Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice

Abstract

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury.

Fig. 1. Tisp40 expression and nuclear translocation are induced by cardiac I/R injury.

a Full-length Tisp40 (370 amino acids) is an endoplasmic reticulum (ER) membrane-resident type II transmembrane protein, and contains an N-terminal acidic transcription activation domain (TAD), a basic zipper (bZIP) domain and a transmembrane (TM) domain, which is cleaved by S1P and S2P proteases to release the N-terminal fragment to the nucleus. b Tisp40 knockout (KO) mice and the wild-type (WT) littermates received sham or cardiac ischemia/reperfusion (I/R) surgery (ischemia for 45 min and reperfusion for 24 h), and then whole-cell lysates together with nuclear lysates from the heart were prepared for western blot (n = 6). c Heart samples were collected for immunofluorescence staining of sarcomeric α-actinin (red) and Tisp40 (green) (n = 6). d usion, and then were stained with Lys–Asp-Glu–Leu (KDEL, an ER marker, red) and Tisp40 (green) (n = 6). e Whole-cell lysates and nuclear lysates from NRCMs were prepared for western blot (n = 6). f Whole-cell lysates and nuclear lysates from the left ventricles of ischemic heart disease (IHD) patients or donors were prepared for western blot (n = 6). g Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to ischemia for 4 h followed by overnight reperfusion, and then whole-cell lysates together with nuclear lysates were prepared for western blot (n = 6).

Fig. 2. Tisp40 deficiency exacerbates oxidative stress, apoptosis, and cardiac I/R injury in vivo.

a Protocol diagram for the study design. b Representative Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC)-stained heart sections from global Tisp40 KO mice and WT littermates (n = 8). c The relative ratios of infarct area (IA, pale) to the area at risk (AAR, not blue) and AAR to left ventricles (LV) were compared between Tisp40 KO and WT hearts 24 h after I/R surgery (n = 8). d Representative TdT-mediated dUTP nick end-labeling (TUNEL) staining images of heart sections and quantitative results from Tisp40 KO mice and WT littermates 24 h after I/R surgery (n = 6). e DNA fragments in the heart (n = 6). f Caspase3 activity in the heart (n = 6). g Representative dihydroethidium (DHE) staining images of heart sections and quantitative results from Tisp40 KO mice and WT littermates 24 h after I/R surgery (n = 6). h Quantitative results of superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) in the heart (n = 6). All data are expressed as the mean ± SD, and analyzed using an unpaired two-tailed Student′s t test. For the analysis in (e, f, h), one-way analysis of variance (ANOVA) followed by Tukey post hoc test was conducted. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. Tisp40 deficiency aggravates cardiac remodeling and dysfunction following I/R injury.

a Heart samples were collected for Wheat Germ Agglutinin (WGA) staining to quantify the cross-sectional area of cardiomyocyte 4 weeks post-I/R surgery (n = 6). b Quantitative results of heart weight/tibial length (HW/TL) 4 weeks post-I/R surgery (n = 6). c Heart samples were collected for picrosirius red (PSR) staining to quantify the collagen deposition 4 weeks post-I/R surgery (n = 6). d Total and insoluble collagen content in the heart 4 weeks post-I/R surgery (n = 6). e–g Cardiac function of Tisp40 KO mice or WT littermates was analyzed by transthoracic echocardiography at the indicated time points, and presented as fractional shortening (FS), left ventricle internal diameters at diastole (LVIDd), systole (LVIDs) or interventricular septal thickness at systole (IVSs) (n = 6). All data are expressed as the mean ± SD, and analyzed using one-way ANOVA followed by Tukey post hoc test. For the analysis in (e–g), repeated measures ANOVA followed by Sidak post hoc test was conducted. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 4. Cardiomyocyte-specific overexpression of full-length Tisp40 prevents I/R-induced acute cardiac injury, remodeling, and dysfunction in mice.

a Representative Evans blue and TTC-stained heart sections, and quantitative data from cardiomyocyte-restricted Tisp40 transgenic (cTG) mice and the matched non-transgenic (NTG) littermates 24 h after I/R surgery (n = 8). b Representative TUNEL staining images of heart sections and quantitative results from Tisp40 cTG mice and WT littermates 24 h after I/R surgery (n = 6). c DNA fragments in the heart (n = 6). d Caspase3 activity in the heart (n = 6). e Representative DHE staining images of heart sections and quantitative results from Tisp40 cTG mice and WT littermates 24 h after I/R surgery (n = 6). f Quantitative results of O₂⁻ and H₂O₂ in the heart (n = 6). g Heart samples were collected for WGA staining to quantify the cross-sectional area of cardiomyocytes 4 weeks post-I/R surgery (n = 6). h Heart samples were collected for PSR staining to quantify the collagen deposition 4 weeks post-I/R surgery (n = 6). i Levels of lysyl oxidase (Lox) mRNA in the heart 4 weeks post-I/R surgery (n = 6). j Total and insoluble collagen content in the heart 4 weeks post-I/R surgery (n = 6). k Cardiac function of Tisp40 cTG mice or NTG littermates was analyzed by transthoracic echocardiography at the indicated time points, and presented as FS, LVIDd and LVIDs (n = 6). All data are expressed as the mean ± SD, and analyzed using one-way ANOVA followed by Tukey post hoc test. For the analysis in (a, b, e), an unpaired two-tailed Student′s t test was conducted. For the analysis in (k), repeated measures ANOVA followed by Sidak post hoc test was conducted. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo.

