Alexis Ascah

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.

Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D.

Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria‐mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca2+‐induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca2+‐induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 versus 475 ± 33 nmol (mg protein)−1 for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca2+ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham‐operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca2+ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.

Increased expression and intramitochondrial translocation of cyclophilin-D associates with increased vulnerability of the permeability transition pore to stress-induced opening during compensated ventricular hypertrophy

Opening of the permeability transition pore (PTP) of mitochondria is a critical permeation event that compromises cell viability and may constitute a factor that participates to the loss of cardiomyocytes in compromised hearts. Mitochondria from hearts with volume overload-induced compensated hypertrophy are more vulnerable to opening of the PTP opening in response to a Ca2+ stress. Several of the factors known to affect PTP opening, including respiratory function, membrane potential, the rate of mitochondrial Ca2+ uptake and endogenous levels of Ca2+ in the mitochondrial matrix, were not altered by volume overload. In contrast, there was an 80% increase in the abundance of the PTP regulating protein cyclophilin-D and a 3.7 fold enhancement of Cyp-D binding to membrane, which all predispose to PTP opening. Mitochondria from volume overloaded animals also displayed elevated rates of production of reactive oxygen species, which may be causally related to both the intramitochondrial translocation of cyclophilin-D and PTP opening, since incubation of cardiac mitochondria with terbutylhydroperoxyde in vitro increased to binding of cyclophilin-D to mitochondrial membranes in a dose-related fashion, except when cyclosporin A (a ligand of cyclophilin D with a known ability to delay PTP opening) was present prior to the addition of terbutylhydroperoxyde. Taken together, these results constitute the first evidence obtained in a pathophysiologic situation that increased abundance of cyclophilin-D within mitochondrial membranes may increase mitochondrial vulnerability to stress, and thus possibly initiate a vicious cycle of cellular dysfunction that may ultimately lead to activation of cell death.

Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil.

Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy, a disease caused by the lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying the disease progression are unclear and treatments are limited. Here, we tested the hypothesis of a greater susceptibility to the opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic (mdx) mice (before the development of overt cardiomyopathy) when subjected to a stress protocol and determined whether the prevention of a PTP opening is involved in the cardioprotective effect of sildenafil, which we have previously reported in mdx mice. Using the 2-deoxy-[(3)H]glucose method to quantify the PTP opening in ex vivo perfused hearts, we demonstrate that when compared with those of controls, the hearts from young mdx mice subjected to ischemia-reperfusion (I/R) display an excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage, and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from nonischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and reactive oxygen species handling, but enhanced Ca(2+) uptake velocity and premature opening of the PTP, which may predispose to I/R-induced injury. The administration of a single dose of sildenafil to mdx mice before I/R prevented excessive PTP opening and its downstream consequences and reduced tissue Ca(2+) levels. Furthermore, mitochondrial Ca(2+) uptake velocity was reduced following sildenafil treatment. In conclusion, beyond our documentation that an increased susceptibility to the opening of the mitochondrial PTP in the mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations and is reported safe and well tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca(2+) loading and mitochondrial Ca(2+) uptake.

Telemetry video-electroencephalography (EEG) in rats, dogs and non-human primates: methods in follow-up safety pharmacology seizure liability assessments.

INTRODUCTION Non-clinical seizure liability studies typically aim to: 1) confirm the nature of EEG activity during abnormal clinical signs, 2) identify premonitory clinical signs, 3) measure plasma levels at seizure onset, 4) demonstrate that drug-induced seizures are self-limiting, 5) confirm that conventional drugs (e.g. diazepam) can treat drug-induced seizures and 6) confirm the no observed adverse effect level (NOAEL) at EEG. Our aim was to originally characterize several of these items in a three species comparative study. METHODS Cynomolgus monkey, Beagle dog and Sprague-Dawley rat with EEG telemetry transmitters were used to obtain EEG using the 10-20 system. Pentylenetetrazol (PTZ) was used to determine seizure threshold or as a positive seizurogenic agent. Clinical signs were recorded and premonitory signs were evaluated. In complement, other pharmacological agents were used to illustrate various safety testing strategies. RESULTS Intravenous PTZ doses required to induce clonic convulsions were 36.1 (3.8), 56.1 (12.7) and 49.4 (11.7) mg/kg, in Beagle dogs, cynomolgus monkeys and Sprague-Dawley rats, respectively. Premonitory clinical signs typically included decreased physical activity, enhanced physiological tremors, hypersalivation, ataxia, emesis (except in rats) and myoclonus. In Sprague-Dawley rats, amphetamine (PO) increased high (approximately 40-120Hz), and decreased low (1-14Hz) frequencies. In cynomolgus monkeys, caffeine (IM) increased power in high (14-127Hz), and attenuated power in low (1-13Hz) frequencies. In the rat PTZ infusion seizure threshold model, yohimbine (SC and IV) and phenobarbital (IP) confirmed to be reliable positive controls as pro- and anticonvulsants, respectively. DISCUSSION Telemetry video-EEG for seizure liability investigations was characterized in three species. Rats represent a first-line model in seizure liability assessments. Beagle dogs are often associated with overt susceptibility to seizure and are typically used in seizure liability studies only if required by regulators. Non-human primates represent an important model in seizure liability assessments given similarities to humans and a high translational potential.

