Untitled

Abstract

At early stages of muscle development, skeletal muscles contract and relax slowly, regardless of whether they are destined to become fast- or slow-twitch. In this study, we have characterised the activation profiles of developing fast- and slow-twitch muscles from a precocial species, the sheep, to determine if the activation profiles of the muscles are characteristically slow when both the fast- and slow-twitch muscles have slow isometric contraction profiles. Single skinned muscle fibres from the fast-twitch flexor digitorum longus (FDL) and slow-twitch soleus muscles from fetal (gestational ages 70, 90, 120 and 140 days; term 147 days) and neonatal (8 weeks old) sheep were used to determine the isometric force-pCa (pCa = -log10[Ca2+]) and force-pSr relations during development. Fast-twitch mammalian muscles generally have a greatly different sensitivity to Ca2+ and Sr2+ whereas slow-twitch muscles have a similar sensitivity to these divalent cations. At all ages studied, the force-pCa and force-pSr relations of the FDL muscle were widely separated. The mean separation of the mid-point of the curves (pCa50-pSr50) was approximately 1.1. This is typical of adult fast-twitch muscle. The force-pCa and force-pSr curves for soleus muscle were also widely separated at 70 and 90 days gestation (pCa50-pSr50 approximately 0.75); between 90 days and 140 days this separation decreased significantly to approximately 0.2. This leads to a paradoxical situation whereby at early stages of muscle development the fast muscles have contraction dynamics of slow muscles but the slow muscles have activation profiles more characteristic of fast muscles. The time course for development of the FDL and soleus is different, based on sarcomere structure with the soleus muscle developing clearly defined sarcomere structure earlier in gestation than the FDL. At 70 days gestation the FDL muscle had no clearly defined sarcomeres. Force (N cm-2) increased almost linearly between 70 and 140 days gestation in both muscle types and there was no difference between the Ca(2+)- and Sr(2+)-activated force throughout development.

Contribution of Stretch to the Change of Activation Properties of Muscle Fibers in the Diaphragm at the Transition from Fetal to Neonatal Life

“…This study was performed on chemically skinned, isolated muscle fibers as described previously for fetal sheep limb muscles (West et al, 1999). Strips of the costal diaphragm were dissected from the diaphragms collected at each pre- and postnatal age described above, and placed directly into a relaxing solution (mM; propionic acid, 150; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20; ethylene glycol bis (2-aminoethyl ether)- N , N , N ′, N ′-tetra acetic acid (EGTA), 10; MgCl 2 , 3; ATP, 2) containing 50% glycerol (v/v) under slight tension, by tying the ends of the muscle strips with surgical silk (Deknatel, 5.0, USA) to small wooden sticks and stored at −20°C until use.…”

“…Solutions used to activate and relax the muscle fibers have been documented elsewhere (Ashley and Moisescu, 1977) and used by us previously (West et al, 1999; Cannata et al, 2009, 2010; see Table 1). Previously determined apparent affinity constants ( K app ) were used to calculate the calcium and strontium ion concentrations of the solutions (expressed as pCa and pSr, the −log 10 ionic concentration), and the amount of free EGTA in each solution determined by titration.…”

“…Fibers were activated using the “jump-technique” (West et al, 1999). Briefly, each fiber was connected to stainless steel pins using surgical silk (Deknatel 10), and one pin was attached to a micromanipulator and the other to a micro force transducer (AE 801, SensoNor, Horten, Norway).…”

“…The first sigmoid curve describing the data at the lower range of Sr 2+ concentrations (F1) is assumed to reflect the activation of slow isoforms because the small separation of the force–pCa and force–pSr curves is typical of that obtained from “pure” slow-twitch fibers (West et al, 1999; Bortolotto et al, 2000; see Figure 1). The second sigmoid (F2) is likely to reflect the activation of fast isoforms, as the greater separation of the force–pCa and force–pSr curves is typical of “pure” fast-twitch fibers.…”

“…It is not known when this “hybrid” property emerges during development, or what effect the transition to air breathing, or the mechanics of regular forceful breathing might have on the activation properties of the diaphragm fibers. In fetal sheep, limb muscle fibers initially have a “fast-twitch” phenotype, whether characterized by the contraction speed of whole muscle groups (Walker and Luff, 1995), or by the Ca 2+ and Sr 2+ activation properties of single “skinned” muscle fibers (West et al, 1999). We hypothesized that, as for hind limb muscle fibers in fetal sheep, all diaphragm fibers would initially have a fast-twitch phenotype, and that slow-twitch characteristics would emerge either late in gestation or at birth.…”