Joshua Goldhaber - US grants

The Biomedical Instrumentation Core, under the direction of Dr. Goldhaber, will provide support for the design, assembly and maintenance of instrumentation used in each of the SCOR projects. The Core's physical plant, the UCLA Biomedical Instrument Facility, is housed in the UCLA Center for the Health Sciences Building and is readily accessible to the SCOR investigators. In its capacity as UCLA's Biomedical Instrument Facility, the Core has extensive experience providing biomedical instrumentation services for scientists and physicians. The Core personnel have extensive experience and have designed and constructed many of the instruments currently being utilized by the SCOR investigators. Consequently, the Core is primed to perform an essential troubleshooting function. A major advantage of the Biomedical Instrument Core is its ability to manufacture instrumentation at substantial savings compared to commercially available equipment, and to provide custom- designed equipment which is not commercially available. The added benefit of in-house consultation with the personnel who designed and fabricated the device is also a significant advantage. The anticipated usage of the Core by the various Projects is as follows: Project 1: 15%, Project 2: 25%, Project 3: 5%, Project 4: 30%, and Project 5: 25%.

Intracellular Ca (Cai) overload is a well-known arrhythmogenic factor which contributes to focal arrhythmias by promoting automaticity and triggered activity, and to reentry by facilitating cellular uncoupling and slow conduction. As a sequela of myocardial ischemia and reperfusion, Cai overload contributes to cellular injury and arrhythmias, including ventricular fibrillation. However, the cellular mechanisms responsible for Cai overload during ischemia/reperfusion remain incompletely understood. The objective of this proposal is to characterize the subcellular mechanisms by which metabolic inhibition and components of the ischemia environment alter Cai regulation in isolated ventricular myocytes. Cai (using fura-2), membrane current and voltage, and cell shortening will be monitored simultaneously in isolated rabbit and guinea pig ventricular myocytes under whole cell patch clamp conditions during exposure to metabolic inhibitors (combined or selective inhibition of glycolysis and oxidative metabolism) and to various components of the ischemic environment (e.g. oxygen free radicals and amphiphiles). Using a rapid extracellular solution exchange device to facilitate pharmacologic interventions and ionic substitutions, we will characterize in detail the effects of these interventions on individual components of excitation-contraction coupling responsible for regulating Cai, including the Ca current (L and T types), Na-Ca exchange, the sarcoplasmic reticulum, mitochondria and the sarcolemmal Ca pump. We will also perform studies in giant excised membrane patches to assess the effects of components of the ischemic environment on Na-Ca exchange. A second major goal is to explore the mechanisms by which Cai overload causes arrhythmias. In particular, we will test a hypothesis predicted from the computer simulations of the cardiac action potential described in Project 3; namely that, by delaying or accelerating diastolic depolarization, delayed afterdepolarizations due to Cai overload are capable of producing a chaotic arrhythmia in a single myocyte. If a chaotic arrhythmia can be produced in the isolated myocyte, it may be amenable to 'chaos control' using the pacing algorithm described in Project 3, which will allow us to study the mechanisms of chaos control at a cellular and subcellular level using patch-clamp and fluorescent indicator techniques. These studies will provide important new information about pathogenesis of abnormal Cai regulation relevant to myocardial ischemia/reperfusion, and its arrhythmogenic consequences.

