Aging-dependent mitochondrial bioenergetic impairment in the skeletal muscle of NNT-deficient mice.

Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt-/-) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt-/- mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt-/- mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt-/- mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.

Aggravation of hepatic lipidosis in red-footed tortoise Chelonoidis carbonaria with age is associated with alterations in liver mitochondria.

The occurrence of hepatic lipidosis is commonly reported in different reptilian species, especially in animals under captivity. Liver accumulation of fat is associated with disorders, better described in mammals as non-alcoholic fatty liver diseases (NAFLD), ranging from simple steatosis, to non-alcoholic steatohepatitis (NASH), and to more severe lesions of cirrhosis and hepatocellular carcinoma. Mitochondria play a central role in NAFLD pathogenesis, therefore in this study we characterized livers of ad libitum fed captive red-footed tortoise Chelonoidis carbonaria through histological and mitochondrial function evaluations of juvenile and adult individuals. Livers from adult tortoises exhibited higher levels of lipids, melanomacrophages centers and melanin than juveniles. The observed high score levels of histopathological alterations in adult tortoises, such as microvesicular steatosis, inflammation and fibrosis, indicated the progression to a NASH condition. Mitochondrial oxygen consumption at different respiratory states and with different substrates was 30 to 58% lower in adult when compared to juvenile tortoises. Despite citrate synthase activity was also lower in adults, cardiolipin content was similar to juveniles, indicating that mitochondrial mass was unaffected by age. Mitochondrial Ca2+ retention capacity was reduced by 70% in adult tortoises. Overall, we found that aggravation of NAFLD in ad libitum fed captive tortoises is associated with compromised mitochondrial function, indicating a critical role of the organelle in liver disease progression in reptiles.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

Cholinergic regulation along the pulmonary arterial tree of the South American rattlesnake: vascular reactivity, muscarinic receptors, and vagal innervation.

Vascular tone in the reptilian pulmonary vasculature is primarily under cholinergic, muscarinic control exerted via the vagus nerve. This control has been ascribed to a sphincter located at the arterial outflow, but we speculated whether the vascular control in the pulmonary artery is more widespread, such that responses to acetylcholine and electrical stimulation, as well as the expression of muscarinic receptors, are prevalent along its length. Working on the South American rattlesnake (Crotalus durissus), we studied four different portions of the pulmonary artery (truncus, proximal, distal, and branches). Acetylcholine elicited robust vasoconstriction in the proximal, distal, and branch portions, but the truncus vasodilated. Electrical field stimulation (EFS) caused contractions in all segments, an effect partially blocked by atropine. We identified all five subtypes of muscarinic receptors (M1-M5). The expression of the M1 receptor was largest in the distal end and branches of the pulmonary artery, whereas expression of the muscarinic M3 receptor was markedly larger in the truncus of the pulmonary artery. Application of the neural tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) revealed widespread innervation along the whole pulmonary artery, and retrograde transport of the same tracer indicated two separate locations in the brainstem providing vagal innervation of the pulmonary artery, the medial dorsal motor nucleus of the vagus and a ventro-lateral location, possibly constituting a nucleus ambiguus. These results revealed parasympathetic innervation of a large portion of the pulmonary artery, which is responsible for regulation of vascular conductance in C. durissus, and implied its integration with cardiorespiratory control.

Heart rate variability in the tegu lizard, Salvator merianae, its neuroanatomical basis and role in the assessment of recovery from experimental manipulation.

Using long-term, remote recordings of heart rate (fH) on fully recovered, undisturbed lizards, we identified several components of heart rate variability (HRV) associated with respiratory sinus arrhythmia (RSA): 1.) A peak in the spectral representation of HRV at the frequency range of ventilation. 2.) These cardiorespiratory interactions were shown to be dependent on the parasympathetic arm of the autonomic nervous system. 3.) Vagal preganglionic neurons are located in discrete groups located in the dorsal motor nucleus of the vagus and also, in a ventro-lateral group, homologous to the nucleus ambiguus of mammals. 4.) Myelinated nerve fibers in the cardiac vagus enabling rapid communication between the central nervous system and the heart. Furthermore, the study of the progressive recovery of fH in tegu following anesthesia and instrumentation revealed that 'resting' levels of mean fH and reestablishment of HRV occurred over different time courses. Accordingly, we suggest that, when an experiment is designed to study a physiological variable reliant on autonomic modulation at its normal, resting level, then postsurgical reestablishment of HRV should be considered as the index of full recovery, rather than mean fH.

Blood flow distribution in embryonic common snapping turtles Chelydra serpentina (Reptilia; Chelonia) during acute hypoxia and α-adrenergic regulation.

