Figure 1.
Effects of tunicamycin and guanabenz on cell viability of neonatal rat cardiomyocytes (NRCM).
(A) Concentration-dependent reduction of cell viability by tunicamycin treatment for 24 hours. (B) Treatment with increasing concentrations of guanabenz for 24 hours did not affect cell viability. Data are means ± SEM (n = 8); ***P<0.0001.
Figure 2.
Effects of tunicamycin and guanabenz on different branches of the UPR in NRCM on the mRNA level.
(A) Tunicamycin (Tm) treatment of NRCM for 12 hours increased levels of the UPR targets GRP78, GADD34, ATF4, and CHOP. (B) Time-dependent increase of UPR targets after treatment with tunicamycin (2.5 µg/ml). (C) Guanabenz (Ga) treatment of NRCM for 24 hours did not affect levels of UPR targets. Data are means ± SEM (n = 3–5); Changes of mRNA expression of ≥1.6 or ≤0.5 compared to DMSO control and a corrected P-value<0.05 were considered significant.
Figure 3.
Effects of tunicamycin and guanabenz on different branches of the UPR in NRCM on the protein level.
Upper panel – representative immunoblots of lysates of NRCMs treated with tunicamycin (left) or guanabenz (right) for 12 and 24 hours, respectively. Lower panel – concentration-dependent increase of UPR targets proteins after treatment with tunicamycin (Tm) or guanabenz (Ga) compared to DMSO control. Please note that the intensity of the upper unspecific band for GRP78 is caused by differences in blotting conditions (10% acrylamide/bisacrylamide gel). Data are means ± SEM (n = 3); *P<0.05.
Figure 4.
Representative immunoblots of ER stress marker proteins related to the loading control calsequestrin (CSQ) in lysates of NRCM treated with tunicamycin (Tm; 2.5 µg/ml) in the presence or absence of guanabenz (Ga; 2.5 µM) for the indicated times (0, 6, 12, 24 and 36 h).
Bar graphs show densitometric and statistical analysis of the immunoblots. Data are means ± SEM (n = 6); *P<0.05 compared to the respective time points.
Figure 5.
Guanabenz protected NRCM from deleterious endoplasmic reticulum stress.
Values are normalized to vehicle control (100%). Data are means ± SEM. (A) Assessment of cell viability after treatment with tunicamycin (Tm; 2.5 µg/ml) and indicated concentrations of guanabenz for 10 h. (n = 8); **P<0.001; ***P<0.0001. (B) Protective effect of guanabenz (Ga; 2.5 µM) in cells treated with tunicamycin (0.1 µg/ml) for 12, 24, and 54 hours. (n = 4); ***P<0.0001. (C) Concentration-dependent protection by guanabenz against cellular stress induced by exposure to H2O2 (80 µM) for 4 hours (n = 7–14); **P<0.001; ***P<0.0001. Cell viability of NRCM treated with doxorubicin (Dox; 100 nM) for 48 hours (D) or with thapsigargin (Tg; 100 nM) for 72 hours (E), with or without indicated concentrations of guanabenz. (n = 6–7); *P<0.05 **P<0.001.
Figure 6.
Impact of guanabenz on EHT contractility during exposure to ER stress.
(A) Force development of EHTs after treatment with tunicamycin (Tm; 2.5 µg/ml) for 12, 24, 36 and 48 hours, with or without guanabenz (Ga; 2.5 µM). Pre-drug values Tm: 0.17±0.01 vs. Ga: 0.17±0.02. (B) Force development of EHTs with or without guanabenz (2.5 µM) during 30 min acute exposure to H2O2 (600 µM) and during following 20 hours recovery time. Pre-drug values Tm: 0.16±0.01 vs. Ga: 0.16±0.01. (C) Representative beating pattern of an EHT treated with thapsigargin (Tg; 500 nM) for 1 hour, with or without guanabenz (3 µM, 10 µM) compared to an untreated control EHT. (D) Force development of EHTs after treatment with doxorubicin (Dox; 100 nM) for 12, 24 and 48 hours, with or without guanabenz (2.5 µM). Pre-drug values Tm: 0.15±0.03 vs. Ga: 0.16±0.02. Data are means ± SEM (n = 4–6). All drugs showed a time-dependent effect on EHT contractility (Two-way ANOVA, row factor, P<0.05), but differences between the groups were not statistically significant.