Intensity-dependent activation of intracellular signalling pathways in skeletal muscle: role of fibre type recruitment during exercise

Physical activity elicits physiological responses in skeletal muscle that result in a number of health benefits, in particular in diseases associated with peripheral metabolic dysfunction such as diabetes, heart failure or chronic obstructive pulmonary disease. These diseases have been associated with altered skeletal muscle metabolism and, in some cases, diminished ATP production, decreased mitochondrial content, and a higher proportion of type II fast glycolytic fibre. Exercise training is one intervention that can increase the percentage of oxidative fibres (type I, slow) and have a beneficial impact on these disease states. Several studies have compared the effects of continuous vs. interval training, and have determined that interval training may be the most effective exercise strategy to promote mitochondrial biogenesis and enhance muscle oxidative capacity. The importance of exercise in regulating skeletal muscle metabolism is well appreciated; however, the molecular mechanisms that underlie the beneficial adaptations to exercise remain to be fully understood. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a key factor involved in the regulation of multiple myocellular signalling pathways such as those implicated in mitochondrial biogenesis and fibre type expression (Russell et al. 2003). Although muscle contraction is known to strongly modulate PGC-1α expression in human skeletal muscle, little is known about the underlying intracellular mechanisms involved. The differential activation across varying training protocols may shed light onto the regulation of signalling pathways upstream of PGC-1α. This Journal Club article discusses the paper of Egan et al. (2010) published recently in The Journal of Physiology and suggests that differences in fibre type recruitment may explain some of the results. Egan et al. compared the effects two isocaloric bouts of exercise performed at either low or high intensity on skeletal muscle signalling. Eight sedentary males performed two trials on a stationary ergocycle: the low- and high-intensity exercise consisted of continuous cycling at 40% or 80% of peak oxygen consumption (), respectively, until the caloric expenditure reached 400 kcal (1674 kJ). Muscle biopsies from the vastus lateralis were taken at rest and at +0, +3 and +19 h after both exercise bouts. The dietary intake during each experimental trial was controlled. PGC-1α mRNA increased 3 h after both exercise bouts; they observed a 3.8-fold increase after low-intensity exercise whereas it increased 10.2-fold after high-intensity training, supporting an intensity-dependent regulation of PGC-1α expression. The authors also explored the signalling pathways upstream of PGC-1α. Protein quantification by immunoblotting revealed a differential activation of multiple signalling pathways involved directly and indirectly in the regulation of PGC-1α transcription. The higher PGC-1α mRNA abundance after high-intensity exercise also coincided with a greater phosphorylation of activating transcription factor-2 (ATF-2) and of class IIa histone deacetylase (HDAC) proteins also suggesting that ATF-2 and HDAC proteins were involved in an intensity-dependent manner. The authors concluded that the intensity during a single bout of exercise regulates PGC-1α mRNA abundance by activating selected upstream signalling pathways in human skeletal muscle with an intensity-dependent response.

Short term training attenuates opening of the mitochondrial permeability transition pore without affecting myocardial function following ischemia-reperfusion.

