Mechanisms of excitation-contraction (EC) coupling have not been defined in the developing heart. We hypothesize that transsarcolemmal Ca/2+ influx is the major source of activator Ca/2+ for contractions in the immature heart. Experiments will be performed using ventricular myocytes isolated from rabbits and humans at four different developmental stages. Confocal laser scanning microscopy will be used to define subcellular Ca/2+ distribution during myocyte contractions (fluo-3) and to quantitate postnatal T-tubule development (di-8-ANEPPS). Complementary approaches will be used to characterize various Ca/2+ transport pathways, including L- and T-type Ca/2+ channels, SR Ca/2+ release (triggered by Ca/2+ influx of depolarization), """"""""reverse"""""""" Na+-Ca/2+ exchange and Ca/2+ entry through Na channels operating in slip mode conductance. A pharmacological approach will be used to individually block the respective Ca/2+ entry pathways (or SR function) while cells are voltage clamped with their own action potential as the command voltage (to eliminate changes in action potential duration). In a second approach, the absolute magnitudes of Ca/2+ influx by each relevant pathway will be established using experimental conditions highly optimized for each pathway (specific experimental solutions and square- step clamping protocols). Since transarcolemmal Ca/2+ influx and the shape of the Ca/2+ transients will be recorded in single myocytes using different action potentials and intracellular Na+ concentrations. Age- related differences in the magnitude and time course of Ca/2+ transients may be influenced by changes in cytosolic Ca/2+ buffering and therefore, Ca/2+ buffer power will be determined in separate experiments. Mathematical models of adult ventricular cells provide relatively accurate descriptions of action-potential configuration and Ca/2+ transients, but no such model exists for immature myocytes. We will construct a mathematical model to describe the electrophysiological and Ca/2+ dynamics of neonatal ventricular cells based on previous reports and new data from the present studies. Results from the proposed experiments will provide important new insights into fundamental aspects of the regulation of [Ca/2+]i and contractions in the immature heart. This new information will help fulfil the longer-term goal of understanding normal and pathological mechanisms involved in controlling contractility during cardiac development. These data will ultimately form the foundation for designing age-appropriate therapeutic strategies for infants and children with cardiac dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058899-04
Application #
6537344
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Schramm, Charlene A
Project Start
1999-04-01
Project End
2003-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
4
Fiscal Year
2002
Total Cost
$270,181
Indirect Cost
Name
New York University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
Srivastava, Shekhar; Cala, Steven E; Coetzee, William A et al. (2007) Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation. Biochem Biophys Res Commun 355:338-41
Srivastava, Shekhar; Collis, Leon; Go, Anita et al. (2005) Paradoxical effect of dofetilide on action potential duration and calcium transient amplitude in newborn rabbit ventricular myocytes. J Cardiovasc Pharmacol 45:165-74
Go, Anita; Srivastava, Shekhar; Collis, Leon et al. (2005) Negative inotropic effect of nifedipine in the immature rabbit heart is due to shortening of the action potential. Pediatr Res 57:399-403
Haddock, P S; Coetzee, W A; Cho, E et al. (1999) Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes. Circ Res 85:415-27