Embryonic turtles have four distinct vascular beds that separately perfuse the developing embryo's body and the extra-embryonic yolk sac, amnion and chorioallantoic membrane (CAM). The mechanisms enabling differential regulation of blood flow through these separate beds, in order to meet the varying demands of the embryo during development, is of current interest. The present investigation followed the changes in blood flow distribution during an acute exposure to hypoxia and after α-adrenergic blockade. We monitored heart rate (fH), mean arterial pressure (Pm), and determined relative blood flow distribution (%Q̇sys) using colored microspheres. At 70% and 90% of the incubation period hypoxia elicited a bradycardia without changing Pm while %Q̇sys was altered only at 70%, increasing to the CAM and liver. Blockade of α-adrenergic responses with phentolamine did not change fH or Pm but increased %Q̇sys to the shell. These results show the capacity of embryos to redistribute cardiac output during acute hypoxia, however α-adrenergic receptors seemed to play a relatively small role in embryonic cardiovascular regulation.

Direct determination of anaerobe contributions to the energy metabolism of Trypanosoma cruzi by chip calorimetry.

We describe a chip calorimetric technique that allows the investigation of biological material under anoxic conditions in a micro-scale and in real time. Due to the fast oxygen exchange through the sample flow channel wall, the oxygen concentration inside the samples could be switched between atmospheric oxygen partial pressure to an oxygen concentration of 0.5% within less than 2 h. Using this technique, anaerobic processes in the energy metabolism of Trypanosoma cruzi could be studied directly. The comparison of the calorimetric and respirometric response of T. cruzi cells to the treatment with the mitochondrial inhibitors oligomycin and antimycin A and the uncoupler FCCP revealed that the respiration-related heat rate is superimposed by strong anaerobic contributions. Calorimetric measurements under anoxic conditions and with glycolytic inhibitors showed that anaerobic metabolic processes contribute from 30 to 40% to the overall heat production rate. Similar basal and antimycin A heat rates with cells under anoxic conditions indicated that the glycolytic rates are independent of the oxygen concentration which confirms the absence of the "Pasteur effect" in Trypanosomes. Graphical abstract.

The electrocardiogram of vertebrates: Evolutionary changes from ectothermy to endothermy.

The electrocardiogram (ECG) reveals that heart chamber activation and repolarization are much faster in mammals and birds compared to ectothermic vertebrates of similar size. Temperature, however, affects electrophysiology of the heart and most data from ectotherms are determined at body temperatures lower than those of mammals and birds. The present manuscript is a review of the effects of temperature on intervals in the ECG of ectothermic and endothermic vertebrates rather than a hypothesis-testing original research article. However, the conclusions are supported by the inclusion of original data (Iguana iguana, N = 4; Python regius, N = 5; Alligator mississippiensis, N = 4). Most comparisons were of animals of approximately 1 kg. Compared to mammals and birds, the reptiles at 35-37 °C had 4 fold lower heart rates, 2 fold slower atrial and ventricular conduction (longer P- and QRS-wave durations), and 4 fold longer PR intervals (atrioventricular delay) and QT intervals (total ventricular repolarization). We conclude that the faster chamber activation in endotherms cannot be explained by temperature alone. Based on histology, we show that endotherms have a more compact myocardial architecture. In mammals, disorganization of the compact wall by fibrosis associates with conduction slowing and we suggest the compact tissue architecture allows for faster chamber activation. The short cardiac cycle that characterizes mammals and birds, however, is predominantly accommodated by shortening of the atrioventricular delay and the QT interval, which is so long in a 1 kg iguana that it compares to that of an elephant.

Convective oxygen transport during development in embryos of the snapping turtle Chelydra serpentina.

This study investigated the maturation of convective oxygen transport in embryos of the snapping turtle (Chelydra serpentina). Measurements included: mass, oxygen consumption (V̇O2 ), heart rate, blood oxygen content and affinity and blood flow distribution at 50%, 70% and 90% of the incubation period. Body mass increased exponentially, paralleled by increased cardiac mass and metabolic rate. Heart rate was constant from 50% to 70% incubation but was significantly reduced at 90% incubation. Hematocrit and hemoglobin concentration were constant at the three points of development studied but arteriovenous difference doubled from 50% to 90% incubation. Oxygen affinity was lower for the early 50% incubation group (stage 19) compared with all other age groups. Blood flow was directed predominantly to the embryo but was highest to the chorioallantoic membrane (CAM) at 70% incubation and was directed away from the yolk as it was depleted at 90% incubation. The findings indicate that the plateau or reduction in egg V̇O2  characteristic of the late incubation period of turtle embryos may be related to an overall reduction in mass-specific V̇O2  that is correlated with decreasing relative heart mass and plateaued CAM blood flow. Importantly, if the blood properties remain unchanged prior to hatching, as they did during the incubation period studied in the current investigation, this could account for the pattern of V̇O2 previously reported for embryonic snapping turtles prior to hatching.

