published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

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

Phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. We hypothesized that SIL-induced protection may be mediated through activation of SIRT1, an enzyme which deacetylates proteins involved in cellular stress response. Adult male ICR mice were treated with SIL (0.7mg/kg ip), Resveratrol (RSV) (5mg/kg ip) (positive control), or saline (0.2 ml ip). The hearts were harvested 24 h later and homogenized for SIRT1 activity analysis. Both SIL and RSV increased cardiac SIRT1 activity (P<0.001) as compared to Saline. Adult mouse ventricular cardiomyocytes pre-treated with either SIL or RSV (1µM) in vitro also upregulated SIRT1 activity (P<0.05). SIL also reduced infarct size following 30 min. ischemia and 24 h reperfusion in vivo. Sirtinol (5mg/kg in 10% DMSO, ip), a SIRT1 inhibitor abolished the infarct-limiting effect of SIL and RSV (P<0.001). In conclusion, activation of SIRT1 by SIL plays an essential role in cardioprotection against I-R injury.

Administration of small interfering RNA (siRNA) specific for prolyl-4 hydroxylase-2 (PHD2) results in PHD2 inhibition, Hypoxia Inducible Factor-I (HIF-1) activation, and cardioprotection versus Ischemia Reperfusion (IR). This study observes the effects of siRNA-mediated PHD2 inhibition on the distribution of cardioprotective proteins by immunofluorescence and basic histology. Fifteen mice were divided into 5 groups: PHD2 Control, Non-Targeting scramble (NTS) Control, IR Control, PHD2 IR, and NTS IR. Histologically, tissue damage was reduced dramatically in the PHD2 IR group compared to the NTS IR and IR control groups. From confocal images, total fluorescent pixels and intensities were quantified. The PHD2 IR group yielded the highest pixel quantity and intensity for HIF-1 and possessed increased pixels and intensity for Inducible Nitric Oxide Synthase, another cardioprotective protein. These results further demonstrate the cardioprotection and HIF-1 activation conferred by PHD2 siRNA administration and supports its role as a potential therapy to alleviate cardiac IR injury.

Recent studies have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor, sildenafil citrate, induces a powerful cardioprotective effect against ischemia-reperfusion (I/R) injury in rabbit and mouse hearts. However, the effect of this drug in inducing long-term protection against I/R injury remains unknown. The goal of this study was to identify the duration of the protective window of sildenafil citrate as well as vardenafil, a more potent PDE-5 inhibitor. Rabbits were treated with sildenafil (0.7 mg/kg, iv), vardenafil (0.143 mg/kg), or an equivalent volume of saline. After 24 hrs, 48 hrs, 96 hrs, or 7 days of sildenafil treatment, the hearts were subjected to I/R. In the vardenafil groups, the hearts were subjected to I/R at 24 hrs and 7 days after administration of the drug. To evaluate the role of nitric oxide (NO) in cardioprotection, a non-selective blocker of nitric oxide synthase, L-NAME (15 mg/kg, iv) was administered 10 minutes prior to I/R. The results show significant reductions in infarct size in hearts treated with sildenafil and vardenafil as compared to the corresponding saline controls at all time points. The protective effects of sildenafil and vardenafil were abrogated in animals treated with L-NAME. L-NAME had no effect on infarct size in saline treated control rabbits. These data suggest that both sildenafil and vardenafil induce a long-term protective effect against myocardial infarction which is mediated via a NO-dependent pathway. These studies are important in exploiting the clinical potential of PDE-5 inhibitors in terms of protection against ischemia/reperfusion injury in patients with coronary artery disease.

