Morphogenetic progression of thigh and lower leg muscles during human embryonic development.

Fetal musculoskeletal movements are first observed at approximately seven to 8 weeks of gestation. However, the separation and formation of skeletal muscles, especially the limbs, have not yet been described in detail. In this study, we elucidate the sequence of events leading to the formation of each thigh and lower leg muscle using serial sections. To observe muscle formation, 26 serial sections (50 legs) of human embryonic specimens ranging from Carnegie stages (CS) 19 to 23 were selected from the Kyoto collection stored at the Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine. As a result, we show the detailed formation and separation processes of the thigh and lower leg muscles. In the thigh, sartorius and tensor fasciae latae are separated at CS19, and the individual muscles observed in adults are identified after CS21. In the lower leg, the tibialis anterior exhibits early separation at CS20, and all muscles are identified at CS22. This study enables future research into the relationship between embryonic development and the evolution of muscle action from quadrupedal to erect bipedal walking.

Three-dimensional visualization and quantitative analysis of embryonic and fetal thigh muscles using magnetic resonance and phase-contrast X-ray imaging.

The musculoskeletal system around the human hip joint has acquired a suitable structure for erect bipedal walking. However, little is known about the process of separation and maturation of individual muscles during the prenatal period, when muscle composition is acquired. Understanding the maturation process of the normal musculoskeletal system contributes to elucidating the acquisition of bipedal walking in humans and to predicting normal growth and detecting congenital muscle disorders and anomalies. In this study, we clarify the process of thigh muscle maturation from the embryonic stage to the mid-fetal stage using serial sections, phase-contrast X-ray computed tomography, and magnetic resonance imaging. We also provide a 4D atlas of human thigh muscles between 8 and 23 weeks of gestation. As a result, we first show that muscle separation in the lower thigh tends to progress from the superficial to the deep layers and that all musculoskeletal components are formed by Carnegie Stage 22. Next, we show that femur and muscle volume grow in correlation with crown-rump length. Finally, we show that the anterior, abductor, and posterior muscle groups in the thigh contain a high percentage of monoarticular muscle volume by the end of the embryonic period. This ratio approaches that of adult muscle composition during normal early fetal development and is typical of bipedal walking. This study of fetal muscle composition suggests that preparation for postnatal walking may begin in early fetal period.

The development of the tensor vastus intermedius during the human embryonic period and its clinical implications.

The tensor vastus intermedius (TVI) is a newly discovered muscle located in the anterolateral thigh area and is considered the fifth component of the quadriceps femoris muscle. There have been several papers describing its anatomical and morphological features in detail; however, many features of this muscle, such as its ontology or kinetic functions, remain unknown. The purpose of this study was to determine the initial appearance of the TVI muscle in human embryonic development and to investigate its growth and development. Histological observations were performed on 30 lower limbs of 15 human embryos from Carnegie stage (CS) 21, 22, and 23 (with crown-rump length ranging from 18.7 to 28.7 mm). Myocyte clusters of the TVI were observed between the vastus lateralis and intermedius muscles in 7 out of 10 limbs in CS 22, indicating that the TVI arises during this stage. In CS 23, the TVI was clearly present in all specimens except one. However, neither the aponeurosis nor the tendonous structure of the TVI were observed in these embryonic stages. Formation of the conventional four components of the quadriceps muscle is completed within CS 21; therefore, our results suggest that the TVI is the last element to develop in the quadriceps femoris complex. It is posited that after the embryonic period, the TVI continues to grow, while forming the tendinous structure toward the patella and receiving vascular supply from certain vascular branches. The clinical significance of these findings is that orthopedists and plastic surgeons who perform surgical procedures within the anterolateral thigh (ALT) area should be aware of the anatomy and development of the TVI in order to reduce surgical complications. Our present research aims to contribute to a deeper understanding of the morphogenesis of the TVI and the other femoral extensor muscles.

Morphometric approach to thermodynamic quantities of solvation of complex molecules: extension to multicomponent solvent.

The morphometric approach (MA) is a powerful tool for calculating a solvation free energy (SFE) and related quantities of solvation thermodynamics of complex molecules. Here, we extend it to a solvent consisting of m components. In the integral equation theories, the SFE is expressed as the sum of m terms each of which comprises solute-component j correlation functions (j = 1,..., m). The MA is applied to each term in a formally separate manner: The term is expressed as a linear combination of the four geometric measures, excluded volume, solvent-accessible surface area, and integrated mean and Gaussian curvatures of the accessible surface, which are calculated for component j. The total number of the geometric measures or the coefficients in the linear combinations is 4m. The coefficients are determined in simple geometries, i.e., for spherical solutes with various diameters in the same multicomponent solvent. The SFE of the spherical solutes are calculated using the radial-symmetric integral equation theory. The extended version of the MA is illustrated for a protein modeled as a set of fused hard spheres immersed in a binary mixture of hard spheres. Several mixtures of different molecular-diameter ratios and compositions and 30 structures of the protein with a variety of radii of gyration are considered for the illustration purpose. The SFE calculated by the MA is compared with that by the direct application of the three-dimensional integral equation theory (3D-IET) to the protein. The deviations of the MA values from the 3D-IET values are less than 1.5%. The computation time required is over four orders of magnitude shorter than that in the 3D-IET. The MA thus developed is expected to be best suited to analyses concerning the effects of cosolvents such as urea on the structural stability of a protein.

Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c: comparison between experimental and theoretical results.

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25 °C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c(551), PH c(552), HT c(552), and AA c(555)) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c(555). However, AA c(555) has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c(555) is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c(555) has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25 °C the α-helix content of the apo state of AA c(555) is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c(551), PH c(552), and HT c(552)).

Effects of side-chain packing on the formation of secondary structures in protein folding.

We have recently shown that protein folding is driven by the water-entropy gain. When the alpha-helix or beta-sheet is formed, the excluded volumes generated by the backbone and side chains overlap, leading to an increase in the total volume available to the translational displacement of water molecules. Primarily by this effect, the water entropy becomes higher. At the same time, the dehydration penalty (i.e., the break of hydrogen bonds with water molecules) is compensated by the formation of intramolecular hydrogen bonds. Hence, these secondary structures are very advantageous units, which are to be formed as much as possible in protein folding. The packing of side chains, which leads to a large increase in the water entropy, is also crucially important. Here we investigate the roles of the side-chain packing in the second structural preference in protein folding. For some proteins we calculate the hydration entropies of a number of structures including the native structure with or without side chains. A hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach is employed in the calculation. Our major findings are as follows. For the structures without side chains, there is an apparent tendency that the water entropy becomes higher as the alpha-helix or beta-sheet content increases. For the structures with side chains, however, a higher content of alpha-helices or beta-sheets does not necessarily lead to larger entropy of water due to the effect of the side-chain packing. The thorough, overall packing of side chains, which gives little space in the interior, is unique to the native structure. To accomplish such specific packing, the alpha-helix and beta-sheet contents are prudently adjusted in protein folding.