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Updated: Jun 17, 2026
Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury
Published on: March 7, 2022
Netrin-1 prevents ischemia/reperfusion-induced myocardial infarction via a DCC/ERK1/2/eNOS s1177/NO/DCC feed-forward
1Division of Molecular Medicine, Cardiovascular Research Laboratories, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles, 650 Charles E. Young Drive, Los Angeles, CA 90095, USA.
Abstract:
We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO()) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO(), the present study investigated whether netrin-1 is cardioprotective via NO(*) signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO(*) production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8+/-4.9% from 42.5+/-3.6% in the controls), which was accompanied by an augmented production of NO(*). Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO(*) scavenger) attenuated protective effects of netrin-1 on infarct size and NO(*) production, indicating upstream roles of DCC and ERK1/2 in NO(*) production, as well as an essential role of NO(*) in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/- mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOS(s1177) phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO(*) and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO(*) production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO(*) production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO(*) production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOS(s1177)/NO(*)/DCC feed-forward mechanism in both cell types.
Insights
Netrin-1 protects the heart from injury by increasing nitric oxide (NO) production through a DCC-ERK1/2 pathway. This mechanism involves a feed-forward loop, reducing cardiac apoptosis and infarct size during ischemia/reperfusion.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Endothelial Cell Biology
Background:
- Nitric oxide (NO) plays a crucial role in cardioprotection.
- Netrin-1, an endothelial mitogen, stimulates NO production via DCC-ERK1/2 signaling.
- The cardioprotective potential of netrin-1 in the heart warrants investigation.
Purpose of the Study:
- To investigate the cardioprotective effects of netrin-1 in the context of ischemia/reperfusion (I/R) injury.
- To elucidate the role of nitric oxide (NO) signaling in mediating netrin-1's cardioprotection.
- To explore the involvement of the netrin-1 receptor DCC and ERK1/2 pathway in this process.
Main Methods:
- Langendorff perfusion system in C57BL/6J mouse hearts.
- Assessment of NO production using DAF-AM staining.
- Measurement of infarct size and cardiac apoptosis.
- Pharmacological inhibition of DCC, ERK1/2, and NO pathways (U0126, L-NAME, PTIO).
- Studies in DCC+/- mice and isolated cardiomyocytes.
Main Results:
- Netrin-1 significantly increased NO production in the heart.
- Netrin-1 reduced infarct size by approximately 50% during I/R injury.
- Inhibition of DCC, ERK1/2, or NO attenuated netrin-1's protective effects.
- Netrin-1 enhanced ERK1/2 and eNOS(s1177) phosphorylation and DCC expression.
- A DCC/ERK1/2/eNOS(s1177)/NO feed-forward mechanism was identified.
- Netrin-1 reduced cardiac apoptosis.
Conclusions:
- Netrin-1 confers potent cardioprotection against I/R injury.
- This protection is mediated by enhanced NO production via a DCC/ERK1/2/eNOS pathway.
- A feed-forward loop involving DCC, NO, and ERK1/2 contributes to netrin-1's efficacy.
- Netrin-1's protective effects extend to reducing cardiac apoptosis.
