Readmission rates following major colorectal surgery.

BACKGROUND: Readmissions following colorectal surgery (CRS) have negative clinical, psychological and financial implications. Identifying patients at risk of readmission remains challenging. AIMS: To determine factors predictive of those likely to require readmission at 40-days following major CRS and to identify novel strategies capable of reducing readmissions. METHODS: Consecutive patients were studied from a prospectively maintained database. All patients were operated on by a single surgeon in a high-volume centre. Where applicable, photography was recorded by patients and emailed directly to the institutional email of the consultant surgeon. Data was recorded and analysed using descriptive statistics. RESULTS: 515 patients were included over a 15-year period (2007-2022). The mean age at surgery was 64 years (18-93). The majority of patients were male (56.9%, n=293) and underwent cancer surgery (58.2%, n=299). Overall, 55 patients were readmitted within 40 days of major CRS (10.7%). Patients with pre-treatment diagnoses of heart failure (P=0.012), ischemic heart disease (P=0.002), renal impairment (P<0.001), atrial fibrillation (P=0.006), hypercholesterolemia (P=0.001), asthma (P=0.013) and hypertension (P=0.001) were more likely to require readmission. The majority of patients were readmitted for definitive management of surgical site issues (SSIs) (43.7% n=24). Other reasons included bowel obstruction (9.1%, n=5), pelvic sepsis (7.3%, n=4) and gastrointestinal upset (7.3%, n=4). CONCLUSION: This series demonstrated that patients with cardiopulmonary comorbidities were more likely to be readmitted following major CRS and most readmissions are SSI related. Readmissions for SSIs can be reduced by patients sending photography to the treating surgeon which could reduce readmissions and A&E attendances.

Evolution and divergence of teleost adrenergic receptors: why sometimes 'the drugs don't work' in fish.

Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and ß-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts.

Evolutionary loss of the ß1-adrenergic receptor in salmonids.

Whole-genome duplications (WGDs) have been at the heart of the diversification of ß-adrenergic receptors (ß-ARs) in vertebrates. Non-teleost jawed vertebrates typically possess three ß-AR genes: adrb1 (ß1-AR), adrb2 (ß2-AR), and adrb3 (ß3-AR), originating from the ancient 2R (two rounds) WGDs. Teleost fishes, owing to the teleost-specific WGD, have five ancestral adrb paralogs (adrb1, adrb2a, adrb2b, adrb3a and adrb3b). Salmonids are particularly intriguing from an evolutionary perspective as they experienced an additional WGD after separating from other teleosts. Moreover, adrenergic regulation in salmonids, especially rainbow trout, has been intensively studied for decades. However, the repertoire of adrb genes in salmonids has not been yet characterized. An exhaustive genome survey of diverse salmonids, spanning five genera, complemented by phylogenetic sequence analysis, revealed each species has seven adrb paralogs: two adrb2a, two adrb2b, two adrb3a and one adrb3b. Surprisingly, salmonids emerge as the first known jawed vertebrate lineage to lack adrb1. adrb1 is nevertheless highly expressed in the hearts of non-salmonid teleosts, indicating that the wealth of data on adrenergic regulation in salmonids should be generalised to other teleost fishes with caution. It is hypothesised that the loss of adrb1 could have been viable because of the evolutionary radiation of adrb2 and adrb3 genes attributable to the salmonid WGD.

Impact of frailty on oncological outcomes in patients undergoing surgery for colorectal cancer - A systematic review and meta-analysis.

