Timothy L. Domeier, Ph.D. - US grants

DESCRIPTION (provided by applicant): The conduction of vasodilation reflects the spread of hyperpolarizing current along the vessel wall through gap junction channels, and serves to coordinate responses in serial and parallel segments of the resistance vasculature. The calcium-activated potassium channels (KCa) initiate hyperpolarizing responses, but their functional role in the initiation of conducted hyperpolarization and vasodilation is unclear. Using isolated, pressurized hamster retractor feed arteries my central aim is to investigate the function of the KCa in the initiation of conducted hyperpolarization and vasodilation. Unlike resistance microvessels isolated from the brain, these feed arteries are long (3-4 mm) and unbranched, and therefore ideal for studying the mechanisms of conducted vasodilation. Utilizing selective KCa blocker application in conjunction with diameter and electrophysiological measurements I will answer: 1) which KCa are necessary for the initiation of a conducted hyperpolarization and vasodilation, and 2) is the functional coupling between muscarinic receptor and KCa activation localized within respective abluminal and luminal domains of endothelial cells. A key factor in diabetes, hypertension, atherosclerosis, and stroke is endothelial dysfunction associated with the impaired ability to oppose vasoconstriction. Understanding the role of specific ion channels and resolving the polarity of endothelial cell-mediated vasodilation will provide new insight that may enhance the prevention and treatment of pathologies associated with vascular disease.

[unreadable] DESCRIPTION (provided by applicant): Cardiac Excitation-contraction coupling (ECC) is mediated by calcium-induced calcium release, where calcium influx through voltage gated calcium channels opens ryanodine receptors and triggers massive release of calcium from intracellular stores. Alterations in the cellular events of ECC predispose the heart to arrhythmia. IP3 receptor (IP3R) activity exerts positive inotropic and arrhythmogenic effects on ECC in atrial myocytes. During heart failure, the expression of IP3R is increased; whether this results in changes in ECC or arrhythmogenesis remains unclear. The goal of this proposal is to investigate the role of IP3R-dependent signaling on ECC (Specific Aim 1) and arrhythmogenesis (Specific Aim 2) during heart failure. Studies will utilize ventricular myocytes isolated from normal rabbits as well as from the well-characterized rabbit heart failure model. IP3R-dependent signaling will be induced using application of the I PS-liberating agonist Endothelin-1 or via direct application of IP3. Inhibition of IP3R-dependent signaling will be achieved using both pharmacological inhibition and the expression of an IP3 affinity trap which binds and buffers intracellular IP3. Fluorescence microscopy with calcium sensitive dyes will monitor intracellular calcium (epifluorescence microscopy, indo-1) and subcellular calcium release events (laser scanning confocal fluorescence microscopy, fluo-4). Further, patch clamp techniques will be used to record membrane potential and ion channel currents. Specific Aim 1 investigates the hypothesis that IP3R-dependent signaling exerts positive inotropic effects on ECC in ventricular myocytes, particularly during heart failure. In this Aim the effects of IP3R-dependent signaling on basal intracellular calcium, action potential-induced calcium transients, and elementary calcium release events (calcium sparks and puffs) will be examined. Specific Aim 2 tests the hypothesis that IP3R-dependent signaling contributes to arrhythmogenesis in heart failure ventricular myocytes. Here, the effects of IP3R-dependent signaling on the frequency of arrhythmogenic calcium signals (spontaneous calcium release, calcium waves, and calcium alternans) and changes in membrane potential (Early and Delayed Afterdepolarizations, spontaneous action potentials) will be investigated. Arrhythmia represents the major cause of sudden death during heart failure. It is expected that results from this proposal will give needed insight into the mechanisms of arrhythmia during heart failure, which may aid treatment of individuals with cardiac disease. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nearly 1 in 8 (12.5%) of Americans are older than 65 years and the number of individuals 65 and over is expected to increase to ~20% over the next 2 decades. Cardiac disease is prevalent in the aged population and therefore represents a major public health concern in America. The heart remodels with advancing age and these remodeling events often result in the impaired ability of the heart to relax between each beat. This period of relaxation is the cardiac diastole, during which time the heart fills with blood and prepares for the next contraction during systole. With aging, diastolic dysfunction is characterized by increased stiffness. Given that greater stiffness makes it harder to fill, the heart is less effective as a pump and operates at elevated filling pressures and volumes with a greater propensity for arrhythmia. Because the mechanism(s) underlying cardiac dysfunction with advancing age are poorly understood, developing new therapeutic treatments for affected individuals requires new mechanistic insight. The rise and fall of calcium concentration within cardiac cells controls their contraction and relaxation, respectively, and is regulated by the sarcoplasmic reticulum (SR). Therefore, this project is focused on understanding how aging alters the relationship between diastolic filling (i.e., passive stretching of cardiac myocytes) an calcium release from the SR. To directly observe well-defined functional elements (e.g., calcium sparks and waves) of calcium release, high resolution confocal imaging will be used with fluorescent calcium indicators (Fluo-4) with reference to the well-defined spacing of contractile proteins (i.e., sarcomere length). More specifically, this research project will compare intact perfused hearts and enzymatically dissociated intact ventricular cardiomyocytes from young (3 month), middle-aged (14 month), and senescent (24 month) C57BL/6 mice to test the central hypothesis that with advancing age cardiomyocytes have increased passive stiffness with highly sensitive coupling between stretch and SR calcium release. Aim 1 will determine the actual changes that occur in cell and sarcomere lengths of young, middle-aged, and senescent hearts in response to defined changes in left ventricular filling pressure. In Aim 2, the length of individual isolated cardiomyocytes will be controlled to determine how passive stretch of the cell alters calcium release in order to identify key age-associated differences in cell stiffness and calcium regulation. In Aim 3, findings from Aims 1 and 2 will be integrated by investigating calcium release and pressure development within the intact, working heart. Overall, this project utilizes innovative methods to study the function of individual cardiomyocytes under highly controlled experimental conditions as well as in their native environment within the intact heart. This mentored award will also provide important technical training in a highly integrative environment at the University of Missouri, which will give the applicant unique experimental skills and valuable perspective into cardiac physiology as it relates to aging populations. It is anticipated that results from this project will yield insight into the mechanisms of cardiac diseas during aging, with the ultimate goal of translating these findings into treatments for the aging population. PUBLIC HEALTH RELEVANCE: The healthy heart exhibits highly coordinated intracellular calcium release in order to function as an effective pump. Alterations in calcium release during diastolic filling (i.e., passive stretch) predispose the heart to impaired filling and arrhythmia, which are commonly associated with aging. This research proposal investigates how aging affects stretch-induced intracellular calcium release with the goal of translating our findings int the development of therapies to improve cardiac function in the aging population.

