1990 — 1994 |
Murray, Katherine T |
K11Activity Code Description: Undocumented code - click on the grant title for more information. |
Metabolic Regulation of the Cardiac Sodium Channel
The cardiac sodium channel plays an essential role in the normal propagation of electrical impulses and in abnormal conduction and arrhythmogenesis. Many ionic channels are regulated by cellular metabolic processes, and it has been shown that the sodium protein channel is an excellent substrate for phosphorylation by both and it has been shown that the sodium protein channel is an excellent substrate for phosphorylation by both adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and protein kinase C. The purpose of this research is to investigate the hypothesis that the cardiac sodium channel is subject to metabolic control. Particular emphasis will be placed on the processes of slow and ultra-slow inactivation of the sodium current (INa), which are more prominent in depolarized or abnormal tissue and are likely targets for regulation. This is supported by preliminary data from our laboratory showing that the kinetics and extent of block of these slow processes by flecainide is modulated by the beta-adrenergic agonist isoproterenol. Examination of this hypothesis has heretofore not been possible because voltage clamp of INa has required lowering experimental temperature, which may obviously affect cellular metabolism. We can now voltage clamp 5-10 mu2 cell- attached patches with successful control of INa at 37degreesC and can measure macroscopic sodium currents in intact cells using physiologic solutions at body temperature for the first time. We will study the effects on macroscopic INa and single channel activity of important cellular signal transduction mechanisms, including cAMP, guanosine 3',5'- cyclic monophosphate (cGMP), phosphatidylinositol turnover, and guanine nucleotide-binding (G) proteins, in guinea pig and neonatal rat and mice ventricular myocytes. We will also determine whether observed pharmacologic effects are due to phosphorylation or to direct modulation of the channel. The approaches we will use in these studies include: 1) elevation of the intracellular concentration of a second messenger (e.g., with isoproterenol, nitrovasodilators, carbachol); 2) direct stimulation of protein kinases (using cAMP/cGMP analogs, phorbol esters); and 3) intracellular administration of specific compounds (e.g., the catalytic subunits of cAMP/cGMP-dependent protein kinases, inositol 1,4,5- trisphosphate). We will also investigate the influence of metabolic processes on the interaction between the cardiac sodium channel and flecainide, an antiarrhythmic agent whose block is a function of slow and ultra-slow inactivation. Ultimately, greater knowledge regarding control of INa and especially the slow inactivation processes could lead to the development of safer, more effective antiarrhythmic agents, since drugs that selectively enhance development of slow inactivation as a primary mechanisms for block would leave normal tissue relatively unaffected. During Phase I (2 years), the candidate will work in the Stahlman Cardiovascular Research Program, (SCRP), directed by Luc M. Hondeghem, M.D., Ph.D., refining her knowledge of the technique of whole-cell voltage clamp and patch clamp as well as intracellular perfusion. For Phase II (3 years), the candidate will continue work on the current proposal in her own laboratory in close collaboration with Dr. Hondeghem and other members of the SCRP.
|
1 |
1997 — 2000 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Cardiac Na+ Channels--Effects of Phosphorylation
While the factors which precipitate cardiac arrhythmias are largely unknown, there is no doubt that acute autonomic stimulation is arrhythmogenic. Voltage-gated sodium channels are a critical determinant of normal and abnormal conduction in the heart, and mutant channels with subtle dysfunction can cause life-threatening arrhythmias. At present, the effects of adrenergic stimuli, which activate protein kinase A and C, on the cardiac sodium current remain controversial. The goal of this proposal is to test the hypothesis that protein kinase activatiOn modulates function of human cardiac sodium channels by phosphorylation of the channel alpha-subunit. Electrophysiologic studies will characterize the functional effects of protein kinase A and C stimulation on the major voltage-gated sodium channel in human heart, hHI, with the channel expressed in two different heterologous systems (Xenopus Laevis oocytes and a mammalian cell line). Substantial preliminary data demonstrate that activation of both kinases causes significant effects on hH1 current. Additional studies will be undertaken to define the biochemical basis for kinase effects on hHI. Immunoprecipitation techniques will be used to determine if the channel is directly phosphorylated by kinase under in Vitro and in vivo conditions. To elucidate the functional role of putative phosphorylation sites in the hHI sequence, experiments will be performed using both chimeric human heart-skeletal muscle channels and site-directed mutagenesis, to pinpoint regions of channel sequence and, ultimately, individual amino acids of functional and biochemical importance. Finally, the protein kinase C isoforms present in both the cellular expression systems used and human myocardium will be identified. The effects of human isoforms which are lacking in the cellular systems on hHI will then be tested to understand more fully the relevance of hHI modulation for human heart. The knowledge gained from these studies will improve our understanding of the nature and molecular basis of sodium channel modulation in human heart, and conditions which could conceivably promote or suppress arrhythmias due to changes in channel function.
