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Heme oxygenase 1 induction protects myocardiac cells against hypoxia/reoxygenation-induced apoptosis

The role of JNK/c-Jun/Caspase-3 inhibition and Akt signaling enhancement

Hämoxygenase-1-Induktion schützt Myokardzellen gegen durch Hypoxie/Reoxygenierung induzierte Apoptose

Bedeutung der JNK/c-Jun/Caspase-3-Inhibition und Akt-Signalverstärkung

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Abstract

Background

Although recent studies have found that heme oxygenase (HO)-1 plays an important role in myocardiac cell survival, the precise mechanisms occurring during hypoxia/reoxygenation (H/R) injury remain unclear. Therefore, the aim of this study was to investigate the cytoprotective mechanisms of HO-1 against H/R-induced myocardiac cell apoptosis and to explore whether the Akt signaling pathway contributed to the protection provided by HO-1.

Methods

Cobalt protoporphyrin (CoPP, a pharmacologic inducer of HO-1) was employed to induce the over-expression of HO-1 before H/R in H9c2 cells. Hoechst staining and flow cytometry were used to examine the extent of apoptosis. Furthermore, the effect of HO-1 on Akt, JNK, and the expression of apoptosis-related proteins (c-JUN and Caspase-3) was determined by Western blotting.

Results

The results showed that over-expressed HO-1 could significantly protect myocardiac cells against H/R-induced apoptosis in H9c2 cells. Furthermore, the protein expression of p‑Akt increased and of p‑JNK decreased in the H/R injury H9c2 cells when treated with CoPP. The apoptosis-related proteins c‑Jun and caspase-3 were both inhibited by over-expression of HO-1. At the same time, retreatment with zinc protoporphyrin (ZnPP, a specific inhibitor of HO-1 enzymatic activity) or LY294002 (an inhibitor of Akt1) reversed the HO-1-induced changes.

Conclusion

In summary, our results suggest that HO-1 can decrease H/R-induced myocardiac cell apoptosis; the mechanism may be related to the activation of the Akt signaling pathway and, furthermore, to the inhibition of the JNK/c-Jun/caspase-3 signaling pathway.

Zusammenfassung

Hintergrund

Aktuellen Studien zufolge spielt die Hämoxygenase(HO)-1 zwar eine wichtige Rolle für das Überleben von Myokardzellen, aber die genauen Abläufe bei einer Schädigung durch Hypoxie/Reoxygenierung (H/R) sind nicht geklärt. Daher war es Ziel der vorliegenden Studie, die zytoprotektiven Wirkungen von HO-1 gegen H/R-induzierte Myokardzellapoptose zu untersuchen und herauszufinden, ob der Akt-Signalweg zum HO-1-vermittelten Schutz beiträgt.

Methoden

Kobaltprotoprophyrin (CoPP, ein pharmakologischer Induktor von HO-1) wurde zur Induktion der Überexpression von HO-1 vor H/R in H9c2-Zellen eingesetzt. Mittels Hoechst-Färbung und Durchflusszytometrie wurde das Ausmaß der Apoptose ermittelt. Außerdem wurde die Wirkung von HO-1 auf Akt, JNK und die Expression von Apoptoseproteinen (c-JUN und Caspase-3) mit dem Western-Blot-Verfahren untersucht.

Ergebnisse

Die Ergebnisse zeigten, dass überexprimierte HO-1 in signifikanter Weise Myokardzellen gegen H/R-induzierte Apoptose bei H9c2-Zellen schützen konnte. Darüber hinaus kam es bei H9c2-Zellen mit H/R-Schädigung zur Zunahme der Proteinexpression von p‑Akt und zur Abnahme der Proteinexpression von p‑JNK, wenn sie mit CoPP behandelt wurden. Die Apoptoseproteine c‑Jun und Caspase-3 wurden beide durch Überexpression von HO-1 inhibiert. Gleichzeitig wurden die HO-1-induzierten Veränderungen durch Behandlung mit Zinkprotoporphyrin (ZnPP, ein spezifischer Inhibitor der HO-1-Enzymaktivität) oder LY294002 (ein Inhibitor von Akt1) aufgehoben.

Schlussfolgerung

Zusammenfassend bedeuten die hier vorliegenden Ergebnisse, dass HO-1 die H/R-induzierte Myokardzellapopotose vermindern kann und dieser Vorgang möglicherweise mit der Aktivierung des Akt-Signalwegs sowie außerdem mit der Inhibition des JNK/c-Jun/Caspase-3-Signalwegs in Zusammenhang steht.

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Funding

This work was supported by the National Natural Science Foundation of China (81500977).

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Authors and Affiliations

  1. Department of Cardiovascular Medicine, The Zoucheng People’s Hospital, 59 Qianquan Road, 273500, Jining, Shandong, P.R. China

    C. Li, C. Zhang, T. Wang, J. Xuan, C. Su & Y. Wang

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  1. C. Li
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  2. C. Zhang
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  3. T. Wang
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  4. J. Xuan
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  5. C. Su
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  6. Y. Wang
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Correspondence to C. Li.

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Conflict of interest

C. Li, C. Zhang, T. Wang, J. Xuan, C. Su, and Y. Wang state that there are no conflicts of interest.

The accompanying manuscript does not include studies on humans or animals.

Additional information

The authors contributed equally to this work. In detail, C.L. designed the study. T.W., C.Z., J.X., and Y.W. performed the experiments and collected the data. C.L. and T.W. analyzed and interpreted the experimental data. C.L. and T.W. prepared the manuscript.

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Li, C., Zhang, C., Wang, T. et al. Heme oxygenase 1 induction protects myocardiac cells against hypoxia/reoxygenation-induced apoptosis. Herz 41, 715–724 (2016). https://doi.org/10.1007/s00059-016-4424-6

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  • DOI: https://doi.org/10.1007/s00059-016-4424-6

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