Fig 2: FNDC4 promotes angiogenesis of endothelial cells through increasing FGF1 secretion from cardiomyocytes.a Human umbilical vein endothelial cells (HUVECs) were cultured with the conditioned medium (NRCMs-ConM) for 24 h, and then EdU+ nuclei were quantified using a commercial kit (n = 6 independent experiments). b HUVECs were cultured with NRCMs-ConM for 24 h, and then exposed to transwell assay. After 12 h, cells in the lower chamber were stained with crystal violet to quantify the migrated cells (n = 6 independent experiments). c HUVECs were cultured with NRCMs-ConM for 24 h, and then exposed to tube formation assay. After 8 h, the branching length and junction number were quantified (n = 6 independent experiments). d The upregulated angiogenic factors in FNDC4-overexpressed hearts were analyzed using the transcriptome data (n = 3). e The mRNA levels of fibroblast growth factor 1 (Fgf1), slit guidance ligand 1 (Slit1) and Slit2 in NRCMs with or without FNDC4 overexpression (n = 6 independent experiments). f The levels of FGF1, SLIT1 and SLIT2 in the medium of NRCMs with or without FNDC4 overexpression (n = 6 independent experiments). g The level of FGF1 in the medium of NRCMs with brefeldin A (BFA) or BAPTA-AM treatment (n = 6 independent experiments). h Single-cell sequencing data of FGF1 in human hearts. i HUVECs were cultured with NRCMs-ConM in the presence of anti-FGF1 or PD173074 for 24 h, and then EdU+ nuclei were quantified using a commercial kit (n = 6 independent experiments). j HUVECs were cultured with NRCMs-ConM in the presence of anti-FGF1 or PD173074 for 24 h, and then exposed to tube formation assay (n = 6 independent experiments). Data were presented as the mean ± S.D., and analyzed using an unpaired two-tailed Student′s t-test. For the analysis in (g–j), one-way ANOVA followed by Tukey post hoc test was used. *P < 0.0001. Source data are provided as a Source Data file.

Fig 3: FNDC4 expression is elevated during cardiac I/R injury in a HIF1α-dependent manner.a The hearts with or without I/R injury were harvested for western blot (n = 6). b The left ventricles from ischemic heart disease (IHD) patients or donors were prepared for western blot (n = 6). c, d Neonatal rat cardiomyocytes (NRCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with or without sI/R injury were prepared for western blot (n = 6 independent experiments). e, f Correlations between cardiac Fndc4 mRNA and serum cardiac isoform of tropnin T (cTnT) or fractional shortening (FS) in I/R-stressed mice (n = 20). g Plasma FNDC4 levels in sham- or I/R-stressed mice (n = 20). h, i Correlations between plasma FNDC4 and serum cTnT or FS in I/R-stressed mice (n = 20). j Plasma FNDC4 levels in acute myocardial infarction (AMI) patients or healthy controls (n = 26 for donors and n = 42 for AMI patients). k Correlation between plasma FNDC4 and high-sensitivity tropnin I (hs-TnI) in AMI patients (n = 42). l Plasma FNDC4 levels in AMI patients at admission and after PCI surgery (n = 42). m FS in AMI patients with low or high FNDC4 levels at 1, 6 and 12 months post-PCI surgery (n = 10 for low FNDC4 group and n = 16 for high FNDC4 group). n The clinical outcome of AMI patients with low or high FNDC4 levels after PCI surgery (n = 21). o The hearts with or without HIF1α inhibition were prepared for western blot (n = 6). p NRCMs with or without HIF1α inhibition were prepared for western blot (n = 6 independent experiments). Data were presented as the mean ± S.D., and analyzed using an unpaired two-tailed Student′s t-test. For the analysis in (e–k), Pearson’s correlation analysis was used. For the analysis in (l), a paired two-tailed Student′s t-test was performed. For the analysis in (m), repeated measures ANOVA was conducted. For the analysis in (n), a two-tailed χ2 test with Yates’ correction was used, χ2 = 2.713, df = 1, z = 1.647. For the analysis in (o, p), one-way ANOVA followed by Tukey post hoc test was used. *P < 0.0001. Source data are provided as a Source Data file.

Fig 4: FNDC4 overexpression prevents sI/R-induced cardiomyocyte injury in vitro.a NRCMs with or without FNDC4 overexpression were harvested for western blot (n = 6 independent experiments). b FNDC4 level in the medium of NRCMs with or without FNDC4 overexpression (n = 6 independent experiments). c Cell viability was determined using the cell counting kit-8 (CCK-8) method (n = 6 independent experiments). d Lactate dehydrogenase (LDH) releases were calculated as (LDH level in ischemia medium + LDH in reperfusion medium)/(LDH in ischemia medium + LDH in reperfusion medium + LDH in cell lysate) (n = 6 independent experiments). e Representative TUNEL staining images of cell coverslips and quantitative results. Arrows indicate TUNEL + nuclei (n = 6 independent experiments). f NRCMs were collected for western blot (n = 6 independent experiments). g Caspase3 activity in NRCMs (n = 6 independent experiments). Data were presented as the mean ± S.D., and analyzed using one-way ANOVA followed by Tukey post hoc test. For the analysis in (a, b), an unpaired two-tailed Student′s t-test was used. *P < 0.0001. Source data are provided as a Source Data file.

Fig 5: FNDC4 ameliorates cardiac I/R injury through activating HIF1α in vivo.a Heart samples with or without FNDC4 overexpression were collected 24 h after I/R surgery and subjected to unbiased transcriptome analysis, and the KEGG analysis was performed using the upregulated differentially expressed genes (DEGs) (n = 3). b Heart samples with or without FNDC4 overexpression were collected 24 h after I/R surgery and subjected to western blot (n = 6). c Heart samples with or without FNDC4 overexpression were collected 24 h after I/R surgery and subjected to immunofluorescence staining of HIF1α (green) and α-actinin (red). Arrows indicate HIF1α expression in the nuclei of cardiomyocytes (n = 6). d, e Mice with or without FNDC4 overexpression were treated with PX-478 to inhibit HIF1α, and heart samples were collected 24 h after I/R surgery for Evans blue and TTC double staining (n = 6). f Cardiac function of FNDC4-overexpressed mice with or without PX-478 treatment was analyzed by transthoracic echocardiography at the indicated time points (n = 6). Data were presented as the mean ± S.D., and analyzed using one-way ANOVA followed by Tukey post hoc test. *P < 0.0001. Source data are provided as a Source Data file.