The consequences of seasonal fasting during the dormancy of tegu lizards (Salvator merianae) on their postprandial metabolic response.

Tegu lizards (Salvator merianae) aestivate for up to 5 months during Brazil's winter, when they retreat to burrows and halt most activities. Dormant tegus reduce their gastrointestinal (GI) mass, which allows a substantial energy economy. This strategy, however, implies that the first post-dormancy digestion would be more costly than subsequent feeding episodes as a result of GI atrophy. To address this, we determined the postprandial metabolic response (SDA) of the first (M1), second (M2) and several (RM) feeding episodes after tegus' dormancy. Another group of tegus (PF) was subjected to an extra 50 day fasting period after arousal. Glucose, triglycerides and uric acid levels were checked before and after feeding. M1 digestion lasted twice as long and cost twofold more when compared with M2 or RM, in agreement with the idea that GI atrophy inflates digestion cost at the first post-dormancy meal. The SDA response was similar in M2 and RM, suggesting that the GI tract was fully reorganized after the first feeding. The SDA cost was equal in PF and RM, implying that the change in state per se (dormant to arousal) triggers the regrowth of GI, independently of feeding. Fasting tegus at M1 presented higher triglyceride and lower uric acid levels than fed tegus, indicating that fasting is mainly sustained by fat storage. Our results show that seasonal fasting imposes an extra digestion cost to tegus following their next feeding, which is fully paid during their first digestion. This surplus cost, however, is negligible compared with the overall energetic savings from GI tract atrophy during the dormancy period.

Cardiorespiratory interactions previously identified as mammalian are present in the primitive lungfish.

The present study has revealed that the lungfish has both structural and functional features of its system for physiological control of heart rate, previously considered solely mammalian, that together generate variability (HRV). Ultrastructural and electrophysiological investigation revealed that the nerves connecting the brain to the heart are myelinated, conferring rapid conduction velocities, comparable to mammalian fibers that generate instantaneous changes in heart rate at the onset of each air breath. These respiration-related changes in beat-to-beat cardiac intervals were detected by complex analysis of HRV and shown to maximize oxygen uptake per breath, a causal relationship never conclusively demonstrated in mammals. Cardiac vagal preganglionic neurons, responsible for controlling heart rate via the parasympathetic vagus nerve, were shown to have multiple locations, chiefly within the dorsal vagal motor nucleus that may enable interactive control of the circulatory and respiratory systems, similar to that described for tetrapods. The present illustration of an apparently highly evolved control system for HRV in a fish with a proven ancient lineage, based on paleontological, morphological, and recent genetic evidence, questions much of the anthropocentric thinking implied by some mammalian physiologists and encouraged by many psychobiologists. It is possible that some characteristics of mammalian respiratory sinus arrhythmia, for which functional roles have been sought, are evolutionary relics that had their physiological role defined in ancient representatives of the vertebrates with undivided circulatory systems.

Rates of oxygen uptake increase independently of changes in heart rate in late stages of development and at hatching in the green iguana, Iguana iguana.

Oxygen consumption (VO2), heart rate (fH), heart mass (Mh) and body mass (Mb) were measured during embryonic incubation and in hatchlings of green iguana (Iguana iguana). Mean fH and VO2 were unvarying in early stage embryos. VO2 increased exponentially during the later stages of embryonic development, doubling by the end of incubation, while fH was constant, resulting in a 2.7-fold increase in oxygen pulse. Compared to late stage embryos, the mean inactive level of VO2 in hatchlings was 1.7 fold higher, while fH was reduced by half resulting in a further 3.6 fold increase in oxygen pulse. There was an overall negative correlation between mean fH and VO2 when data from hatchlings was included. Thus, predicting metabolic rate as VO2 from measurements of fH is not possible in embryonic reptiles. Convective transport of oxygen to supply metabolism during embryonic incubation was more reliably indicated as an index of cardiac output (COi) derived from the product of fH and Mh. However, a thorough analysis of factors determining rates of oxygen supply during development and eclosion in reptiles will require cannulation of blood vessels that proved impossible in the present study, to determine oxygen carrying capacity by the blood and arteriovenous oxygen content difference (A-V diff), plus patterns of blood flow.

The progressive onset of cholinergic and adrenergic control of heart rate during development in the green iguana, Iguana iguana.

