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Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes.

Neuber C, Uebeler J, Schulze T, Sotoud H, El-Armouche A, Eschenhagen T - PLoS ONE (2014)

Bottom Line: During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, α2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in prolonged eIF2α phosphorylation, inhibition of protein synthesis and protection from ER stress.We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2α, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz.The data confirm that guanabenz interferes with ER stress-signalling and has protective effects on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Center for Cardiovascular Research), partner site, Hamburg/Kiel/Luebeck, Germany.

ABSTRACT
Endoplasmic reticulum (ER) stress has been implicated in a variety of cardiovascular diseases. During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, α2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in prolonged eIF2α phosphorylation, inhibition of protein synthesis and protection from ER stress. In this study we assessed whether guanabenz protects against ER stress in cardiac myocytes and affects the function of 3 dimensional engineered heart tissue (EHT). We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2α, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz. The data confirm that guanabenz interferes with ER stress-signalling and has protective effects on cell survival. Data show for the first time that this concept extends to cardiac myocytes. The modest protection in EHTs points to more complex mechanisms of force regulation in intact functional heart muscle.

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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.
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pone-0098893-g001: 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.

Mentions: To induce ER stress in neonatal rat cardiomyocytes (NRCM) we treated cells with increasing concentrations of tunicamycin (0.1–2.5 µg/ml) and measured cell survival after 24 hours. As expected, tunicamycin reduced cell viability in a concentration-dependent manner (Figure 1A), whereas guanabenz (0.5–50 µM) did not affect cell survival (Figure 1B). Additionally, we measured the effect of the treatment on different branches of UPR on mRNA and protein level. Tunicamycin strongly upregulated mRNA concentrations of the ER-chaperone GRP78 and activated the PERK pathway, which is reflected by the increase of the typical UPR targets ATF4, GADD34 and CHOP in a concentration- and time-dependent manner (Figure 2A and B). Accordingly, protein amounts of GRP78, ATF4 and CHOP were elevated as well (Figure 3 left panel). Increased eIF2α phosphorylation (Figure 3 left panel) indicated inhibiton of the translational machinery. Besides activation of the PERK pathway, we could detect activation of a second UPR pathway that is initiated by IRE1α resulting in concomitant elevation of its protein amount (Figure 3 left panel). The third UPR pathway that is reflected by initiation of ATF6 was not activated by tunicamycin (data not shown).


Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes.

Neuber C, Uebeler J, Schulze T, Sotoud H, El-Armouche A, Eschenhagen T - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4044035&req=5

pone-0098893-g001: 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.
Mentions: To induce ER stress in neonatal rat cardiomyocytes (NRCM) we treated cells with increasing concentrations of tunicamycin (0.1–2.5 µg/ml) and measured cell survival after 24 hours. As expected, tunicamycin reduced cell viability in a concentration-dependent manner (Figure 1A), whereas guanabenz (0.5–50 µM) did not affect cell survival (Figure 1B). Additionally, we measured the effect of the treatment on different branches of UPR on mRNA and protein level. Tunicamycin strongly upregulated mRNA concentrations of the ER-chaperone GRP78 and activated the PERK pathway, which is reflected by the increase of the typical UPR targets ATF4, GADD34 and CHOP in a concentration- and time-dependent manner (Figure 2A and B). Accordingly, protein amounts of GRP78, ATF4 and CHOP were elevated as well (Figure 3 left panel). Increased eIF2α phosphorylation (Figure 3 left panel) indicated inhibiton of the translational machinery. Besides activation of the PERK pathway, we could detect activation of a second UPR pathway that is initiated by IRE1α resulting in concomitant elevation of its protein amount (Figure 3 left panel). The third UPR pathway that is reflected by initiation of ATF6 was not activated by tunicamycin (data not shown).

Bottom Line: During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, α2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in prolonged eIF2α phosphorylation, inhibition of protein synthesis and protection from ER stress.We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2α, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz.The data confirm that guanabenz interferes with ER stress-signalling and has protective effects on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Center for Cardiovascular Research), partner site, Hamburg/Kiel/Luebeck, Germany.

ABSTRACT
Endoplasmic reticulum (ER) stress has been implicated in a variety of cardiovascular diseases. During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, α2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in prolonged eIF2α phosphorylation, inhibition of protein synthesis and protection from ER stress. In this study we assessed whether guanabenz protects against ER stress in cardiac myocytes and affects the function of 3 dimensional engineered heart tissue (EHT). We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2α, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz. The data confirm that guanabenz interferes with ER stress-signalling and has protective effects on cell survival. Data show for the first time that this concept extends to cardiac myocytes. The modest protection in EHTs points to more complex mechanisms of force regulation in intact functional heart muscle.

Show MeSH
Related in: MedlinePlus