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Published on: February 13, 2019
Nox4 Knockout Does Not Prevent Diaphragm Atrophy, Contractile Dysfunction, or Mitochondrial Maladaptation in the
Dongwoo Hahn1, Ravi A Kumar1, Derek R Muscato1
1Department of Applied Physiology and Kinesiology, College of Health and Human Performance, University of Florida, Gainesville, FL, USA.
Background/Aims:
Diaphragm dysfunction with increased reactive oxygen species (ROS) occurs within 72 hrs post-myocardial infarction (MI) in mice and may contribute to loss of inspiratory maximal pressure and endurance in patients.
Methods:
We used wild-type (WT) and whole-body Nox4 knockout (Nox4KO) mice to measure diaphragm bundle force in vitro with a force transducer, mitochondrial respiration in isolated fiber bundles with an O2 sensor, mitochondrial ROS by fluorescence, mRNA (RT-PCR) and protein (immunoblot), and fiber size by histology 72 hrs post-MI.
Results:
MI decreased diaphragm fiber cross-sectional area (CSA) (~15%, p = 0.015) and maximal specific force (10%, p = 0.005), and increased actin carbonylation (5-10%, p = 0.007) in both WT and Nox4KO. Interestingly, MI did not affect diaphragm mRNA abundance of MAFbx/atrogin-1 and MuRF-1 but Nox4KO decreased it by 20-50% (p < 0.01). Regarding the mitochondria, MI and Nox4KO decreased the protein abundance of citrate synthase and subunits of electron transport system (ETS) complexes and increased mitochondrial O2 flux (JO2) and H2O2 emission (JH2O2) normalized to citrate synthase. Mitochondrial electron leak (JH2O2/JO2) in the presence of ADP was lower in Nox4KO and not changed by MI.
Conclusion:
Our study shows that the early phase post-MI causes diaphragm atrophy, contractile dysfunction, sarcomeric actin oxidation, and decreases citrate synthase and subunits of mitochondrial ETS complexes. These factors are potential causes of loss of inspiratory muscle strength and endurance in patients, which likely contribute to the pathophysiology in the early phase post-MI. Whole-body Nox4KO did not prevent the diaphragm abnormalities induced 72 hrs post-MI, suggesting that systemic pharmacological inhibition of Nox4 will not benefit patients in the early phase post-MI.
Insights
Myocardial infarction (MI) causes diaphragm atrophy and dysfunction in mice, linked to mitochondrial changes. Whole-body Nox4 knockout did not prevent these early post-MI diaphragm abnormalities.
Area of Science:
- Cardiovascular Research
- Skeletal Muscle Physiology
- Mitochondrial Biology
Background:
- Myocardial infarction (MI) can lead to diaphragm dysfunction, characterized by increased reactive oxygen species (ROS), potentially impairing respiratory muscle strength and endurance.
- Early post-MI diaphragm dysfunction may be a significant factor in patient recovery and overall pathophysiology.
Purpose of the Study:
- To investigate the impact of MI on diaphragm structure and function in mice.
- To explore the role of Nox4, a key enzyme in ROS production, in early post-MI diaphragm alterations.
- To assess the potential of whole-body Nox4 knockout (Nox4KO) as a therapeutic strategy for post-MI diaphragm dysfunction.
Main Methods:
- Diaphragm bundle force, mitochondrial respiration, mitochondrial ROS production, and gene/protein expression were measured in wild-type and Nox4KO mice 72 hours post-MI.
- Histological analysis was performed to determine diaphragm fiber size (cross-sectional area).
- Mitochondrial function was assessed by measuring oxygen flux and hydrogen peroxide emission relative to citrate synthase activity.
Main Results:
- MI induced diaphragm atrophy (decreased fiber CSA) and reduced maximal specific force in both wild-type and Nox4KO mice.
- Actin carbonylation, an indicator of oxidative stress, increased post-MI.
- Mitochondrial alterations included decreased citrate synthase and electron transport system (ETS) complex subunits, alongside increased mitochondrial oxygen and hydrogen peroxide flux. Nox4KO did not prevent these changes.
Conclusions:
- Early post-MI leads to diaphragm atrophy, contractile dysfunction, and mitochondrial changes, contributing to reduced inspiratory muscle strength.
- Whole-body Nox4 knockout did not ameliorate the diaphragm abnormalities observed 72 hours post-MI.
- These findings suggest that systemic pharmacological inhibition of Nox4 may not be beneficial for patients in the acute phase following myocardial infarction.