Opening of the mitochondrial permeability transition pore (PTP) is known to occur during reperfusion of the ischemic heart and to cause dysfunction and injury. The purpose of the present study was to determine whether short-term training (treadmill dunning for 5 days, 30m.min−1, 0%) in male Sprague Dawley rats reduces the occurrence of PTP opening in the ischemic-reperfused heart. Hearts from control (C) and trained (T) rats perfused in the Langendorff mode were submitted to ischemia-reperfusion (I-R: 30 and 40min respectively). In situ PTP opening was quantified using the mitochondrial 2-deoxy [3H]glucose ([3H]DOG) entrapment method. Following I-R, the recovery of intact mitochondria upon isolation was significantly greater in T vs C hearts (11.7 ± 0.5 vs 9.1 ± 0.4mU citrate synthase.g−1 wet ventricles, p ≤ 0.01). Training also reduced the entrapment of mitochondrial [3H]DOG normalized for the loss of intact mitochondria (14.4 ± 1.4 vs 9.6 ± 0.8 [3H]DOG ratio units, p ≤ 0.01). However, under the experimental conditions used the recovery of contractile function, coronary flow and release of LDH in the coronary effluent were similar in both experimental groups. Taken together, these results suggest that short-term training can confer mitochondrial protection and reduce PTP opening.

Non-invasive measure of respiratory mechanics and conventional respiratory parameters in conscious large animals by high frequency Airwave Oscillometry

INTRODUCTION A number of drugs in clinical trials are discontinued due to potentially life-threatening airway obstruction. As some drugs may not cause changes in core battery parameters such as tidal volume (Vt), respiratory rate (RR) or minute ventilation (MV), including measurements of respiratory mechanics in safety pharmacology studies represents an opportunity for design refinement. The present study aimed to test a novel non-invasive methodology to concomitantly measure respiratory system resistance (Rrs) and conventional respiratory parameters (Vt, RR, MV) in conscious Beagle dogs and cynomolgus monkeys. METHODS An Airwave Oscillometry system (tremoFlo; THORASYS Inc., Montreal, Canada) was used to concomitantly assess Rrs and conventional respiratory parameters before and after intravenous treatment with a bronchoactive agent. Respiratory mechanics measurements were performed by applying a short (i.e. 16s) single high frequency (19Hz) waveform at the subjects airway opening via a face mask. During measurements, pressure and flow signals were recorded. After collection of baseline measurements, methacholine was administered intravenously to Beagle dogs (n=6) and cynomolgus monkeys (n=4) at 8 and 68μg/kg, respectively. RESULTS In dogs, methacholine induced significant increases in Vt, RR and MV while in monkeys, it only augmented RR. A significant increase in Rrs was observed after methacholine administration in both species with mean percentage peak increases from baseline of 88 (53)% for dogs and 28 (16)% for cynomolgus monkeys. CONCLUSION Airwave Oscillometry appears to be a promising non-invasive methodology to enable respiratory mechanics measurements in conscious large animals, a valuable refinement in respiratory safety pharmacology.

Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle

Non‐technical summary Activation of apoptotic cell death signalling by mitochondria is involved in mediating skeletal muscle atrophy following denervation. However, the underlying molecular mechanisms remain unclear, in particular the role of the permeability transition pore (PTP), a high conductance non specific channel of the mitochondrial inner membrane involved in cell death. Using Ppif−/− mice, which are devoid of the PTP sensitizing protein cyclophilin‐D and thus more resistant to pore opening, we directly tested the hypothesis that this pore is involved in atrophy and activation of apoptotic proteolytic signalling following denervation. Our results demonstrate that cyclophilin‐D, and by extension opening of the PTP, is dispensable for atrophy and activation of apoptotic proteolytic signalling induced by denervation.

Rat cardiovascular telemetry: Marginal distribution applied to positive control drugs

Cardiovascular effects are considered frequent during drug safety testing. This investigation aimed to characterize the pharmacological response of the conscious telemetered rat in vivo model to known cardiovascular active agents. These effects were analyzed using statistical analysis and cloud representation with marginal distribution curves for the contractility index and heart rate as to assess the effect relationship between cardiac variables. Arterial blood pressure, left ventricular pressure, electrocardiogram and body temperature were monitored. The application of data cloud with marginal distribution curves to heart rate and contractility index provided an interesting tactic during the interpretation of drug-induced changes particularly during selective time resolution (i.e. marginal distribution curves restricted to Tmax). Taken together, the present data suggests that marginal distribution curves can be a valuable interpretation strategy when using the rat cardiovascular telemetry model to detect drug-induced cardiovascular effects. Marginal distribution curves could also be considered during the interpretation of other inter-dependent parameters in safety pharmacology studies.