Background: Heart disease remains the leading cause of morbidity and mortality worldwide. Typical symptoms of heart disease are lacking in nearly one-third of patients with acute myocardial infarction (AMI). Simplified ECG assessment via lead I by various handheld and smartphone-based electrocardiogram (ECG) devices may be used for rapid screening without the traditional delays, privacy concerns, or costs of 12-lead ECG recording in patients who are asymptomatic or have atypical symptoms. The purpose of this DNP project was to compare ECG data from lead I to the standard 12-lead ECG to determine its potential efficacy as an early screening tool for AMI. Methods: This project compared ECGs in 84 patients with cardiac diagnoses, 66 (78.6%) had acute myocardial infarction with abnormal 12-lead ECGs and 18 (22.6 %) were without AMI or abnormal findings on the standard 12-lead ECG. ST-segment and T-wave amplitude and characteristics were compared between infarction territories. Results: Lead I in those with abnormal ECGs had a mean ST-segment deviation from baseline of 1.0 ± 0.7 mm, which was significantly different than those with normal ECGs (mean 0.1 ± 0.3 mm)(p = .000). The mean T-wave amplitude in patients with abnormal ECGs was 0.7 ± 1.3 mm, which was a significant reduction compared to those with normal ECGs group of 2.2 ± 1.5 mm (p = .000). Conclusion: ST-segment deviations and reduction in T-wave amplitude are significant indicators of acute myocardial infarction in lead I ECG, the same vector used in handheld single-lead ECG devices.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Anandamide (AEA), a well characterized endocannabinoid that has actions at multiple targets in the eye, may have potential as a novel therapeutic in the treatment of retinal disease. However, AEA is rapidly degraded by fatty acid amide hydrolase (FAAH). Therefore this study examined the drug URB597, that inhibits FAAH degradation of AEA, to assess AEA effects in experimental models of retinal damage. The objectives were to: 1) evaluate changes present in the aging retina, 2) determine whether the aging retina is more susceptible to tissue damage, and 3) investigate whether increasing AEA can provide retinal neurovascular protection in young and aged retina following damage. The results from this study showed that URB597 had protective effects on retinal ganglion cells and retinal capillaries and inhibited phagocytotic MG in models of retinal damage in young, but not the aged retina.

Insulin and cellular stressors both activate p38 MAPK. Insulin protects cardiac tissue in a p38 MAPK-dependent manner. Paradoxically, inhibiting p38 MAPK is also protective. Hsp27 phosphorylation is regulated by p38 MAPK. Insulin was tested in H9c2 cardiomyocytes subjected to media exchange, 6 hours of oxygen-glucose deprivation, and reoxygenation. Insulin suppressed stress-induced phosphorylation of Hsp27 due to media-exchange or oxygen-glucose deprivation. Surprisingly, insulin increased Hsp27 phosphorylation during reoxygenation. Insulin also reduced total p38 MAPK levels. Insulin before oxygen-glucose deprivation prevented both localization of Hsp27 to the nucleus and localization of phospho-p38 MAPK to the cytoplasm. Insulin during oxygen-glucose deprivation caused the localization of phospho-p38 MAPK in the cytoplasm, but did not increase Hsp27 phosphorylation until reoxygenation. In conclusion, insulin may protect before oxygen-glucose deprivation by redirecting phospho-p38 MAPK to the nucleus away from damaging pathways in the cytoplasm and protects during oxygen-glucose deprivation by priming phospho-p38 MAPK to phosphorylate Hsp27. / Insulin was used on a model on H9c2 myotubes to determine the effect of oxygen-glucose deprivation and reoxygenation on the localization and phosphorylation of Hsp27 and p38 MAPK

