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Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia–reperfusion in vivo

  • Martinus I. F. J. Oerlemans
  • Jia Liu
  • Fatih Arslan
  • Krista den Ouden
  • Ben J. van Middelaar
  • Pieter A. Doevendans
  • Joost P. G. SluijterEmail author
Original Contribution

Abstract

Accumulating evidence indicatesthat programmed necrosis plays a critical role in cell death during ischemia–reperfusion. Necrostatin-1 (Nec-1), a small molecule capable of inhibiting a key regulator of programmed necrosis (RIP1), was shown to prevent necrotic cell death in experimental models including cardiac ischemia. However, no functional follow-up was performed and the action of Nec-1 remains unclear. Here, we studied whether Nec-1 inhibits RIP1-dependent necrosis and leads to long-term improvements after ischemia–reperfusion in vivo. Mice underwent 30 min of ischemia and received, 5 min before reperfusion, 3.3 mg/kg Nec-1 or vehicle treatment, followed by reperfusion. Nec-1 administration reduced infarct size to 26.3 ± 1.3 % (P = 0.001) compared to 38.6 ± 1.7 % in vehicle-treated animals. Furthermore, Nec-1 inhibited RIP1/RIP3 phosphorylation in vivo and significantly reduced necrotic cell death, while apoptotic cell death remained constant. By using MRI, cardiac dimensions and function were assessed before and 28 days after surgery. Nec-1-treated mice displayed less adverse remodeling (end-diastolic volume 63.5 ± 2.8 vs. 74.9 ± 2.8 μl, P = 0.031) and preserved cardiac performance (ejection fraction 45.81 ± 2.05 vs. 36.03 ± 2.37 %, P = 0.016). Nec-1 treatment significantly reduced inflammatory influx, tumor necrosis factor-α mRNA levels and oxidative stress levels. Interestingly, this was accompanied by significant changes in the expression signature of oxidative stress genes. Administration of Nec-1 at the onset of reperfusion inhibits RIP1-dependent necrosis in vivo, leading to infarct size reduction and preservation of cardiac function. The cardioprotective effect of Nec-1 highlights the importance of necrotic cell death in the ischemic heart, thereby opening a new direction for therapy in patients with myocardial infarction.

Keywords

Myocardial infarction Reperfusion Cell death Necrostatin-1 

Notes

Acknowledgments

We thank the following persons for their excellent assistance: Maringa Emons, Sebastian Baars, Jerry Oduro, Esther van Eeuwijk, Sridevi Jaksani, Corina Metz, Chaylendra Strijder and Arjan Schoneveld (all at the University Medical Center Utrecht, The Netherlands). This work was supported by the Novartis Foundation for Cardiovascular Excellence (JS), a Bekalis price (PD), the “Wijnand M. Pon Stichting” (MO) and “Stichting Swaeneborgh” (MO).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

395_2012_270_MOESM1_ESM.pdf (363 kb)
Supplementary Fig. 1 Overview of the experimental design and tissue collection obtained at different times of reperfusion, including MRI measurements. For both vehicle- and Nec-1-treated animals, a similar design was used. (PDF 362 kb)
395_2012_270_MOESM2_ESM.pdf (299 kb)
Supplementary Fig. 2 Cardiac mRNA levels of RIP3 and RIP1 at 1 day after I/R. (a) RIP3 mRNA levels increased significantly after I/R in both groups when compared to baseline. (b) RIP1 mRNA levels increased slightly after I/R, which was similar in both groups. N = 6/group, *P < 0.05 vs. baseline. (c) Control western blot showing a strong depletion of RIP1 after immunoprecipitation (post IP), while RIP1 was not present in the negative control (IgG). n = 5/group. (PDF 299 kb)
395_2012_270_MOESM3_ESM.pdf (1 mb)
Supplementary Fig. 3 TUNEL staining in vivo after I/R. Representative fluorescent pictures of TUNEL (green) and nuclear (Hoechst, blue) staining 1d after I/R of vehicle-treated (a) and Nec-1–treated (b) animals (scale bar 20 μm). (c) Quantification of TUNEL-positive cells representing apoptotic cell death 1 day after I/R (n = 6/group), showing no difference between treatments. (PDF 1033 kb)
395_2012_270_MOESM4_ESM.pdf (3 mb)
Supplementary Fig. 4 Nec-1 reduces reactive oxygen species in vivo after I/R. Representative fluorescent pictures of ROS detection using dihydroethidium (DHE) of vehicle-treated (a) and Nec-1–treated (b) animals (scale bar 20 μm). (c) Quantification of DHE intensity, showing a significant decrease after Nec-1 treatment 1 day after reperfusion (n = 6/group, *P < 0.05). (d) Cardiac mRNA levels of PYGL and GLUL increased significantly in both groups after I/R, while GLUD1 mRNA levels decreased significantly when compared to baseline (n = 6/group, *P < 0.05 vs. baseline). (PDF 3084 kb)
395_2012_270_MOESM5_ESM.pdf (366 kb)
Supplementary Fig. 5 Nec-1 changes the expression of oxidative stress genes after I/R. (a) Scatter plot showing the relative gene expression of 84 genes involved in oxidative stress in Nec-1 vs. vehicle treatment after 1 day of reperfusion. Lines on both sides mark a twofold difference. (b) List of differentially expressed genes (> twofold difference when compared to vehicle treatment) related to cardiac injury with representative references. (PDF 366 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Martinus I. F. J. Oerlemans
    • 1
  • Jia Liu
    • 1
    • 2
  • Fatih Arslan
    • 1
  • Krista den Ouden
    • 1
  • Ben J. van Middelaar
    • 1
  • Pieter A. Doevendans
    • 1
    • 3
  • Joost P. G. Sluijter
    • 1
    • 3
    • 4
    Email author
  1. 1.Department of CardiologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  2. 2.Department of EndocrinologyProvincial Hospital Affiliated to Shandong UniversityJinanChina
  3. 3.Interuniversity Cardiology Institute Netherlands (ICIN)UtrechtThe Netherlands
  4. 4.Laboratory of Experimental Cardiology, G02.523University Medical Centre UtrechtUtrechtThe Netherlands

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