Poly (ADP-ribose) polymerases 16 triggers pathological cardiac hypertrophy via activating IRE1α–sXBP1–GATA4 pathway

Fig. 1. PARP16 expresses at high level in heart tissues and cardiomyocytes. a The high expression of PARP16 protein in the heart tissue. Eleven tissues including heart, liver, spleen, kidney, brain, lung, lymph, skeletal muscle and adipose tissue were collected from adult C57BL/6J mice and extracted proteins. Western blot was performed to examined the protein expression of PARP16 in each tissue. b The expression of PARP16 was compared in NRCMs and NRCFs separated from newborn rats by western blot analysis. Data are presented as mean ± SD, and analyzed using Student t test. n = 3, ***p < 0.001

Fig. 2. PARP16 increases in pathological cardiac hypertrophy both in vivo and in vitro. a The protein expressions of PARP16, fibrotic marker MMP9 and hypertrophic marker (ANP, BNP) in heart tissues from mice subjected to sham or TAC surgery were detected by western blot. b Representative images of immunohistochemistry staining with anti-PARP16 in heart sections from sham and TAC groups. n = 3/group. Scale bar: 100 μm. c The protein expressions of PARP16, the myocardial hypertrophy marker BNP, myocardial fibrotic markers (Collagen 1 and MMP2/9) and the inflammatory marker VCAM-1 in heart tissues from mice subjected to sham or Ang II infusion were detected by western blot. d The immunofluorescence staining with anti-α-actinin in PE (100 µM, 48 h)-induced H9C2 cells. Scale bar: 50 μm. Quantification of cell surface area as shown on the right. e The protein expressions of PARP16 and myocardial hypertrophic markers (ANP, BNP) in PE-induced H9C2 cells were detected by western blot. f The mRNA level of PARP16 in PE-treated H9C2 cells were detected by RT-qPCR. g, h Representative immunofluorescence staining images of ANP or BNP in PE-treated H9C2 cells. Scale bar: 50 μm. Data are presented as…

Fig. 3. Depletion of PARP16 alleviates PE-induced cardiomyocytes hypertrophy in vitro. H9C2 cells were transfected with PARP16-specific short interfering RNA (siPARP16) and then treated with PE (100 µM, 48 h). a Representative western blot (left) and quantification (right) of PARP16, ANP and BNP expressions. b The mRNA levels of hypertrophic marker genes (Nppa, Nppb, Myh7) were detected by RT-qPCR analysis. c, d Representative immunofluorescence staining images of ANP or BNP. Scale bar: 50 μm. e Representative immunofluorescence images of α-actinin staining. Scale bar: 50 μm. Quantification of cell surface area as shown on the right. Data are presented as mean ± SD, and analyzed using one-way ANOVA followed by Tukey Post-hoc tests. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4. PARP16 overexpression activates hypertrophic responses in vitro. H9C2 cells were transfected with PARP16 OE lentivirus. a Representative western blots of PARP16, ANP and BNP. b Representative immunofluorescence images of PARP16 staining. Scale bar: 50 μm. c The mRNA levels of Parp16 and hypertrophic marker genes (Nppa, Nppb, Myh7) were detected by RT-qPCR analysis. d Representative immunofluorescence images of α-actinin staining. Scale bar: 50 μm. Quantification of cell surface area as shown on the right. e, f Representative immunofluorescence double staining images of PARP16 and ANP or BNP. Scale bar: 50 μm. Data are presented as mean ± SD, and analyzed using Student t test. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5. PARP16 deficiency ameliorates pathological cardiac hypertrophy in vivo. Adeno-associated viruses AAV9-PARP16 shRNA (AAV9-shPARP16) were injected into the left ventricle of mice for 4–5 dots to drive the knockdown of PARP16, then followed by TAC surgery for 4 weeks, AAV9-GFP was used as the negative control. a The schematic diagram depicting the design of the animal study. b The heart weight/tibia length (HW/TL) ratios. n = 9 or 11/group. c The ejection fraction (EF%). n = 9 or 11/group. d The fractional shortening (FS%). n = 9 or 11/group. e LVESV (LV volume of systole), n = 9 or 11/group. f LV mass. n = 9 or 11/group. g Representative images of heart sections by the gross morphologic examination, hematoxylin and eosin (H&E) staining, wheat germ agglutinin (WGA) staining as well as echocardiography images results from WT mice injected with AAV9-GFP or AAV9-shPARP16 subsequently challenged to sham or TAC surgery. h The quantitative cross-sectional diameter of cardiomyocytes by WGA staining. n = 5/group. i, j Histological analysis of the Masson staining (i) and Sirius Red Staining (j) of heart sections from WT mice injected with AAV9-GFP or AAV9-shPARP16 subsequently challenged to…

