Fig 2: Model for endoplasmic reticulum (ER) stress–mediated upregulation of T-box transcription factor 20 (Tbx20) resulting in increased cardiomyocyte survival. ER stress induction for short interval results in upregulation of activating transcription factor 6 (Atf6) pathway of unfolded protein response (UPR). Full-length Atf6 is cleaved (Atf6-p50), and it translocates to the nucleus where it increases the transcription of Tbx20. Increase in the level of Tbx20 results in upregulation of its downstream signaling cascade consisting of bone morphogenetic protein 2 (Bmp2)–pSmad1/5/8. Increased expression of Bmp2–pSmad1/5/8 further results in increased cardiomyocyte proliferation, thereby restoring homeostasis and increasing the survival of cardiomyocyte post ER stress induction. Tbx20 also helps in restoration of homeostasis by decreasing apoptosis, fibrosis, and reactive oxygen levels (ROS) levels during ER stress. Increased expression of Tbx20 during ER stress also helps to maintain the total pool of Atf6 during ER stress. A prolonged ER stress however results in decreased expression of cleaved Atf6 (Atf6-p50). This in turn results in decrease in the expression of Tbx20, and it downstreams Bmp2–pSmad1/5/8 signaling molecules resulting in decreased cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Ectopic administration of recombinant Bmp2 protein results in upregulation of Bmp2, which in turn upregulates Tbx20 in a feed-forward mechanism, thereby restoring homeostasis by increasing cardiomyocyte proliferation.

Fig 3: Prolonged endoplasmic reticulum (ER) stress in adult murine heart results in altered cardiac function with increased cardiomyocyte size and collagen deposition in adult murine heart.A, the heart weight to body weight ratios indicative of cardiac hypertrophy is increased significantly during prolonged (2 days) ER stress as compared with ER stress induction for short duration (8 h) and control group. The change in heart weight to body weight ratio between 8 h ER stress and control group was however negligible (n = 9). Scale bar represents 20 µm. B, quantitative RT–PCR (qRT–PCR) analysis showed that prolonged (2 days) ER stress induction resulted in significant increase in the expression of cardiac function test markers bnp and ß-mhc and significant decrease in the expression of serca2 as compared with 8 h ER stress induction group. C, prolonged (2 days) ER stress results in increased collagen deposition indicative of cardiac fibrosis as shown in Masson’s trichrome-stained adult heart sections as compared with 8 h ER stress group and control group (n = 6). D, in prolonged (2 days) ER stress–induced adult mice, cardiomyocyte cell size is increased marked by wheat-germ agglutinin (WGA) staining (green) in comparison to 8 h ER stress group and control group, respectively (n = 6). E, Western blot analysis of Tbx20 showed a significant increase in its expression in 8 h Tun-treated group as compared with control. The expression of Tbx20 later decreased significantly in the 2-day group. The expression of Bmp2 increased significantly during 2 days as compared with 8 h Tun-treated group. The expression of apoptotic marker Chop increased significantly in the 2-day group as compared with 8 h and control group. F, quantitative representation by ImageJ software of the proteins using three biological replicates from E. Scale bar represents 50 µm. Error bars represent SD from at least three independent biological replicates. Statistical significance was calculated by one-way ANOVA. ns, p: nonsignificant, *p < 0.05, **p < 0.005, ***p < 0.0005, ##p < 0.0001; n = 3 independent experiments. Tbx20, T-box transcription factor 20.

