La sonde d’intubation est le facteur de risque principal des microinhalations. Sa présence empêche la fermeture des cordes vocales, favorisant ainsi la progression des sécrétions oropharyngées vers les voies respiratoires inférieures. Le ballonnet de la sonde trachéale prévient en partie cette microinhalation, mais la présence de microsillons sur les ballonnets en polyvinyl chloride (PVC) et la souspression du ballonnet (<20 cmH2O) favorisent les microinhalations autour du ballonnet. Alors que lésions ischémiques trachéales sévères liées à l’intubation ne sont pas fréquentes, elles sont associées à une morbidité et une mortalité élevées. Leurs principaux facteurs de risque sont la surpression du ballonnet trachéal (>30 cmH2O) et la durée de l’intubation. Hypothèse Malgré les précautions habituelles consistant à réguler la Pbal 3 fois par jour avec un manomètre manuel, les souspressions et surpressions du ballonnet trachéal sont probablement fréquentes. Si tel est le cas la régulation continue de la Pbal avec un régulateur de pression pneumatique permettrait de prévenir les microinhalations et l’ischémie trachéale liées à l’intubation. 1.Déterminer l’incidence et les facteurs de risque de souspression et de surpression du ballonnet trachéal chez les patients de réanimation. 2.Déterminer l’impact du matériau et de la forme du ballonnet sur les variations de la Pbal. 3.Déterminer l’impact de la régulation continue de la Pbal sur la survenue de complications liées à l’intubation sur un modèle animal d’abord puis chez le patient de réanimation. Incidence et facteurs de risque Tout d’abord nous avons réalisé une étude prospective observationnelle sur une cohorte de 101 patients intubés et ventilés afin de déterminer l’incidence des souspressions et surpressions du ballonnet et de déterminer leurs facteurs de risque. La Pbal a été ajustée manuellement toutes les 8h. Les Pbal et pressions des voies aériennes ont été enregistrées en continu sur 8h. Seuls 18% des patients avaient une Pbal constamment normale (20-30 cmH2O). 54% des patients ont présenté des souspressions, 73% des surpressions. De plus, 33% des patients ont présenté une souspression ou une surpression >30 minutes. Les facteurs de risque indépendamment associés à la survenue de souspressions étaient la durée d’intubation (OR=1,1(ICà95 %)1-1,2, p=0,039) et l’absence de sédation (2,5(1-6), p<0,01). Nous n’avons pas pu identifier de facteurs de risques de surpressions. 2.Impact du matériau et de la forme du ballonnet Nous avons réalisé ensuite une autre étude prospective observationnelle afin de déterminer l’impact du polyuréthane (PU) et de la forme du ballonnet sur les variations de la Pbal. 76 patients intubés et ventilés (26 ballonnets en PVC, 22 ballonnets en PU de forme cylindrique [Cy] et 28 ballonnets en PU de forme conique [Co]) ont été inclus. La Pbal a été ajustée manuellement toutes les 8h. Les Pbal et pressions des voies aériennes ont été enregistrées en continu sur 24h. Aucune différence significative n’a été retrouvée entre les 3 groupes quant au pourcentage du temps passé avec une souspression du ballonnet (moy±SD 26±22, 28±12, 30±13% dans les groupes PVC, PUCy et PUCo; respectivement) ou au pourcentage du temps passé avec une surpression (med[25è-75è centiles] 7[2-14], 6[3-14], 11%[5-20]). 3.Impact de la régulation continue de la Pbal sur la survenue de complications •Etude animale Il s’agit d’une étude randomisée contrôlée portant sur 12 porcs intubés et ventilés pendant 48h. L’objectif principal était de déterminer l’impact de la régulation continue de la Pbal sur les lésions ischémiques trachéales. / Microaspiration of contaminated oropharyngeal secretions and gastric contents represents the main pathophysiologic mechanism responsible for VAP. Tracheal tube prevents normal closure of vocal cords resulting in microaspiration of secretions towards lower respiratory tract. The cuff of tracheal tube protects, at least in part, the lower airway from microaspiration. However, folds in polyvinyl-chloride (PVC) cuffs and underinflation of tracheal cuff (<20 cmH2O) favour microaspiration around the cuff. Although, severe tracheal ischemic lesions are not common, they are associated with substantial morbidity and mortality. Their major risk factors include overinflation of tracheal cuff (>30 cmH2O), and prolonged intubation. Hypothesis In spite of routine care of cuff pressure (Pcuff) including manual regulation thrice a day using a manometer, underinflation and overinflation of tracheal cuff are probably common in critically ill patients. In that case, continuous regulation of Pcuff using a pneumatic device would allow preventing intubation-related complications such as microaspiration and tracheal ischemia. To determine incidence of and risk factors for underinflation and overinflation of tracheal cuff in critically ill patients. 