INTRODUCTION: Frailty describes patients who are at an extreme risk of vulnerability to stressors that may lead to adverse clinical outcomes. The impact of frailty on clinical, oncological and survival outcomes in colorectal cancer (CRC) remains unclear. AIM: To determine the anticipated oncological and survival outcomes for patients who are frail when diagnosed and undergo treatment with curative intent for CRC. METHODS: A systematic review and meta-analysis was performed as per PRISMA guidelines. Descriptive statistics were used to determine associations between frailty and survival outcomes. The impact of frailty on disease-free and overall survival were expressed as hazard Ratios (HRs) and 95% confidence intervals (CIs) were estimated using the time-to-effect generic inverse variance and Mantel-Haenszel method. RESULTS: Nine studies including 15,555 patients were included, of whom 8.1% were frail (1206/14,831). The mean age was 77.1 years (range: 42-94 years), 61.1% were female (9510/15,555) and mean follow-up was 48.0 months. Overall, frailty was associated with an increased risk of mortality (HR: 2.95, 95% CI: 1.64-5.29, P < 0.001) and worse disease-free survival (HR: 1.80, 95% CI: 1.34-2.41, P < 0.001). Frailty was also associated with an increased risk of mortality at 1-year (HR: 3.70, 95% CI: 1.00-13.66, P = 0.050) and 5-years (HR: 2.79, 95% CI: 1.65-4.71, P < 0.001) follow-up respectively. CONCLUSION: Frailty is associated with poorer oncological and survival outcomes in patients diagnosed and treated with curative intent for CRC. CRC multidisciplinary team meetings should incorporate these findings into the management paradigm for these patients and patient counselling should be tailored to include these findings.

No evidence for pericardial restraint in the snapping turtle (Chelydra serpentina) following pharmacologically induced bradycardia at rest or during exercise.

Most animals elevate cardiac output during exercise through a rise in heart rate (fH), whereas stroke volume (VS) remains relatively unchanged. Cardiac pacing reveals that elevating fH alone does not alter cardiac output, which is instead largely regulated by the peripheral vasculature. In terms of myocardial oxygen demand, an increase in fH is more costly than that which would incur if VS instead were to increase. We hypothesized that fH must increase because any substantial rise in VS would be constrained by the pericardium. To investigate this hypothesis, we explored the effects of pharmacologically induced bradycardia, with ivabradine treatment, on VS at rest and during exercise in the common snapping turtle (Chelydra serpentina) with intact or opened pericardium. We first showed that, in isolated myocardial preparations, ivabradine exerted a pronounced positive inotropic effect on atrial tissue but only minor effects on ventricle. Ivabradine reduced fH in vivo, such that exercise tachycardia was attenuated. Pulmonary and systemic VS rose in response to ivabradine. The rise in pulmonary VS largely compensated for the bradycardia at rest, leaving total pulmonary flow unchanged by ivabradine, although ivabradine reduced pulmonary blood flow during swimming (exercise × ivabradine interaction, P < 0.05). Although systemic VS increased, systemic blood flow was reduced by ivabradine both at rest and during exercise, despite ivabradine's potential to increase cardiac contractility. Opening the pericardium had no effect on fH, VS, or blood flows before or after ivabradine, indicating that the pericardium does not constrain VS in turtles, even during pharmacologically induced bradycardia.

Absence of atrial smooth muscle in the heart of the loggerhead sea turtle (Caretta caretta): a re-evaluation of its role in diving physiology.

Contraction of atrial smooth muscle in the hearts of semi-aquatic emydid turtles regulates ventricular filling, and it has been proposed that it could regulate stroke volume during characteristic rapid transitions in cardiac output associated with diving. For this hypothesis to be supported, atrial smooth muscle should be widely distributed in diving Testudines. To further understand the putative function and evolutionary significance of endocardial smooth muscle in Testudines, we studied the hearts of loggerhead sea turtles, Caretta caretta (n=7), using immunohistochemistry and histology. Surprisingly, we found no evidence of prominent atrial smooth muscle in C. caretta. However, smooth muscle was readily identified in the sinus venosus. Our results suggest that atrial smooth muscle does not contribute to the diving capabilities of C. caretta, indicating that the possible roles of smooth muscle in emydid turtle hearts require a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling.

The evolutionary and physiological significance of the Hif pathway in teleost fishes.

The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems.