Transplantation is the optimal form of therapy for patients with end-stage liver disease; however, the use of organs with hepatic steatosis is often associated with increased risks for poor function and graft loss. In addition, ischemia reperfusion (IR) injury leads to cellular damage that can culminate in functional impairment and loss of graft. Furthermore, IR injury is aggravated by pre-existing steatosis and may involve additional mechanisms and mediators of cellular damage. Current models to study IR in vitro are not well defined and may overlook periods of injury that are involved in transplantation. In this thesis, I present an in vitro model for IR injury that includes multiple phases of injury and leads to the upregulation of heme oxygenase-1 (HO-1), and possibly enhances the expression of matrix metalloproteinase-9 (MMP-9). As graft HO-1 expression correlates positively with reduced injury, but MMP-9 expression is associated with increased injury, I therefore examined the utility of in vitro gene therapies to affect the expression of these proteins. We conclude that the in vitro model of ischemia and reperfusion is a promising tool to study the cellular response to IR and may provide a platform for the development of future therapies which could have clinical applications.

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.

The purpose of this study was to determine if del Nido cardioplegia provides superior protection for aged and young adult hearts. We used our isolated working heart model of cardioplegic arrest and reperfusion to compare functional recovery in both senescent and young adult rat hearts, with delivery of del Nido or our standard cardioplegia. In the aged hearts, use of del Nido cardioplegia prevented spontaneous contractions during arrest, reduced troponin release, and provided superior functional recovery during working heart. In contrast, in the young adult hearts, although stroke work was higher in the del Nido group, there were no significant differences in spontaneous activity, troponin release, and cardiac output between del Nido and standard cardioplegia, suggesting that del Nido cardioplegia did not provide superior functional recovery in the young adult heart. Del Nido cardioplegia has the potential to provide superior myocardial protection for elderly patients undergoing cardiac surgery.

CapZ is an actin capping protein that locates at cardiac Z-discs and anchors sarcomeric actin [1]. Transgenic (TG) mice overexpressing CapZ in cardiac myocytes develop a lethal cardiac hypertrophy [2], while a large reduction in CapZ protein causes severe myofibrillar disarray and death [2]. However, a TG model that contains a modest reduction in cardiac CapZ protein levels is viable and is associated with decreased PKC-dependent regulation of myofilament function [3]. Given the well known role of PKC in myocardial pathogenesis, the general aim of this thesis was to investigate how the modest reduction in CapZ protein affects cardiac function in models of cardiac stress. I found that PKC-translocation to cardiac myofilaments during cold cardioplegic arrest impairs myofilament activation, and that decreased cardiac CapZ protein disrupts this pathway and provides cardioprotective benefit. Using an in vivo model of ischemia-reperfusion (IR), I made the novel discovery that myofilament-associated PKC is altered during prolonged global ischemia, and found that a CapZ deficiency affects the translocation of PKC to myofilaments in a time-dependent manner. Furthermore, I found that TG mice deficient in CapZ demonstrate significant reductions in IR injury, while providing enhanced cardioprotection following ischemic preconditioning. The cardioprotected phenotype of CapZ-deficient TG mice is associated with altered translocation of several PKC-isoforms to cardiac myofilaments. Finally, having uncovered new information about the activation of protein phosphatase type 2A (PP2A) in IR, I investigated the role of CapZ in PP2A-dependent myofilament regulation. I found that reductions in CapZ may affect cardiac contractility by interrupting the association of PP2A with myofilaments. Together these findings expand the role of CapZ as a regulator of intracellular signaling molecules and demonstrate the novel ability of reduced CapZ to protect the heart against significant pathological threats. / Canadian Institutes of Health Research (CIHR), Heart and Stroke Foundation of Ontario (HSFO), Heart and Stroke Foundation of Canada (HSFC), The Premier's Research Excellence Award (PREA), Ontario Graduate Scholarship Program (OGS).