2. To determine the impact of polyurethane (PU) and shape of tracheal cuff on variations of Pcuff. To determine the impact of continuous control of Pcuff on the incidence of intubation-related complications first in an animal model, and then in critically ill patients. Methods and results 1.Incidence and risk factors We performed a prospective observational cohort study on 101 intubated patients in order to determine incidence of and risk factors for underinflation and overinflation of tracheal cuff. After manual adjustment of Pcuff at 25 cmH2O, continuous recording of Pcuff and airway pressure was performed for 8 h. Only 18% of study patients spent 100% of recording time with normal (20-30 cmH2O) Pcuff. 54% of study patients developed cuff underinflation, 73% developed cuff overinflation. 33% of study patients developed underinflation or overinflation for >30 min. Absence of sedation [odds ratio (95% confidence interval)=2.5 (1-6), P=0.03] and duration of prior intubation [1.1(1-1.2), P<0.01] were independently associated with cuff underinflation. No risk factor for overinflation could be determined. 2.Impact of polyurethane and tracheal cuff shape We performed another prospective observational before-after study to determine the impact of PU, and tracheal cuff shape on variations in Pcuff in intubated critically ill patients. Pcuff was continuously recorded for 24 h in 76 intubated patients, including 26 with PVC, 22 with cylindrical (C) PU, and 28 with tapered (T) PU-cuffed tracheal tubes. Pcuff was manually adjusted every 8 h by nurses and was maintained around 25 cmH2O. Time spent with cuff underinflation and overinflation was continuously recorded. No significant difference was found in percentage of time spent with underinflation (mean±SD, 26±22, 28±12, 30±13% in PVC, CPU, and TPU groups, respectively) and overinflation [median (IQR), 7(2-14), 6(3-14), 11%(5-20)] among the three groups. 3.Impact of continuous regulation of Pcuff on intubation-related complications. •Animal study This was a prospective randomized controlled study aiming to determine the impact of continuous control of Pcuff on tracheal ischemic lesions. Twelve piglets were intubated and mechanically ventilated for 48 hours.

The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE(soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo. The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.

Global cerebral ischemia is an established animal model mimicking the effects of cardiac arrest in humans. It is characterized by selective neuronal damage in the hippocampus and significant behavioural and cognitive impairments. In this light, novel therapeutic compounds with numerous physiological targets as well as neuroprotective capabilities and the capacity to lessen residual cognitive deficits pose as great candidates in the treatment of ischemic pathology. The current thesis investigates the possible therapeutic properties of resveratrol (3, 4, 5´trihydroxystilbene), a naturally occurring phytoalexin present in the skin of grapes, against cerebral ischemia-induced neuronal degeneration and cognitive impairments, as well as elaborate on possible mechanisms of action of the compound in male Wistar rats. In Article 1, neuronal density assessment and behavioural testing following chronic pretreatment with resveratrol at two doses (1 and 10 mg/kg) revealed that the compound has important neuroprotective properties at short and long post-ischemic intervals. Despite comparable neuronal protection, the two resveratrol doses showed distinct behavioural effects, highlighting independent actions of the polyphenol on discrete physiological systems mediating cellular survival and behavioural recovery. Articles 2 and 3 investigated possible mechanisms of action of the polyphenol that have not yet been explored with regards to cerebral ischemia. Specifically, Article 2 demonstrated that resveratrol influences markers of plasticity in both ischemic and control animals as well as promotes angiogenesis in the hippocampal region postischemia. Further elaborating on documented effects attributing non-neuronal mechanisms of action of resveratrol in reducing glial activation postischemia, Article 3 highlighted important regulatory effects of resveratrol on mediating glial type-1 glutamate transporter expression at a short reperfusion interval. These findings support the notion of multiple biological targets by resveratrol and highlight its potential role in attenuating forebrain ischemia-induced neuronal degeneration through multiple physiological targets, while cautioning against possible dose-related effects on behaviour and in healthy controls.

