Some of our current graduate students have provided descriptions of their research projects that offer insights into the work taking place in their laboratories.
The Schulz lab studies ischemic and inflammatory heart diseases and investigates the role of specific molecules contributing to oxidative stress injury of the heart. Being in the first laboratory to discover that matrix metalloproteinase-2 (MMP-2) proteolyses specific proteins inside cardiomyocytes, my main project is to investigate further novel intracellular targets of MMP-2 in myocardial cell injury and/or death.
In the Schulz lab we use various pharmacological and genetic techniques to address our hypotheses including: Isolated perfused heart preparations, Gel Electrophoresis techniques, (SDS-PAGE, Native Agarose gel (for DNA/RNA), Agarose-SDS gel (for giant proteins; Titin, Neulin...etc), Immunoblotting & Immunoprecipitation techniques. Gelatin & Casien Zymography, In-vitro degradation assay, shRNA technique to knockdown MMP-2 expression in cardiac myocytes, DNA/RNA isolation, PCR & cDNA construction and cloning, Eukaryotic cells transfection for protein expression, Immunocytochemistry-Confocal microscopy analysis.
One of my exciting findings is discovering the unique role of MMP-2 in the degradation of titin, the largest known protein (3000-4000 kD) inside the heart responsible for heart muscle contraction. This effect particularly explains the poor contractile function of the heart following ischemia-reperfusion and suggests that increased MMP-2 activity inside heart muscle may be a fundamental problem in ischemic heart disease. Accordingly, the results from this research can form the basis for the development of novel therapies (e.g. specifically targeted MMP-2 inhibitors) which may ultimately prevent or reduce ischemic heart disease including reperfusion injury.Recent Publications
The sensation of pain is initiated by activation of nociceptors and transmission of information into the dorsal horn of the spinal cord via primary afferent neurons. The cell bodies of these neurons lie in the dorsal root ganglia (DRG) and their terminals reside primarily in the superficial laminae of the dorsal horn.
Classical analgesics such as morphine are thought to reduce the release of excitatory neurotransmitters from these primary afferent terminals and to thereby impair the transmission of noxious information into the central nervous system. Studying classical analgesics forms one aspect of my project. In particular I investigate the hypothesis that suppression of Ca2+ current in primary afferent terminals does not completely account for the ability of opioids to suppress neurotransmitter release. (This hypothesis is based on recent work in the Smith lab.)
The other part of my project involves studying a specific type of pain known as 'neuropathic pain' that is caused by damage to the central nervous system. Neuropathic pain is a major clinical problem as it is almost always chronic and frequently intractable. It is estimated that 2 to 3% of the population in the developed world suffers from neuropathic pain, which equates to approximately one million Canadians that have this devastating condition. The onset of many types of neuropathic pain involves altered expression of ion channels in DRG neurons and altered synaptic transmission in the spinal dorsal horn. Since the relationship between altered expression of Ca2+ channels in DRG cell bodies and alterations in neurotransmitter release are poorly understood, I monitor Ca2+ dynamics in primary afferent terminals under conditions of neuropathic pain such chronic constriction injury (CCI) in rats.
My experiments involve the use of 1) Ca2+ imaging in live tissue using confocal microscopy and the genetically encoded fluorescent Ca2+ indicator protein gCaMP or AM dyes such as Fluo-4 and 2) whole cell patch clamp recording from DRG and substantia gelatinosa neurons in the spinal dorsal horn.
