Lincoln E. Ford, Ph.D. - US grants

We have recently discovered that the contractile elements of the myocardium contain a viscoelastic element which provides a length independent restoring force to the muscle. This element enables the ventricle to store some of the energy liberated during contraction, and use the energy to hasten diastolic relaxation. It also greatly complicates studies of the mechanical properties of heart muscle. To overcome this complication, we plan to make a "striation follower" that will enable us to measure, and control in a servo-system, the length of a central segment of isolated cardiac muscle. Servo-controlled length steps will be applied to both stimulated and relaxed papillary muscles. The force responses of the relaxed muscle will be subtracted from the responses of the stimulated muscles to define the tension transients produced by the cross-bridges formed during contraction. In addition, the calculated increases in force in the passive elements during isotonic shortening will be calculated to obtain an accurate estimate of the force-velocity capabilities of the cross-bridges. The corrected tension transients and force-velocity relations will be measured during various inotropic interventions to determine whether any of the interventions influence the actomyosin cross-bridges directly, or whether all inotropic interventions act simply by increasing activation of the thin filaments. The inotropic interventions include increased stimulation rate, pair pacing, cardiac glycosides, epinephrine, and caffeine. The proposed analysis will attempt to distinguish between the four following reactions in the cross-bridge cycle: 1) attachment of bridges; 2) movement of attached bridges; 3) detachment of bridges; 4) recovery of recently detached bridges. In addition, the rate of rise of thin filament activation will be inferred from the rise of extrapolated isometric force. This value is determined by extrapolating force-velocity curves, measured during the rise of activation, to zero velocity. The results should provide more accurate data to design better tests for rassessing ventricular contractility and more rational clinical therapies.

This project will test three specific hypotheses that in tracheal smooth muscle: I. the long functioning range of length results from plastic rearrangements of the filament array; II. the slowing of velocity during sustained contraction is due to thick filament lengthening; and III. the activation of contraction is regulated by a second process in addition to myosin phosphorylation. Hypothesis I derives from the preliminary finding that over a 3-fold range of length the muscle has a 2.3-fold change in shortening velocity, a 1.9- fold change in compliance, with less than 25% change in isometric force. These findings indicate that the number of contractile units in series varies directly and almost in proportion to the overall muscle length. The findings by others that thick filaments can be evanescent suggests that this plasticity in structure may result from the dissolution and reformation of thick filaments. This consideration leads to hypothesis II, that the lengthening of thick filaments slows velocity. The additional findings by others that phosphorylation of myosin, which is required for activation, causes folded, inactive myosin to unfold in a way that allows it to form thick filaments suggests that the primary role of myosin phosphorylation may be to control the structural changes. This consideration leads to hypothesis II, because there must be an additional mechanism that will distinguish phosphorylated myosin in thick filaments from phosphorylated myosin free in the cytoplasm. The three hypotheses will be tested by measuring and controlling length of the central segment of the muscle, and using the mechanical responses of the central segment (its compliance, shortening velocity, and transient tension responses to sudden length changes) to indicate changes in the number of contractile units in series and parallel. In studies of activation the instantaneous maximum power will be used to signal changes in the number of activated myosin crossbridges, which will be compared with changes in myosin phosphorylation, and tension transients will be used to signal changes in the number and arrangement of attached crossbridges. Length perturbations of the fully activated muscle will be used to detach crossbridges and reduce isometric force. The time course of redevelopment of force, stiffness, and power will be compared with the rate of rise of the same parameters at the onset of stimulation to distinguish activation processes that occur early in contraction. The results will provide new insights into the contractile mechanisms of smooth muscle. These insights will further our understanding of patho-physiological processes in diseases, such asthma and hypertension, where muscle tone is increased.

musculoskeletal system; magnetic resonance imaging; human tissue; nuclear magnetic resonance spectroscopy; carbohydrates; biomedical resource; Chordata;

An apparatus has been built for the servo-control of working contractions of isolated frog skeletal muscle within an NMR spectrometer. 31P and 1H NMR spectra are obtained of repetitively contracting muscle. During the past year efforts have been directed at (1) determining intracellular buffering power as a function of pH from the 1H and 31P spectra, and (2) obtaining and validating anisotropic water diffusion coefficients from GSLIM processed diffusion images.

An apparatus has been built for the servo-control of working contractions of isolated frog skeletal muscle within an NMR spectrometer. 31P and 1H NMR spectra are obtained of repetitively contracting muscle. During the past year efforts have been directed at (1) determining intracellular buffering power as a function of pH from the 1H and 31P spectra, and (2) obtaining and validating anisotropic water diffusion coefficients from GSLIM processed diffusion images.

Our finding that airway smooth muscle operates over a broad length range suggests 3 related hypotheses: I. Smooth muscle adapts to longer lengths by increasing the number of contractile elements in series. II. Thick-filament lengthening produces the well-known velocity slowing during the rise of activation. III. A thin-filament regulatory mechanism selectively engages filamentous myosin. We have obtained much evidence supporting all three hypotheses, and the results suggest the following Specific Aims to further test these hypotheses and to define the relevant mechanisms: Specific Aim I. Our experiments show that muscles adapt to longer lengths by placing more thick filaments in series, but that the filament increase is only two-thirds of the length increase, suggesting that structures other than thick filaments also contribute to muscle lengthening. Aim I is to quantify the relative contributions of the following four factors likely to increase muscle length: a) longer thick filaments, b) more thick filaments in series, c) lattice tilt, and d) more structures in series with contractile elements. Specific Aim 2. Our experiments show that velocity slowing during the rise of activation is closely correlated with thick-filament formation at an intermediate length and that thick-filament formation is greater at shorter adapted muscle lengths. Aim IIA is to further test the correlation between velocity slowing and filament formation by measuring velocity slowing at different lengths. Aim IIB is to distinguish between 3 possible mechanisms for the slowing, a) longer but fewer thick filaments in series, b) inactivation of whole thick filaments, and c) cytoskeletal rearrangements. Aim IIC will assess the effect of force-altering length manipulations to test whether force changes during the rise of activation alter velocity slowing. Specific Aim 3. The dual role of myosin light-chain phosphorylation, promoting thick-filament formation from myosin monomers and activating myosin[unreadable]s interaction with actin, suggests that another mechanism exists to prevent phosphorylated myosin monomers from interacting with actin before they join filaments. Our observation that calcium promotes crossbridge movement away from thick filaments in the absence of phosphorylation suggests that calcium may regulate this second mechanism. Aim III will use a permeabilized muscle preparation with permanently thiophosphorylated crossbridges to test whether the posited second mechanism exists, how it is regulated, and what other substances affect the interaction of myosin with actin. Relevance. Although asthma and hypertension are usually ascribed to increased smooth muscle tone, the diseases only occur when the muscles are too short. Our finding that smooth muscle undergoes structural adaptations to new lengths suggests that an understanding of the mechanisms of length adaptation would provide a much better understanding of these diseases and suggest new therapeutic targets.