Stroke results from disturbance in blood flow to an area of the brain, leading to neuronal dysfunction and loss. Mitochondrial dysfunction and oxidative stress are critical factors in neuropathology of stroke. They have also been implicated in Parkinson's disease (PD). Select cases of PD are caused by homozygous mutations in the PINK1 gene. Critically, this gene works with another PD gene, Parkin, to regulate mitochondrial quality control (MQC) mechanisms. Additionally, initial studies of the PINK1 protein have suggested that it plays a critical role in cellular pro-survival responses to oxidative stress though the mechanism by which it does so is unclear. In this dissertation, I explored the potential mechanisms through which PINK1 confers neuroprotection, particularly in the case of ischemic insult. I found that PINK1 deficiency sensitizes neurons to glutamate-induced excitotoxicity. I also found that the PINK1 kinase domain, but not the mitochondrial targeting motif, is essential for its protective effect. Additionally, PINK1 or Parkin deficiency significantly increases the infarct volume after middle cerebral artery occlusion, in vivo. Importantly, expression of Parkin reduces the sensitivity of neurons to cytotoxicity induced by PINK1 deficiency indicating that Parkin functionally interacts with PINK1 either through the same or on parallel survival pathways. Moreover, I investigated if PINK1 and Parkin confer neuroprotection against ischemia through PINK1/Parkin MQC pathways. However, I did not find any evidence indicating Parkin mitochondrial translocation following stroke insult suggesting that PINK1/Parkin MQC pathways are not involved in the protective functions of PINK1/Parkin. Interestingly, I found that PINK1 or Parkin deficiency decreases the level of phosphorylation of pro-survival protein AKT (pAKT) whereas expression of these genes enhances pAKT following glutamate treatment. My data also indicate that the mTORC2/AKT pathway partially mediates the neuroprotective effect of PINK1. Taken together, my data indicate that both PINK1 and Parkin play a critical neuroprotective role against ischemia and Ca2+ dysregulation in a fashion independent of mitochondrial control but dependent on AKT function.