Fig 4: Tbx20–Bmp2 signaling acts in a feed-forward loop mechanism to protect cultured H9c2 cardiomyocytes against endoplasmic reticulum (ER) stress.A, Western blot analysis of H9c2 cells treated with 50 µg/ml tunicamycin (Tun) followed by administration of recombinant Bmp2 (RecBmp2) showed significant increase in the expression of Bmp2 even during increased ER stress. Administration of RecBmp2 protein following ER stress induction resulted in significant increase in the expression of Tbx20 as compared with 50 µg/ml Tun-treated group. Treatment of the ER-stressed cells with RecBmp2 also resulted in decrease in the expression of apoptotic marker Chop. B, quantitative representation by ImageJ software of the proteins using three biological replicates from A. C, immunofluorescence staining showed that RecBmp2 treatment following ER stress induction (50 µg/ml Tun) resulted in increase in the expression of Bmp2 (a’’) and Tbx20 (b’’) in comparison to 50 µg/ml Tun treatment alone (a’ and b’) and control cells (a and b), respectively. Increase in Bmp2 expression is accompanied by increased expression of Ki67 (c’’) and decreased expression of Chop (d’’) as compared with 50 µg/ml Tun treatment alone (c’ and d’) and control cells (c and d), respectively. D, quantitative representation of panels in C. E, Western blot analysis of H9c2 cells treated with Bmp2 inhibitor Noggin followed by ER stress induction (10 µg/ml Tun) caused significant decrease in the expression of Bmp2 as compared with ER stress induction group alone. Treatment with Noggin followed by ER stress induction however caused no significant change in the expression of Tbx20 from ER stress induction-alone group. F, quantitative representation by ImageJ software of the proteins using three biological replicates from E. G, immunostaining of H9c2 cells treated with Bmp2 inhibitor Noggin followed by Tun treatment (10 µg/ml Tun) resulted in decrease in the expression of Bmp2 (a’’) with reference to 10 µg/ml Tun treatment alone (a’) and control cells (a). Noggin treatment followed by ER stress induction caused no significant change in the expression of Tbx20 (b’’) as compared with ER stress induction alone (b’). Noggin administration followed by Tun treatment resulted in significant decrease in the expression of proliferative marker Ki67 (c’’) as compared with Tun treatment alone group (c’). The expression of apoptosis inducer Chop however remained unchanged between Noggin administered group (d’’) as compared with Tun treatment alone (d’). H, quantitative representation of panels in G. Scale bar represents 50 µm. Statistical significance was calculated by one-way ANOVA. Error bars represent SD from three independent biological replicates (n = 3); ns, p: nonsignificant, *p < 0.05, **p < 0.005, ***p < 0.0005, ##p < 0.0001. Bmp2, bone morphogenetic protein 2; Tbx20, T-box transcription factor 20.

Fig 5: During endoplasmic reticulum (ER) stress, activating transcription factor 6 (Atf6)-mediated upregulation of T-box transcription factor 20 (Tbx20) promotes cardiomyocyte proliferation and limits cardiomyocyte apoptosis in cultured H9c2 cardiomyocytes.A, the rat tbx20 gene contains conserved canonical Atf6 DNA-binding sequence TGACGTG in the tbx20 promoter region. B, chromatin immunoprecipitation (ChIP) analysis showed direct binding of Atf6 in tbx20 promoter region. C, ChIP assay followed by quantitative RT–PCR (qRT–PCR) showed ~19-fold enrichment of Atf6 binding to the tbx20 promoter region during ER stress induction. D, Western blot analysis revealed pretreatment of H9c2 cardiomyocytes with 300 µM AEBSF followed by ER stress induction resulted in significant decrease in the expression of Atf6-p50. Inhibition of nuclear translocation of Atf6 was accompanied by concomitant decrease in the expression of Tbx20. E, quantitative representation by ImageJ software of the proteins using three biological replicates from D. F, level of ROS generation upon induction of ER stress showed a gradual increase in the level of ROS production by DCFDA method. Decrease in the expression of Tbx20 in 50 µg/ml tunicamycin (Tun)-treated cells was accompanied by significant rise in ROS levels as compared with 20 µg/ml Tun-treated cells. G, immunofluorescence staining revealed that increase in expression of Tbx20 during ER stress is accompanied with increased expression of proliferative marker Ki67 (a’’, a’’’, and a’’’’) as compared with control (a). However, a Tun concentration of 50 µg/ml resulted in decrease in cardiomyocyte proliferation marked by decreased expression of Ki67 (a’’’’’). Decrease in expression of Tbx20 is accompanied by increased cardiomyocyte apoptosis. The expression of apoptosis inducer Chop and proapoptotic marker Bax is increased upon 50 µg/ml Tun treatment (b’’’’’ and c’’’’’) in comparison to lower concentrations of Tun (b’, b’’, b’’’, and b’’’’) and (c’, c’’, c’’’, and c’’’’). The expression of antiapoptotic marker BclXL is decreased during 50 µg/ml Tun treatment (d’’’’’) in comparison to lower concentrations of Tun (d’, d’’, d’’’, and d’’’’). Insets in G show single-channel cropped images of indicated areas (white arrows). Scale bar of main images represents 50 µm. Scale bar of inset represents 20 µm. H–K, quantitative representation of G. Statistical significance was calculated by one-way ANOVA. Error bars represent SD from n = 3 independent biological replicates. ns, p: nonsignificant, *p < 0.05, **p < 0.005, ***p < 0.0005, ##p < 0.0001. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; DCFDA, 2',7'-dichlorofluorescin diacetate; ROS, reactive oxygen species.