|
1 |
1998 — 2001 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Voltage Gated Cardiac Potassium Channels
DESCRIPTION (Adapted from Applicant's abstract): The long term goal of this project is to gain an understanding of the molecular basis of cardiac excitability, concentrating on molecular structure-function relationships of cardiac K+ channels. The hypotheses to be tested are that specific regions and even specific amino acid residues are implicated in activation and deactivation gating and rectification of cardiac K channels. The specific aims to be investigated include: (1) test of whether or not an extracellular ring of positive charges in hKv1.5 is a molecular determinant of outward rectification, (2) test whether or not the sixth transmembrane domain (S6) comprises part of the activation gate, This information should advance our understanding of the molecular physiology and pharmacology of cardiac K channels and further help in correlating cloned subunits with membrane currents in native cardiac cells. Finally, the information gained will expand our knowledge of the function of this important class of K channels, which may ultimately result in improved understanding of the genesis of cardiac arrhythmias and the development of better antiarrhythmic agents.
|
1 |
1999 — 2002 |
Murray, Katherine T |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. |
Genetic Susceptibility in Acquired Long Qt Syndrome
Many commonly used antiarrhythmic drugs prolong ventricular repolarization and the qt interval on the electrocardiogram by blocking conductance of cardiac potassium channels. In a small percentage of patients receiving these drugs, a life-threatening polymorphic ventricular tachycardia called Torsades de Pointes can occur in association with excessive QT prolongation. Recently, it was found that the congenital form of this syndrome results from mutations in genes which encole voltage-gated ion channels, including K+ and Na+ channels. There is considerable precendent for genetically- determined diseases that become manifested only upon exposure to drugs. Therefore the discovery of ion channel gene mutations in the congenital long QT syndrome raises the hypothesis that some patients in the acquire long QT syndrome harbor subclinical genetic mutations in these or other genes but require drug exposure for expression of the phenotype. This study will compare drug-induced QT prolongation in first degree relatives of ALQT probands to that of non-alqt controls using the antiarrjythmic drug ibutilide. An abnormal QT response detected in the relatives of ALQT patients would provide evidence of a genetic predisposition for drug-induced long QT syndrome, and additional studies would continue in the attempt to identify candidate genes. The ultimate goal of this research is to identify susceptible genotypes which would be predictive of the development of Torsades de Pointes in patients treated with QT-prolonging drugs.
|
1 |
2001 — 2004 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Modulation of Cardiac Na+ Channel Function
Activation of cellular signaling systems is increasingly recognized as a potential modulator of not only the function of plasma membrane proteins, but also their trafficking into and out of the cell membrane. In the heart, Na+ channels are pivotal to both normal electrophysiology and the genesis of life-threatening cardiac arrhythmias. While cardiac Na+ current is modulated by protein kinase(pKA), the nature of this regulation is controversial and the mechanisms unknown. Based on data we have generated, the goal of this research is to test the hypothesis that PKA activation regulates trafficking of cardiac Na+ channels and to investigate the molecular mechanisms. In cells expressing the human cardiac Na+ channel hHl and in rat ventricular myocytes, the effects of pKA activation on N a+ current, plasma membrane channel density, and the cellular distribution of Na+ channels will be determined using electrophysiologic and biochemical methods, immunolocalization, and a fluorescent channel fusion protein to visualize trafficking in living cells. Our preliminary data have implicated the I-II interdomain linker as a region contributing to this effect. To investigate the structural and molecular components required for channel recycling under basal and stimulated states, structure- function hypotheses will be explored for this region as well as the carboxy terminus, which interacts with other proteins (e.g., those with PDZ-domains). Evidence for interacting or adaptor proteins will be sought by both overexpression and binding studies using the channel region(s) involved in the PKA effect. To explore the cellular mechanisms that mediate regulated trafficking of Na+ channels, the role of specific molecular components in both exocytotic and endocytotic pathways will be investigated. The knowledge gained from these studies will improve our understanding of the molecular mechanisms whereby cardiac cells regulate Na+ channel activity and could identify novel strategies to modulate cardiac Na+ currents.