The autonomic control of heart rate was studied throughout development in embryos of the green iguana, Iguana iguana by applying receptor agonists and antagonists of the parasympathetic and sympathetic systems. Acetylcholine (Ach) slowed or stopped the heart and atropine antagonized the response to Ach indicating the presence of muscarinic cholinoceptors on the heart of early embryos. However, atropine injections had no impact on heart rate until immediately before hatching, when it increased heart rate by 15%. This cholinergic tonus increased to 34% in hatchlings and dropped to 24% in adult iguanas. Although epinephrine was without effect, injection of propranolol slowed the heart throughout development, indicating the presence of ß-adrenergic receptors on the heart of early embryos, possibly stimulated by high levels of circulating catecholamines. The calculated excitatory tonus varied between 33% and 68% until immediately before hatching when it fell to 25% and 29%, a level retained in hatchlings and adults. Hypoxia caused a bradycardia in early embryos that was unaffected by injection of atropine indicating that hypoxia has a direct effect upon the heart. In later embryos and hatchlings hypoxia caused a tachycardia that was unaffected by injection of atropine. Subsequent injection of propranolol reduced heart rate both uncovering a hypoxic bradycardia in late embryos and abolishing tachycardia in hatchlings. Hypercapnia was without effect on heart rate in late stage embryos and in hatchlings.

An appraisal of the use of an infrared digital monitoring system for long-term measurement of heart rate in reptilian embryos.

Measurement of heart rate (fH) in embryonic reptiles has previously imposed some degree of invasive treatment on the developing embryo. Recently a non-invasive technique of fH detection from intact eggs was developed for commercial avian breeders and has since been used in biological research. This device uses infrared light, enabling it to detect heartbeats in very early embryos. However, infrared light is a source of heat and extended enclosure of an egg in the device is likely to affect temperature with consequent effects on physiological processes, including fH. We studied the effect of use of the monitor on the temperature of eggs and on fH in two species of reptiles, the snapping turtle (Chelydra serpentina) and the green iguana (Iguana iguana). Egg temperature increased from a room temperature of 27-28 °C, by 26% in turtles and 14% in iguanas over 1h of enclosure, resulting in an increase in fH of 76-81% in turtles and 35-50% iguanas. These effects on fH can either be avoided by brief enclosure of each egg in the monitor or measured and accounted for during the design of long-term experiments.

The phylogeny and ontogeny of autonomic control of the heart and cardiorespiratory interactions in vertebrates.

Heart rate in vertebrates is controlled by activity in the autonomic nervous system. In spontaneously active or experimentally prepared animals, inhibitory parasympathetic control is predominant and is responsible for instantaneous changes in heart rate, such as occur at the first air breath following a period of apnoea in discontinuous breathers like inactive reptiles or species that surface to air breathe after a period of submersion. Parasympathetic control, exerted via fast-conducting, myelinated efferent fibres in the vagus nerve, is also responsible for beat-to-beat changes in heart rate such as the high frequency components observed in spectral analysis of heart rate variability. These include respiratory modulation of the heartbeat that can generate cardiorespiratory synchrony in fish and respiratory sinus arrhythmia in mammals. Both may increase the effectiveness of respiratory gas exchange. Although the central interactions generating respiratory modulation of the heartbeat seem to be highly conserved through vertebrate phylogeny, they are different in kind and location, and in most species are as yet little understood. The heart in vertebrate embryos possesses both muscarinic cholinergic and ß-adrenergic receptors very early in development. Adrenergic control by circulating catecholamines seems important throughout development. However, innervation of the cardiac receptors is delayed and first evidence of a functional cholinergic tonus on the heart, exerted via the vagus nerve, is often seen shortly before or immediately after hatching or birth, suggesting that it may be coordinated with the onset of central respiratory rhythmicity and subsequent breathing.

A role for histamine in cardiovascular regulation in late stage embryos of the red-footed tortoise, Chelonoidis carbonaria Spix, 1824.

A chorioallantoic membrane artery in embryos of the red-footed tortoise, Chelonoidis carbonaria was occlusively cannulated for measurement of blood pressure and injection of drugs. Two age groups of embryos in the final 10 % of incubation were categorized by the ratio of embryonic body to yolk mass. All embryos first received cholinergic and ß-adrenergic blockade. This revealed that ß-adrenergic control was established in both groups whereas cholinergic control was only established in the older group immediately prior to hatching. The study then progressed as two series. Series one was conducted in a subset of embryos treated with histamine before or after injection of ranitidine, the antagonist of H2 receptors. Injection of histamine caused an initial phasic hypertension which recovered, followed by a longer lasting hypertensive response accompanied by a tachycardia. Injection of the H2 receptor antagonist ranitidine itself caused a hypotensive tachycardia with subsequent recovery of heart rate. Ranitidine also abolished the cardiac effects of histamine injection while leaving the initial hypertensive response intact. In series, two embryos were injected with histamine after injection of diphenhydramine, the antagonist to H1 receptors. This abolished the whole of the pressor response to histamine injection but left the tachycardic response intact. These data indicate that histamine acts as a non-adrenergic, non-cholinergic factor, regulating the cardiovascular system of developing reptilian embryos and that its overall effects are mediated via both H1 and H2 receptor types.