Abstract 345: R403Q Mutation Increases the Rate of Force Redevelopment in 2 Month Mice

Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy which progresses towards heart failure. Along with deteriorating cardiac function, these hearts experience an overall change in metabolic landscape, suggesting altered energetic function in hearts that express the R403Q mutation. We tested the hypothesis that the R403Q mutation intrinsically increases the energetic cost of contraction. To do this, we determined myofilament function in demembranated cardiac trabeculae from male wild-type (WT) and R403Q mice at 2 months of age, prior to overt signs of cardiac pathology. Firstly, steady-state Ca2+ sensitivity of force generation was not significantly different between male R403Q (n=4) and WT counterparts (n=2) consistent with previous findings. Secondly, the rate of force redevelopment (ktr) in skinned cardiac tissue was measured following unloaded isotonic shortening and a rapid re-stretch to 15% of the original muscle length at a sarcomere length of 2.0μm. R403Q mice display an increased rate of force redevelopment (49.89 s-1 ± 8.13, n = 4) compared to WT counterparts (24.52 ± 4.29, n = 6) at maximal activation indicating an increase in the apparent rates of crossbridges entering and leaving force-generating states (p < 0.05). In conclusion, the R403Q mutation does not impact steady-state Ca2+ sensitivity of force but increases total crossbridge cycling rate suggesting a higher energy cost of force generation. Future studies are aimed at determining the energetic cost of contraction in R403Q hearts and how this increased energetic cost leads to hypertrophic cardiomyopathy.

Time course of isotonic shortening and the underlying contraction mechanism in airway smooth muscle

Although the structure of the contractile unit in smooth muscle is poorly understood, some of the mechanical properties of the muscle suggest that a sliding-filament mechanism, similar to that in striated muscle, is also operative in smooth muscle. To test the applicability of this mechanism to smooth muscle function, we have constructed a mathematical model based on a hypothetical structure of the smooth muscle contractile unit: a side-polar myosin filament sandwiched by actin filaments, each attached to the equivalent of a Z disk. Model prediction of isotonic shortening as a function of time was compared with data from experiments using ovine tracheal smooth muscle. After equilibration and establishment of in situ length, the muscle was stimulated with ACh (100 μM) until force reached a plateau. The muscle was then allowed to shorten isotonically against various loads. From the experimental records, length-force and force-velocity relationships were obtained. Integration of the hyperbolic force-velocity relationship and the linear length-force relationship yielded an exponential function that approximated the time course of isotonic shortening generated by the modeled sliding-filament mechanism. However, to obtain an accurate fit, it was necessary to incorporate a viscoelastic element in series with the sliding-filament mechanism. The results suggest that a large portion of the shortening is due to filament sliding associated with muscle activation and that a small portion is due to continued deformation associated with an element that shows viscoelastic or power-law creep after a step change in force.

Causes of fatigue in slow-twitch rat skeletal muscle during dynamic activity

Skeletal muscle fatigue is most often studied in vitro at room temperature and is classically defined as a decline in maximum force production or power output, exclusively linked to repeated isometric contractions. However, most muscles shorten during normal use, and we propose that both the functional correlate of fatigue, as well as the fatigue mechanism, will be different during dynamic contractions compared with static contractions. Under isoflurane anesthesia, fatigue was induced in rat soleus muscles in situ by isotonic shortening contractions at 37°C. Muscles were stimulated repeatedly for 1 s at 30 Hz every 2 s for a total of 15 min. The muscles were allowed to shorten isotonically against a load corresponding to one-third of maximal isometric force. Maximal unloaded shortening velocity (V0), maximum force production (Fmax), and isometric relaxation rate (−dF/d t) was reduced after 100 s but returned to almost initial values at the end of the stimulation protocol. Likewise, ATP and creatine phosphate (CrP) were reduced after 100 s, but the level of CrP was partially restored to initial values after 15 min. The rate of isometric force development, the velocity of shortening, and isotonic shortening were also reduced at 100 s, but in striking contrast, did not recover during the remainder of the stimulation protocol. The regulatory myosin light chain (MLC2s) was dephosphorylated after 100 s and did not recover. Although metabolic changes may account for the changes of Fmax, −dF/d t, and V0, dephosphorylation of MLC2s may be involved in the fatigue seen as sustained slower contraction velocities and decreased muscle shortening.

Sensitized airway smooth muscle plasticity and hyperreactivity: a reviewThis article is one of a selection of papers published in the Special Issue on Recent Advances in Asthma Research.

