MSc (Med), University of the Witwatersrand, Faculty of Health Sciences, School of Physiology / The role of metabolic remodelling in heart failure is not fully understood, significant evidence has accumulated to suggest that it may be central to the development of left ventricular (LV) remodelling and LV dysfunction. Heart failure is also characterized by sustained neurohumoral activation. We have previously demonstrated that chronic low dose administration of isoproterenol contributes to cardiac structural and functional changes, however, little is known about metabolic and mitochondrial changes that may accompany the development of isoproterenol-mediated heart failure. In the current study, we hypothesised that metabolic dysregulation and loss of mitochondrial integrity mediates left ventricular hypertrophy (LVH) and left ventricular (LV) systolic dysfunction in the isoproterenol model of heart failure. Furthermore, modulation of expression of key metabolic genes and mitochondrial transcription factors by fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, will preserve left ventricular function. To achieve this, male Sprague-Dawley rats weighing between 250-300g were injected with low dose isoproterenol (0.04 mg.kg-1.day-1) and/or administered with fenofibrate (100 mg.kg-1.day-1) for five weeks. Thereafter, metabolic substrates such as glucose, FFAs and TG concentrations were obtained. Left ventricular hypertrophy (LVH) and cardiac function were assessed using echocardiography. Expressions of metabolic and mitochondrial genes such as PPARα, AMP-activated protein kinase alpha 2 (AMPKα2), PPARγ coactivator-1 (PGC-1α), mitochondrial transcription factor (TFAM) and nuclear respiratory factor-1 (NRF-1) were determined using real-time polymerase chain reaction. Mitochondrial integrity was assessed using transmission electron microscopy. Administration of isoproterenol significantly increased left ventricular mass (LVM) and decreased endocardial fractional shortening (FSend); isoproterenol also induced myofibrillar iv derangement, mitochondrial derangement and cristae disruption. Fenofibrate prevented isoproterenol-induced increase in LVM and improved FSend. Fenofibrate co-administration prevented loss of mitochondrial integrity possibly via TFAM. Furthermore, fenofibrate may have induced metabolic remodelling via upregulation of AMPKα2 and downregulation of cardiac PPARα and PGC-1α. Therefore our data suggests that fenofibrate-mediated cardioprotection against isoproterenol-induced LVH and LV systolic dysfunction was accompanied by metabolic switching and preservation of mitochondrial integrity. While isoproterenol did not induce any changes in metabolic genes, fenofibrate-mediated cardioprotection could have been through changes in metabolic genes.

The role of lipids and ROS in cardioprotective mechanism of chronic hypoxia Cardiovascular diseases, mainly ischemic heart disease is one of the most frequently cause of morbidity and mortality in developed countries. Therefore effective protection of the heart against ischemia and reperfusion injury is the crucial aim of experimental and clinical cardiology. One of the main streams of cardiovascular research is looking for possibilities of natural heart resistance augmentation. Adaptation to chronic hypoxia is one possibility how to protect the heart against ischemia-reperfusion injury. Chronic hypoxia increases resistance of the myocardium to acute deficiency of oxygen leading to vetricular arrythmias, postischemic contractile dysfunction and necrotic changes in the tissue. Recently, it has been shown that reactive oxygen species (ROS) play an important role in the cardioprotective mechanism of chronic hypoxia. It is known that oxidative stress has a harmful effect in acute ischemia-reperfusion however ROS generated during the adaptation to hypobaric intermittent chronic hypoxia play a role in the induction of cardioprotection. In this study, we demonstrated that adaptation of adult rats to chronic hypoxia increased the activity and protein abundance of manganese superoxide dismutase (MnSOD) in the...

Hypoxia-inducible factor- 1α (HIF-1α) is a transcription factor that directs many of the cellular responses to hypoxia. In these studies, we have used a mouse model containing a cardiac-specific, oxygen-stabilized, doxycycline (Dox)-off regulated HIF-1α transgene to probe the role of HIF-1α in cardioprotection. Hearts used in these studies were derived from wild-type (WT), noninduced (Non-I), and 2 day (2D) and 6 day (6D) Dox-deprived mice. Whereas HIF-1α protein is undetectable in WT mice, it is present in heart tissue of "noninduced" transgenic mice, presumably because of leakiness of the promoter construct. In mice denied Dox for 2 or 6 days, HIF-1α is overexpressed to a much greater extent than Non-I or WT animals, as expected. WT and HIF-1α- expressing hearts (Non-I, 2D and 6D induced) were subjected to 30 min of ischemia, and functional recovery was measured upon reperfusion. Recovery of preischemic left ventricular developed pressure was 14% for WT, 67% for Non-I hearts, 64% for 2D-induced, and 62% for 6D-induced hearts. 6D-induced HIF hearts have increased preischemic glycogen reserves, higher glycogen synthase protein levels, and significantly higher lactic acid release during ischemia. 6D-induced HIF hearts were also better able to maintain ATP levels during ischemia compared with WT and Non-I hearts. Interestingly, Non-I hearts showed no significant increase in glycogen reserves, glycolytic flux, or greater ATP preservation during ischemia and yet were protected to a similar extent as the 6D-induced hearts. Finally, the mitochondrial membrane potential of isolated adult myocytes was monitored during anoxia or treatments with cyanide and 2-deoxyglucose. HIF-1α expression was shown to protect mitochondrial polarization during both stress treatments. Taken together these data indicate that, while HIF-1α expression in heart does induce increases in compensatory glycolytic capacity, these changes are not necessarily required for cardioprotection, at least in this model of ischemic stress.