The nature and extent of myocardial ischemic injury (Mil) produced either by coronary artery ligation/reperfusion or by injection of isoproterenol (ISO) was studied in the 10-week alloxan-diabetic rabbit. Prior to the induction of ischemic injury, investigation of the left ventricles of the diabetic rabbit after 10-weeks revealed significant magnesium depletion and inhibition of myofibrillar and sarcoplasmic reticulum ATPase activities. In addition, the activity of the lysosomal enzyme, N-acetyl-β-glucosaminidase was significantly increased in diabetic left ventricular homogenates. Ultrastructural studies revealed significant lipid and glycogen accumulation, dilatation of the sarcoplasmic reticulum and damage to the mitochondria in left ventricles of the diabetic animals. Administration of ISO to both control and diabetic animals resulted in atrial tachycardias and ventricular fibrillation. The severity of the arrhythmias and the overall mortality was the same in both groups of animals. Serum analyses revealed significantly greater increases in blood glucose, free fatty acids, total cholesterol and creatine kinase activity in the ISO-treated diabetic animals relative to ISO-treated controls. ISO treatment of both control and diabetic animals produced similar increases in heart weight, left ventricular weight and myocardial water content. Analyses of various subcellular organelle marker enzyme activities indicated a significantly greater decrease in the K⁺ ,Ca²⁺ -stimulated sarcoplasmic reticulum ATPase of ISO-treated diabetic animal hearts. In addition, significantly greater increases in Ca and hydroxyproline and decreases in the levels of ATP were evident in the ISO-treated diabetic animal hearts. Ultra-structural studies revealed significant damage to the mitochondria in both ISO-treated control and diabetic hearts, the magnitude of the damage being greater in the diabetic animals. Mitochondria from both groups of animals showed swelling and fragmentation, myofibrils appeared as a homogeneous mass and did not show the characteristic Z-lines. Glycogen depletion and lipid accumulation was observed in both groups of animals. In addition, both groups of animals showed amorphous dense bodies in the mitochondria after ISO-treatment. After 40-minutes occlusion of the left circumflex coronary artery followed by 60-minutes of reperfusion, hemodynamic measurements revealed significant decreases in the left ventricular and systemic arterial pressures in the diabetic animals relative to controls. Analyses of subcellular organelle enzymes from the ischemic tissue revealed that sarcolemmal Na⁺ ,K⁺ -ATPase, mitochondrial ATPase and sarcoplasmic reticulum ATPase activities were decreased after coronary occlusion in both control and diabetic animals. However, upon reperfusion, unlike the control, no recovery of the mitochondrial ATPase was observed in the diabetic animals. In addition, a further depression of both the sarcolemmal and sarcoplasmic reticulum ATPase activities were seen in the diabetic animals compared to controls on reperfusion. Ion measurements revealed a significant accumulation of calcium in both control and diabetic animals, the magnitude of the increase being greater in the diabetic animals. Similarly, both tissue ATP levels and the ability of the mitochondria to generate ATP were depressed in the diabetic animals as compared to controls following coronary artery occlusion and reperfusion. Following coronary artery ligation and reperfusion, the diabetic animals showed a significantly higher incidence of ventricular fibrillation and cardiogenic shock as compared to controls. Ultrastructural studies revealed myocardial damage to both control and diabetic hearts following coronary artery ligation and reperfusion. However, the diabetic myocardium showed a higher incidence and frequency of hypercontraction bands, an increase in the amorphous dense bodies and slightly greater damage to the mitochondria. Coronary artery ligation in conscious control, 6 and 12 week-diabetic rats resulted in post-ligation arrhythmias (especially ventricular fibrillation), the incidence of which was much greater in the diabetic animals. The mortality rate of 12-week diabetic rats undergoing coronary ligation was 100% within 1-7 minutes following ligation. No differences in occluded or infarcted zones of the surviving 6-week diabetic and control rats were detected. Analyses of ionic composition revealed a significant magnesium deficiency in the diabetic hearts as compared to controls. These data indicate that the diabetic animals show a greater susceptibility of the myocardium to ischemic injury. Although numerous metabolic and chemical alterations are present in the diabetic myocardium, it is possible that magnesium deficiency may be a factor determining the higher incidence of arrhythmias and ischemic injury in diabetic animals. / Pharmaceutical Sciences, Faculty of / Graduate