Elevated cortisol lowers thermal tolerance but results in limited cardiac remodelling in rainbow trout (Oncorhynchus mykiss) experiencing chronic social stress.

Juvenile rainbow trout (Oncorhynchus mykiss) held in pairs form dominance hierarchies in which subordinate individuals experience chronic social stress accompanied by lowered thermal tolerance (assessed as the critical thermal maximum, CTmax). Here, we tested the hypothesis that chronic elevation of circulating cortisol levels reduces thermal tolerance in subordinate trout. In support of this hypothesis, subordinate trout that recovered from social stress for 48 h, a period sufficient to return cortisol to normal baseline levels, no longer showed reduced CTmax. Further, thermal tolerance was not restored in subordinates treated with cortisol during recovery from social stress. To explore possible mechanisms underlying the effect of chronic stress on CTmax, we also tested the hypothesis that chronic cortisol elevation induces cardiac remodelling in subordinate trout, as previously reported for cortisol-treated rainbow trout. Ventricle mass and cardiac hypertrophy markers were unaffected by social stress. Picrosirius Red staining revealed a trend for lower collagen levels in the ventricles of subordinate relative to dominant trout. However, collagen type I transcript and protein levels, and markers of collagen turnover were unaffected. Indicators of cardiac function, including ventricle passive stiffness and intrinsic heart rate (fH), similarly were unaffected. In vivo fH was also similar between subordinate and dominant fish. Nevertheless, in keeping with their lower CTmax, subordinate fish exhibited cardiac arrhythmia at significantly lower temperatures than dominant fish during CTmax trials. Thus, high baseline cortisol levels in subordinate trout result in lowered thermal tolerance, but 5 days of social stress did not greatly affect cardiac structure or function.

Resilience of cardiac performance in Antarctic notothenioid fishes in a warming climate.

Warming in the region of the Western Antarctic Peninsula is occurring at an unprecedented rate, which may threaten the survival of Antarctic notothenioid fishes. Herein, we review studies characterizing thermal tolerance and cardiac performance in notothenioids - a group that includes both red-blooded species and the white-blooded, haemoglobinless icefishes - as well as the relevant biochemistry associated with cardiac failure during an acute temperature ramp. Because icefishes do not feed in captivity, making long-term acclimation studies unfeasible, we focus only on the responses of red-blooded notothenioids to warm acclimation. With acute warming, hearts of the white-blooded icefish Chaenocephalus aceratus display persistent arrhythmia at a lower temperature (8°C) compared with those of the red-blooded Notothenia coriiceps (14°C). When compared with the icefish, the enhanced cardiac performance of N. coriiceps during warming is associated with greater aerobic capacity, higher ATP levels, less oxidative damage and enhanced membrane integrity. Cardiac performance can be improved in N. coriiceps with warm acclimation to 5°C for 6-9 weeks, accompanied by an increase in the temperature at which cardiac failure occurs. Also, both cardiac mitochondrial and microsomal membranes are remodelled in response to warm acclimation in N. coriiceps, displaying homeoviscous adaptation. Overall, cardiac performance in N. coriiceps is malleable and resilient to warming, yet thermal tolerance and plasticity vary among different species of notothenioid fishes; disruptions to the Antarctic ecosystem driven by climate warming and other anthropogenic activities endanger the survival of notothenioids, warranting greater protection afforded by an expansion of marine protected areas.

How cardiac output is regulated: August Krogh's proto-Guytonian understanding of the importance of venous return.

August Krogh pioneered a view of cardiac output that emphasised the rate of venous return, and stressed the importance of changing the filling of the 'inadequately' supplied heart, instead of changes in heart rate or cardiac inotropy. Krogh, by and large, presented this concept in three manuscripts published in 1912, which relied on innovative experimental techniques (developed in collaboration with Johannes Lindhard) as well as mathematical and physical models. In this graphical review, we revisit Krogh's original articles, demonstrate how they have stood the test of time, and show how they are relevant to comparative cardiovascular physiology. In doing so, we present an overview of the fundamental, but sometimes counterintuitive, principle that peripheral factors are at least as important as cardiac function in determining cardiac output.

