Nox4 Knockout Does Not Prevent Diaphragm Atrophy, Contractile Dysfunction, or Mitochondrial Maladaptation in the Early Phase Post-Myocardial Infarction in Mice

Abstract

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.

Skeletal muscle Nox4 knockout prevents and Nox2 knockout blunts loss of maximal diaphragm force in mice with heart failure with reduced ejection fraction

“…One interpretation is that the mechanisms of muscle weakness are different in the early (up to 72 h) vs. late/chronic stage of HFrEF, with XO playing predominant role in the early phase. Indeed, a recent study from our lab investigated diaphragm weakness 72 h post-MI and found no protection with whole-body KO of Nox4 [ 59 ], contrasting with our findings with skmNox4 KO in chronic HFrEF.…”

The Importance of the Diaphragm Muscle in Cardiac Rehabilitation after Myocardial Infarction

“…Carbonylation negatively affects the myosin heavy chain (MHC) and actin, altering contractile expression, proteins within the mitochondrion (aconitase, creatine kinase) and within the cytoplasm (aldolase, enolase, carbonic anhydrase III, glyceraldehyde 3), making the diaphragm less resistant [37]. Oxidation stimulates the activation of nicotinamide adenine dinucleotide phosphate type 4 (NADPH oxidase-4 or NOX-4), which is found in the sarcoplasmic reticulum and mitochondria; NOX-4 impairs mitochondrial function and calcium release within the cytoplasm, contributing, in part, to diaphragmatic dysfunction [38]. It seems that the dysfunction particularly affects the IIb/x fibers or anaerobic fibers [38].…”

“…Oxidation stimulates the activation of nicotinamide adenine dinucleotide phosphate type 4 (NADPH oxidase-4 or NOX-4), which is found in the sarcoplasmic reticulum and mitochondria; NOX-4 impairs mitochondrial function and calcium release within the cytoplasm, contributing, in part, to diaphragmatic dysfunction [38]. It seems that the dysfunction particularly affects the IIb/x fibers or anaerobic fibers [38]. Oxidation is accentuated by the fact that there is an accumulation of iron within the fibers of the diaphragm and a concomitant decrease in the gene that synthesizes transferrin [34].…”

Skeletal muscle atrophy, regeneration, and dysfunction in heart failure: Impact of exercise training

“…Interestingly, only certain isoforms of Nox seem to play a role in diaphragm abnormalities in HF; knockout of a subunit necessary for Nox2 activity restored diaphragm function, 88 whereas Nox4 knockout had no impact on acute MI. 93 While complex, ROS may also facilitate the dissociation of FKBP12 from the RYR1, destabilizing the closed state and perpetuating further Ca 2+ leaks. 94 , 95 …”