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A small molecule activator of AKT does not reduce ischemic injury of the rat heart.

Moreira JB, Wohlwend M, Alves MN, Wisløff U, Bye A - J Transl Med (2015)

Bottom Line: Similarly, mitochondrial enzyme activity was not affected by SC79.Finally, SC79 failed to reduce infarct size or release of cardiac injury biomarkers at reperfusion.We conclude that selective AKT activation by the synthetic molecule SC79 does not protect the rat heart against ischemic injury, indicating that acute pharmacological activation of AKT is not sufficient for cardioprotection.

View Article: PubMed Central - PubMed

Affiliation: K.G. Jebsen Center of Exercise in Medicine, Department of Circulation and Medical Imaging, St. Olavs Hospital, Norwegian University of Science and Technology (NTNU), Prinsesse Kristinas gt. 3, 7006, Trondheim, Norway. jose.moreira@ntnu.no.

ABSTRACT

Background: Activation of protein kinase AKT is required for cardioprotection by ischemic preconditioning, and transgenic overexpression of AKT protects the heart against ischemia. However, it is unknown whether acute pharmacological activation of AKT alone, using a therapeutically relevant strategy, induces cardioprotection. In this study we provide the first evidence to clarify this question.

Methods: We used a recently described specific activator of AKT, the small molecule SC79, to treat rat hearts submitted to ischemia and reperfusion. Initially, isolated rat hearts were perfused with increasing doses of SC79 to verify the magnitude of AKT activation. Low and high doses were determined and used to treat hearts submitted to ischemia (35 minutes) and reperfusion (60 minutes), in a randomized and blinded design. AKT activation was verified by western immunobloting. Metabolic profile was determined by cardiac ATP content and mitochondrial enzyme activity, while cytosolic levels of cytochrome C and caspase-3 activity were used as markers of apoptosis. Ischemic injury was assessed by quantification of infarct size and cardiac release of creatine kinase and lactate dehydrogenase.

Results: SC79 activated cardiac AKT within 30 minutes in a dose-dependent fashion. ATP content was largely reduced by ischemia, but was not rescued by SC79. Similarly, mitochondrial enzyme activity was not affected by SC79. SC79 administered before ischemia or at reperfusion did not prevent cytosolic accumulation of cytochrome C and overactivation of caspase-3. Finally, SC79 failed to reduce infarct size or release of cardiac injury biomarkers at reperfusion.

Conclusion: We conclude that selective AKT activation by the synthetic molecule SC79 does not protect the rat heart against ischemic injury, indicating that acute pharmacological activation of AKT is not sufficient for cardioprotection.

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Related in: MedlinePlus

AKT activation in ischemic hearts.A: Illustrative representation of all experimental groups. “IR”, Ischemia-reperfusion. “Pre”, SC79 administered before ischemia. “Post”, SC79 administered at reperfusion. “Both”, SC79 administered before ischemia and at reperfusion. “Low” indicates 100nM SC79 and “High” indicates 300nM SC79. B: AKT phosphorylation in normoxic-perfused (N) or ischemic hearts (IR) treated with DMSO or SC79. C: Total AKT expression; D: Correlation between AKT phosphorylation and GSK-3β phosphorylation. E: Correlation between AKT phosphorylation and Ribosomal-S6 phosphorylation. The Pearson correlation test was used to analyze the correlations shown in panels D and E. Number of animals analyzed per group is displayed within the bars. Western blot bands displayed in panels D and E are disposed in the same group order as in panels B and C. Data are shown as mean ± SE. #, p < 0.05 vs. IR-Control; &, p < 0.05 vs. all other groups.
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Fig2: AKT activation in ischemic hearts.A: Illustrative representation of all experimental groups. “IR”, Ischemia-reperfusion. “Pre”, SC79 administered before ischemia. “Post”, SC79 administered at reperfusion. “Both”, SC79 administered before ischemia and at reperfusion. “Low” indicates 100nM SC79 and “High” indicates 300nM SC79. B: AKT phosphorylation in normoxic-perfused (N) or ischemic hearts (IR) treated with DMSO or SC79. C: Total AKT expression; D: Correlation between AKT phosphorylation and GSK-3β phosphorylation. E: Correlation between AKT phosphorylation and Ribosomal-S6 phosphorylation. The Pearson correlation test was used to analyze the correlations shown in panels D and E. Number of animals analyzed per group is displayed within the bars. Western blot bands displayed in panels D and E are disposed in the same group order as in panels B and C. Data are shown as mean ± SE. #, p < 0.05 vs. IR-Control; &, p < 0.05 vs. all other groups.

