Roles of nitric oxide, nitrite and myoglobin on myocardial efficiency in trout (Oncorhynchus mykiss) and goldfish (Carassius auratus): Implications for hypoxia tolerance

The roles of nitric oxide synthase activity (NOS), nitrite and myoglobin (Mb) in the regulation of myocardial function during hypoxia were examined in trout and goldfish, a hypoxia-intolerant and hypoxia-tolerant species, respectively. We measured the effect of NOS inhibition, adrenaline and nitrite on the O2 consumption rate and isometric twitch force development in electrically paced ventricular preparations during hypoxia, and measured O 2 affinity and nitrite reductase activity of the purified heart Mbs of both species. Upon hypoxia (9% O2), O2 consumption and developed force decreased in both trout and goldfish myocardium, with trout showing a significant increase in the O2 utilization efficiency, i.e. the ratio of twitch force to O2 consumption, suggesting an increased anaerobic metabolism. NOS inhibition enhanced myocardial O2 consumption and decreased efficiency, indicating that mitochondrial respiration is under a tone of NOS-produced NO. When trout myocardial twitch force and O2 consumption are enhanced by adrenaline, this NO tone disappears. Consistent with its conversion to NO, nitrite reduced O2 consumption and increased myocardial efficiency in trout but not in goldfish. Such a difference correlates with the lower O2 affinity measured for trout Mb that would increase the fraction of deoxygenated heme available to catalyze the reduction of nitrite to NO. Whereas low-affinity trout Mb would favor O 2 diffusion within cardiomyocytes at high in vivo O2 tensions, goldfish Mb having higher O2 affinity and higher nitrite reductase activity appears better suited to facilitate O2 diffusion and nitrite reduction in the heart during severe hypoxia, a condition particularly well tolerated by this species

Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

<div><p>The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O₂ affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated <i>in vitro</i> by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O₂ affinity (<i>P</i>₅₀ = 2.7 torr) that was increased by S-nitrosation (<i>P</i>₅₀ = 1.7 torr), whereas in tuna Mb, O₂ affinity (<i>P</i>₅₀ = 0.9 torr) was independent of S-nitrosation. O₂ dissociation rates (<i>k</i>_off) of trout and salmon Mbs were not altered when Cys were in the SNO or <i>N</i>-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O₂ affinity by raising the O₂ association rate (<i>k</i>_on). Taken together, these results indicate that O₂-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O₂ entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb <i>in vivo</i> is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.</p></div

Amino acid sequence alignment of salmon, trout, tuna, carp Mb1 and Mb2 and human Mb shows variable number and position of Cys residues (highlighted).

<p>Mb sequences have been retrieved from Pub Med (Atlantic salmon: Atlantic salmon: GenBank, ACM09229.1, rainbow trout: GenBank, BAI45225.1, yellowfin tuna: GenBank: AAG02112.1, common carp Mb1: UniProtKB/Swiss-Prot, P02204.2, common carp Mb2: GenBank: ABC69306.1, human: NCBI Reference Sequence: NP_976311.1). The positions of the α-helices (A–C, E–H) are indicated and are based on the structure of yellowfin tuna Mb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097012#pone.0097012-Schreiter1" target="_blank">[18]</a>.</p

Salmon and tuna Mbs have faster reacting Cys than human Mb.

<p>Time course of the reaction of 4-PDS with accessible free thiols of salmon (black dots), tuna (white circles) and human Mb (grey circles) were measured in 50 mM Hepes, pH 7.2 at 20°C at a ratio of 4∶1 4-PDS/heme. Data fittings by double (salmon) or single (tuna and human) exponential equations are indicated.</p

S-nitrosation increases O₂ affinity of salmon and trout Mbs but not of tuna Mb and is functionally equivalent to modification by <i>N</i>-ethylmaleimide.

<p>A) O₂ equilibrium curves for tuna and salmon Mb and Mb-SNO and B) O₂ equilibrium curves for trout Mb, Mb-NEM and Mb-SNO, as indicated, measured in 50 mM Tris, 0.5 mM EDTA, pH 8.3 at 20°C. Mb-SNO data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097012#pone.0097012-Helbo1" target="_blank">[9]</a>.</p