Skeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury. / text

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Pharmacology / Doctoral / Doctor of Philosophy

Contraction of cardiac muscle is a highly regulated event that relies on a delicate balance of ions entering and leaving the cell through ion channels. In particular, voltage gated sodium channels are responsible for the rapid depolarization that leads to a contraction. During an oxidative challenge, sodium channels rapidly activate, but do not fully turn off. This alters the rate of cardiac repolarization and can induce cardiac arrhythmias. It is currently unknown whether the most common sodium channel isoform found in the heart, NaV1.5, generates this oxidant-induced persistent current or if other isoforms are responsible. Therefore, I sought to further explore the biophysical properties NaV1.5, and determine if it can enter this persistent mode. I tested the biophysical properties of native INa in cardiac myocytes and in NaV1.5 transfected HEK293 cells under macro cell-attached voltage-clamp. I used a sodium channel enhancer (Anemonia sulcata toxin II; 10 nM), a sodium channel blocker (tetrodotoxin; 10 nM) and a model of oxidative stress (H2O2; 100 µM, 200 µM, 1000 µM) to compare and contrast the cellular responses between both cell types. I observed that transfected HEK293 cells and cardiac myocytes were unaffected by H2O2 at various concentrations. Given the lack of other isoforms in transfected HEK293 cells, and the low abundance (<5%) of other isoforms in cardiac myocytes, I propose that NaV1.5 function is unaffected by H2O2. Furthermore, ATX II prolonged the inactivation process in both HEK293 cells and cardiac myocytes in a voltage-dependent manner, indicating that NaV1.5 can give rise to persistent sodium current. Finally, by comparing both cell types under control settings, I found that transfected HEK293 cells inactivated at a much slower rate and at more negative potentials compared to the current in cardiac myocytes. My results suggest that NaV1.5 does not underlie oxidant-induced persistent current and that β subunits likely play a significant role in the inactivation process. / Thesis (Master, Physiology) -- Queen's University, 2011-08-19 14:46:42.665

No description available.

ABSTRACT To understand the mechanisms underlying cardiac dysfunction during ischemia reperfusion (I/R) injury, we tested the hypothesis that oxidative stress and defects in endothelium play a critical role in the activation of calpain and matrix metalloproteinases (MMP), inhibition of sarcolemmal (SL) Na+-K+-ATPase, and induction of cardiac dysfunction during I/R injury. It was observed that I/R induced depression in cardiac function and SL Na+-K+-ATPase activity was greater in hearts perfused at constant flow than in hearts perfused at constant pressure. Such a difference was associated with increased calpain activity as well as decreased endothelial nitric oxide synthase protein content and in nitric oxide production. The depression in Na+-K+-ATPase activity and decrease in protein content of Na+-K+-ATPase isoforms in I/R hearts were associated with an increase in calpain activity and translocation of calpain isoforms (I and II) from the cytosol to SL membrane as well as changes in the distribution of calpastatin. I/R induced alterations were Ca2+-dependent and were prevented by treatment with calpain inhibitors, MDL28170 and Leupeptin (Leu). These results suggest that depressions in cardiac function and SL Na+-K+-ATPase activity in the I/R hearts may be due to endothelial dysfunction as well as changes in the activity and translocation of calpain. In another set of experiments, we examined the role of oxidative stress in activation of calpain during I/R and its association with changes in the activity of MMP. Our results show depression of cardiac function and Na+-K+-ATPase activity in I/R hearts were associated with increased calpain and MMP activities. These alterations due to I/R were attenuated by ischemic preconditioning as well as treatment with antioxidant, N-acetylcysteine (NAC) and mercaptopropionylglycine (MPG). Treatment of I/R hearts with MMP inhibitor doxycycline (Dox) improved I/R-induced changes in cardiac function and Na+-K+-ATPase activity without affecting the calpain activation while treatment with calpain inhibitors, Leu and MDL 28170, reduced the MMP activity significantly in addition to attenuating the I/R-induced depression in Na+-K+-ATPase activity. These results suggests that alterations in Na+-K+-ATPase activity in I/R hearts are associated with oxidative stress and intracellular Ca2+ overload induced activation of calpain and MMP.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population and can result in various neurological sequalae including seizures, coma, and brain death. There is still a limited understanding of the generation and propagation of hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal damage. Moreover, glucose reperfusion after a period of transient hypoglycemia has been shown to cause neuronal hyperexcitability which can have further damaging effects. Gap junctional communication can be involved in the spread of hypoglycemic injury in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily propagate and 2) by engaging the astrocytic network in metabolic compensation as well as enhancing astrocytic buffering of K+. This study aims to investigate the role that gap junctions play during brain energy deprivation. Results from these experiments show that gap junction blockade can have a neuroprotective role during hypoglycemia and glucose reperfusion.

ABSTRACT To understand the mechanisms underlying cardiac dysfunction during ischemia reperfusion (I/R) injury, we tested the hypothesis that oxidative stress and defects in endothelium play a critical role in the activation of calpain and matrix metalloproteinases (MMP), inhibition of sarcolemmal (SL) Na+-K+-ATPase, and induction of cardiac dysfunction during I/R injury. It was observed that I/R induced depression in cardiac function and SL Na+-K+-ATPase activity was greater in hearts perfused at constant flow than in hearts perfused at constant pressure. Such a difference was associated with increased calpain activity as well as decreased endothelial nitric oxide synthase protein content and in nitric oxide production. The depression in Na+-K+-ATPase activity and decrease in protein content of Na+-K+-ATPase isoforms in I/R hearts were associated with an increase in calpain activity and translocation of calpain isoforms (I and II) from the cytosol to SL membrane as well as changes in the distribution of calpastatin. I/R induced alterations were Ca2+-dependent and were prevented by treatment with calpain inhibitors, MDL28170 and Leupeptin (Leu). These results suggest that depressions in cardiac function and SL Na+-K+-ATPase activity in the I/R hearts may be due to endothelial dysfunction as well as changes in the activity and translocation of calpain. In another set of experiments, we examined the role of oxidative stress in activation of calpain during I/R and its association with changes in the activity of MMP. Our results show depression of cardiac function and Na+-K+-ATPase activity in I/R hearts were associated with increased calpain and MMP activities. These alterations due to I/R were attenuated by ischemic preconditioning as well as treatment with antioxidant, N-acetylcysteine (NAC) and mercaptopropionylglycine (MPG). Treatment of I/R hearts with MMP inhibitor doxycycline (Dox) improved I/R-induced changes in cardiac function and Na+-K+-ATPase activity without affecting the calpain activation while treatment with calpain inhibitors, Leu and MDL 28170, reduced the MMP activity significantly in addition to attenuating the I/R-induced depression in Na+-K+-ATPase activity. These results suggests that alterations in Na+-K+-ATPase activity in I/R hearts are associated with oxidative stress and intracellular Ca2+ overload induced activation of calpain and MMP.