Fig 2: 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.

Fig 3: 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 4: 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 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.