FNDC4 alleviates cardiac ischemia/reperfusion injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis in male mice

Abstract

Fibronectin type III domain-containing (FNDC) proteins play critical roles in cellular homeostasis and cardiac injury, and our recent findings define FNDC5 as a promising cardioprotectant against doxorubicin- and aging-related cardiac injury. FNDC4 displays a high homology with FNDC5; however, its role and mechanism in cardiac ischemia/reperfusion (I/R) injury remain elusive. Here, we show that cardiac and plasma FNDC4 levels are elevated during I/R injury in a hypoxia-inducible factor 1α (HIF1α)-dependent manner. Cardiac-specific FNDC4 overexpression facilitates, while cardiac-specific FNDC4 knockdown inhibits cardiomyocyte survival and angiogenesis in I/R-stressed hearts of male mice through regulating the proteasomal degradation of HIF1α. Interestingly, FNDC4 does not directly stimulate angiogenesis of endothelial cells, but increases the expression and secretion of fibroblast growth factor 1 from cardiomyocytes to enhance angiogenesis in a paracrine manner. Moreover, therapeutic administration of recombinant FNDC4 protein is sufficient to alleviate cardiac I/R injury in male mice, without resulting in significant side effects. In this work, we reveal that FNDC4 alleviates cardiac I/R injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis, and define FNDC4 as a promising predictive and therapeutic target of cardiac I/R injury.

Fig. 1. 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. 2. Cardiac-specific FNDC4 overexpression facilitates cardiomyocyte survival and angiogenesis during cardiac I/R injury.

a Evans blue and TTC double staining was performed to identify the infarct area (IA), area at risk (AAR) and remote area of heart samples 24 h after I/R surgery (n = 6). b Heart samples with or without FNDC4 overexpression were collected 24 h after I/R surgery and subjected to TUNEL staining, and TUNEL+ nuclei were quantified. Arrows indicate TUNEL+ nuclei (n = 6). c Heart samples with or without FNDC4 overexpression were collected 24 h after I/R surgery and subjected to unbiased transcriptome analysis, and the expressions of angiogenesis-related genes were presented using a heatmap (n = 3). d, e Heart samples with or without FNDC4 overexpression were collected 4 weeks after I/R surgery and subjected to immunofluorescence staining, and the numbers of capillaries as well as arterioles were quantified. Arrows indicate α-SMA⁺ arterioles (n = 6). f, g Heart samples with or without FNDC4 overexpression were collected 4 weeks after I/R surgery and subjected to hematoxylin-eosin (HE) and picrosirius red (PSR) staining, and cell size as well as fibrotic area were quantified. Circles indicate the cross-sectional area of cardiomyocytes (n = 6). h Total, soluble and insoluble collagen content in the heart 4 weeks post-I/R surgery (n = 6). i Cardiac function of FNDC4-overexpressed and control mice was analyzed by transthoracic echocardiography at the indicated time points (n = 6). Data were presented as the mean ± S.D., and analyzed using an unpaired two-tailed Student′s t-test. For the analysis in (f–h), one-way ANOVA followed by Tukey post hoc test was used. For the analysis in (i), repeated measures ANOVA was performed. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. Cardiac-specific FNDC4 knockdown inhibits cardiomyocyte survival and angiogenesis during cardiac I/R injury.

a Evans blue and TTC double staining was performed to identify the IA, AAR and remote area of heart samples 24 h after I/R surgery (n = 6). b Heart samples with or without FNDC4 knockdown were collected 24 h after I/R surgery and subjected to TUNEL staining, and TUNEL+ nuclei were quantified. Arrows indicate TUNEL+ nuclei (n = 6). c–e Heart samples with or without FNDC4 knockdown were collected 4 weeks after I/R surgery and subjected to immunofluorescence staining, and the numbers of capillaries as well as arterioles were quantified. Arrows indicate α-SMA⁺ arterioles (n = 6). f, g Heart samples with or without FNDC4 knockdown were collected 4 weeks after I/R surgery and subjected to HE and PSR staining, and cell size as well as fibrotic area were quantified. Circles indicate the cross-sectional area of cardiomyocytes (n = 6). h Cardiac function of FNDC4-silenced and control mice was analyzed by transthoracic echocardiography at the indicated time points (n = 6). Data were presented as the mean ± S.D., and analyzed using an unpaired two-tailed Student′s t-test. For the analysis in (f, g), one-way ANOVA followed by Tukey post hoc test was used. For the analysis in (h), repeated measures ANOVA was performed. *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 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. 6. 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.

Fig. 7. FNDC4 elevates HIF1α protein level through suppressing its proteasomal degradation.

a, b NRCMs with FNDC4 overexpression or knockdown were treated with cycloheximide (CHX) to inhibit protein synthesis, and western blot was performed to analyze HIF1α protein levels at the indicated time points (n = 6 independent experiments). c NRCMs with FNDC4 knockdown were treated with proteasomal inhibitors (BZM and CFZ) or lysosomal inhibitors (E-64d and leupeptin), and western blot was performed to measure HIF1α protein level (n = 6 independent experiments). d The top 10 downregulated GO terms of the transcriptome analysis (n = 3). e The expressions of proteasome-related genes were presented using a heatmap (n = 3). f Heart samples were collected from von Hippel-Lindau protein (Vhl) cKO mice with or without FNDC4 knockdown 24 h after I/R surgery, and then exposed to Evans blue and TTC double staining (n = 6). g Cardiac function of Vhl cKO mice with or without FNDC4 knockdown was analyzed by transthoracic echocardiography at the indicated time points (n = 6). Data were presented as the mean ± S.D., and analyzed using repeated measures ANOVA. For the analysis in (c), one-way ANOVA followed by Tukey post hoc test was used. For the analysis in (f), an unpaired two-tailed Student′s t-test was used. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 8. Therapeutic administration of rFNDC4 protein is sufficient to attenuate cardiac I/R injury.

a Evans blue and TTC double staining was performed to identify the IA, AAR and remote area of heart samples 24 h after I/R surgery. Circles indicate the cross-sectional area of cardiomyocytes (n = 6). b Serum levels of cTnT, creatine kinase isoenzymes (CK-MB) and LDH in I/R-stressed mice with or without recombinant FNDC4 (rFNDC4) protein treatment (n = 6). c Heart samples with or without rFNDC4 protein treatment were collected 4 weeks after I/R surgery and subjected to HE and PSR staining, and cell size as well as fibrotic area were quantified (n = 6). d Cardiac function of rFNDC4- or vehicle-treated mice was analyzed by transthoracic echocardiography at the indicated time points (n = 6). (e) Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cr) (n = 6 biological samples). 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–e), an unpaired two-tailed Student′s t-test was used. For the analysis in (d), repeated measures ANOVA was performed. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 9. Schematic diagram of the molecular mechanisms underlying FNDC4-mediated protection against cardiac I/R injury.

FNDC4 is elevated during cardiac I/R injury, which blocks the proteasomal degradation of HIF1α and subsequently activates HIF1α signaling pathway to inhibit cardiomyocyte apoptosis. In addition, FNDC4-mediated HIF1α activation increases the expression and secretion of FGF1 from cardiomyocytes to enhance the angiogenic capacity of endothelial cells in a paracrine manner to facilitate cardiac repair during I/R stress.