Hypoxia-inducible factor- 1α (HIF-1α) is a transcription factor that directs many of the cellular responses to hypoxia. In these studies, we have used a mouse model containing a cardiac-specific, oxygen-stabilized, doxycycline (Dox)-off regulated HIF-1α transgene to probe the role of HIF-1α in cardioprotection. Hearts used in these studies were derived from wild-type (WT), noninduced (Non-I), and 2 day (2D) and 6 day (6D) Dox-deprived mice. Whereas HIF-1α protein is undetectable in WT mice, it is present in heart tissue of "noninduced" transgenic mice, presumably because of leakiness of the promoter construct. In mice denied Dox for 2 or 6 days, HIF-1α is overexpressed to a much greater extent than Non-I or WT animals, as expected. WT and HIF-1α- expressing hearts (Non-I, 2D and 6D induced) were subjected to 30 min of ischemia, and functional recovery was measured upon reperfusion. Recovery of preischemic left ventricular developed pressure was 14% for WT, 67% for Non-I hearts, 64% for 2D-induced, and 62% for 6D-induced hearts. 6D-induced HIF hearts have increased preischemic glycogen reserves, higher glycogen synthase protein levels, and significantly higher lactic acid release during ischemia. 6D-induced HIF hearts were also better able to maintain ATP levels during ischemia compared with WT and Non-I hearts. Interestingly, Non-I hearts showed no significant increase in glycogen reserves, glycolytic flux, or greater ATP preservation during ischemia and yet were protected to a similar extent as the 6D-induced hearts. Finally, the mitochondrial membrane potential of isolated adult myocytes was monitored during anoxia or treatments with cyanide and 2-deoxyglucose. HIF-1α expression was shown to protect mitochondrial polarization during both stress treatments. Taken together these data indicate that, while HIF-1α expression in heart does induce increases in compensatory glycolytic capacity, these changes are not necessarily required for cardioprotection, at least in this model of ischemic stress.

Background: Erythropoietin (EPO) can reduce myocardial ischemia/reperfusion (I/R) injury. However, the cellular mechanisms have not been elucidated entirely. The present study was to investigate whether transcription factor GATA-4 could be involved in EPO-induced cardioprotection when it was administered after ischemia, immediately before reperfusion. Methods and results: Male Balb/c mice treated with or without EPO were subjected to ischemia (45 min) followed by reperfusion (4 h). TTC staining showed that the infarct size in EPO-treated mice was significantly reduced compared with untreated I/R mice (P < 0.05). Echocardiography examination suggested that EPO administration significantly improved cardiac function following I/R. TUNEL assay indicated that EPO treatment decreased apoptosis. EPO administration also significantly increased the level of nuclear GATA-4 phosphorylation in the myocardium which was positively correlated with the reduction of myocardial infarction. In vitro hypoxia/re-oxygenation study showed that EPO treatment increased the levels of phospho-GATA-4 and decreased cardiomyocyte apoptosis. More significantly, blocking GATA-4 by transfection of a dominant-negative form of GATA-4 (dnGATA-4) abolished EPO-induced cardioprotective effects. Conclusion: EPO administration after ischemia, just before reperfusion induced cardioprotection and stimulated GATA-4 phosphorylation. Activation of GATA-4 may be one of the mechanisms by which EPO induced protection against myocardial I/R injury.

