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TNF-α induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury
Yue-Liang Shen1, Ying-Zhou Shi1, Gai-Ge Chen1
1Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, P.R. China.
Abstract:
Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. Sprague‑Dawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylated‑Drp1 (p‑Drp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL‑6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNF‑α (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and p‑Drp1 Ser616. Following TNF‑α treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y‑27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNF‑α‑induced increase in p‑Drp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y‑27632 and fasudil may also attenuate the TNF‑α‑induced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNF‑α is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.
Insights
Tumor necrosis factor-alpha (TNF-α) significantly increases dynamin-related protein 1 (Drp1) phosphorylation and mitochondrial fission during inflammatory heart injury. The RhoA/ROCK pathway mediates this process, impacting cell viability.
Area of Science:
- Cardiovascular Biology
- Mitochondrial Dynamics
- Cellular Signaling
Background:
- Dynamin-related peptide 1 (Drp1)-mediated mitochondrial fission is crucial in cardiac dysfunction.
- Understanding Drp1 regulation during inflammatory myocardial injury is essential.
Purpose of the Study:
- To investigate the expression and role of Drp1 in inflammatory myocardial injury.
- To elucidate the specific inflammatory mediators and signaling pathways involved in Drp1 activation.
Main Methods:
- Lipopolysaccharides (LPS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were used to induce inflammatory injury in Sprague-Dawley rats and H9C2 cardiomyocytes.
- Western blotting was employed to analyze the expression of Drp1, phosphorylated-Drp1 (p-Drp1 Ser616), and RhoA.
- Confocal laser microscopy was used to assess mitochondrial morphology, and Rho kinase (ROCK) inhibitors (Y-27632, fasudil) were utilized to explore pathway involvement.
Main Results:
- LPS injection increased mitochondrial Drp1 and p-Drp1 Ser616 levels in rats.
- In vitro, TNF-α significantly enhanced mitochondrial Drp1 and p-Drp1 Ser616 levels, alongside increased RhoA expression.
- ROCK inhibitors attenuated TNF-α-induced p-Drp1 Ser616, mitochondrial Drp1, mitochondrial fragmentation, and improved cell viability.
Conclusions:
- TNF-α is a primary inducer of Drp1 S616 phosphorylation in sepsis-induced myocardial inflammation.
- The RhoA/ROCK pathway plays a significant role in mediating TNF-α-induced Drp1 phosphorylation and mitochondrial translocation.
- Targeting the RhoA/ROCK pathway may offer therapeutic potential for mitigating Drp1-mediated mitochondrial fragmentation in cardiac injury.
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