|
1 |
2002 — 2005 |
Murray, Katherine T |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Targets in Atrial Fibrillation
DESCRIPTION (provided by applicant): Atrial fibrillation is the most common sustained cardiac arrhythmia and a major source of morbidity and mortality in the US. Available antiarrhythmic drugs are often ineffective and create serious proarrhythmia because channels in the ventricle are affected. In addition, electrical remodeling due to rapid stimulation in the atrium further perpetuates the arrhythmia, contributing to its refractory nature. The goal of this proposal is to identify novel targets for the treatment of atrial fibrillation by investigating the molecular basis of an atrial-specific ultra-rapid K+ current, IKur, and the early intracellular events that trigger the remodeling process. While the Kv1.5 gene product is an important component of IKur, our preliminary data indicate that this -subunit cannot fully recapitulate IKur, and we will test the hypothesis that co-assembly of additional channel subunits and/or signaling proteins occurs in vivo. The Kv1.5 complex will be isolated from human atrium and coassembled K+ channel lpha and/or etasubunits will be identified using antibody-based methods. Following heterologous expression of the proteins identified, electrophysiologic techniques will be used to confirm if the resultant K+ current phenotype is that of IKur. An analogous strategy will be used to determine the role of A-kinase anchoring proteins (AKAPs) in the Kv1.5 signaling complex. We will also test the hypothesis generated by our preliminary data that a Kv eta subunit can function as an AKAP. Finally, our initial results indicate that chronic rapid stimulation of atrial cells in culture leads to electrical remodeling, and this system will be used to test the hypothesis that the molecular events that trigger remodeling resemble those of cardiac hypertrophy, with activation of specific intracellular signaling cascades. The knowledge gained from these studies will improve our understanding of the molecular components of atrial electrophysiology, and should lead to the development of novel targets to treat atrial fibrillation.
|
1 |
2003 — 2007 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Molecular Target For Atrial Fibrillation
DESCRIPTION (provided by applicant): Atrial fibrillation is the most common sustained cardiac arrhythmia in the United States, and it remains a major source of morbidity and mortality in this country. Antiarrhythmic drugs currently available to treat this arrhythmia are often ineffective, and they can create serious proarrhythmia because ion channels in the ventricle are affected. The goal of this proposal is to investigate the molecular basis of an atrial-specific ultrarapid K+ current, IKur, a potential target for pharmacologic therapy of atrial fibrillation. While the Kv 1.5 gene product is an important component of IKur, our preliminary data indicate that this alpha-subunit cannot fully recapitulate the native K+ current. In the proposed specific aims, we will test the hypothesis that IKur is a macromolecular complex composed of multiple channel subunits, signaling molecules, and additional proteins that can modify channel function. The Kv 1.5 complex will be isolated from human atrium, and associated K+ channel alpha and/or beta-subunits will be identified using antibody-based methods. Following heterologous expression of the proteins identified, electrophysiologic techniques will be used to confirm if the resultant K+ current phenotype is that of IKur. Additional experiments will determine whether chamber and disease-specific alterations in the channel complex occur. An analogous strategy will be used to determine the role of A-kinase anchoring proteins (AKAPs) in the Kv 1.5 signaling complex. We will also test the hypothesis generated by our preliminary data that a Kv beta subunit can function as an AKAP. Finally, a proteomics approach will be employed to identify previously unknown protein partners in the Kv 1.5 complex, with protein-protein interactions validated using standard biochemical approaches. This technology will also be used to confirm associated Kv subunits and signaling molecules. The knowledge gained from these studies will improve our understanding of the molecular components of atrial electrophysiology and facilitate the development of novel strategies in the treatment of atrial fibrillation.
