Instantaneous force-velocity-length relationship in diaphragmatic sarcomere

Coirault, Catherine, Denis Chemla, Jean-Claude Pourny,Francine Lambert, and Yves Lecarpentier. Instantaneousforce-velocity-length relationship in diaphragmatic sarcomere.J. Appl. Physiol. 82(2): 404-412, 1997.The simultaneous analysis of muscle force, length, velocity, andtime has been shown to precisely characterize the mechanicalperformance of isolated striated muscle. We tested the hypothesis thatthe three-dimensional force-velocity-length relationship reflectsmechanical properties of sarcomeres. In hamster diaphragm strips,instantaneous sarcomere length (SL) and muscle length were simultaneously measured during afterloaded twitches. SL was measured by means of laser diffraction. Wealso studied the influence of initialSL, abrupt changes in total load, and2 × 10⁷ M dantrolene.Baseline resting SL at the apex of thelength-active tension curve was 2.2 ± 0.1 µm, whereasSL at peak shortening was 1.6 ± 0.1 µm in the preloaded twitch and 2.1 ± 0.1 µm in the "isometric" twitch. Over the whole load continuum and at anygiven level of isotonic load, there was a unique relationship between instantaneous sarcomere velocity and instantaneousSL. Part of this relationship was timeindependent and initial SL independent and was markedly downshifted after dantrolene. When five different muscle regions were considered, there were no significant variations ofSL and sarcomere kinetics along themuscle. These results indicate that the time- and initiallength-independent part of the instantaneous force-velocity-lengthrelationship previously described in muscle strips reflects intrinsicsarcomere mechanical properties.

     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.     

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Background

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR α and β/δ) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.

Methodology/Principal Findings

Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR α and β/δ gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (−14%, P<0.05) associated with a decrease in myosin ATPase activity (−49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARβ/δ (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.

Conclusions/Significance

Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARβ/δ that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms.     

Effects of load and tone on the mechanics of isolated human bronchial smooth muscle

Isotonic and isometric properties of nine human bronchial smoothmuscles were studied under various loading and tone conditions. Freshlydissected bronchial strips were electrically stimulated successively atbaseline, after precontraction with10⁷ M methacholine (MCh),and after relaxation with10⁵ M albuterol (Alb).Resting tension, i.e., preload determining optimal initial length(L_o) atbaseline, was held constant. Compared with baseline, MCh decreasedmuscle length to 93 ± 1%L_o(P < 0.001) before any electricalstimulation, whereas Alb increased it to 111 ± 3%L_o(P < 0.01). MCh significantlydecreased maximum unloaded shortening velocity (0.045 ± 0.007 vs.0.059 ± 0.007 L_o/s), maximalextent of muscle shortening (8.4 ± 1.2 vs. 13.9 ± 2.4%L_o), and peakisometric tension (6.1 ± 0.8 vs. 7.2 ± 1.0 mN/mm²). Alb restored all thesecontractile indexes to baseline values. These findings suggest that MChreversibly increased the number of active actomyosin cross bridgesunder resting conditions, limiting further muscle shortening and activetension development. After the electrically induced contraction,muscles showed a transient phase of decrease in tension below preload.This decrease in tension was unaffected by afterload levels but wassignificantly increased by MCh and reduced by Alb. These findingssuggest that the cross bridges activated before, but not during, theelectrically elicited contraction may modulate the phase of decrease intension below preload, reflecting the active part of resting tension.     

Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin

Green fluorescent proteins (GFP) are widely used in biology for tracking purposes. Although expression of GFP is considered to be innocuous for the cells, deleterious effects have been reported. We recently demonstrated that expression of eGFP in muscle impairs its contractile properties (Agbulut, O., Coirault, C., Niederlander, N., Huet, A., Vicart, P., Hagege, A., Puceat, M., and Menasche, P. (2006) Nat. Meth. 3, 331). This prompted us to identify the molecular mechanisms linking eGFP expression to contractile dysfunction and, particularly, to test the hypothesis that eGFP could inhibit actin-myosin interactions. Therefore, we assessed the cellular, mechanical, enzymatic, biochemical, and structural properties of myosin in the presence of eGFP and F-actin. In vitro motility assays, the maximum actin-activated ATPase rate (V(max)) and the associated constant of myosin for actin (K(m)) were determined at 1:0.5, 1:1, and 1:3 myosin:eGFP molar ratios. At a myosin:eGFP ratio of 1:0.5, there was a nearly 10-fold elevation of K(m). As eGFP concentration increased relative to myosin, the percentage of moving filaments, the myosin-based velocity, and V(max) significantly decreased compared with controls. Moreover, myosin co-precipitated with eGFP. Crystal structures of myosin, actin, and GFP indicated that GFP and actin exhibited similar electrostatic surface patterns and the ClusPro docking model showed that GFP bound preferentially to the myosin head and especially to the actin-binding site. In conclusion, our data demonstrate that expression of eGFP in muscle resulted in the binding of eGFP to myosin, thereby disturbing the actin-myosin interaction and in turn the contractile function of the transduced cells. This potential adverse effect of eGFP should be kept in mind when using this marker to track cells following transplantation.     

Increased entropy production in diaphragm muscle of PPAR alpha knockout mice

Peroxisome proliferator activated receptor alpha (PPAR alpha) regulates fatty acid beta-oxidation (FAO) and plays a central role in the metabolic and energetic homeostasis of striated muscles. The thermodynamic consequences of the absence of PPAR alpha were investigated in diaphragm muscle of PPAR alpha knockout mice (KO). Statistical mechanics provides a powerful tool for determining entropy production, which quantifies irreversible chemical processes generated by myosin molecular motors and which is the product of thermodynamic force A/T (chemical affinity A and temperature T) and thermodynamic flow (myosin crossbridge (CB) cycle velocity upsilon). The behavior of both wild type (WT) and KO diaphragm was shown to be near-equilibrium and in a stationary state, but KO was farther from equilibrium than WT. In KO diaphragm, a substantial decrease in contractile function was associated with an increase in both A/T and upsilon and with profound histological injuries such as contraction band necrosis. There were no changes in PPAR delta and gamma expression levels or myosin heavy chain (MHC) patterns. In KO diaphragm, a marked increase in entropy production (A/T x upsilon) accounted for major thermodynamic dysfunction and a dramatic increase in irreversible chemical processes during the myosin CB cycle.     

Short-term variability of pulse pressure and systolic and diastolic time in heart transplant recipients

In heart transplant recipients (HTR), short-term systolic blood pressure variability is preserved, whereas heart rate variability is almost abolished. Heart period is the sum of left ventricular ejection time (LVET) and diastolic time (DT). In the present time-domain prospective study, we tested the hypothesis that short-term fluctuations in aortic pulse pressure (PP) in HTR were related to fluctuations in LVET. Seventeen male HTR (age 48 +/- 6 yr) were studied 16 +/- 11 mo after transplantation. Aortic root pressure was obtained over a 15-s period using a micromanometer both at rest (n = 17) and following the cold pressor test (CPT, n = 14). There was a strong positive linear relationship between beat-to-beat LVET and beat-to-beat PP in all patients at rest and in 13 of 14 patients following CPT (each P < 0.01). The slope of this relationship showed little scatter both at rest (0.34 +/- 0.07 mmHg/ms) and following CPT (0.35 +/- 0.09 mmHg/ms, P = not significant). Given the essentially fixed heart period, DT varied inversely with LVET. As a result, in 13 of 17 HTR at rest and in 12 of 14 HTR following CPT, there was a negative linear relationship between beat-to-beat PP and DT. In conclusion, our short-term time-domain study demonstrated a strong positive linear relationship between LVET and blood pressure variability in male HTR. We also identified a subgroup of HTR in whom there was a mismatch between PP and DT.