Intracellular Ca(2+)-handling differs markedly between intact human muscle fibers and myotubes.

Karl Olsson,Thomas Gustafsson,Joseph D Bruton,Mamdoh Al-Ameri,Håkan Westerblad,Seher Alam,Eric Rullman,Johanna T Lanner,Arthur J Cheng

doi:10.1186/s13395-015-0050-x

Karl Olsson, Thomas Gustafsson + Show 7 more

Open Access

https://doi.org/10.1186/s13395-015-0050-x

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Abstract

BackgroundIn skeletal muscle, intracellular Ca2+ is an important regulator of contraction as well as gene expression and metabolic processes. Because of the difficulties to obtain intact human muscle fibers, human myotubes have been extensively employed for studies of Ca2+-dependent processes in human adult muscle. Despite this, it is unknown whether the Ca2+-handling properties of myotubes adequately represent those of adult muscle fibers.MethodsTo enable a comparison of the Ca2+-handling properties of human muscle fibers and myotubes, we developed a model of dissected intact single muscle fibers obtained from human intercostal muscle biopsies. The intracellular Ca2+-handling of human muscle fibers was compared with that of myotubes generated by the differentiation of primary human myoblasts obtained from vastus lateralis muscle biopsies.ResultsThe intact single muscle fibers all demonstrated strictly regulated cytosolic free [Ca2+] ([Ca2+]i) transients and force production upon electrical stimulation. In contrast, despite a more mature Ca2+-handling in myotubes than in myoblasts, myotubes lacked fundamental aspects of adult Ca2+-handling and did not contract. These functional differences were explained by discrepancies in the quantity and localization of Ca2+-handling proteins, as well as ultrastructural differences between muscle fibers and myotubes.ConclusionsIntact single muscle fibers that display strictly regulated [Ca2+]i transients and force production upon electrical stimulation can be obtained from human intercostal muscle biopsies. In contrast, human myotubes lack important aspects of adult Ca2+-handling and are thus an inappropriate model for human adult muscle when studying Ca2+-dependent processes, such as gene expression and metabolic processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0050-x) contains supplementary material, which is available to authorized users.

Highlights

In skeletal muscle, intracellular Ca2+ is an important regulator of contraction as well as gene expression and metabolic processes
While about half of the myotubes responded to electrical stimulation with a transient increase in fluo-3 signal, none of the myoblasts showed any change in fluo-3 signal upon electrical stimulation (Fig. 1a)
Despite the marked increases in [cytosolic free [Ca2+] (Ca2+]i) upon electrical stimulation, no contraction of any myotube was observed. This was further investigated in myotubes exposed to 4-CmC which acts on the ryanodine receptor (RyR) to facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR) [31] in order to fully activate SR Ca2+ release, and again, no contraction was observed even when [Ca2+]i was maximally increased (Fig. 2a, b)

Summary

Introduction

Intracellular Ca2+ is an important regulator of contraction as well as gene expression and metabolic processes. Human myotubes can obtain contractile ability under specific culture conditions, generated contractile forces are Olsson et al Skeletal Muscle (2015) 5:26 lower, fused tetani are attained at lower stimulation frequencies, and kinetic parameters are slower than those in adult muscle fibers [12,13,14]. This suggests an immature intracellular Ca2+-handling in myotubes, a notion supported by findings in previous studies investigating the Ca2+-handling properties of human myotubes [15,16,17]. There is a lack of knowledge regarding the quantitative aspects of these differences since no direct comparison between human intact muscle fibers and myotubes has been previously performed

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