|
1 |
2010 — 2014 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Preamyloid Oligomers and Susceptibility to Atrial Fibrillation
DESCRIPTION (provided by applicant): Atrial fibrillation (AF) is the most common cardiac arrhythmia, resulting in substantial morbidity and mortality. An important risk factor for developing AF is age, with a lifetime risk of 1 in 6 for the condition. The incidence of AF is increasing in epidemic proportion as the US population ages, and currently available treatment is often ineffective. The clinical course of AF is typically progressive, due to electrical and structural remodeling in the atria with rapid stimulation that increases arrhythmia susceptibility. Oxidative stress and inflammation play an important role in generating the AF substrate and promoting this remodeling process. Recently, we showed that atrial cells rapidly stimulated in culture undergo remodeling very similar to that observed in human AF. Importantly, transcriptional profiling in paced cells exhibited striking concordance with changes seen in vivo. Unexpectedly, we observed conserved transcriptional upregulation in proteins involved in amyloidosis, a process associated with protein misfolding and deposition in multiple neurodegenerative diseases, notably Alzheimer's disease. Substantial evidence indicates that the toxic species in these disorders are soluble preamyloid oligomer intermediates, rather than the mature fibrillar, amyloid-positive deposits. Indeed, our preliminary data demonstrate striking accumulation of preamyloid oligomers in rapidly-paced atrial cells, with similar results in experimental and human AF. Taken together, these data form a strong rationale for the proposed studies. The goal of this proposal is to test the hypothesis that atrial preamyloid oligomers are pathophysiologically linked to the development of AF in humans. In Specific Aim 1, human atrial samples obtained during routine cardiac surgery at multiple centers will be used to examine the relationship of preamyloid oligomer formation to age, the risk of postoperative AF, and established AF in humans. Indicators of oxidative stress will also be investigated in these samples. In Specific Aim 2, we will explore the effects of potent antioxidant/anti-inflammatory compounds that are also known to inhibit soluble oligomer formation, on the generation of atrial preamyloid oligomers in response to rapid stimulation in vitro and during experimental AF. Atrial natriuretic peptide (ANP) is known to form amyloid fibrils, and it is present in isolated atrial amyloidosis, a process that increases with aging in humans. Recently, mutations in ANP were causally linked to familial AF. In Specific Aim 3, we will determine whether these ANP mutations promote the formation of preamyloid oligomers as a potential mechanism to increase AF susceptibility. The proposed studies have substantial significance, since preamyloid oligomers may not only provide a mechanistic link between oxidative stress, aging, and AF, but they may also provide a novel therapeutic target in the treatment of this common and difficult to treat arrhythmia. PUBLIC HEALTH RELEVANCE: The studies described in this proposal will improve our understanding of the basic mechanisms that cause a heart rhythm disturbance known as atrial fibrillation. This is important because atrial fibrillation is common in the general population, and it causes a substantial number of strokes each year, as well as weakened heart function, or heart failure. We anticipate that we will identify new mechanisms that increase a person's susceptibility to atrial fibrillation, and thus new approaches should develop to prevent its occurrence.
|
1 |
2012 — 2013 |
Murray, Katherine T |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Azithromycin and Sudden Cardiac Death: Electrophysiologic Mechanisms
DESCRIPTION (provided by applicant): Sudden cardiac death is the single most common cause of death in industrialized countries, accounting for more than half of deaths from cardiovascular diseases. Most sudden cardiac deaths are caused by ventricular tachyarrhythmias, and an important risk factor for such arrhythmias is medications. A number of non- cardiovascular drugs have been shown to alter cardiac electrophysiology, and in some cases, the occurrence of serious ventricular arrhythmias has led to their withdrawal from the market. The macrolide antibiotics erythromycin and clarithromycin have been linked to life-threatening arrhythmias, while azithromycin is considered to have minimal adverse cardiac effects. However, recent case reports of serious ventricular arrhythmias, along with data from the FDA's Adverse Events Reporting System, have challenged this assumption. We have performed a large pharmacoepidemiologic study using the Tennessee Medicaid database, demonstrating that azithromycin increases the risk of sudden cardiac death by several fold. Moreover, case reports indicate that while QT prolongation is rarely causative, polymorphic ventricular tachycardia can occur in the absence of ECG abnormalities, implying novel pharmacologic effects, a concept also supported by our preliminary data. The mechanism of this unusual proarrhythmic syndrome is currently not known, and the electrophysiologic effects of azithromycin remain understudied. The goal