Intracellular oxygen tension limits muscle contraction-induced change in muscle oxygen consumption under hypoxic conditions during Hb-free perfusion.

Under acute hypoxic conditions, the muscle oxygen uptake (mV˙O2) during exercise is reduced by the restriction in oxygen-supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the mV˙O2 under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in mV˙O2 is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PmbO2) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin-free Krebs-Henseleit buffer equilibrated with three different fractions of O2 gas: 95.0%O2, 71.3%O2, and 47.5%O2 The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near-infrared spectroscopy. The ∆[deoxy-Mb] kinetics were converted to oxygen saturation of myoglobin (SmbO2), and the PmbO2 was then calculated based on the SmbO2 and the O2 dissociation curve of the Mb. The SmbO2 and PmbO2 at rest decreased with the decrease in O2 supply, and the muscle contraction caused a further decrease in SmbO2 and PmbO2 under all O2 conditions. The net increase in mV˙O2 from the muscle contraction (∆mV˙O2) gradually decreased as the ∆PmbO2 decreased during muscle contraction. The results of this study suggest that ΔPmbO2 is a key determinant of the ΔmV˙O2

Endurance training facilitates myoglobin desaturation during muscle contraction in rat skeletal muscle.

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O2 to the mitochondria. Accordingly, intracellular O2 tension (PmbO2) markedly declines in order to increase muscle O2 uptake (mVO2). However, whether the change in PmbO2 during muscle contraction modulates mVO2 and whether the O2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O2 saturation of Mb (SmbO2) and PmbO2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min × 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the VmO2peak significantly increased by 32%. Although the PmbO2 during muscle contraction did not affect the increased mVO2 in endurance-trained muscle, the O2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in SmbO2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster VO2 kinetics in endurance-trained muscle

NIRS measurement of O(2) dynamics in contracting blood and buffer perfused hindlimb muscle.

金沢大学人間社会研究域人間科学系In order to obtain evidence that Mb releases O(2) during muscle contraction, we have set up a buffer-perfused hindlimb rat model and applied NIRS to detect the dynamics of tissue deoxygenation during contraction. The NIRS signal was monitored on hindlimb muscle during twitch contractions at 1 Hz, evoked via electrostimulator at different submaximal levels. The hindlimb perfusion was carried out by perfusion of Krebs Bicarbonate buffer. The NIRS still detected a strong signal even under Hb-free contractions. The deoxygenation signal (Delta[deoxy]) was progressively increased at onset of the contraction and reached the plateau under both blood- and buffer-perfused conditions. However, the amplitude of Delta[deoxy] during steady state continued to significantly increase as tension increased. The tension-matched comparison of the Delta[deoxy] level under buffer-perfused and blood perfused conditions indicate that Mb can contribute approximately 50% to the NIRS signal. These results clarify the Mb contribution to the NIRS signal and show a falling intracellular PO(2) as workload increases

Muscle contraction increases carnitine uptake via translocation of OCTN2

Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL uptake) of l-[ 3H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL uptake of [ 14C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL uptake of l-[ 3H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL uptake of [ 14C]IAP was much higher than that of l-[ 3H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane. © 2012 Elsevier In

Intracellular oxygen tension limits muscle contraction-induced change in muscle oxygen consumption under hypoxic conditions during Hb-free perfusion

Under acute hypoxic conditions, the muscle oxygen uptake (m (Formula presented.) O2) during exercise is reduced by the restriction in oxygen-supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the m (Formula presented.) O2 under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in m (Formula presented.) O2 is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PmbO2) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin-free Krebs–Henseleit buffer equilibrated with three different fractions of O2 gas: 95.0%O2, 71.3%O2, and 47.5%O2. The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near-infrared spectroscopy. The ∆[deoxy-Mb] kinetics were converted to oxygen saturation of myoglobin (SmbO2), and the PmbO2 was then calculated based on the SmbO2 and the O2 dissociation curve of the Mb. The SmbO2 and PmbO2 at rest decreased with the decrease in O2 supply, and the muscle contraction caused a further decrease in SmbO2 and PmbO2 under all O2 conditions. The net increase in m (Formula presented.) O2 from the muscle contraction (∆m (Formula presented.) O2) gradually decreased as the ∆PmbO2 decreased during muscle contraction. The results of this study suggest that ΔPmbO2 is a key determinant of the Δm (Formula presented.) O2. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society

Endurance training facilitates myoglobin desaturation during muscle contraction in rat skeletal muscle

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O2 to the mitochondria. Accordingly, intracellular O2 tension (PmbO2) markedly declines in order to increase muscle O2 uptake (mVO2). However, whether the change in PmbO2 during muscle contraction modulates mVO2 and whether the O2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O2 saturation of Mb (SmbO2) and PmbO2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min x 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the mVO2peak significantly increased by 32%. Although the PmbO2 during muscle contraction did not affect the increased mVO2 in endurance-trained muscle, the O2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in SmbO2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster VO2 kinetics in endurance-trained muscle. © 2015, Nature Publishing Group. All rights reserved

Mechanical guidance of self-condensation patterns of differentiating progeny

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.Matsuzaki T., Shimokawa Y., Koike H., et al. Mechanical guidance of self-condensation patterns of differentiating progeny. iScience 25, 105109 (2022); https://doi.org/10.1016/j.isci.2022.105109

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).Matsuzaki T., Kawano Y., Horikiri M., et al. Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells. STAR Protocols 4, 102471 (2023); https://doi.org/10.1016/j.xpro.2023.102471