Expression of the Plasma Membrane Ca2+-ATPase in Myogenic Cells

Annette Hammes,Silke Oberdorf-Maass,Susanne Jenatschke,Theo Pelzer,Alexander Maass,Frank Gollnick,Rainer Meyer,Jörn Afflerbach,Ludwig Neyses

doi:10.1074/jbc.271.48.30816

Annette Hammes, Silke Oberdorf-Maass + Show 7 more

Open Access

https://doi.org/10.1074/jbc.271.48.30816

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Abstract

To study the physiological function of the plasma membrane calmodulin-dependent calcium ATPase (PMCA) in intact cells, L6 myogenic cell lines stably overexpressing the human PMCA isoform 4CI (= human PMCA isoform 4b) were generated. Several independent L6 clones and controls stably transfected with the empty expression vector were analyzed in detail. The resting cytosolic calcium level in hPMCA4CI-overexpressing muscle cells (measured by the Fura-2 method) was significantly reduced by 20-30% compared with controls. This was shown in a cytosolic window of 1322 single cells (p < 0.01). Furthermore, the differentiation process of these cells was remarkably accelerated compared with control myoblasts and parental nontransfected L6 cells as assessed by multinucleated myotube formation and creatine phosphokinase activity elevation. After 4 and 6 days of differentiation, PMCA-overexpressing L6 cells from four independent clones displayed a 3- and 4-fold higher creatine phosphokinase activity compared with controls (n = 5, p < 0.02). These results may extend the concept of the function of the PMCA from simple prevention of calcium overload to an active involvement in intracellular calcium regulation with potentially important consequences for cellular functions.

Highlights

The calmodulin-dependent plasma membrane calcium ATPase [1, 2] is a system for extrusion of Ca2ϩ from the cell
20 neomycin-resistant L6 myoblast clones were isolated by G418 selection after transfecting the cells with a vector carrying the human plasma membrane calcium ATPase isoform 4CI cDNA and the neomycin resistance gene both driven by the CMV promoter
The question addressed in the present work was whether the plasma membrane calmodulin-dependent calcium ATPase (PMCA) is involved in the regulation of intracellular calcium in intact muscle cells and whether changes in the calcium concentration result in alterations of a major physiological cellular function

Summary

Introduction

The calmodulin-dependent plasma membrane calcium ATPase [1, 2] is a system for extrusion of Ca2ϩ from the cell. L6 myoblasts can be induced to differentiate into multinucleated myotubes under low serum conditions and are a suitable model to study terminal myogenic differentiation In this model recent results from our group have shown that expression of endogenous PMCA isoforms is regulated in a differentiation-specific manner and forced expression of the myogenic determination factor myogenin in fibroblasts is sufficient to induce the muscle-specific PMCA expression pattern [16, 17]. These results showed the complex and precise regulation of expression of PMCA isoforms and splice variants and suggested their importance for differentiation in this model of myogenesis

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