Regulation of splenic contraction persists as a vestigial trait in white-blooded Antarctic fishes.

In fishes, the spleen can function as an important reservoir for red blood cells (RBCs), which, following splenic contraction, may be released into the circulation to increase haematocrit during energy-demanding activities. This trait is particularly pronounced in red-blooded Antarctic fishes in which the spleen can sequester a large proportion of RBCs during rest, thereby reducing blood viscosity, which may serve as an adaptation to life in cold environments. In one species, Pagothenia borchgrevinki, it has previously been shown that splenic contraction primarily depends on cholinergic stimulation. The aim of the present study was to investigate the regulation of splenic contraction in five other Antarctic fish species, three red-blooded notothenioids (Dissostichus mawsoni Norman, 1937, Gobionotothen gibberifrons Lönnberg, 1905, Notothenia coriiceps Richardson 1844) and two white-blooded "icefish" (Chaenocephalus aceratus Lönnberg, 1906 and Champsocephalus gunnari Lönnberg, 1905), which lack haemoglobin and RBCs, but nevertheless possess a large spleen. In all species, splenic strips constricted in response to both cholinergic (carbachol) and adrenergic (adrenaline) agonists. Surprisingly, in the two species of icefish, the spleen responded with similar sensitivity to red-blooded species, despite contraction being of little obvious benefit for releasing RBCs into the circulation. Although the icefish lineage lost functional haemoglobin before diversifying over the past 7.8-4.8 millions of years, they retain the capacity to contract the spleen, likely as a vestige inherited from their red-blooded ancestors.

Low incidence of atrial septal defects in nonmammalian vertebrates.

The atrial septum enables efficient oxygen transport by separating the systemic and pulmonary venous blood returning to the heart. Only in placental mammals will the atrial septum form by the coming-together of the septum primum and the septum secundum. In up to one of four placental mammals, this complex morphogenesis is incomplete and yields patent foramen ovale. The incidence of incomplete atrial septum is unknown for groups with the septum primum only, such as birds and reptiles. We found a low incidence of incomplete atrial septum in 11 species of bird (0% of specimens) and 13 species of reptiles (3% of specimens). In reptiles, there was a trabecular interface between the atrial septum and the atrial epicardium which was without a clear boundary between left and right atrial cavities. In developing reptiles (four squamates and one crocodylian), the septum primum initiated as a sheet that acquired perforations and the trabecular interface developed late. We conclude that atrial septation from the septum primum only results in a low incidence of incompleteness. In reptiles, the atrial septum and atrial wall develop a trabecular interface, but previous studies on atrial hemodynamics suggest this interface has a very limited capacity for shunting.

What determines systemic blood flow in vertebrates?

In the 1950s, Arthur C. Guyton removed the heart from its pedestal in cardiovascular physiology by arguing that cardiac output is primarily regulated by the peripheral vasculature. This is counterintuitive, as modulating heart rate would appear to be the most obvious means of regulating cardiac output. In this Review, we visit recent and classic advances in comparative physiology in light of this concept. Although most vertebrates increase heart rate when oxygen demands rise (e.g. during activity or warming), experimental evidence suggests that this tachycardia is neither necessary nor sufficient to drive a change in cardiac output (i.e. systemic blood flow, Q̇sys) under most circumstances. Instead, Q̇sys is determined by the interplay between vascular conductance (resistance) and capacitance (which is mainly determined by the venous circulation), with a limited and variable contribution from heart function (myocardial inotropy). This pattern prevails across vertebrates; however, we also highlight the unique adaptations that have evolved in certain vertebrate groups to regulate venous return during diving bradycardia (i.e. inferior caval sphincters in diving mammals and atrial smooth muscle in turtles). Going forward, future investigation of cardiovascular responses to altered metabolic rate should pay equal consideration to the factors influencing venous return and cardiac filling as to the factors dictating cardiac function and heart rate.

