Contractility and kinetics of human fetal and human adult skeletal muscle

Abstract

Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non-human mammalian muscle. Data on human fetal skeletal muscle contraction are lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development and a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterised the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation in human fetal when compared to human adult skeletal muscle. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F-actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) (apparent (fitted) Michaelis-Menten constant) of F-actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy-ATP (dATP) increased F-actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.

Figure 2. Representative traces of myofibril kinetics

A, typical myofibril force trace for a human fetal myofibril. The inset is a close-up of the slow phase of relaxation of the fetal myofibril, in response to a change from maximum [Ca²⁺] (pCa 4.0) to minimum [Ca²⁺] (pCa 9.0). B, activation kinetic differences between human adult and human fetal myofibrils; y-axis is force normalised to each myofibril's maximum force (F_MAX). C, relaxation kinetics between human adult and human fetal myofibrils; y-axis is force normalised to each myofibril's F_MAX. Inset features slow phase kinetics.

Figure 1. Structure of human fetal skeletal muscle of 12–15 weeks gestation with adult human skeletal muscle as a comparison

A, cross-sectional images of tissue stained with H&E (the yellow arrow points to an example of a central nucleus), subjected to alkaline (type II, fast-twitch are dark, pH 10.3) or acidic (type I, slow-twitch are dark, pH 4.3) conditions in an ATPase assay, or hybridised with an anti-embryonic myosin heavy chain or an anti-adult fast/perinatal heavy chain antibody in an immunohistochemistry assay are shown. B, longitudinal images stained with the Richardson stain or EM images at two magnifications demonstrating sarcomeric structure are shown.

Figure 3. In vitro motility unregulated F-actin speeds for increasing [ATP] or [dATP]

Adult rabbit (open triangle with dashed line regression for ATP, fitted K_m(app)= 0.10 mm; filled triangle for dATP) vs. human fetal (open circle for ATP with continuous line for regression, fitted K_m= 0.03 mm; filled circle with dotted line for regression for dATP, fitted K_m(app)= 0.07 mm). Regression fitted curves are hyberbolic fits to the Michaelis–Menten kinetics equation: V_f=V_MAX×[ATP]/(K_m(app)+[ATP]).

Figure 4. In vitro motility adult rabbit (triangle) vs. human fetal (circle) unregulated product (ADP) inhibition while [NTP] is held constant at 2 mm

A, myosin's response to increasing ratio of ADP to ATP, demonstrating product inhibition. B, normalised myosin reponse to increasing the ratio of ADP to ATP, where normalisation is to the top speed of each myosin type individually. Curves are fitted to a linear fit: V_f=a×[ADP]+V_MAX.