a Tisp40 KO mice received a single intravenous injection of AAV9ΔTM-HA or AAV9Ctrl, and heart samples were collected for western blot 4 weeks post-AAV9 injection (n = 6). b Heart samples were collected for immunofluorescence staining of sarcomeric α-actinin (red) and Tisp40 (green) (n = 6). c Representative Evans blue and TTC-stained heart sections, and quantitative data from Tisp40 KO mice with AAV9ΔTM-HA or AAV9Ctrl injection 24 h after I/R surgery (n = 6). d Circulating levels of cTnT, CK-MB and LDH in Tisp40 KO mice with AAV9ΔTM-HA or AAV9Ctrl injection 4 h after I/R surgery (n = 6). e Heart samples were collected for HE or PSR staining 4 weeks post-I/R surgery (n = 6). f Quantitative results of the cross-sectional area of cardiomyocyte (n = 6). g, h Total, insoluble and soluble collagen content in the heart 4 weeks post-I/R surgery (n = 6). i Quantitative results of the collagen deposition (n = 6). j, k Cardiac function was presented as FS, LVIDd, and LVIDs (n = 6). All data are expressed as the mean ± SD, and analyzed using one-way ANOVA followed by Tukey post hoc test. For the analysis in (c), an unpaired two-tailed Student′s t test was conducted. For the analysis in (j, k), repeated measures ANOVA followed by the Sidak post hoc test was conducted. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 6. Tisp40 ameliorates cardiac I/R injury through stimulating HBP flux and protein O-GlcNAcylation.

a KEGG analysis of GSE7223 dataset. b An overview of the hexosamine biosynthetic pathway (HBP). c–e Analysis of GlcNAc-6P and UDP-GlcNAc in I/R-injured Tisp40 KO or WT hearts by UHPLC-MS/MS (n = 12). f, g Protein O-GlcNAc levels in I/R-injured Tisp40 KO or WT hearts were evaluated by western blot or by immunofluorescence staining of sarcomeric α-actinin (red) and O-GlcNAc (green) (n = 6). h To inhibit OGA, Tisp40 KO mice were intraperitoneally injected with NButGT daily for 14 consecutive days or TMG every other day for 20 consecutive days, and the last injections of NButGT or TMG were done 30 min before cardiac I/R surgery. Next, Evans blue and TTC staining were performed to demarcate IA and AAR (n = 8). i Quantitative results of FS in mice (n = 6). All data are expressed as the mean ± SD, and analyzed using one-way ANOVA followed by Tukey post hoc test. For the analysis in (e), an unpaired two-tailed Student′s t test was conducted. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 7. Tisp40 facilitates HBP flux and cardioprotection through transcriptionally upregulating GFPT1.

a Volcano map of GSE7223 dataset. b Sham- or I/R-operated hearts were collected for immunofluorescence staining of sarcomeric α-actinin (red) and GFPT1 (green) 24 h after I/R surgery (n = 6). c, d Heart samples were collected for western blot and quantitative real-time PCR 24 h after I/R surgery (n = 6). e A conserved UPRE was identified in the GFPT1 promoter across different species. f Tisp40-deficient neonatal mouse cardiomyocytes were infected with AdΔTM-HA for 4 h and cultured in fresh medium for an additional 48 h, which were then cross-linked with 1% formaldehyde and immunoprecipitated with anti-HA or anti-IgG isotype control. PCR amplification was performed using primers spanning the UPRE in the GFPT1 promoter (n = 6). g To knock down endogenous GFPT1 in the heart, Tisp40 cTG mice were intravenously injected with shGfpt1 4 weeks before I/R surgery. Next, Evans blue and TTC staining were performed to demarcate IA and AAR (n = 6). h Quantitative results of TUNEL staining in the heart (n = 6). i DNA fragments in the heart (n = 6). j Quantitative results of the cross-sectional area of cardiomyocyte 4 weeks post-I/R surgery (n = 6). k Quantitative results of HW/TL 4 weeks post-I/R surgery (n = 6). l Quantitative results of the collagen deposition 4 weeks post-I/R surgery (n = 6). m, n Cardiac function was presented as FS, LVIDd and LVIDs (n = 6). All data are expressed as the mean ± SD, 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. 8. Tisp40 is a cardiomyocyte-enriched UPR-associated transcription factor.

a–c NRCMs were stimulated with thapsigargin (THA), tunicamycin (TUN) or dithiothreitol (DIT) for 6 h, and then whole-cell lysates and nuclear lysates from NRCMs were prepared for western blot (n = 6). d To suppress ER stress in NRCMs, 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid sodium salt (TUDCA) were added during sI/R injury. Next, whole-cell lysates and nuclear lysates from NRCMs were prepared for western blot (n = 6). e To inhibit ER stress in mice, 4-PBA or TUDCA was administered by a single intraperitoneal injection 15 min before reperfusion, and then whole-cell lysates together with nuclear lysates from the heart were prepared for western blot (n = 6). f Schematic diagram of the molecular mechanisms underlying Tisp40-regulated cardiac I/R injury. ER membrane-resident Tisp40 in I/R-injured hearts is upregulated and cleaved under ER stress, and then released to the nucleus, where it directly binds to the promoter of GFPT1 and subsequently facilitates HBP flux and protein O-GlcNAcylation, thereby mitigating cardiac I/R injury.