Cycling Cross-Bridges Augment Passive Stiffness Componenets During Diastole

Diastolic dysfunction afflicts 1.9 million Americans and is a major cause of morbidity and mortality. The condition forms the primary diagnosis for at least one-third of all heart failure patients and is characterized by diminished cardiac output in the absence of impaired systolic performance. It is particularly prevalent in elderly populations. The proposed experiments examine the general hypothesis that diastolic dysfunction reflects low levels of contractile activity that persist inappropriately throughout the filling phase of the cardiac cycle. Specific Aim 1: Compare the active and passive components of myocardial stiffness in pigs, dogs, rats and mice. Experiments will test the hypotheses that (1) cycling cross-bridges augment the basal passive stiffness of myocardial samples isolated from both large mammals (pigs and dogs) and small rodents (rats and mice) and that (2) the relative size of the cross-bridge stiffness component increases with the N2BAlN2B titin isoform expression ratio and is greatest in large mammals. Specific Aim 2: Identify the molecular mechanisms underpinning age-related changes in myocardial stiffness. Approach: Age-dependent effects on myocardial stiffness will be assessed by stretching intact trabeculae isolated from young, middle-aged and senescent Fischer 344 x Brown Norway F1 rats in the presence and absence of the cross-bridge inhibitor BDM. Potential changes in intracellular free calcium transients will be evaluated using indo-1 fluorescence measurements. Specific Aim 3: Determine how altered cross-bridge kinetics affect dynamic myocardial mechanical properties. Approach: Myocardial stiffness will be measured under conditions where the rates of different acto-myosin state transitions have been altered by changing the concentrations of hydrogen ions, phosphate ions and ADP in the bathing solution. Experiments will utilize (1) porcine preparations as a useful model of human myocardium and (2) enzymatically digested single rat myocytes which exhibit minimal extracellular structural stiffness.