This work will provide important new insights into the action of classical analgesics and into the etiology of neuropathic pain.Recent Publications
Two common genetic variants, E23K and S1369A, found in ATP-sensitive potassium (KATP) channel, are associated with type 2 diabetes (T2D) such that people carrying K23/A1369 haplotype are more susceptible to T2D. Indeed, homozygous of K23/A1369 haplotype is found in ~20% of T2D patients and 10% of non-diabetic people. KATP channels play an important role in many tissues including pancreatic β-cells where they couple glucose metabolism to insulin secretion. Also, KATP channel inhibitors, such as sulfonylurea and glinide drugs, are clinically prescribed extensively as insulin secretagogues to treat T2D. Several clinical studies have shown that T2D patients with K23/A1369 risk haplotype show different responses to sulfonylurea therapies.Pharmacogenomics is the study of how individual genetic variation affects drug response. Therefore the aim of my research is to determine the pharmacogenomic profile of clinically used sulfonylurea and glinide drugs on KATP channels with K23/A1369 risk haplotype by using the excised inside-out patch-clamp technique. I patched on tsA201 cells transfected with K23/A1369 or E23/S1369 haplotype to record the macroscopic KATP channel current with or without drug perfusion. We found out that gliclazide and mitiglinide have increased potencies on K23/A1369 variant channels, whereas glimepiride tolbutamide and chlorpropamide have decreased potencies on K23/A1369 variant channels. The clinical benefit of this study will be used to personalize treatment strategies to insure optimal glucose control in diabetic patients who possess E23K and S1369A in such a way to adjust proper dosage of certain drugs that are LESS effective in certain genotypes.Recent Publications
I. Introduction: Heart failure is associated with changes in energy metabolism, but there is no agreement on the nature and consequences of these alterations. One theory suggests that the failing heart is in an energetic crisis where rates of energy metabolism are insufficient to meet normal energy demands, while a second theory concerns the inefficient utilization of energy for mechanical function. Recently, modulation of energy substrate metabolism has been considered as a new approach in the treatment of heart failure.
II. Hypothesis: Optimizing energy substrate metabolism in failing hearts will 1) improve cardiac mechanical function, 2) limit ischemia reperfusion injury, and 3) slow the progression of adverse remodeling in heart.
III. Objectives: a) To compare energy metabolism in normal and failing hearts. We will characterize the molecular basis for these differences in mouse hearts subjected to coronary artery ligation and then relate them to the observed changes in mechanical function. This will help us determine whether metabolic changes in heart failure help improve left ventricular function or are maladaptive and worsen heart function. b) To examine the ability of drug-induced metabolic modulation to improve mechanical function of failing hearts ex vivo. Metabolic modulators will be used to assess the impact of improving the coupling between glycolysis and glucose oxidation and whether this lessens left ventricular dysfunction.
IV. Methodology: We use echocardiography to assess cardiac function in vivo in mouse hearts that are either normal or that have been remodeled following coronary artery ligation. Left ventricular work and rates of energy metabolism are assessed in vitro using hearts perfused in working mode.
V. Significance: These studies will provide new information about whether left ventricular dysfunction in heart failure is due to energy deficiency and/or inefficiency in energy utilization. By examining the mechanisms and consequences of altered substrate metabolism, we hope to identify novel targets for pharmacological manipulation that via future in vivo validation, will translate to clinical practice to aid in the clinical management of heart failure.
GABAA receptors are ion channels found in the CNS that, when activated by agonist, inhibit neuronal activity by allowing chloride ions to flow across the cell membrane. These receptors exist as assemblies of five subunits, and can consist of up to three different subunit types (e.g. 2α, 2β and 1γ subunit). Since there are 19 different subunits in most mammals, this produces a massive number of possible receptor subtypes, each of which can have significantly different functions. Therefore, it is important to know which receptor subtypes are actually formed throughout the nervous system. Unfortunately, while there have been many studies showing where individual subunits are expressed, showing that they are actually interacting with each other is much more difficult.
One of the goals of my project is to develop an assay that can one day be used to solve this problem by using fluorescently labeled antibodies directed against GABAA receptor subunits. If the dyes used to tag the antibodies are chosen properly, they can actually interact with each other in a distant-dependent manner, so that when two or more antibodies are bound to the same receptor, they will produce a positive signal. This would be useful for a lot of areas of research, a few examples being studies looking at basic GABAA receptor physiology, developmental biology, mechanisms of drug tolerance and the influence of hormones on neurotransmission, all of which are believed to involve significant changes in GABAAreceptor expression.
The second goal of my project is to determine how the structures of different receptor subtypes produce their unique characteristics. For instance, a1b2g2 GABAA receptors, the most common subtype, produce large currents when activated, but also desensitize quickly. However, a4b3d receptors desensitize slowly in the presence of agonist, and bind to GABA with a higher affinity than a1b2g2 receptors. To determine why these receptors have such different characteristics, we probe the structure of the subunits by introducing mutations at regions believed to be most important for receptor activity. To look at receptor activity, we inject frog eggs with RNA and set a particular voltage across their cell membrane using glass electrodes. By measuring the current required to maintain a constant voltage inside the cell, we get an indirect measure of any currents passing through the receptors, allowing us to perform dose-response testing on mutated and normal receptors, which will tell us if the mutation has had the effect we expected.