Acute kidney injury (AKI) is a major health burden associated with a 50% mortality rate. Of particular concern, the incidence of AKI has increased dramatically over the last decade. Yet, there is a paucity of available treatments to prevent AKI or to reduce the high rate of AKI-associated mortality. A common cause of AKI, especially in hospital settings, is prolonged decreases in renal blood flow (i.e., renal ischemia). Recent studies have demonstrated that activating the cholinergic anti-inflammatory pathway via vagal stimulation can mitigate AKI severity in rodent models of renal ischemia-reperfusion (IR) injury. While vagal stimulation is not a practical approach to prevent AKI in patients due its invasive nature and numerous side effects, recent studies have identified non-neuronal cholinergic cells within the kidney that could be targeted to reduce the severity of AKI. The overarching goal of this project is to examine the potential role of the renal cholinergic system in modulating the severity of and recovery from AKI in transgenic mice expressing green fluorescent protein (GFP) under control of the choline acetyl-transferase (ChAT) promoter, a protein involved in the synthesis of acetylcholine. The objectives of this study were to develop a clinically relevant model of renal IR-induced AKI in mice by identifying the duration of ischemia required for manifestation of the effects of AKI and to determine whether differences in susceptibility to AKI exists between male and female mice. Initially, male mice underwent 20 (n=3), 22 (n=3), or 25 (n=4) minutes of bilateral renal IR under isoflurane anesthesia with body temperature controlled at 37°C. Ischemia was achieved by careful placement of vascular clamps on the renal artery and vein supplying each kidney. The severity of AKI was determined by measuring serum creatinine (SCr) at 3 days post-AKI. Compared to SCr of mice that were 3 days post-sham AKI (SCr = 0.47 mg/dl, n=2), SCr of male mice from all three ischemia time categories was substantially elevated (SCr > 3 mg/dl, n=10). However, mortality associated with 22 and 25 minutes IR was striking (>90%) making studies of long-term AKI effects difficult. In contrast, 20 minutes IR resulted in AKI manifest by elevated SCr (3.43±0.7 mg/dl, n=3), widespread acute tubular necrosis and a clinically relevant mortality rate of 50%. Next, male (n=10) and female (n=5) mice were subjected to 20 minutes of IR. The mortality rate in male mice (n=10) was 50% (n=10) through 7 days post-AKI; however, all female mice survived. Additional studies showed that female mice had lower SCr 3 days post-AKI (0.63±0.1 mg/dl, n=2) with very modest levels of acute tubular necrosis as compared to the higher SCr (1.92±0.1 mg/dl, n=2) and extensive acute tubular necrosis observed in male mice. The differences observed in AKI severity and mortality rates suggest that female mice are protected against AKI as compared to male mice and future studies will explore the potential role of the renal cholinergic system in contributing to these sex differences in AKI.

Hypothesis: Glucose is known to be protective in moderate low flow ischaemia due to the production of glycolytic ATP. However, it is questioned whether glucose would still be protective in ultra-low flow ischaemia. Firstly, glycolysis is thought to be inhibited, and secondly, deleterious glycolytic metabolites accumulate. Our hypothesis was that in ultra-low flow ischaemia, glucose utilisation is not inhibited at the level of glycolysis, but by delivery. Increased delivery of glucose should result in increased production of protective glycolytic ATP, but the rate of metabolite accumulation would also increase. Using ultra low flow rates, I wished to investigate how to achieve optimal rates of glycolysis, and how such rates would be balanced by any detrimental component of metabolite accumulation. Methods: The isolated Langendorff-perfused rat heart, with a left ventricular balloon to record ischaemic contracture and reperfusion stunning, was used, with severe flow restriction. Glucose concentrations were changed and pre-ischaemic glycogen contents were altered by perfusion with different substrates (acetate - depletion~ glucose + insulin - loading) or by preconditioning, with 5 min ischaemia and 5 min reperfusion prior to sustained ischaemia. Results: Analysis of glucose uptake relative to delivery showed that in severe low flow ischaemia, the extraction of glucose was increased, and glycolysis was thus limited more by substrate supply than by enzyme inhibition. Analysis of metabolites confirmed this concept. The optimal glucose concentration during severe low flow ischaemia was 11 mM, giving maximal recovery on reperfusion. Both lower and higher glucose concentrations increased ischaemic contracture. Changes in pre-ischaemic glycogen levels correlated with the time to onset of contracture, such that a reduction in glycogen accelerated contracture. Prior glycogen depletion or loading did not improve functional recovery. The benefits of preconditioning on reperfusion function following sustained total global ischaemia could not be related to glycogen depletion. If preconditioning were followed by sustained low flow ischaemia, glucose uptake was increased, but no benefit was found, possibly because a low residual flow abolished the effects of preconditioning. Many of the above results are consistent with the hypothesis that too low a rate of glycolysis results. in insufficient ATP production for protection, while excess glycolytic rates lead to excess metabolite accumulation with detrimental effects. Conclusions: Provision of glucose at the correct concentration, when the benefit associated with glycolytic ATP outweighs the detriment associated with moderate metabolite accumulation, is protective to the low-flow ischaemic myocardium, which can upregulate its ability to extract glucose. Improved residual flow enhances this benefit. Prior glycogen depletion is not beneficial, despite a reduced metabolite accumulation. This mechanism cannot be related to the protective effect of preconditioning.