Aims: The ability of lipopolysaccharide (LPS) pre-treatment to induce cardioprotection following ischaemia/reperfusion (I/R) has been well documented; however, the mechanisms have not been fully elucidated. LPS is a Toll-like receptor 4 (TLR4) ligand. Recent evidence indicates that there is cross-talk between the TLR and phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathways. We hypothesized that activation of PI3K/Akt signalling plays a critical role in LPS-induced cardioprotection. Methods and results: To evaluate this hypothesis, we pre-treated mice with LPS 24 h before the hearts were subjected to ischaemia (45 min) and reperfusion (4 h). We examined activation of the PI3K/Akt/GSK-3β signalling pathway. The effect of PI3K/Akt inhibition on LPS-induced cardioprotection was also evaluated. LPS pre-treatment significantly reduced infarct size (71.25%) compared with the untreated group (9.3 ± 1.58 vs. 32.3 ± 2.92%, P < 0.01). Cardiac myocyte apoptosis and caspase-3 activity in LPS-pre-treated mice were significantly reduced following I/R. LPS pre-treatment significantly increased the levels of phospho-Akt, phospho-GSK-3β, and heat shock protein 27 in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase-defective Akt transgenic mice abolished the cardioprotection induced by LPS. Conclusion: These results indicate that LPS-induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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L’infarctus du myocarde, problème majeur de santé publique, est caractérisé par une nécrose cardiomyocytaire. Des séries d’occlusions-reperfusions courtes, réalisées avant l’ischémie (Préconditionnement (PréC) ischémique) ou au moment de la reperfusion (Postconditionnement (PostC) ischémique), protègent le coeur contre des lésions d’ischémie-reperfusion (IR). Les mécanismes intracellulaires impliqués restent obscurs. Nous avons étudié la signalisation intracellulaire du PréC et du PostC, et la cardioprotection qui en découle, sur un modèle de coeur isolé perfusé de souris. Le PréC ischémique peut être mimé par une activation directe du canal potassique mitochondrial ATP dépendant (mitoKATP), entraînant la mise en place d’une boucle d’auto-amplification incluant l’activation d’Akt, l’inhibition de GSK-3β et l’ouverture du mitoKATP. Cette réponse est liée à la production modérée d’espèces dérivées de l’oxygène par le mitoKATP et aboutie à une cardioprotection. La voie de développement Wnt est capable de moduler le PréC via GSK-3β. La voie de survie mTOR, cible de GSK-3β est aussi impliquée et pourrait induire des modifications traductionnelles lors de la réponse adaptative à l’IR. Le PostC ischémique peut également être mimé par activation directe du mitoKATP lors de la reperfusion, engendrant une protection du coeur et la mise en place d’une boucle d’auto-amplification similaire à celle du PréC, comprenant Akt, GSK-3β et le mitoKATP. Le PostC est dépendant de GSK-3β, mais contrairement au PréC, il n’impliquerait pas les voies Wnt et mTOR. Cette étude est la première démontrant que le PréC implique les voies de survie mTOR et de développement Wnt avec un rôle central de GSK-3β. / Myocardial infarction is a major problem of public health, whose prognosis is related to the extent of the infarcted territory. Transient episodes of ischemia/reperfusion before ischemia (ischemic PreConditioning (PreC)), or at the onset of reperfusion (ischemic PostConditioning (PostC)) confer myocardium resistance to lethal ischemia. However the exact mechanism of PreC and PostC remains obscure. Our objectives were to examine signaling events during PreC and PostC and their effects on cardioprotection in an isolated mouse heart model. We provide evidence that pharmacological PreC by direct activation of mitoKATP, like ischemic PreC, involve an amplification loop involving ROS production and resulting in a sustained down-regulation of GSK-3β via Akt activation and a constant opening of mitoKATP. The mTOR pathway is a target of this loop and participates to cardioprotection. Disruption of Wnt pathway by sFRP1 modulates this loop inducing GSK-3β activation. This is the first evidence that PreC involves both a pro-survival mTOR pathway and an embryonic developmental Wnt pathway targeting GSK-3β. During ischemic and pharmacological PostC, the same amplification loop is activated, including Akt, GSK-3β and the mitoKATP. Unlike PreC, PostC did not induce the mTOR survival pathway: neither phosphorylation of mTOR nor of its targets p70S6K and 4E-BP1 were observed. However, cardiac overexpression of a Wnt antagonist, impairing PreC through GSK3-β, was unable to abolish cardioprotection afforded by PostC. PostC signaling differs from the preC pathway. Despite these discrepancies, GSK-3β plays a key role in both types of cardioprotection.

