bims-noxint 2021-08-22 papers

Issue of 2021‒08‒22
three papers selected by
Laia Caja Puigsubira
Uppsala University

Mol Med Rep. 2021 Oct;pii: 712. [Epub ahead of print]24(4):

Protective effects of metformin against myocardial ischemia‑reperfusion injury via AMPK‑dependent suppression of NOX4.

Numerous studies have demonstrated that metformin can reduce the incidence of myocardial infarction and improve the prognosis of patients. However, its specific mechanism has not been determined. Using a rat model of myocardial ischemia‑reperfusion injury (MIRI), it was observed that metformin significantly reduced infarct size, and decreased the levels of plasma lactate dehydrogenase and creatine kinase‑MB form. A TTC‑Evans blue staining was used to detect the infarct size and MTT assay was used to evaluate the cell viability. TUNEL assay was performed to evaluate apoptosis. Furthermore, 4‑hydroxynonenal was detected by immunohistochemical staining. mRNA expression levels were detected by reverse transcription‑quantitative PCR; protein expression levels were detected by immunoblotting. When treated with metformin, the number of TUNEL‑positive cells was significantly decreased. Reduced 4HNE immunoreactivity was observed in metformin‑treated rats as determined via immunohistochemistry. Furthermore, NADPH oxidase 4 (NOX4) was downregulated by metformin at both the mRNA and protein levels, and adenosine 5'‑monophosphate‑activated protein kinase (AMPK) phosphorylation was increased by metformin. In a primary myocardial hypoxia‑reoxygenation cell model, metformin increased the viability of cardiomyocytes and reduced the content of malondialdehyde. It was also found that metformin upregulated the phosphorylation of AMPK and decreased the expression of NOX4. Furthermore, pre‑treatment with AMPK inhibitor compound‑C could block the effect of metformin, indicated by increased NOX4 compared with metformin treatment alone. These results suggested that metformin was capable of reducing the oxidative stress injury induced by MIRI. In conclusion, the present study indicated that metformin activated AMPK to inhibit the expression of NOX4, leading to a decrease in myocardial oxidative damage and apoptosis, thus alleviating reperfusion injury.

Keywords: NADPH oxidase 4; adenosine 5'‑monophosphate‑activated protein kinase; apoptosis; metformin; myocardial reperfusion injury; oxidative stress

DOI: https://doi.org/10.3892/mmr.2021.12351

Cell Physiol Biochem. 2021 Aug 20. 55(4): 489-504

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

Dongwoo Hahn, Ravi A Kumar, Derek R Muscato, Terence E Ryan, Katrin Schröder, Leonardo F Ferreira.

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.

Keywords: Atrophy; Oxidants; Force; Respiration; Heart failure

DOI: https://doi.org/10.33594/000000400

Metab Brain Dis. 2021 Aug 16.

Therapeutic hypothermia augments the restorative effects of PKC-β and Nox2 inhibition on an in vitro model of human blood-brain barrier.

Rais Reskiawan A Kadir, Mansour Alwjwaj, Zoe McCarthy, Ulvi Bayraktutan.

To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood-brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.

Keywords: Blood–brain barrier; Hyperglycaemia; Oxidative stress; Protein kinase C; Stroke; Therapeutic hypothermia

DOI: https://doi.org/10.1007/s11011-021-00810-8