Mentions: Low- and high-activating doses of SC79 were used to treat hearts submitted to ischemia and reperfusion (IR). Treatments were performed either before ischemia, at reperfusion or both. Same concentration of DMSO (0.01%) was added to the buffer of normoxic perfused hearts and ischemic control hearts. Experimental groups are illustrated in Figure 2A and described as: (group “N”) normoxic hearts, perfused continuously for 125 min; (IR-Control) ischemic DMSO-treated control group, hearts perfused for 30 min with normoxic buffer, followed by 35 min of no-flow ischemia (37°C) and 60 min reperfusion; (IR-Low-Pre) hearts received low-dose SC79 for 30 min before ischemia; (IR-Low-Post) hearts received low-dose SC79 for the initial 30 min of reperfusion; (IR-Low-Both) hearts received low-dose SC79 before ischemia and at reperfusion; (IR-High-Pre) hearts received high-dose SC79 for 30 min before ischemia; (IR-High-Post) hearts received high-dose SC79 for the initial 30 min of reperfusion; (IR-High-Both) hearts received high-dose SC79 before ischemia and at reperfusion. Each group consisted of 11 rats (see “Statistical analysis” for details on power calculation). Experiments were conducted in a randomized and blinded fashion. DMSO and SC79 were aliquoted before the start of the study, when the aliquots received unique five-digit codes and were kept frozen until the experiment. Each code was randomly assigned to one rat. The identity of the vials was controlled by two colleagues not involved in the study, and was revealed to the investigators after data were collected from all animals. No animal was arbitrarily excluded from the study.Figure 2


A small molecule activator of AKT does not reduce ischemic injury of the rat heart.

Moreira JB, Wohlwend M, Alves MN, Wisløff U, Bye A - J Transl Med (2015)

AKT activation in ischemic hearts.A: Illustrative representation of all experimental groups. “IR”, Ischemia-reperfusion. “Pre”, SC79 administered before ischemia. “Post”, SC79 administered at reperfusion. “Both”, SC79 administered before ischemia and at reperfusion. “Low” indicates 100nM SC79 and “High” indicates 300nM SC79. B: AKT phosphorylation in normoxic-perfused (N) or ischemic hearts (IR) treated with DMSO or SC79. C: Total AKT expression; D: Correlation between AKT phosphorylation and GSK-3β phosphorylation. E: Correlation between AKT phosphorylation and Ribosomal-S6 phosphorylation. The Pearson correlation test was used to analyze the correlations shown in panels D and E. Number of animals analyzed per group is displayed within the bars. Western blot bands displayed in panels D and E are disposed in the same group order as in panels B and C. Data are shown as mean ± SE. #, p < 0.05 vs. IR-Control; &, p < 0.05 vs. all other groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4352273&req=5