Introduction : La prévention de la mort de cellules cardiaques contractiles suite à un épisode d'infarctus du myocarde représente le plus grand défi dans la récupération de la fonction cardiaque. On a démontré à maintes reprises que l'ocytocine (OT), l'hormone bien connue pour ses rôles dans le comportement social et reproductif et couramment utilisée dans l’induction de l’accouchement, diminue la taille de l'infarctus et améliore la récupération fonctionnelle du myocarde blessé. Les mécanismes de cette protection ne sont pas totalement compris. Objectif : Étudier les effets d'un traitement avec de l'ocytocine sur des cardiomyocytes isolés en utilisant un modèle in vitro qui simule les conditions d'un infarctus du myocarde. Méthodes : La lignée cellulaire myoblastique H9c2 a été utilisée comme modèle de cardiomyocyte. Pour simuler le dommage d'ischémie-reperfusion (IR), les cellules ont été placées dans un tampon ischémique et incubées dans une chambre anoxique pendant 2 heures. La reperfusion a été accomplie par la restauration du milieu de culture régulier dans des conditions normales d'oxygène. L'OT a été administrée en présence ou en absence d'inhibiteurs de kinases connues pour être impliquées dans la cardioprotection. La mortalité cellulaire a été évaluée par TUNEL et l'activité mitochondriale par la production de formazan pendant 1 à 4 heures de reperfusion. La microscopie confocale a servie pour localiser les structures cellulaires. Résultats : Le modèle expérimental de l'IR dans les cellules H9c2 a été caractérisé par une diminution dans la production de formazan (aux alentours de 50 à 70 % du groupe témoin, p < 0.001) et par l'augmentation du nombre de noyaux TUNEL-positif (11.7 ± 4.5% contre 1.3 ± 0.7% pour le contrôle). L'addition de l'OT (10-7 a 10-9 M) au commencement de la reperfusion a inversé les effets de l'IR jusqu'aux niveaux du contrôle (p < 0.001). L'effet protecteur de l'OT a été abrogé par : i) un antagoniste de l'OT ; ii) le knockdown de l'expression du récepteur à l'OT induit par le siRNA ; iii) la wortmannin, l'inhibiteur de phosphatidylinositol 3-kinases ; iv) KT5823, l'inhibiteur de la protéine kinase dépendante du cGMP (PKG); v) l'ODQ, un inhibiteur du guanylate cyclase (GC) soluble, et A71915, un antagoniste du GC membranaire. L'analyse confocale des cellules traitées avec OT a révélé la translocation du récepteur à l'OT et la forme phosphorylée de l'Akt (Thr 308, p-Akt) dans le noyau et dans les mitochondries. Conclusions : L'OT protège directement la viabilité des cardiomyocytes, lorsqu'elle est administrée au début de la reperfusion, par le déclenchement de la signalisation du PI3K, la phosphorylation de l'Akt et son trafic cellulaire. La cytoprotection médiée par l'OT implique la production de cGMP par les deux formes de GC. / Introduction: The prevention of the death of contractile cardiac cells following an episode of myocardial infarction represents the largest challenge in the recovery of myocardial function. Oxytocin, the hormone best known for its roles in reproduction and social behaviour and used commonly for the induction of parturition, has been repeatedly demonstrated to decrease the infarct size and to ameliorate the functional recovery of the injured myocardium. The mechanisms for this protection are incompletely understood. Objective: To study the effects of oxytocin treatment on isolated cardiomyocytes using an in vitro model simulating the conditions of a myocardial infarction. Methods: The cardiomyoblastic cell line H9c2 was used as a model of cardiomyocyte. For IR injury, the cells were placed in ischemic buffer and incubated in an anoxic chamber for 2 hours. Reperfusion was achieved by restoring cell media under normoxic conditions. OT was administered in the presence or absence of enzyme inhibitors. Cell death was evaluated by TUNEL and mitochondrial activity by formazan production during 1-4 hours of reperfusion. Confocal microscopy served for localization of cell structures. Results. The experimental model of IR in H9c2 cells was characterized by decreased formazan production (at the range of 50-70% of normoxic control, p < 0.001) and by the increased number of TUNEL-positive nuclei (11.7±4.5 vs. 1.3±0.7% in normoxic control). The addition of OT (10-7 to 10-9 M) at the onset of reperfusion reversed the effects of IR to the control levels (p < 0.001). The protective effect of OT was abrogated by: i) an OT antagonist, OTA and siRNA-mediated OT receptor knockout; ii) the phosphatidylinositol 3-kinases inhibitor wortmannin; iii) the cGMP-dependent protein kinase (PKG) inhibitor, KT5823. Soluble guanylate cyclase (GC) inhibitor ODQ and particulate GC antagonist A71915 only partially blocked the protective effects of OT. Confocal analysis of OT-treated cells revealed translocation of OT receptor and the phosphorylated form of Akt (Thr 308, p-Akt) into the nucleus and mitochondria. Conclusions: OT directly protects cardiomyocyte viability if administered at the onset of reperfusion by triggering signaling of Pi3K, Akt phosphorylation and its cellular trafficking. OT-mediated cytoprotection involves cGMP production by both forms of GC.