Although there is a clear relationship between the duration of myocardial ischemia and the extent of contractile failure and cell injury upon reperfusion, the cellular basis of these abnormalities is not well understood. Intracellular calcium (Ca2+) overload and abnormal intracellular Ca2+ handling are thought to underlie the contractile abnormalities and cell damage associated with reperfusion injury. However, the causes of altered Ca2+ homeostasis are unknown. One possible explanation is that potentially injurious compounds, ordinarily absent or at extremely low levels in the cellular environment under physiologic conditions, increase to toxic concentrations during ischemia and reperfusion. Three classes of compounds are of particular interest in this regard: oxygen derived free radicals, inflammatory cytokines, and amphiphiles. Agents from each of these classes have been found to accumulate in cardiac tissue during ischemia and reperfusion, and each has been implicated in the pathogenesis of abnormal Ca2+ handling and altered excitation-contraction coupling. The objective of this proposal is to investigate the mechanisms whereby these mediators of cardiac injury disrupt excitation-contraction coupling, with particular emphasis on the individual components of Ca2+ regulation. Experiments will be performed on patch clamped single ventricular myocytes from rabbits loaded with the Ca2+ sensitive fluorescent indicator FURA-2. Alterations in intracellular Ca2+ will be correlated with membrane current and voltage, as well as cell edge motion using a video motion detector. A rapid extracellular solution exchange device will facilitate ionic and pharmacologic interventions. The individual components of Ca2+ regulation to be studied in this fashion will include the Ca2+ current (both T and L components), sarcoplasmic reticulum, sodium-calcium exchanger, sarcolemmal Ca2+ ATPase, and mitochondria. Sodium-calcium exchange will also be studied using caged Ca2+ and, in other experiments, the giant excised patch technique in xenopus oocytes. We will also investigate the potential roles of Ca2+ leak channels in altered intracellular Ca2+ homeostasis. We believe these studies will improve our understanding of the cellular mechanisms of reperfusion injury and may also provide a basis for developing new strategies aimed at limiting contractile failure and irreversible injury following reperfusion in the setting of myocardial infarction.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine how metabolic abnormalities common to ischemia and congestive heart failure produce defects in cellular excitation-contraction (E-C) coupling. These defects are responsible for the contractile abnormalities that typify cardiogenic shock in patients sustaining a large myocardial infarction or suffering from end-stage dilated and ischemic cardiomyopathies. We have three specific aims: 1) We will investigate the metabolic regulation of cardiac E-C coupling gain and subcellular Ca2+ release events in adult ventricular myocytes. A major goal is to determine whether E-C coupling is preferentially dependent upon ATP derived from glycolysis versus oxidative metabolism; 2) We will determine how single Ca2+ channel properties are regulated by glycolytic versus oxidative metabolism. We will also determine the relative roles of the Ca2+ current, and the ryanodine receptor, on changes in Ca2+ spark probability during metabolic inhibition; 3) We will study alterations in total transmembranous Ca2+ flux produced by metabolic inhibition, and determine the extent to which Ca2+ current activates sodium-calcium exchange under these conditions. Our general approach is to study the response of subcellular Ca2+ movements and transmembranous Ca2+ fluxes to metabolic inhibitors, in patch clamped isolated ventricular cardiac myocytes from rats and rabbits loaded with fluorescent Ca2+ indicators. Metabolic inhibitors will be chosen to block, alternatively, glycolytic metabolism, oxidative metabolism, or both glycolytic and oxidative metabolism simultaneously. We will use novel confocal imaging strategies to record subcellular Ca2+ movements during metabolic stress with unusually high spatial and temporal resolution. We will, for the first time, assess the effects of metabolic inhibition on the single channel properties of L-type Ca2+ channels in cell-attached patches on rat and rabbit ventricular myocytes. We will use a novel epifluorescence approach to sort out the effects of metabolic inhibition on the complex interaction between L-type Ca2+ channels and the sodium-calcium exchanger. A better understanding of these issues will assist in the development of new therapies to restore contractile function in patients with cardiac failure.

Project Summary/Abstract The applicant[unreadable]s long-term aims are to continue studies on calcium signaling, excitation-contraction (EC) coupling, and the dependence of these processes on energy metabolism in cardiac muscle. The specific aims are to study ventricular cells from rabbits and mice (including cardiac-specific sodium-calcium exchanger knock-out mice) to: 1) investigate calcium signaling and EC coupling in remodeled cells from the peri-infarct zone in rabbits. This will include an assessment of whether loss of L-type calcium channel function can account for failure of EC coupling and whether significant alterations in transverse-tubules, ryanodine and dihydropyridine receptors are involved in the failure of these cells; 2) investigate the effect of metabolic inhibition on the function and structure of couplons in rabbit ventricular myocytes. This will include a measurement of the minimum number of L-type calcium channels in a couplon and the way that metabolic inhibition affects their function. In particular, alterations in calcium spark and spike formation and cellular microarchitecture of the transverse-tubule system as a cause of the functional loss of couplons during metabolic inhibition will be considered; 3) study the resistance of sodium-calcium exchanger knock-out mice to metabolic stress. This will include an investigation of the hypothesis that metabolic inhibition prevents activation of reverse sodium-calcium exchange in wild-type mice, resulting in disruption of the calcium-induced calcium release mechanism of EC coupling. In contrast, it is hypothesized that sodium-calcium exchanger knock-out mice do not require sodium-calcium exchange for EC coupling and are therefore resistant to the effects of metabolic inhibition. The consequences of inhibiting sodium-calcium exchange activation on calcium spike latency will be examined. These experiments are, among other things, designed to explain the importance of diadic cleft calcium in the trigger process. Methods include measuring calcium spike probabilities and their latency distributions in rabbits and mice before and after treatment with metabolic inhibitors. In addition the methods include recently developed procedures for reconstructing the 3-dimensional architecture of the transverse[unreadable]tubule system and the 3-dimensional distribution of ryanodine and dihydropyridine receptors in peri-infarct cells and cells treated with metabolic inhibitors.