This thesis concerns the investigation of the neuroprotective effects of the peptides relaxin-2 and relaxin-3. Previous studies have shown that intracerebral relaxin-2 reduces brain lesion size in an in vivo model of stroke, thereby providing evidence of a neuroprotective action of relaxin-2. This thesis set out to extend this work to determine whether or not relaxin-2 and relaxin-3 protected neural tissues from stroke in vivo and to determine the mechanisms by which relaxin-2 and relaxin-3 may protect astrocytes from injury by affecting migration, resistance to hypoxia and prevention of apoptosis. The first set of experiments show that relaxin-2 and relaxin-3 pre- and post-treatments following stroke induction protect neural tissues from cerebral damage in vivo. The next experiments show that relaxin-2 and relaxin-3 increase astrocyte migration in vitro through nitric oxide, phosphoinositide 3-kinase and matrix metalloproteinase-mediated pathways. A third set of experiments show that relaxin-2 and relaxin-3 treated astrocytes exhibited a higher viability compared to untreated astrocytes when exposed to oxygen glucose deprivation for 24 hours. Astrocytes that were cultured with relaxin-2 or relaxin-3 also showed a lower production of reactive oxygen species compared to astrocytes that were exposed to oxygen glucose deprivation alone. Finally, relaxin-2 and relaxin-3 protected astrocytes from 24-hour apoptosis injury that was induced by tumor necrosis factor alpha and hydrogen peroxide. Taken together these experiments provide evidence that relaxin-2 and relaxin-3 peptides protect neural tissues from the deleterious effects of cerebral ischemia in vivo and help elucidate some of the cellular mechanisms by which relaxin peptides might protect the brain. Furthermore, these data show that relaxin-2 and relaxin-3 act directly on astrocytes, the most numerous cell type in the brain, to increase astrocyte migration and to protect these cells from some of the deleterious effects of stroke, namely hypoxia and apoptosis.

Background/Objective: Reperfusion injury is a serious consequence of blood flow reestablishment after myocardial infarction (MI) mediated by reactive oxygen species and neutrophilic cellular damage. Following MI, the left ventricle (LV) undergoes remodeling characterized by progressive wall thinning and cavity dilatation. Heme-Oxygenase-1 (HO-1) dependent decrease in oxidative stress may attenuate injury in part by inhibiting transcription factor NFκB-mediated inflammation. Hypothesis: I hypothesized that upregulation of HO-1 by hemin administration confers acute and chronic cardioprotection against I/R injury in rats and attenuates LV remodeling post-MI. I proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic myocardial injury in part by inhibiting NFκB-mediated inflammation. Methods: Six week old male Wistar rats were randomly assigned to sham, vehicle, or hemin-treated groups. Vehicle and hemin were administered intraperitoneally once daily for 3 consecutive days prior to left anterior descending (LAD) coronary artery occlusion. Administration resumed 48 hours post-operatively and continued once every 3 days. Infarct size was determined by H&E histological analysis and fibrosis was quantified by Masson’s Trichrome staining. Transthoracic echocardiography was used to assess LV parameters and wall motion. Results: Hemin increased HO-1 expression, decreased infarct size and fibrosis, and attenuated LV remodeling in the short-term (4 days post-infarction). The decrease in infarct size and area of fibrosis in the hemin group was accompanied by a decrease in NFκB activity. No significant difference in infarct size and area of fibrosis between hemin and vehicle-treated groups was observed at 3 months. LV diameter and cardiac function did not differ significantly between the two groups at 3 months despite an attenuation of anterior wall thinning in the hemin group. Conclusion: HO-1 upregulation by hemin administration conferred acute cardioprotection and attenuated LV remodeling, possibly by inhibiting NFκB-mediated inflammation. However, chronic treatment with hemin did not prevent long-term post-infarction LV remodeling. It is possible that cardioprotection afforded by HO-1 upregulation is strong enough to curtail inflammation post-reperfusion and prevent LV remodeling acutely, but is not robust enough to protect the myocardium to the same degree in the long-term. Future research should focus on optimal HO-1 upregulation to attenuate long-term LV remodeling due to reperfusion injury. / Thesis (Master, Physiology) -- Queen's University, 2007-09-25 19:01:33.87