-Nrf1 injection at P4; experiments were repeated independently twice with similar results. b Illustration of a timeline showing the experimental design for AAV9 injection and time points of sample collections. c TTC staining showing infarct size on transverse heart sections collected at the end of reperfusion; Scale bar, 500 μm. d Quantification of infarct area in sections from (c); AAV9-TdTomato, n = 6; AAV9-Nrf1, n = 5; *p = 0.0183, by Student’s t-test two-tailed. e–g Fractional shortening (e), ejection fraction (f), and systolic volume (g) of left ventricles from AAV9-TdTomato and AAV9-Nrf1 hearts at baseline (before I/R), and 1–3 weeks (wk) after I/R; n = 5 animals for each group. e 2wk, *p = 0.0388; 3wk, *p = 0.034. f 2wk, *p = 0.0422; 3wk, *p = 0.0336. g 1wk, *p = 0.0207, 2wk, *p = 0.0188; 3wk, *p = 0.0155. h Masson’s trichrome staining showing fibrotic scarring on transverse sections of hearts collected at 600 μm below the ligation suture; Scale bar, 500 μm. i Quantification of fibrotic…

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  Fig. 4. Nrf1 confers protection by regulating the proteasome and antioxidant response. a Gene ontology analysis of genes upregulated in Nrf1 samples compared to LacZ control samples. Upregulated genes are displayed in semantic space as clusters; the color and proportional size of circles indicate log10 (P value). P values were obtained using Metascape (see the “Methods” section). b Left: Venn diagram showing a number of genes upregulated by Nrf1, Nrf2, or both; Right: top enriched GO terms for each gene group. c Log2 fold-change (FC) of selected proteasome subunit genes (blue) and antioxidant genes (pink) in NRVMs overexpressing Nrf1 (x-axis) or Nrf2 (y-axis). d Proteasomal activity in arbitrary units (arb. units) of NRVMs overexpressing LacZ, Nrf1 or Nrf2; n = 6 biologically independent experiments for groups LacZ and Nrf1; n = 3 biologically independent experiments for group Nrf2; Trypsin-like, **p = 0.0030; Chemotrypsin-like, ***p = 0.000009; Caspase-like, **p = 0.000031. e qPCR measurement of proteasome subunit genes and antioxidant genes in Nrf1 cKO and control hearts at P14; n = 3 for each group. Psma1, *p = 0.0266; Psma5, *p = 0.0261; Psmd1, *p = 0.0120; Cat, *p = 0.0364; Sod1,*p…

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  Fig. 5. NRF1 is an essential stress regulator in human iPSC-derived cardiomyocytes. a Expression (TPM, transcript per million reads) of NRF1 in 431 human heart left ventricle samples from the GTEx database. Samples were collected from 293 males and 138 females. b Expression of NRF1 grouped by age. Age 20–29, n = 22; Age 30–39, n = 26; Age 40–49, n = 66; Age 50–59, n = 154; Age 60–69, n = 149; Age 70–79, n = 14; *p = 0.0106, **p = 0.0027, one-way ANOVA test. c–f Expression correlation plots showing that PSMD7 (c), PSMC1 (d), SOD1 (e), and CAT (f) are highly co-expressed with NRF1 in the 431 human heart left ventricle samples from the GTEx database. Spearman’s correlation coefficient (ρ) and p-values are depicted; one-tailed t-test. g Top enriched GO terms for the 1167 NRF1 co-expressed genes. h–j NRF1 expression is significantly reduced in heart ventricular tissue from patients with heart failure compared to healthy hearts. Box-and-whisker plots are provided in which the central line denotes the median value, the edges of the boxes represent the upper and lower quartiles, and whiskers indicate the minimum and maximum values; *p < 0.05 with exact p-value depicted. h RNAseq data from…