Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment

Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Crossbridge Recruitment Capacity of Wild-Type and Hypertrophic Cardiomyopathy-Related Mutant Troponin-T Evaluated by X-ray Diffraction and Mechanical Study of Cardiac Skinned Fibers

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without “troponin-T treatment,” which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm−1 was decreased significantly by ATP addition after treatment with wild-type or mutant troponin-T, indicating that crossbridge formation affected the conformation of troponin-T. In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy.

Diversity of left-right symmetry breaking strategy in animals

Left-right (L-R) asymmetry of visceral organs in animals is established during embryonic development via a stepwise process. While some steps are conserved, different strategies are employed among animals for initiating the breaking of body symmetry. In zebrafish (teleost), Xenopus (amphibian), and mice (mammal), symmetry breaking is elicited by directional fluid flow at the L-R organizer, which is generated by motile cilia and sensed by mechanoresponsive cells. In contrast, birds and reptiles do not rely on the cilia-driven fluid flow. Invertebrates such as Drosophila and snails employ another distinct mechanism, where the symmetry breaking process is underpinned by cellular chirality acquired downstream of the molecular interaction of myosin and actin. Here, we highlight the convergent entry point of actomyosin interaction and planar cell polarity to the diverse L-R symmetry breaking mechanisms among animals.

Acidosis decreases the Ca2+ sensitivity of thin filaments by preventing the first actomyosin interaction

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca2+) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca2+ sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly ( P < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8–pCa 6) but not at maximal Ca2+ concentration (pCa 5–pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin’s powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca2+]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca2+]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca2+ concentrations that might contribute to the loss of force during muscle fatigue.

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke

Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

Development and maintenance of force and stiffness in airway smooth muscle

Airway smooth muscle (ASM) plays a central role in the excessive narrowing of the airway that characterizes the primary functional impairment in asthma. This phenomenon is known as airway hyper-responsiveness (AHR). Emerging evidence suggests that the development and maintenance of ASM force involves dynamic reorganization of the subcellular filament network in both the cytoskeleton and the contractile apparatus. In this review, evidence is presented to support the view that regulation of ASM contraction extends beyond the classical actomyosin interaction and involves processes within the cytoskeleton and at the interfaces between the cytoskeleton, the contractile apparatus, and the extracellular matrix. These processes are initiated when the muscle is activated, and collectively they cause the cytoskeleton and the contractile apparatus to undergo structural transformation, resulting in a more connected and solid state that allows force generated by the contractile apparatus to be transmitted to the extracellular domain. Solidification of the cytoskeleton also serves to stiffen the muscle and hence the airway. Oscillatory strain from tidal breathing and deep inspiration is believed to be the counter balance that prevents hypercontraction and stiffening of ASM in vivo. Dysregulation of this balance could lead to AHR seen in asthma.