The applicant's long-term aims are to continue his studies on excitation contraction coupling in the heart.
The specific aims are to study rabbit ventricular cells and 1. Investigate the dependence of spark probability (Ps) on [Ca]o 2. Investigate the dependence of spark probability on [Ca]o in the absence of Na gradient; 3. Investigate the fluorescence produced by Ca current and its relationship to (Ps) and, 4. Investigate the relationship between spark sites and structural morphology. The application is aimed at adducing evidence that clusters of L-type Ca channels are required to trigger sparks at high probability during a Ca transient. It is argued that spark probability is steeply dependent upon the size of the clusters of L-type Ca channels that trigger sparks. Since L-type Ca channels are reduced in diseased heart this phenomenon could explain the extinction of Ca transients in disease and as such establishes an aspect of the health relatedness of this project. Experiments are designed to show that a) the recruitment of spark sites will depend on [Ca]o and that sites triggered by smaller clusters of L-type channels will appear at lower [Ca]o. b) Sparks sites triggered by larger clusters of L-type Ca channels should show a steeper dependence of Ps on [Ca]o. These experiments will be repeated on cells in the absence of a Na gradient to establish the effect of the Na gradient on Ps. The experimental design includes a demonstration that sparks exhibit a lower Ps in the absence of a Na gradient when the SR content is matched to its value in the presence of a Na gradient. Finally the binomial distribution will be used to test the hypothesis that with the SR Ca content matched more L-type Ca channels are available to gate RyRs in the presence of a Na gradient than in its absence. As a corollary the hypothesis that not all open L-type Ca channels gate RyRs will be tested, c) The probability of spark production (Ps) is expected to correlate with the intensity of fluorescent lines that is attributable to Ca passing through clusters of L-type Ca channels. The greater the intensity of these lines, the larger the cluster of L-type Ca channels and hence the greater Ps. Finally, d) the relationship between sparks sites and local cell morphology will be established by measuring sparks and then investigating the distribution of RyRs and DHPRs in the same cell. Methods include measuring sparks and cell morphology with a combination of antibodies and mathematical deconvolution techniques. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062690-06
Application #
6884079
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Przywara, Dennis
Project Start
1999-05-01
Project End
2009-04-30
Budget Start
2005-05-18
Budget End
2006-04-30
Support Year
6
Fiscal Year
2005
Total Cost
$336,375
Indirect Cost
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Zahradníková, Alexandra; Gaburjáková, Marta; Bridge, John H B et al. (2010) Challenging quantal calcium signaling in cardiac myocytes. J Gen Physiol 136:581-3
Larbig, Robert; Torres, Natalia; Bridge, John H B et al. (2010) Activation of reverse Na+-Ca2+ exchange by the Na+ current augments the cardiac Ca2+ transient: evidence from NCX knockout mice. J Physiol 588:3267-76
Torres, Natalia S; Larbig, Robert; Rock, Alex et al. (2010) Na+ currents are required for efficient excitation-contraction coupling in rabbit ventricular myocytes: a possible contribution of neuronal Na+ channels. J Physiol 588:4249-60
Sachse, Frank B; Savio-Galimberti, Eleonora; Goldhaber, Joshua I et al. (2009) Towards computational modeling of excitation-contraction coupling in cardiac myocytes: reconstruction of structures and proteins from confocal imaging. Pac Symp Biocomput :328-39
Sobie, Eric A; Cannell, Mark B; Bridge, John H B (2008) Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes. Biophys J 94:L54-6
Savio-Galimberti, Eleonora; Frank, Joy; Inoue, Masashi et al. (2008) Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images. Biophys J 95:2053-62
Bridge, John H B; Savio-Galimberti, Eleonora (2008) What are the consequences of phosphorylation and hyperphosphorylation of ryanodine receptors in normal and failing heart? Circ Res 102:995-7
Sachse, Frank B; Savio-Galimberti, Eleonora; Goldhaber, Joshua I et al. (2008) Sub-micrometer anatomical models of the sarcolemma of cardiac myocytes based on confocal imaging. Pac Symp Biocomput :390-401
Bridge, John H B; Davidson, Christopher J; Savio-Galimberti, Eleonora (2006) A novel mechanism of pacemaker control that depends on high levels of cAMP and PKA-dependent phosphorylation: a precisely controlled biological clock. Circ Res 98:437-9
Inoue, Masashi; Bridge, John H B (2005) Variability in couplon size in rabbit ventricular myocytes. Biophys J 89:3102-10

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