verbatim): One of the factors which enables the heart to supply enough blood to the body to supply its metabolic needs is the strong effect of diastolic ventricular filling volume on the subsequent systolic ejection of blood from the Heart. In individual myocardial cells increased diastolic volume translates into increased cell length and stretching of the functional units of contraction, the sarcomeres. The strong dependence of contractile force on sarcomere length (SL) is the cellular basis of Starling's Law of the Heart, yet the reason that contraction is so strongly SL dependent in cardiac muscle is not fully understood. In this study we will test the hypothesis that this strong dependence results from a limited ability of intracellular Ca2+ and the force-generating acto-myosin interaction, alone or in combination, to activate cardiac thin filaments. As a result, in cardiac muscle contractile force is more sensitive to the effects of SL (or accompanying changes in myo-filament lattice spacing (LS)) on either the number of attached XBr's or Ca2+ bound to thin filaments. If this idea is correct interventions that enhance or diminish the ability of Ca2+ or XBr's to active contraction should predictably decrease or increase the SL dependence of maximal force and force-Ca2+ relations in chemically skinned cardiac muscle. To test this we will measure the SL dependence of maximal force and force-Ca2+ relations when strong XBr binding is either enhanced or inhibited or when the properties of myosin are altered. To support this approach we will also measure the relation between SL and LS, and monitor myosin structure with low angle X-ray diffraction in skinned cardiac muscle. To determine if SL (or LS) dependent changes in the affinity of thin filament regulatory proteins for Ca2+ also contribute to the Starling mechanism, we will alter the ability of Ca2+ to activate thin filaments by (1) phosphorylation of troponin I, which decreases Ca2+-sensitivity of force (2) interventions which alter the numbers of thin filament regulatory units that can be activated and (3) adding the drug calmidazolium, which increases Ca2+-binding affinity of cardiac troponin C. We will also determine the roles that specific contractile protein isoforms play in regulation by comparing the properties of skinned slow skeletal fibers from soleus with cardiac muscle. The importance of understanding the cellular/molecular basis of Starling's Law is emphasized by the recent findings that the Starling mechanism is not operative in end stage cardiac disease and that mutations in cardiac regulatory proteins are involved in Familial Hypertrophic Cardio-myopathy. The data to be obtained during this study will be of fundamental importance in designing rational drug and gene therapy approaches to intervention in myocardial disease.