Hypoxia inducible factor-1α knockout does not impair acute thermal tolerance or heat hardening in zebrafish.

The rapid increase in critical thermal maximum (CTmax) in fish (or other animals) previously exposed to critically high temperature is termed 'heat hardening', which likely represents a key strategy to cope with increasingly extreme environments. The physiological mechanisms that determine acute thermal tolerance, and the underlying pathways facilitating heat hardening, remain debated. It has been posited, however, that exposure to high temperature is associated with tissue hypoxia and may be associated with the increased expression of hypoxia-inducible factor-1 (Hif-1). We studied acute thermal tolerance in zebrafish (Danio rerio) lacking functional Hif-1α paralogs (Hif-1aa and Hif-1ab double knockout; Hif-1α-/-), which are known to exhibit markedly reduced hypoxia tolerance. We hypothesized that Hif-1α-/- zebrafish would suffer reduced acute thermal tolerance relative to wild type and that the heat hardening ability would be lost. However, on the contrary, we observed that Hif-1α-/- and wild-type fish did not differ in CTmax, and both genotypes exhibited heat hardening of a similar degree when CTmax was re-tested 48 h later. Despite exhibiting impaired hypoxia tolerance, Hif-1α-/- zebrafish display unaltered thermal tolerance, suggesting that these traits are not necessarily functionally associated. Hif-1α is accordingly not required for short-term acclimation in the form of heat hardening.

Contraction of atrial smooth muscle reduces cardiac output in perfused turtle hearts.

Unusual undulations in resting tension (tonus waves) were described in isolated atria from freshwater turtles more than a century ago. These tonus waves were soon after married with the histological demonstration of a rich layer of smooth muscle on the luminal side of the atrial wall. Research thereafter waned and the functional significance of this smooth muscle has remained obscure. Here, we provide evidence that contraction of the smooth muscle in the atria may be able to change cardiac output in turtle hearts. In in situ perfused hearts of the red-eared slider turtle (Trachemys scripta elegans), we demonstrated that activation of smooth muscle contraction with histamine (100 nmol kg-1 bolus injected into perfusate) reduced cardiac output by decreasing stroke volume (>50% decrease in both parameters). Conversely, inhibition of smooth muscle contraction with wortmannin (10 µmol l-1 perfusion) approximately doubled baseline stroke volume and cardiac output. We suggest that atrial smooth muscle provides a unique mechanism to control cardiac filling that could be involved in the regulation of stroke volume during diving.

Weighing the evidence for using vascular conductance, not resistance, in comparative cardiovascular physiology.

Vascular resistance and conductance are reciprocal indices of vascular tone that are often assumed to be interchangeable. However, in most animals in vivo, blood flow (i.e. cardiac output) typically varies much more than arterial blood pressure. When blood flow changes at a constant pressure, the relationship between conductance and blood flow is linear, whereas the relationship between resistance and blood flow is non-linear. Thus, for a given change in blood flow, the change in resistance depends on the starting point, whereas the attendant change in conductance is proportional to the change in blood flow regardless of the starting conditions. By comparing the effects of physical activity at different temperatures or between species - concepts at the heart of comparative cardiovascular physiology - we demonstrate that the difference between choosing resistance or conductance can be marked. We also explain here how the ratio of conductance in the pulmonary and systemic circulations provides a more intuitive description of cardiac shunt patterns in the reptilian cardiovascular system than the more commonly used ratio of resistance. Finally, we posit that, although the decision to use conductance or resistance should be made on a case-by-case basis, in most circumstances, conductance is a more faithful portrayal of cardiovascular regulation in vertebrates.

Adrenergic and adenosinergic regulation of the cardiovascular system in an Antarctic icefish: Insight into central and peripheral determinants of cardiac output.