of this proposal is to test the hypothesis that azithromycin causes novel electrophysiologic effects in the heart that increase susceptibility to serious ventricular arrhythmias. In Specific Aim 1, we will obtain intracellular calcium fluorescence measurements in intact cardiomyocytes to test the hypothesis that azithromycin increases the risk of sudden cardiac death by promoting abnormal spontaneous calcium release from the sarcoplasmic reticulum. This mechanism has been linked to polymorphic ventricular tachycardia in the setting of a normal QT interval in inherited arrhythmia syndromes. In Specific Aim 2, we will perform electrophysiologic studies to test the hypothesis that azithromycin alters ionic currents other than hERG to prolong repolarization and increase arrhythmia susceptibility. Currents will be studied following heterologous expression of recombinant human channels, as well as in native ventricular myocytes. The studies outlined in this proposal will improve our understanding of proarrhythmic mechanisms in humans, and specifically for the widely-prescribed antibiotic azithromycin. This knowledge would enable the screening and identification of additional compounds in drug development with similar properties that could also have the potential for serious adverse effects. Thus, an improved understanding of the basic mechanisms causing azithromycin-induced sudden cardiac death should lead to safer pharmacotherapy. PUBLIC HEALTH RELEVANCE: The studies described in this proposal will improve our understanding of the basic mechanisms that cause a commonly used antibiotic, azithromycin, to increase the risk of sudden death in the general community. We anticipate that we will identify new mechanisms that increase a person's susceptibility to serious heart rhythm disturbances that can cause sudden death. This information can be used to screen and identify other drugs, either before or after they are marketed, that may cause a similar public health problem.
|
1 |
2017 — 2020 |
Murray, Katherine T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Pathophysiological Targets in Atrial Fibrillation Susceptibility @ Vanderbilt University Medical Center
SUMMARY Atrial fibrillation (AF) is the most common cardiac arrhythmia of clinical significance, and it often results in devastating outcomes. Because current treatment is frequently ineffective, there is a critical need for an improved understanding of the mechanisms causing AF and novel strategies to treat it. Abundant evidence has linked oxidative stress to the pathogenesis and progression of AF, yet upstream therapy to target these processes has been ineffective. Lipid aldehydes are a major component of oxidative stress-related injury, and the most reactive products generated, isolevuglandins (IsoLGs), react almost instantaneously with proteins to cause dysfunction. Dicarbonyl scavengers have been developed that preemptively bind IsoLGs before they can interact with biologic targets. Using these tools, IsoLGs were recently identified as critical mediators in angiotensin II-mediated hypertension and Alzheimer's disease. Diseases of oxidative stress are also linked to proteotoxicity, or cellular dysfunction caused by misfolded proteins. In amyloid diseases like Alzheimer's, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species that correlates with disease progression. Notably, IsoLGs markedly accelerate PAO formation for amyloidogenic proteins. Based on our preliminary data, the goal of this proposal is to test the hypotheses that both IsoLGs and PAOs are biologically-relevant mediators that promote AF susceptibility, making them potential therapeutic targets. We have acquired compelling preliminary data to support the concept that IsoLGs and PAOs are drivers of the AF substrate: PAOs are commonly detected in human atrium, with the fibrillogenic protein atrial natriuretic peptide (ANP) a major component, and they associate with hypertension; IsoLGs and PAOs are formed in cellular and in vivo models associated with AF susceptibility, including rapidly-stimulated atrial cells, hypertension, obesity, and familial AF; and there is a beneficial effect of scavenging IsoLGs to reduce atrial PAO and AF burden. Moreover, a mutant form of ANP linked to familial AF markedly enhances the formation of cytotoxic ANP oligomers, and these PAOs accumulate in the atria of mice modeling the human disease. The first Aim will test the hypothesis that in hypertension and obesity, oxidative stress-mediated IsoLGs promote atrial cell injury and AF susceptibility. Aim 2 will test the hypothesis that mutant ANP oligomers alter atrial myocyte homeostasis to generate AF susceptibility, and they promote oxidative stress/IsoLG formation that can feed-forward to perpetuate the pathologic process. Finally, Aim 3 will test the hypothesis that in addition to hypertension, other AF risk factors linked to oxidative stress are also associated with accumulation of IsoLGs and/or cytotoxic PAOs, supporting their role in human disease. The proposed studies have major significance, given that IsoLG and PAOs may provide not only common mechanistic links between oxidative stress, proteotoxicity, common clinical risk factors, and AF, but also novel therapeutic targets in the prevention and/or treatment of this common arrhythmia.
|
1 |