Fig. 6. Ablation of PARP16 downregulated the hypertrophic and fibrotic marker expressions. a, b The protein expressions of hypertrophic markers (ANP, BNP) and fibrotic markers (Collagen 1, FN, MMP2/9 and α-SMA) in heart sections from WT mice injected with AAV9-GFP or AAV9-shPARP16 subsequently subjected to sham or TAC surgery by immunoblot analysis. The quantification results were listed below. n = 3. c The mRNA levels of fibrotic marker gene (Fn, Mmp9 and Ctgf) and hypertrophic marker gene (Nppb) in heart sections from WT mice injected with AAV9-GFP or AAV9-shPARP16 subsequently subjected to sham or TAC surgery by RT-qPCR analysis. n = 3. Data are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey Post-hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7. PARP16 regulated the IRE1α–sXBP1 pathway in response to the hypertrophic stimuli. a The activation of IRE1α–sXBP1 pathway in PE-treated NRCMs. b PARP16 knockdown abolished the activation of IRE1α–sXBP1 pathway by western blot assay. c PARP16 OE directly activated the IRE1α–sXBP1 pathway in NRCMs. d The IRE1α–sXBP1 pathway was also regulated in heart sections from WT mice injected with AAV9-GFP or AAV9-shPARP16 subsequently subjected to sham or TAC surgery by immunoblot analysis. e The interaction between PARP16 and IRE1α was determined by co-IP assay in 293T cells transfected by Flag-tagged PARP16 overexpressed lentiviruses. f, g The ADP-ribosylation level of IRE1α was detected by co-IP assay using the ADP-ribose binding agent. Data are presented as mean ± SD and analyzed using Student t test or one-way ANOVA followed by Tukey Post-hoc tests. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8. PARP16 activated GATA4 via IRE1α–sXBP1 pathway in cardiac hypertrophy. a The protein expression of GATA4 in PE-treated NRCMs. b Knockdown of PARP16 abolished the activation of GATA4 in PE-treated NRCMs by western blot assay. c The protein expression of GATA4 was also regulated in heart sections from WT mice injected with AAV9-GFP or AAV9-shPARP16 and subsequently subjected to sham or TAC surgery by immunoblot analysis. d Representative immunofluorescence staining images of GATA4 in PARP16 knockdown followed by PE treatment. scale bar: 50 μm. e PARP16 OE directly activated the expression of GATA4 in NRCMs. f Representative immunofluorescence double staining images of PARP16 and GATA4 in PARP16 OE-transfected H9C2 cells. Scale bar: 50 μm. g The effects of PARP16 knockdown on the nuclear and cytoplasm distribution and expression of GATA4 by western blot. h, i The effects of the ER stress inhibitor (TUDCA) and the specific IRE1α inhibitor STF-083010 on the expressions of GATA4 as well as the hypertrophic markers ANP and BNP in response to PE treatment. j Knockdown of IRE1α abolished the activation of GATA4 or hypertrophic marker BNP in PE-treated NRCMs. k The effects of the specific…