To help elucidate the mechanisms underlying asthmatic bronchospasm, the goal of our research has been to determine whether airway smooth muscle (ASM) hyperreactivity was the responsible factor. We reported that in a canine model of asthma, the shortening capacity (ΔLmax) and velocity (Vo) of in vitro sensitized muscle were significantly increased. This increase was of sufficient magnitude to account for 75% narrowing of the in vivo airway, but maximal isometric force was unchanged. This last feature has been reported by others. Under lightly loaded conditions, ASM completes 75% of its isotonic shortening within the first 2 s. Furthermore, 90% of the increased shortening of ragweed pollen-sensitized ASM (SASM), compared with control (CASM), is complete within the first 2 s. The study of shortening beyond this period will apparently not yield much useful information, and studies of isotonic shortening should be focused on this interval. Although both CASM and SASM showed plasticity and adaptation with respect to isometric force, neither muscle type showed a difference in the force developed in these phases. During isotonic shortening, no evidence of plasticity was seen, but the equilibrated SASM showed increased ΔLmax and Vo of shortening. Molecular mechanisms of changes in Vo could result from changes in the kinetics of the myosin heavy chain ATPase. Motility assay, however, showed no changes between CASM and SASM in the ability of the purified myosin molecule (SF1) to translocate a marker actin filament. On the other hand, we found that the state of activation of the ATPase by phosphorylation of smooth muscle myosin light chain (molecular mass 20 000 Da) was greater in the SASM. This would account for the increased Vo. Investigating the signalling pathway, we found that whereas [Ca2+]i increased in both isometric and isotonic contraction, there was no significant difference between CASM and SASM. The content and activity of calmodulin were also not different between the 2 muscles. Nevertheless, we did find that content and total activity of smooth muscle myosin light chain kinase (smMLCK) and the abundance of its message were greater; this would explain the increased MLC20 phosphorylation. The binding affinity between Ca2+ and calmodulin and between 4 Ca2+ calmodulin and smMLCK remains to be studied. We conclude that SASM shows increased isotonic shortening capacity and velocity. It also shows increased content and total activity of smMLCK, which is consistent with the increased shortening. Plasticity produced by oscillation is not seen in the shortening muscle, although it is seen with respect to force development. It did not modulate the behaviour of the sensitized muscle.

Endurance exercise training attenuates cardiac β2-adrenoceptor responsiveness and prevents ventricular fibrillation in animals susceptible to sudden death

Enhanced cardiac β2-adrenoceptor (β2-AR) responsiveness can increase susceptibility to ventricular fibrillation (VF). Exercise training can decrease cardiac sympathetic activity and could, thereby, reduce β2-AR responsiveness and decrease the risk for VF. Therefore, dogs with healed myocardial infarctions were subjected to 2 min of coronary occlusion during the last minute of a submaximal exercise test; VF was observed in 20 susceptible, but not in 13 resistant, dogs. The dogs were then subjected to a 10-wk exercise-training program ( n = 9 susceptible and 8 resistant) or an equivalent sedentary period ( n = 11 susceptible and 5 resistant). Before training, the β2-AR antagonist ICI-118551 (0.2 mg/kg) significantly reduced the peak contractile (by echocardiography) response to isoproterenol more in the susceptible than in the resistant dogs: −45.5 ± 6.5 vs. −19.2 ± 6.3% . After training, the susceptible and resistant dogs exhibited similar responses to the β2-AR antagonist: −12.1 ± 5.7 and −16.2 ± 6.4%, respectively. In contrast, ICI-118551 provoked even greater reductions in the isoproterenol response in the sedentary susceptible dogs: −62.3 ± 4.6%. The β2-AR agonist zinterol (1 μM) elicited significantly smaller increases in isotonic shortening in ventricular myocytes from susceptible dogs after training ( n = 8, +7.2 ± 4.8%) than in those from sedentary dogs ( n = 7, +42.8 ± 5.8%), a response similar to that of the resistant dogs: +3.0 ± 1.4% ( n = 6) and +3.2 ± 1.8% ( n = 5) for trained and sedentary, respectively. After training, VF could no longer be induced in the susceptible dogs, whereas four sedentary susceptible dogs died during the 10-wk control period and VF could still be induced in the remaining seven animals. Thus exercise training can restore cardiac β-AR balance (by reducing β2-AR responsiveness) and could, thereby, prevent VF.

Active and passive components in the length-dependent stiffness of tracheal smooth muscle during isotonic shortening

Contraction of smooth muscle tissue involves interactions between active and passive structures within the cells and in the extracellular matrix. This study focused on a defined mechanical behavior (shortening-dependent stiffness) of canine tracheal smooth muscle tissues to evaluate active and passive contributions to tissue behavior. Two approaches were used. In one, mechanical measurements were made over a range of temperatures to identify those functions whose temperature sensitivity (Q10) identified them as either active or passive. Isotonic shortening velocity and rate of isometric force development had high Q10 values (2.54 and 2.13, respectively); isometric stiffness showed Q10 values near unity. The shape of the curve relating stiffness to isotonic shortening lengths was unchanged by temperature. In the other approach, muscle contractility was reduced by applying a sudden shortening step during the rise of isometric tension. Control contractions began with the muscle at the stepped length so that properties were measured over comparable length ranges. Under isometric conditions, redeveloped isometric force was reduced, but the ratio between force and stiffness did not change. Under isotonic conditions beginning during force redevelopment at the stepped length, initial shortening velocity and the extent of shortening were reduced, whereas the rate of relaxation was increased. The shape of the curve relating stiffness to isotonic shortening lengths was unchanged, despite the step-induced changes in muscle contractility. Both sets of findings were analyzed in the context of a quasi-structural model describing the shortening-dependent stiffness of lightly loaded tracheal muscle strips.