Timothy G. West

Physiological properties of human diaphragm muscle fibres and the effect of chronic obstructive pulmonary disease

The contractile and actomyosin ATPase properties of single fibres were examined in human diaphragm muscle obtained from patients with and without chronic obstructive pulmonary disease (COPD). Costal diaphragm biopsies were taken from five patients without evidence of COPD and from 11 age‐matched individuals with varying degrees of the disease. Our aim was to establish whether changes in contractile properties of COPD diaphragm could be fully explained by the previously documented shift towards a greater proportion of type I myosin heavy chain isoform in COPD. The relative proportion of type I diaphragm fibres from non‐COPD and COPD patients was measured by gel electrophoresis, and was negatively correlated with FEV1 over the full range of values investigated. There was also significant atrophy of the type I fibre population in COPD diaphragms. Isometric tension was similar among the fibre types and between the COPD and non‐COPD patients. The intrinsic energetic properties of diaphragm fibres were examined by monitoring the time‐resolved actomyosin ATPase activity in COPD and non‐COPD fibres that produced similar isometric forces. The isometric ATPase rate in COPD fibres was reduced to 50% of the rate in non‐COPD fibres; hence, the cost of isometric contraction in type I and type IIA COPD fibres was reduced to between one‐third and one‐half of the tension cost calculated for non‐COPD fibres. The rate of force development in type I COPD fibres was reduced to 50% of the rate seen in non‐COPD type‐I fibres. No difference in the rate of ATP consumption between COPD and non‐COPD fibres was evident during isovelocity shortening. These data extend previous findings showing that aspects of breathing mechanics during progressive COPD are associated with remodelling of the diaphragm fibre‐type distribution; on top of the increase in type I fibres there are fibre‐specific reductions in force development rate (type I fibres) and ATPase rate that are consistent with the impairment of cross‐bridge cycling kinetics.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function. Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening. Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease. Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease. Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Actomyosin energy turnover declines while force remains constant during isometric muscle contraction

Energy turnover was measured during isometric contractions of intact and Triton‐permeabilized white fibres from dogfish (Scyliorhinus canicula) at 12°C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (Pi) release by permeabilized fibres was recorded using the fluorescent protein MDCC‐PBP, N‐(2‐[1‐maleimidyl]ethyl)‐7‐diethylamino‐coumarin‐3 carboxamide phosphate binding protein. The steady‐state ADP release rate was measured using a linked enzyme assay. The rates decreased five‐fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 ±s.e.m. 28 μW mg−1 (n= 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the Pi release rate decreased from 5.00 ± 0.39 mmol l−1 s−1 at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady‐state ADP release rate at 15 s was 21% of the Pi rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and Pi release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing Pi concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres.

Millisecond-Scale Biochemical Response to Change in Strain

Muscle fiber contraction involves the cyclical interaction of myosin cross-bridges with actin filaments, linked to hydrolysis of ATP that provides the required energy. We show here the relationship between cross-bridge states, force generation, and Pi release during ramp stretches of active mammalian skeletal muscle fibers at 20°C. The results show that force and Pi release respond quickly to the application of stretch: force rises rapidly, whereas the rate of Pi release decreases abruptly and remains low for the duration of the stretch. These measurements show that biochemical change on the millisecond timescale accompanies the mechanical and structural responses in active muscle fibers. A cross-bridge model is used to simulate the effect of stretch on the distribution of actomyosin cross-bridges, force, and Pi release, with explicit inclusion of ATP, ADP, and Pi in the biochemical states and length-dependence of transitions. In the simulation, stretch causes rapid detachment and reattachment of cross-bridges without release of Pi or ATP hydrolysis.

Power output of skinned skeletal muscle fibres from the cheetah ( Acinonyx jubatus )

SUMMARY Muscle samples were taken from the gluteus, semitendinosus and longissimus muscles of a captive cheetah immediately after euthanasia. Fibres were ‘skinned’ to remove all membranes, leaving the contractile filament array intact and functional. Segments of skinned fibres from these cheetah muscles and from rabbit psoas muscle were activated at 20°C by a temperature-jump protocol. Step and ramp length changes were imposed after active stress had developed. The stiffness of the non-contractile ends of the fibres (series elastic component) was measured at two different stress values in each fibre; stiffness was strongly dependent on stress. Using these stiffness values, the speed of shortening of the contractile component was evaluated, and hence the power it was producing. Fibres were analysed for myosin heavy chain content using gel electrophoresis, and identified as either slow (type I) or fast (type II). The power output of cheetah type II fibre segments was 92.5±4.3 W kg−1 (mean ± s.e., 14 fibres) during shortening at relative stress 0.15 (the stress during shortening/isometric stress). For rabbit psoas fibre segments (presumably type IIX) the corresponding value was significantly higher (P<0.001), 119.7±6.2 W kg−1 (mean ± s.e., 7 fibres). These values are our best estimates of the maximum power output under the conditions used here. Thus, the contractile filament power from cheetah was less than that of rabbit when maximally activated at 20°C, and does not account for the superior locomotor performance of the cheetah.

Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.

Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l−1 (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P3‐1‐(2 nitrophenyl) ethyl ester of ATP (NPE‐caged ATP), and time‐resolved phosphate (Pi) release was detected with the fluorescent protein MDCC‐PBP, N‐(2[1‐maleimidyl]ethyl)‐7‐diethylamino‐coumarin‐3‐carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of Pi release at standard and high IS values were not significantly different. A five‐step reaction mechanism was used to account for the observed time courses of force and Pi release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and Pi with the contractile proteins varied with IS, thus suggesting that the actin–myosin interactions are largely non‐ionic. Our reaction scheme also fits previous results for intact fibres.

Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula

Key points  •  Muscle force and shortening are generated by a structural change called the working stroke in myosin motor proteins that cross‐link the myosin and actin filaments in muscle. •  Precise values for two key parameters of the myosin motor – its mechanical stiffness and the size of the working stroke at low load – were previously only available from one type of muscle in one species, fast twitch muscles of the frog, so it was not clear how generally applicable these values were. •  We show that in dogfish fast muscle the low‐load working stroke is the same as in frog muscle, but the myosin motor stiffness is smaller. •  The results provide new insights into how the molecular properties of myosin motors in different muscle types and species may be adapted for different muscle functions.

Time Course and Strain Dependence of ADP Release during Contraction of Permeabilized Skeletal Muscle Fibers

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P approximately NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P(i) transient during force recovery at 20 degrees C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.

Biomechanics of predator–prey arms race in lion, zebra, cheetah and impala

The fastest and most manoeuvrable terrestrial animals are found in savannah habitats, where predators chase and capture running prey. Hunt outcome and success rate are critical to survival, so both predator and prey should evolve to be faster and/or more manoeuvrable. Here we compare locomotor characteristics in two pursuit predator–prey pairs, lion–zebra and cheetah–impala, in their natural savannah habitat in Botswana. We show that although cheetahs and impalas were universally more athletic than lions and zebras in terms of speed, acceleration and turning, within each predator–prey pair, the predators had 20% higher muscle fibre power than prey, 37% greater acceleration and 72% greater deceleration capacity than their prey. We simulated hunt dynamics with these data and showed that hunts at lower speeds enable prey to use their maximum manoeuvring capacity and favour prey survival, and that the predator needs to be more athletic than its prey to sustain a viable success rate.