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.

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm⁻¹, and of myosin reflection at 1/14.4 nm⁻¹. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.

The specific heat capacity of skinned muscle in an adhering rigor solution was studied with differential scanning calorimetry (DSC) heating runs to search for a heat sink in the sarcomere of the muscle. To elucidate the contribution of major myoproteins to heat capacity, myosin and actin were partially removed by high-KCl and gelsolin treatments, respectively. Differential heat denaturation of myosin (together with α-actinin) and actin was induced to confirm their contributions. On the DSC curve, aside from the endothermic peaks representing ice melting and protein denaturation, the steady baseline level showed a significant increase in basal heat capacity in the presence of skinned muscle compared to the rigor solution alone. In the physiological temperature range from 10 to 25 °C, untreated skinned muscle in the native state (non-denatured) introduced an extra basal heat capacity of 0.4 J K⁻¹ (g evaporable weight)⁻¹, which was diminished by both removing and denaturing actin and was additionally increased by removing myosin; myosin denaturation had little effect on the basal heat capacity. Based on these results, we considered actin to be the fundamental source of extra basal heat capacity, which was partly suppressed by the thermally stable region of myosin under rigor conditions. This extra basal heat capacity was roughly preserved at sub-zero temperatures, suggesting the involvement of non-freezing water molecules. The extra basal heat capacity may have contributed to thermal buffering during muscle function via actin-associated hydration. As a supplemental result, we found a small reversible endothermic peak around −21 °C, which was suppressed in the presence of skinned muscle. Heating beyond the denaturing temperatures reduced this suppression effect.

Leptin receptor-deficient WBN/Kob-Lepr fa/fa (WKF) rats spontaneously developed chronic pancreatitis and severe diabetes with obesity. Here, we evaluated the protective effects of diet restriction and chronic exercise against fatty and inflammation-induced disorders in the vulnerable pancreas of WKF rats. Six-week-old male WKF rats were divided into obese control (Obese), diet restriction (DR), and diet restriction + exercise (DR + Ex) groups. WBN/Kob (WK) rats were used as lean control (Lean). Lean and Obese rats had free access to food, whereas food intake for DR and DR + Ex rats was restricted to 69% and 70% of the Obese level, respectively. The DR + Ex rats exercised voluntarily on a wheel ergometer daily. After six weeks, the rats were euthanized with isoflurane after overnight fasting. Obese rats exhibited diabetes, early stages of pancreatitis, diffuse pancreatic islets, and ultrastructural deteriorations in the pancreatic acinar cells, such as lipid droplet accumulation and swollen mitochondria with destroyed cristae, whereas Lean rats did not. DR rats exhibited improved glucose metabolism and serum triglyceride levels, effectively preventing inflammatory processes in the pancreas. However, DR rats exhibited no amelioration in the serum free fatty acids (FFAs) level, and limited improvements in ultrastructural deterioration in pancreatic cells. Chronic exercise combined with diet restriction (DR + Ex) improved serum FFA levels, the boundary of pancreatic islets, and the ultrastructure of subcellular organelles. These results demonstrate that diet restriction suppresses pancreatic inflammation, and further additional exercise effectively improves fatty pancreas-related deterioration by skeletal muscle activity linked through the circulatory network in WKF rats.