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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

SC79 activates cardiac AKT.A: Dose–response experiment in normoxic perfused hearts treated with DMSO or SC79. SC79 activates AKT in a dose-dependent fashion, as demonstrated by phosphorylation at serine 473. B: Total AKT expression is not changed by SC79. C: Phosphorylation of GSK-3β at serine 9 (AKT-phosphorylated site). D: Total expression of GSK-3β. E: ATP levels are preserved in SC79-perfused hearts. F: LDH activity was not detected in SC79-treated hearts. Perfusion buffer from hearts submitted to ischemia-reperfusion (IR) were used as positive control for the LDH activity experiment. “ua/min”, units of absorbance per minute. Data are shown as mean ± standard error (SE). *, p < 0.05 vs. DMSO; &, p < 0.05 vs. all other groups.
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Fig1: SC79 activates cardiac AKT.A: Dose–response experiment in normoxic perfused hearts treated with DMSO or SC79. SC79 activates AKT in a dose-dependent fashion, as demonstrated by phosphorylation at serine 473. B: Total AKT expression is not changed by SC79. C: Phosphorylation of GSK-3β at serine 9 (AKT-phosphorylated site). D: Total expression of GSK-3β. E: ATP levels are preserved in SC79-perfused hearts. F: LDH activity was not detected in SC79-treated hearts. Perfusion buffer from hearts submitted to ischemia-reperfusion (IR) were used as positive control for the LDH activity experiment. “ua/min”, units of absorbance per minute. Data are shown as mean ± standard error (SE). *, p < 0.05 vs. DMSO; &, p < 0.05 vs. all other groups.

Mentions: The small molecule activator of AKT (referred to as SC79) was purchased from Tocris Biosciences (product #4635). For dose–response experiments, hearts were perfused with increasing doses of SC79 for 30 min, in a randomized fashion. The same concentration of DMSO (vehicle, 0.01%) was administered to the control group. The investigators conducting the experiments (J.B.N.M. and M.W.) were blinded to treatment assignment (DMSO or SC79). After perfusion, hearts were removed from the rig and snap-frozen in liquid nitrogen. A sample from the left ventricle was used to determine AKT activation by western immunobloting (protocol below). 100nM was adopted as low-activating dose and 300nM was adopted as high-activating dose (Figure 1A).Figure 1


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)

SC79 activates cardiac AKT.A: Dose–response experiment in normoxic perfused hearts treated with DMSO or SC79. SC79 activates AKT in a dose-dependent fashion, as demonstrated by phosphorylation at serine 473. B: Total AKT expression is not changed by SC79. C: Phosphorylation of GSK-3β at serine 9 (AKT-phosphorylated site). D: Total expression of GSK-3β. E: ATP levels are preserved in SC79-perfused hearts. F: LDH activity was not detected in SC79-treated hearts. Perfusion buffer from hearts submitted to ischemia-reperfusion (IR) were used as positive control for the LDH activity experiment. “ua/min”, units of absorbance per minute. Data are shown as mean ± standard error (SE). *, p < 0.05 vs. DMSO; &, p < 0.05 vs. all other groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: SC79 activates cardiac AKT.A: Dose–response experiment in normoxic perfused hearts treated with DMSO or SC79. SC79 activates AKT in a dose-dependent fashion, as demonstrated by phosphorylation at serine 473. B: Total AKT expression is not changed by SC79. C: Phosphorylation of GSK-3β at serine 9 (AKT-phosphorylated site). D: Total expression of GSK-3β. E: ATP levels are preserved in SC79-perfused hearts. F: LDH activity was not detected in SC79-treated hearts. Perfusion buffer from hearts submitted to ischemia-reperfusion (IR) were used as positive control for the LDH activity experiment. “ua/min”, units of absorbance per minute. Data are shown as mean ± standard error (SE). *, p < 0.05 vs. DMSO; &, p < 0.05 vs. all other groups.
Mentions: The small molecule activator of AKT (referred to as SC79) was purchased from Tocris Biosciences (product #4635). For dose–response experiments, hearts were perfused with increasing doses of SC79 for 30 min, in a randomized fashion. The same concentration of DMSO (vehicle, 0.01%) was administered to the control group. The investigators conducting the experiments (J.B.N.M. and M.W.) were blinded to treatment assignment (DMSO or SC79). After perfusion, hearts were removed from the rig and snap-frozen in liquid nitrogen. A sample from the left ventricle was used to determine AKT activation by western immunobloting (protocol below). 100nM was adopted as low-activating dose and 300nM was adopted as high-activating dose (Figure 1A).Figure 1

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