Dexrazoxane (DEX) is clinically used to reduce cardiotoxic efects of anthracycline cytostatics. Its cardioprotective efect is caused by chelatation of free iron and defends myocard against dangerous hydroxyl radicals. This research finds out how dexrazoxane works in ischemic-reperfusion damages of rat's heart. Each rat was infused by DEX (50, 150, 450 mg/kg) or by control solution. Isolated perfused rat's hearts were exposed to local ischemia for 30 minutes than 10 minutes of reperfusion for studing ischemic arrhythmias followed by 15 minutes of local ischemia and 10 minutes of reperfusion to examine reperfusion arrhythmias. For evaluation of EKG (ventricular arrhythmias) was used software CAR and Lambeth convention. Global ischemias (15 min.) were induced in rat's hearts (DEX 150 mg/kg) and left ventricules were used for HPLC to determinate concentration of glutathion. In vivo experiments rats were infused by DEX 50, 150 mg/kg or control solution and were exposed for 20 minutes to local ischemia and for 3 hours to reperfusion. Infarct size was evaluated based on the cross section of heart (GIMP, Ellipse). Maximum total number of ischemic arrhytmias decreased by DEX 150 mg/kg (64% comparing to controls). Reperfusion score was reduced by DEX 150 to 48% and percents of ventricular fibrilation was...

Recent studies have shown that Toll-like receptors (TLRs) are involved in cerebral ischemia/reperfusion (I/R) injury. This study was to investigate the role of TLR2 and TLR4 in acute focal cerebral I/R injury. Cerebral infarct size, neurological function and mortality were evaluated. NFk{cyrillic}B binding activity, phosphorylation of Ik{cyrillic}Bα, Akt and ERK1/2 were examined in ischemic cerebral tissue by EMSA and Western blots. Compared to wild type (WT) mice, in TLR4 knockout (TLR4KO) mice, brain infarct size was decreased (2.6 ± 1.18% vs 11.6 ± 1.97% of whole cerebral volume, p < 0.05) and neurological function was maintained (7.3 ± 0.79 vs 4.7 ± 0.68, p < 0.05). However, compared to TLR4KO mice, TLR2 knockout (TLR2KO) mice showed higher mortality (38.2% vs 13.0%, p < 0.05), decreased neurological function (2.9 ± 0.53 vs 7.3 ± 0.79, p < 0.05) and increased brain infarct size (19.1 ± 1.33% vs 2.6 ± 1.18%, p < 0.05). NFk{cyrillic}B activation and Ik{cyrillic}Bα phosphorylation were attenuated in TLR4KO mice (1.09 ± 0.02 and 1.2 ± 0.04) compared to TLR2KO mice (1.31 ± 0.02 and 2.2 ± 0.32) after cerebral ischemia. Compared to TLR4KO mice, in TLR2KO mice, the phosphorylation of Akt (0.2 ± 0.03 vs 0.9 ± 0.16, p < 0.05) and ERK1/2 (0.8 ± 0.06 vs 1.3 ± 0.17) evoked by cerebral I/R was attenuated. The present study demonstrates that TLR2 and TLR4 play differential roles in acute cerebral I/R injury. Specifically, TLR4 contributes to cerebral I/R injury, while TLR2 appears to be neuroprotective by enhancing the activation of protective signaling in response to cerebral I/R.

Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca2+-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H2O2 generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca2+ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca2+ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca2+ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.