👤 Nozomi Tokuhara

Regulation of myosin motor extension and conformation is central to cardiac muscle contraction-relaxation, with myosin playing a critical role in mechanosensing during the cardiac cycle. Direct assessment of in vivo dynamic interplay between myosin head position, cross-bridge cycling, sarcomere shortening (filament sliding), muscle stress-strain rates and pressure-volume (PV) relationships is key to understanding both normal cardiac function and ventricle pathological states. This work aims to demonstrate that in vivo temporal regulation of myosin head transfer to actin filaments in systole and diastole has important points of difference from current models based on in vitro and ex vivo muscle studies, particularly in settings of diastolic dysfunction. The first study investigated myosin activation-deactivation in a mouse model of diet-induced obesity (high-fat high-sugar diet) with moderate contraction-relaxation impairment. In a second study myosin regulation was investigated in a novel hypertrophic cardiomyopathy mouse model due to a truncation mutation in the sarcomeric gene encoding cardiac myosin binging protein-C, Mybpc3 (Exon 33 deletion). We demonstrate with in vivo small-angle X-ray scattering (SAXS) simultaneous with PV loop analysis in the beating heart that dynamic regulation of myosin is often non-uniform across the left ventricle from the epicardium to subendocardium, with large differences in myosin head behaviour in both systole and diastole, at least in rodents. Our findings underscore that myosin activation-deactivation is intricately tuned to the mechanical demands of the heart and the work of each myocardial layer. Regional myosin filament dysregulation underpins muscle relaxation impairment, offering new insights into potential therapeutic targets. KEY POINTS: This small-angle X-ray scattering (SAXS) study demonstrates that in vivo myosin activation-deactivation and myosin interfilament spacing vary across myocardial layers and are influenced by diet, exercise and pathological conditions in vivo in the murine heart. During the isovolumetric contraction phase, myosin heads exhibit strong cross-bridge binding in response to mechanical load, which increases in the ejection phase, highlighting the critical role of mechanosensing in early force development. In the absence of myosin binding protein-C within the thick filament complex this strong cross-bridge formation is not sustained during ejection. Impaired myosin cross-bridge detachment likely contributes to sustained cross-bridge activation in the isovolumetric relaxation phase and prolongation of relaxation in hypertrophic cardiomyopathy. This study highlights how disturbed myosin mechanosensing evoked by metabolic stress and genetic mutations can impair myosin motor function and correlates with global cardiac dysfunction. Show less

Peyer's patches (PPs), which are covered by specialized follicle-associated epithelium (FAE) including M cells, play a central role in immune induction in the gastrointestinal tract. This study is to investigate a new molecule to characterize PPs. We generated a monoclonal antibody (mAb 10-15-3-3) that specifically reacts to the epithelium of PPs and isolated lymphoid follicles. Target antigen was analyzed by immunoprecipitation and mass spectrometry. Localization and expression of target antigen were evaluated by immunofluorescence, in situ hybridization and real-time PCR. Immunoprecipitation and mass spectrometry revealed that mAb 10-15-3-3 recognized apolipoprotein A-IV (ApoA-IV), a well-known lipid transporter; this finding was confirmed by the specific reactivity of mAb 10-15-3-3 to cells transfected with the murine ApoA-IV gene. Immunofluorescence using mAb 10-15-3-3 showed intestinal localization of ApoA-IV, in which strong expression of the ApoA-IV protein occurred throughout the entire intestinal epithelium during developing period before weaning but was restricted to the FAE in adult mice. In support of these findings, in situ hybridization showed strong expression of the ApoA-IV gene throughout the entire intestinal epithelium during developing period before weaning, but this expression was restricted to the FAE predominantly and the tips of villi to a lesser extent in adult mice. Deficiency of ApoA-IV had no effect on the organogenesis of PP in mice. Our current results reveal ApoA-IV as a novel FAE-specific marker especially in the upper small intestine of adult mice. Show less