Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation
Norbert Frey, … , Hugo A. Katus, Eric N. Olson
Norbert Frey, … , Hugo A. Katus, Eric N. Olson
Published October 9, 2008
Citation Information: J Clin Invest. 2008;118(11):3598-3608. https://doi.org/10.1172/JCI36277.
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Research Article

Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation

Abstract

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle–specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2–deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

Authors

Norbert Frey, Derk Frank, Stefanie Lippl, Christian Kuhn, Harald Kögler, Tomasa Barrientos, Claudia Rohr, Rainer Will, Oliver J. Müller, Hartmut Weiler, Rhonda Bassel-Duby, Hugo A. Katus, Eric N. Olson

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Figure 1

Generation of Myoz1 mutant mice.

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Generation of Myoz1 mutant mice. (A) Schematic protein structure of ...
(A) Schematic protein structure of calsarcin-2 highlights the calsarcin homology domain (CHD), which is highly conserved between calsarcin isoforms, the α-actinin binding domain (ABD), and the C terminus, which is highly homologous throughout the calsarcin family. The targeting vector and the targeted mouse Myoz1 allele are shown. Homologous recombination resulted in the deletion of exon 2 and 3 in the Myoz1 gene and their replacement with a neomycin-lacZ reporter cassette. CS2, calsarcin-2. (B) Southern blot analysis of EcoRI-digested genomic DNA of the F1 generation of Myoz1–/+ mice. The 3.5-kb band represents the WT allele, and the 2.5-kb band corresponds to the targeted allele. (C) Western blot of muscle protein homogenate (50 and 150 μg) from gastrocnemius of WT and Myoz1 KO mice. (D) β-Galactosidase staining of tissues from WT and Myoz1 KO animals. The calsarcin-2–deficient tissues showed strong staining in predominantly fast-twitch fiber–containing muscles (e.g., white vastus [WV], gastrocnemius [gastroc]). The soleus, a predominantly slow-twitch muscle, displayed staining in a minor subset of fibers. The heart showed no significant expression of Myoz1. Original magnification, ×4. (E) H&E staining (top) and van Giesson staining (bottom) of gastrocnemius from Myoz1 KO mice and WT littermates. Scale bars: 50 μm. (F) Electron microscopy photographs of gastrocnemius (original magnification, ×19,000). (G) Quantification of 100 Z-disc widths from n = 4 mice per genotype. ‡P < 0.001.