Regulation of K+ in cellular and extracellular compartments is of central importance to volume regulation, fluid transport, the resting membrane potential and electrical activity in excitable cells. In heart, extracellular [K+] in narrow clefts between cells and in T-tubules is not always in diffusional equilibrium with external K+ ([K+]o ) and can be depleted or accumulated and thereby differ from [K+]o during a wide range of physiological/pathological conditions. The central goal of the project is to elucidate the consequences of extracellular K+ accumulation ([K+]a) on cardiac electrical activity in physiological and pathological conditions. To do so, we developed new K+ sensitive fluorescent probes that can be anchored to plasma membranes and upon binding to K+ produce a fluorescence change that reports the local [K+]. We can calibrate the probe, measure rapid (1 ms) changes in [K+]a in ventricular myocytes and Langendorff hearts.
The Specific Aims are:
Aim 1 : Improve our new K+ sensitive probes consisting of: i) hydrophobic groups to anchor the probe to lipid bilayers, ii) hydrophilic groups to dissolve and prevent the probe from diffusing across the membrane into cells, iii) a fluorescent dye that senses iv) the binding of K+ to a K+- selective organic crown ether ring and exhibits a fluorescence change as a function of [K+] in the range of 1-50 mM.
Aim 2 : To test the hypothesis that different K+ currents contribute differently to [K+]a under different physiological conditions( ?? heartrate, ?? 2-adrenergic activity) and metabolic states. [K+]a responses will be compared during pharmacological interventions of channels responsible for K+ efflux (IK1, It,o, IK,slow, IKr, IKs, IKATP) and during manipulations of K+ re-uptake via the Na/K pumps. [K+]a will be compared in atrial and ventricular myocytes to test the effects of differences in APDs, K+ currents and T-tubules. In perfused hearts, APs and [K+]a will be simultaneously mapped, and changes in [K+]a will be measured on a beat-to-beat basis.
Aim 3 : To test the hypothesis that in pathological conditions, the rise of [K+]a can lead to regions of myocardium with an increase in excitability and a greater propensity to focal activity or premature beats that initiate arrhythmias. Simultaneous optical mapping of APs and [K+]a in perfused hearts will be used to measure APs and beat-to- beat changes in [K+]a transients to elucidate the role of [K+]a during arrhythmias elicited by ischemia/reperfusion applied either globally (zero flow) or locally via an LAD ligation. [K+]a elevation will be correlated with coronary vessels, fiber orientation and the origins of premature impulses. Such a project will develop K+ sensitive dyes for applications to cardiac electrophysiology and for the first time characterize [K+]a on a beat-to-beat basis. Dual optical mapping of [K+]a and APs will determine the changes in [K+]a that occur during physiological and pathological interventions, will validate methods to reduce [K+]a to offer new targets to treat cardiac diseases.

Public Health Relevance

Extracellular K+ accumulation in T-tubules and narrow invaginations of heart muscle has been implicated as a determinant of normal cardiac physiology and pathology. However, [K+] concentrations in narrow regions that are not in diffusional equilibrium with the surrounding aqueous medium have never been measured directly. The project will synthesize new K+ indicator probes, measure [K+] in t-tubules, and interstitial spaces and characterize local [K+] accumulation in normal physiological conditions and various pathologies. The role of K+ accumulation may lead to a new understanding of mechanisms that enhanced the likelihood of arrhythmias, provide novel approaches to reduce defibrillation threshold, reduce the recurrence of VF after defibrillation, improve the success rate of defibrillation past 5 min of fibrillation and may lead to new therapeutic targets for the treatment of ischemic heart disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL093074-02
Application #
8013813
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Lathrop, David A
Project Start
2010-01-15
Project End
2013-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
2
Fiscal Year
2011
Total Cost
$473,980
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Němec, Jan; Kim, Jong J; Salama, Guy (2016) The link between abnormal calcium handling and electrical instability in acquired long QT syndrome--Does calcium precipitate arrhythmic storms? Prog Biophys Mol Biol 120:210-21
Henry, Brian L; Gabris, Beth; Li, Qiao et al. (2016) Relaxin suppresses atrial fibrillation in aged rats by reversing fibrosis and upregulating Na+ channels. Heart Rhythm 13:983-91
Kim, Jong J; Němec, Jan; Li, Qiao et al. (2015) Synchronous systolic subcellular Ca2+-elevations underlie ventricular arrhythmia in drug-induced long QT type 2. Circ Arrhythm Electrophysiol 8:703-12
Kim, Jong J; Yang, Lei; Lin, Bo et al. (2015) Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells. J Mol Cell Cardiol 81:81-93
Tanha, Matteus; Chakraborty, Subhasish K; Gabris, Beth et al. (2014) Computational and experimental characterization of a fluorescent dye for detection of potassium ion concentration. J Phys Chem A 118:9837-43
Salama, Guy; Bett, Glenna C L (2014) Sex differences in the mechanisms underlying long QT syndrome. Am J Physiol Heart Circ Physiol 307:H640-8
Kim, Jong J; Nemec, Jan; Papp, Rita et al. (2013) Bradycardia alters Ca(2+) dynamics enhancing dispersion of repolarization and arrhythmia risk. Am J Physiol Heart Circ Physiol 304:H848-60
Parikh, Ashish; Patel, Divyang; McTiernan, Charles F et al. (2013) Relaxin suppresses atrial fibrillation by reversing fibrosis and myocyte hypertrophy and increasing conduction velocity and sodium current in spontaneously hypertensive rat hearts. Circ Res 113:313-21
Parikh, Ashish; Mantravadi, Rajkumar; Kozhevnikov, Dmitry et al. (2012) Ranolazine stabilizes cardiac ryanodine receptors: a novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2. Heart Rhythm 9:953-60
Salama, Guy; Akar, Fadi G (2011) Deciphering Arrhythmia Mechanisms - Tools of the Trade. Card Electrophysiol Clin 3:11-21

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