Genetic Loss of Calcineurin Blocks Mechanical Overload-induced Skeletal Muscle Fiber Type Switching but Not Hypertrophy

Abstract

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/ calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin A␤ gene-targeted mice, which show a Ϸ50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin A␤؊/؊ and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin A␤؊/؊ mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of genetargeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overloadmediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1-or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.

Effect of passive stretching on the immobilized soleus muscle fiber morphology

“…Calcineurin, a cytoplasmic calcium-regulated phosphatase, has emerged as a possible candidate in the signaling of skeletal muscle hypertrophy (34), but is not likely to directly regulate skeletal muscle hypertrophy. A prominent role has been suggested for insulin-like growth factor 1 by activating the phosphatidyl-inositol 3-kinase-Akt (a serinethionine kinase) signaling circuit in this process (27,35).…”

Microarray analysis of Tbx5‐induced genes expressed in the developing heart

“…Protein lysate preparation and Western blot analysis was performed as previously described in (Parsons et al, 2004). Briefly, protein was isolated from NIH 3T3 cells 48 hr after infection with Ad-␤-gal or Ad-Tbx5.…”

“…The blot was incubated with a 1:1,000 dilution of a custom synthesized, affinity purified rabbit ␣-Tbx5 antibody raised to the peptide corresponding to amino acids 10 -23 of the murine Tbx5 protein (Zymed Laboratories, Inc.). The blot was subsequently incubated with a 1:1,000 dilution of a goat ␣-rabbit alkaline phosphatase (AP) antibody (Santa Cruz Biotechnology, Inc.) and visualized using chemifluorescence as described in (Parsons et al, 2004).…”

Forced Expression of α-Myosin Heavy Chain in the Rabbit Ventricle Results in Cardioprotection Under Cardiomyopathic Conditions

“…12 To determine cardiomyocyte cross-sectional area, 5-m paraffinembedded sections were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-labeled wheat germ agglutinin (Sigma). 13 Quantification was accomplished by confocal microscopy and SimplePCI imaging software (Compix Inc). Ten fields per section were captured for analysis, and all elliptical myocytes with complete sarcolemmal delineation were traced.…”