Fig2: AKT activation in ischemic hearts.A: Illustrative representation of all experimental groups. “IR”, Ischemia-reperfusion. “Pre”, SC79 administered before ischemia. “Post”, SC79 administered at reperfusion. “Both”, SC79 administered before ischemia and at reperfusion. “Low” indicates 100nM SC79 and “High” indicates 300nM SC79. B: AKT phosphorylation in normoxic-perfused (N) or ischemic hearts (IR) treated with DMSO or SC79. C: Total AKT expression; D: Correlation between AKT phosphorylation and GSK-3β phosphorylation. E: Correlation between AKT phosphorylation and Ribosomal-S6 phosphorylation. The Pearson correlation test was used to analyze the correlations shown in panels D and E. Number of animals analyzed per group is displayed within the bars. Western blot bands displayed in panels D and E are disposed in the same group order as in panels B and C. Data are shown as mean ± SE. #, p < 0.05 vs. IR-Control; &, p < 0.05 vs. all other groups.
Mentions: Low- and high-activating doses of SC79 were used to treat hearts submitted to ischemia and reperfusion (IR). Treatments were performed either before ischemia, at reperfusion or both. Same concentration of DMSO (0.01%) was added to the buffer of normoxic perfused hearts and ischemic control hearts. Experimental groups are illustrated in Figure 2A and described as: (group “N”) normoxic hearts, perfused continuously for 125 min; (IR-Control) ischemic DMSO-treated control group, hearts perfused for 30 min with normoxic buffer, followed by 35 min of no-flow ischemia (37°C) and 60 min reperfusion; (IR-Low-Pre) hearts received low-dose SC79 for 30 min before ischemia; (IR-Low-Post) hearts received low-dose SC79 for the initial 30 min of reperfusion; (IR-Low-Both) hearts received low-dose SC79 before ischemia and at reperfusion; (IR-High-Pre) hearts received high-dose SC79 for 30 min before ischemia; (IR-High-Post) hearts received high-dose SC79 for the initial 30 min of reperfusion; (IR-High-Both) hearts received high-dose SC79 before ischemia and at reperfusion. Each group consisted of 11 rats (see “Statistical analysis” for details on power calculation). Experiments were conducted in a randomized and blinded fashion. DMSO and SC79 were aliquoted before the start of the study, when the aliquots received unique five-digit codes and were kept frozen until the experiment. Each code was randomly assigned to one rat. The identity of the vials was controlled by two colleagues not involved in the study, and was revealed to the investigators after data were collected from all animals. No animal was arbitrarily excluded from the study.Figure 2

Bottom Line: Similarly, mitochondrial enzyme activity was not affected by SC79.Finally, SC79 failed to reduce infarct size or release of cardiac injury biomarkers at reperfusion.We conclude that selective AKT activation by the synthetic molecule SC79 does not protect the rat heart against ischemic injury, indicating that acute pharmacological activation of AKT is not sufficient for cardioprotection.

View Article: PubMed Central - PubMed

Affiliation: K.G. Jebsen Center of Exercise in Medicine, Department of Circulation and Medical Imaging, St. Olavs Hospital, Norwegian University of Science and Technology (NTNU), Prinsesse Kristinas gt. 3, 7006, Trondheim, Norway. jose.moreira@ntnu.no.

ABSTRACT

Background: Activation of protein kinase AKT is required for cardioprotection by ischemic preconditioning, and transgenic overexpression of AKT protects the heart against ischemia. However, it is unknown whether acute pharmacological activation of AKT alone, using a therapeutically relevant strategy, induces cardioprotection. In this study we provide the first evidence to clarify this question.

Methods: We used a recently described specific activator of AKT, the small molecule SC79, to treat rat hearts submitted to ischemia and reperfusion. Initially, isolated rat hearts were perfused with increasing doses of SC79 to verify the magnitude of AKT activation. Low and high doses were determined and used to treat hearts submitted to ischemia (35 minutes) and reperfusion (60 minutes), in a randomized and blinded design. AKT activation was verified by western immunobloting. Metabolic profile was determined by cardiac ATP content and mitochondrial enzyme activity, while cytosolic levels of cytochrome C and caspase-3 activity were used as markers of apoptosis. Ischemic injury was assessed by quantification of infarct size and cardiac release of creatine kinase and lactate dehydrogenase.

Results: SC79 activated cardiac AKT within 30 minutes in a dose-dependent fashion. ATP content was largely reduced by ischemia, but was not rescued by SC79. Similarly, mitochondrial enzyme activity was not affected by SC79. SC79 administered before ischemia or at reperfusion did not prevent cytosolic accumulation of cytochrome C and overactivation of caspase-3. Finally, SC79 failed to reduce infarct size or release of cardiac injury biomarkers at reperfusion.

Conclusion: We conclude that selective AKT activation by the synthetic molecule SC79 does not protect the rat heart against ischemic injury, indicating that acute pharmacological activation of AKT is not sufficient for cardioprotection.

Show MeSH
Related in: MedlinePlus