STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice

Abstract

Immediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.

Fig 1

Skeletal muscle-specific deletion of STIM1. (A) Photograph of mouse (P14) lacking STIM1 in skeletal muscle. Mice were generated by crossing STIM1 exon 1-floxed mice with mice carrying Cre recombinase under the control of the myogenin promoter. (B) Protein lysates prepared from mSTIM1^−/− and WT mice were immunoblotted for STIM1 using a polyclonal antibody. (C) Kaplan-Meyer curves for survival of WT (n = 66) and mSTIM1^−/− (n = 18) mice. (D) Growth curves for mSTIM1^−/− mice reveal severe growth failure after postnatal day 3 (P3). n = 30 for each genotype.

Fig 2

Skeletal muscle-specific deletion of STIM1 limits skeletal muscle growth. (A) Fiber numbers in WT and mSTIM1^−/− mice at P21. (B) Myofiber sizes (μm²) in P1 and P21 mice. (C to F) Demonstration of fiber size using dystrophin staining (red) of cross sections of WT and mSTIM1^−/− skeletal muscle at P1 and P21. (C) WT at P1, (D) mSTIM1^−/− at P1, (E) WT at P21, (F) mSTIM1^−/− at P21. (G and H) PAX7 (red) labeling of satellite cell nuclei in WT (G) and mSTIM1^−/− (H) muscle cross sections at P21. Nuclei are labeled with DAPI (blue). (I) Percentages of Pax 7-positive (+ve) nuclei at P21. (J) Numbers of nuclei per fiber cross section at P1 and P21. Data shown represent the means ± SEM. *, P < 0.01. n = 3. Scale bars = 50 μm.

Fig 3

Reduced myonuclear domain and altered ultrastructure in WT and mSTIM1^−/− skeletal muscle. (A) Representative images of WT and mSTIM1^−/− FDB fibers stained with DAPI, for the measurement of myonuclear domain size. Scale bar = 50 μm. (B) Comparison of myonuclear domain (total fiber volume/myonuclei) in FDB fibers from WT and mSTIM1^−/− littermates at P21 (n = 3). Data shown represent the means ± SEM. *, P < 0.05. (C) Immunoblotting for STIM1, p-ERK1/2, ERK1/2, p-AKT, AKT, p-p38, p38, and GAPDH in three pairs of WT and mSTIM1^−/− muscles. (D) Transmission electron micrographs of hind-limb muscles from P10 WT and mSTIM1^−/− (SMKO) mice.

Fig 4

Abnormalities of Ca²⁺ signaling in mSTIM1^−/− muscle fibers. Ca²⁺ signals were measured in single FDB myofibers from WT and mSTIM1^−/− mice using Fura-2. (A) Store-operated calcium entry (SOCE) in WT and mSTIM1^−/− fibers. Representative traces from individual myofibers illustrate a significant decrease in SOCE in mSTIM1^−/− myofibers compared to that in myofibers from WT littermates. (B) The rate of SOCE, calculated by the first derivative of the 340-/380-nm ratio during the maximal rate of rise of the Ca²⁺ increase, was significantly higher in WT myofibers (n = 15) than in mSTIM1^−/− myofibers (n = 9). *, P < 0.005. (C) The relative Ca²⁺ store contents were determined for WT (n = 15; from 2 mice) and mSTIM1^−/− (n = 18; from 2 mice) myofibers. *, P < 0.0005. (D) Relative basal Ca²⁺ levels in WT (n = 62; 3 mice) and mSTIM1^−/− (n = 61; 4 mice) myofibers. Images of 10 fields of myofibers were obtained from each dish. (E) ECC was evaluated by monitoring Ca²⁺ signals produced in myofibers by electrical field stimulation. Left, representative Ca²⁺ responses to stimulus bursts of different durations (100 ms to 5 s at 50 Hz) in WT and mSTIM1^−/− fibers. Right, stimulus-response plot of average Ca²⁺ transient amplitudes versus burst duration in WT (n = 26) and mSTIM1^−/− myofibers (n = 22). (F) Ca²⁺ currents (I_Ca^S) were measured using patch clamp recording from WT (n = 9; from 3 mice) and mSTIM1^−/− (n = 15; from 4 mice) myotubes. I_Ca^S were elicited by a test potential of 10 mV for 250 ms from a holding potential of −40 mV (left). Current-voltage relationship of I_Ca in myotubes from WT and mSTIM1^−/− myotubes (middle). Summary data are provided for WT and mSTIM1^−/− myotubes (right). pA/pF, inward current amplitude normalized to cell capacitance.

Fig 5

mSTIM1^−/− myofiber and muscle responses to sustained electrical activity. (A) Ca²⁺ transients generated by repeated bursts of electrical activity in WT (top) and mSTIM1^−/− (bottom) FDB myofibers. Fibers were repeatedly stimulated with bursts (2 s at 50 HZ, every 5 s [long bar]) for 13 min. (B) The phosphorylation of AKT and p44/42 was measured by immunoblotting of hind-limb muscles following repeated electrical stimulation (500 ms at 50 Hz, every 5 s) of hind limbs for 30 min (ES) and in the absence of electrical stimulation (Con [control]). (C) Relative ratios of phosphorylated to total AKT and p44/42 in hind-limb muscles. Protein loading was normalized by immunoblotting for actin. *, P < 0.05.

Fig 6

Impaired hypertrophic signaling in muscles of mSTIM1^−/− mice. (A) Representative immunoblots for p-NFATC3 and NFATC3 in protein lysates from WT and mSTIM1^−/− muscle (top). Ratio between phospho-NFATc3 and total NFATc3 in WT and mSTIM1^−/− muscles reveals decreased phosphorylation in mSTIM1^−/− muscle (bottom). (B) Representative immunoblots for MEF-2, PPAR-β/δ, PGC-1α, NFATC1, SERCA1, and myoglobin protein levels in WT and mSTIM1^−/− muscle lysates. (C) Overexpression of constitutively active calcineurin (CnA) in the primary cultured STIM1^−/− myotubes rescues the hypertrophic signaling pathway. Expression levels of p-ERK1/2, ERK1/2, p-AKT, AKT, myoglobin, and MEF-2 and actin were measured in WT myotubes, mSTIM1^−/− myotubes (transduced with adenovirus carrying beta-galactosidase), and mSTIM1^−/− myotubes (transduced with an adenovirus that overexpresses constitutively active calcineurin) (MOI = 50 for each). Each experiment was repeated three times using four animals from each genotype. *, P < 0.05.