Icefishes characteristically lack the oxygen-binding protein haemoglobin and therefore are especially reliant on cardiovascular regulation to augment oxygen transport when oxygen demand increases, such as during activity and warming. Using both in vivo and in vitro experiments, we evaluated the roles for adrenaline and adenosine, two well-established cardio- and vasoactive molecules, in regulating the cardiovascular system of the blackfin icefish, Chaenocephalus aceratus. Despite increasing cardiac contractility (increasing twitch force and contraction kinetics in isometric myocardial strip preparations) and accelerating heart rate (ƒH), adrenaline (5 nmol kg-1 bolus intra-arterial injection) did not significantly increase cardiac output (Q̇) in vivo because it elicited a large decrease in vascular conductance (Gsys). In contrast, and despite preliminary data suggesting a direct negative inotropic effect of adenosine on isolated atria and little effect on isolated ventricle strips, adenosine (500 nmol kg-1) generated a large increase in Q̇ by increasing Gsys, a change reminiscent of that previously reported during both acute warming and invoked activity. Our data thus illustrate how Q̇ in C. aceratus may be much more dependent on peripheral control of vasomotor tone than direct regulation of the heart.

Maximum heart rate does not limit cardiac output at rest or during exercise in the American alligator ( Alligator mississippiensis).

In most vertebrates, increases in cardiac output result from increases in heart rate (fH) with little or no change in stroke volume (Vs), and maximum cardiac output (Q̇) is typically attained at or close to maximum fH. We therefore tested the hypothesis that increasing maximum fH may increase maximum Q̇. To this end, we investigated the effects of elevating fH with right atrial pacing on Q̇ in the American alligator ( Alligator mississippiensis) at rest and while swimming. During normal swimming, Q̇ increased entirely by virtue of a tachycardia (29 ± 1 to 40 ± 3 beats/min), whereas Vs remained stable. In both resting and swimming alligators, increasing fH with right atrial pacing resulted in a parallel decline in Vs that resulted in an unchanged cardiac output. In swimming animals, this reciprocal relationship extended to supraphysiological fH (up to ~72 beats/min), which suggests that maximum fH does not limit maximum cardiac output and that fH changes are secondary to the peripheral factors (for example vascular capacitance) that determine venous return at rest and during exercise.

Suppression of reactive oxygen species generation in heart mitochondria from anoxic turtles: the role of complex I S-nitrosation.

Freshwater turtles (Trachemys scripta) are among the very few vertebrates capable of tolerating severe hypoxia and re-oxygenation without suffering from damage to the heart. As myocardial ischemia and reperfusion causes a burst of mitochondrial reactive oxygen species (ROS) in mammals, the question arises as to whether, and if so how, this ROS burst is prevented in the turtle heart. We find that heart mitochondria isolated from turtles acclimated to anoxia produce less ROS than mitochondria from normoxic turtles when consuming succinate. As succinate accumulates in the hypoxic heart and is oxidized when oxygen returns, this suggests an adaptation to lessen ROS production. Specific S-nitrosation of complex I can lower ROS in mammals and here we show that turtle complex I activity and ROS production can also be strongly depressed in vitro by S-nitrosation. We detect in vivo endogenous S-nitrosated complex I in turtle heart mitochondria, but these levels are unaffected upon anoxia acclimation. Thus, while heart mitochondria from anoxia-acclimated turtles generate less ROS and have a lower aerobic capacity than those from normoxic turtles, this is not due to decreases in complex I activity or expression levels. Interestingly, in-gel activity staining reveals that most complex I of heart mitochondria from normoxic and anoxic turtles forms stable super-complexes with other respiratory enzymes and, in contrast to mammals, these are not disrupted by dodecyl maltoside. Taken together, these results show that although S-nitrosation of complex I is a potent mechanism to prevent ROS formation upon re-oxygenation after anoxia in vitro, this is not a major cause of the suppression of ROS production by anoxic turtle heart mitochondria.