Cardiac injury is increased following ischemia-reperfusion. Mitochondria are the “effector organelles” that are damaged during ischemia (ISC) when there is no blood flow. Resumption of metabolism by damaged mitochondria during reperfusion (REP) results in increased cell injury. Current therapeutic interventions to pre-condition and post-condition the heart during ISC are ineffective during certain conditions like aging and diabetes due to defects in the signaling cascades. In contrast, mitochondrial-based strategies are effective in protecting the heart during ISC-REP. Hence direct therapeutic targeting of dysfunctional mitochondria will provide the potential to bypass the upstream signaling defects and intervene directly upon the effector organelle. Novel mitochondrial-targeted therapy relies on understanding the sites in the electron transport chain (ETC) that are damaged by ISC and produce cell-injury during REP. This project identifies a novel pathological role of cytochrome c in depleting cardiolipin during ischemia after which the mitochondria are in a defective condition that leads to additional cell death during reperfusion. During ischemia oxidants from complex III oxidize cytochrome c, forming a peroxidase, which causes oxidative damage and depletion of cardiolipin. Depletion of cardiolipin disrupts normal physiology and augments cell death. Identification of the innovative pathobiology during ISC-REP recognizes a novel therapeutic target, cytochrome c peroxidase, which can be a focal point for new therapeutic interventions to decrease cardiac injury. In order to maintain homeostatis, living organisms have the methionine sulfoxide reductase system, which reduce both free and protein bound Met(O) back to methionine (Met) in the presence of thioredoxin. Oxidized Trx is inactive and unable to bind to ASK1 thereby activating ASK1 and causing cell death via p38/JNK pathways thereby contributing to the pathogenesis of myocardial ISC-REP injury. In this study we have shown that inhibition of ASK1 protects the heart during REP via the modulation of mitochondria that sustained damage during ISC. The mitochondrial-based mechanism of cardioprotection with ASK1 inhibition enhanced the functional integrity of the inner mitochondrial membrane retaining cytochrome c thereby decreasing cell death. This therapeutic intervention is a key step to achieve the ultimate goal to improve clinical outcomes in patients that suffer an acute myocardial infarction.

In resting muscle, blood flow is regulated to meet the demand for O2 by the tissue. A modified ischemia (I)/reperfusion(R) investigation was systematically run and PISFO2, PaO2, Q and VO2 were observed. Twenty-nine spinotrapezius muscles from male Sprague-Dawley rats (284±20 grams) were surgically exteriorized for intravital microscopy to test a model relating blood flow, O2 supply and O2 demand. The model can aid in the understanding of the regulation of tissue PO2. The interstitial PO2 (PISFO2) and perivascular PO2 (PaO2) measurements were made using phosphorescence quenching microscopy (PQM). O2 consumption (VO2) values were obtained with a quasi-continuous, flash-synchronized, pressurized airbag to initiate ischemia and sample the rate of O¬2 change (dPO2/dt). Centerline red blood cell velocity was measured with an Optical Doppler Velocimeter and converted to flow using vessel diameter. 5-, 15-, 30-, 60-, 300- and 600-second ischemic durations were used to observe changes in PISFO2, Q, and VO2. A critical point was observed following 30 seconds of (I) where dPISFO2/dt during recovery was the fastest (4.25±0.72 mmHg/s) and was 1.00±0.16 mmHg/s following 600 seconds. Flow recovery, dQ/dt, peaked to 3.88±0.64 (µl•min-1)/s after 60 seconds of (I) but significantly dropped to 2.83±0.55 (µl•min-1)/s following 300 seconds of (I) but increased to 2.92±0.45 (µl•min-1)/s following 600 seconds. This gives evidence to a no-reflow phenomenon occurring in the extended periods of ischemia. A peak in VO¬2 to 309.2±45.0 nl O2/cm3•s with a time course of 160 seconds occurred following 600 seconds of ischemia. As the ischemic duration decreased, the time course and peak VO2 also decreased. VO2 following 300 seconds of (I) was significantly higher than 5-60 seconds of (I) (p <0.05) but was not significantly different from 600 seconds of (I). The information collected during the Q and VO2 studies can be incorporated into a factor, M, that relates VO2, Q and ∆PO2. M calculated for the recovery of 5- through 60-second (I) groups reasonably relates the three variables due to consistency and little variability. However, recovery in 600- and especially 300-second (I) groups showed higher variability in M which requires more consideration.

Acute myocardial infarction (AMI) is the leading cause of death worldwide. Currently, the best method of treating cardiac ischemia is early reperfusion which, itself, induces myocardial damage. The mTOR complex is a key regulator of cardioprotection against cell stressors. We hypothesized that reperfusion therapy with Rapamycin, a potent mTOR inhibitor, would reduce infarct size in adult mouse hearts. Rapamycin was administered at the onset of reperfusion following 30 min in situ LAD ligation. After 24 hours of reperfusion, myocardial infarct size and apoptosis were significantly reduced in rapamycin-treated mice compared to control. Rapamycin inhibited pro-apoptotic protein Bax and phosphorylation of ribosomal protein S6 (target of mTORC1), while it induced phosphorylation of AKT (target of mTORC2). Rapamycin also induced phosphorylation of ERK, while significantly reduced phosphorylation of p38. Thus, our study shows that reperfusion therapy with Rapamycin provides cardioprotection through induction of the phosphorylation of Akt and ERK.

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