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  • br Materials and methods br Results We examined the

    2021-11-30


    Materials and methods
    Results We examined the cardiac mitochondrial density of HKI and markers of oxidative and metabolic stress in trout exposed to an aerated control exposure, an aerated (normoxic) thermal insult, or a hyperoxic thermal insult. Should mtHK provide a cardioprotective effect in the face of combined hyperoxia and thermal stress, a higher relative mitochondrial fraction of HK would be expected. Indeed, immunoblotting of the mitochondrial fraction revealed a significant difference in mtHKI binding (p=0.037, Fig. 1A), with higher mtHKI levels in the hyperoxic than both the aerated heat exposure (p=0.021) and the control (p=0.029), but there was no difference in ventricular MDA levels (p=0.176, Fig. 1B). Higher levels of ubiquitinylated conjugates were observed in the hyperoxic exposure than either the aerated exposure (p=0.041, Fig. 2A) or control (p=0.0021), while there was no difference between the latter two (p=0.318), strongly suggesting changes to protein turnover. There was a significant effect of treatment on plasma lactate levels (p=0.032, Fig. 2B) with the aerated exposure tending higher than either hyperoxic (p=0.054) or control (p=0.053) treatments, suggesting a minor reliance on anaerobism. To confirm the cardioprotective role of mtHK, we carried out in vitro experiments using a LND concentration which dissociates HK from the hiv protease inhibitor without inhibiting HK activity (Chambers et al., 2008). If mtHK is essential for maintained CM health due to prevention of ROS production and blockage of mPTP, then LND-mediated dissociation of mtHK with minimal inhibitory impact on enzymatic activity would be expected to have little effect on muscular force production. Furthermore, the LND-opened mPTP would be expected to decrease oxidative metabolic efficiency due to dissipation of the accumulated H+ gradient. Finally, the administration of LND would be expected to decrease cell viability, ostensibly through oxidative damage and mPTP-related apoptotic induction. Ventricle strip preparations did not differ in peak force production (p=0.54, Fig. 3A), resting tension (p=0.079, Fig. 3B), or in contractile kinetics (time to peak tension and time to half-relaxation; not shown). Incubation of isolated cardiomyocytes with LND increased their ṀO2 by 60% (p=0.00015, Fig. 3C) and mortality by 330% (p=0.00037, Fig. 3D). We expected that exposure to the ionophore FCCP would have a lower associated rate of mortality as it would not allow for an unguarded mPTP (i.e. FCCP-increased mortality would be due to metabolic insufficiency alone). Separate or co-administration of FCCP would be expected to have a similar but non-cumulative effect on respiration (as a complete dissipation of the proton motive force cannot be exacerbated). The application of FCCP did not change ṀO2 on its own or in combination with LND (p=0.67, Fig. 3E), but induced cumulative mortality when compounded with LND exposure (p=0.0072, Fig. 3F), demonstrating identical mitochondrial uncoupling effects between LND and FCCP through different mechanisms. Alignment of the whole sequences of Atlantic salmon and human HKI and HKII revealed expected levels of variation between isoforms and species; the same isoform across species shared higher positive homology than different isoforms within the same species (Suppl. Table 1). However, one of the regions of the lowest homology and conserved residue identity is found in the regulatory subunit (N-terminal half) centered around human HKII D341, with no other sequence sharing even a homologous residue (Fig. 4). Furthermore, the human HKI N345 residue, important in the regulation of VDAC (Rosano, 2011), had homologs in salmonid HKI and HKII, but no analogous residues in human HKII. These patterns of homology in this key area suggest a discrepancy between the domain's functionality between human and salmonid HKII similarly to that between human HKI and HKII.
    Discussion A hyperoxic acute heat exposure allows both higher maximum ṀO2 and aerobic scope in fish (Brijs et al., 2015), potentially allowing for greater ROS production at the mitochondria. Increasing mtHK activity, as seen in the hyperoxic thermal exposure, may facilitate the maintenance of low ATP/ADP ratios in the mitochondria (Treberg et al., 2007) and thereby prevent excessive mitochondrial ROS production by promoting electron transport chain efficiency (Arora and Pedersen, 1988, Calmettes et al., 2016, Da-Silva et al., 2004, Sun et al., 2008). A similar phenomenon is observed in the heart of armored catfish exposed to hypoxia, where the mitochondrial HK fraction increases in density (Treberg et al., 2007). This may represent a rapid and sensitive cardioprotective response in the teleost heart before any oxidative damage accumulates, as evidenced by the lack of difference between treatments in ventricular MDA levels. The increase in ventricular ubiquitinylated conjugates provides further evidence of disrupted homeostasis. Ubiquitinylation, as a correlative marker of protein damage, supports the evidence of hyperoxia inducing an additional cellular stress at high temperatures.