Project Summary/Abstract Advancing age is the primary risk factor for coronary artery disease and myocardial infarction (MI). A major cause of arrhythmia and tissue damage following MI is reperfusion injury (ischemia-reperfusion or I-R) which associates with excessive calcium concentration within cardiomyocytes, a process termed calcium overload. Our laboratory recently showed that the transient receptor potential vanilloid, member 4 (TRPV4) cation channel is highly expressed within cardiomyocytes of the aged (but not young) heart and contributes to intracellular calcium overload following hypoosmotic stress. Osmotic changes in cardiac tissue are pronounced during I-R, and reperfusion associates with substantial hypoosmotic stress on cardiomyocytes. Therefore, this proposal tests the central hypothesis that TRPV4 contributes to calcium overload and cardiac dysfunction following I-R. To test this hypothesis we utilize young (3-6 months, with low TRPV4 expression) and aged (24-26 months, with high TRPV4 expression) C576BL/6 mice, young cardiomyocyte-specific TRPV4 transgenic mice (?overexpressors? with high TRPV4 expression), and aged TRPV4 knock-out mice (with no TRPV4 expression). To complement this genetic approach we also utilize a pharmacological approach with specific antagonists of TRPV4. Specific Aim 1 tests the hypothesis that TRPV4 produces cardiomyocyte calcium overload, excessive contractility, and arrhythmia following I-R. Isolated cardiomyocytes subjected to simulated I-R (combined metabolic and osmotic stress) will be used to examine the effect of TRPV4 on calcium signaling modalities using high-resolution confocal fluorescence microscopy. Similarly, transgenic mouse hearts which encode the GCaMP6f calcium sensor will be subjected to ex vivo I-R with pro-arrhythmic calcium signaling monitored within the intact organ. Langendorff-perfused hearts will be utilized to test the role of TRPV4 on contractility and arrhythmia following I- R. Specific Aim 2 tests the hypothesis that TRPV4 contributes to cardiomyocyte death, tissue damage, and adverse cardiac remodeling following I-R. Fluorescence imaging approaches of mitochondrial membrane potential and plasma membrane integrity will elucidate the role of TRPV4 on cardiomyocyte dysfunction and death in real time following simulated I-R or in ex vivo perfused hearts following I-R. Translational studies using orally bioavailable TRPV4 inhibitors will be used to examine the role of TRPV4 in adverse cardiac remodeling and tissue damage following I-R in vivo. The goal of this project is to rigorously examine the role of the TRPV4 ion channel in cardiac dysfunction and tissue damage following I-R, with the long-term goal of translating our research findings into new treatments for aged individuals following MI.