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Ca(2+) Permeation and/or Binding to CaV1.1 Fine-Tunes Skeletal Muscle Ca(2+) Signaling to Sustain Muscle Function

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Ca(2+) Permeation and/or Binding to CaV1.1 Fine-Tunes Skeletal Muscle Ca(2+) Signaling to Sustain Muscle Function

Chang Seok Lee et al. Skelet Muscle.

Abstract

Background: Ca(2+) influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca(2+) permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed.

Methods: We generated a mouse with a Ca(2+) binding and/or permeation defect in the voltage-dependent Ca(2+) channel, CaV1.1, and used Ca(2+) imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca(2+) imaging techniques to define pathways modulated by Ca(2+) binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles.

Results: Using mice with a pore mutation in CaV1.1 required for Ca(2+) binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca(2+) stores during sustained activity. Decreases in these Ca(2+)-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca(2+) binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers.

Conclusions: While not essential for excitation-contraction coupling, Ca(2+) binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

Keywords: CaM kinase II; CaV1.1; Fatigue; Fiber type; Protein synthesis and Skeletal muscle.

Figures

Figure 1
Figure 1
Effects of the EK mutation on Ca V 1.1 currents, ECC, and ECCE. Voltage dependence of peak CaV1.1 current density for FDB fibers from (A) WT mice (n = 5) and (B) EK mice (n = 9). (C and D) Representative traces of electrically-evoked mag-fluo-4 transients in FDB fibers obtained from (C) WT and (D) EK mice. Insets: The first transient for each condition on an expanded time scale in FDB fibers obtained from WT and EK mice. (E) Average amplitude and (F) decay constant of the recovery phase in FDB fibers from WT and EK mice. (G) Representative Mn2+ quench of fura-2 fluorescence in myotubes from WT and EK mice. (H) Average rate of Mn2+ quench in myotubes from WT and EK mice. (I) Scheme of ECC changes altered in EK muscle. Data are shown as mean ± SEM. **P <0.01 and ***P <0.001.
Figure 2
Figure 2
Effects of repetitive stimulation. (A) Representative traces for mag-fluo-4 fluorescence in WT and EK FDB fibers subjected to a single 50 Hz train of stimulation for one second and the response was acquired in the line scan mode of a confocal microscope. (B) Tetanic calcium response averaged from first to 50th peak (50 pulses for one second) and the calculated averaged response was plotted. (C) Effects of fatiguing stimulation (100 Hz) on the amplitude of the Ca2+ transients in WT and EK fibers measured with mag-fluo-4. (D) Average amplitude of the 4CmC releasable Ca2+ stores after repetitive stimulation (100 Hz) measured with mag-fluo-4. (E) Cytosolic Ca2+ concentrations measured with Fura-2 before and after electrical stimulation using the same stimulation protocol as in (C). Values represent the average values over one second at 30 seconds after stimulation. Data are shown as mean ± SEM. *P <0.05, **P <0.01, and ***P <0.001.
Figure 3
Figure 3
Ca 2+ handling proteins. (A) Representative western blot images of CaV1.1, RyR1, SERCA1, SERCA2, calsequestrin (1 and 2), and sarcolipin. To allow the use of data in multiple western blots each band within a single western blot was normalized to GAPDH for that sample as a loading control and then normalized to the average WT values for that particular gel to give %WT. (B) Analysis of muscle levels of CaV1.1 normalized to GAPDH. (C) Analysis of muscle levels of RyR1 normalized to GAPDH. (D) Analysis of muscle levels of SERCA1 normalized to GAPDH. (E) Analysis of muscle levels of SERCA2 normalized to GAPDH. (F) Analysis of muscle levels of CSQ normalized to GAPDH. (G) Analysis of sarcolipin normalized to GAPDH. (H) SERCA activity as a function of Ca2+ concentration. (I) Scheme of changes in Ca2+ handling proteins. Values are shown as mean ± SEM. *P <0.05.
Figure 4
Figure 4
Activity-dependent CaMKII translocation and activation. (A) KN-93 decreases the height of the Ca2+ transient during repetitive stimulation. Tetanic calcium response averaged from first to 50th peak (50 pulses for one second) and the calculated averaged response was plotted as in Figure 2B. (B) Ratio of p-CaMKII to CaMKII in muscles of EK and WT mice (%WT). Inset: Representative western blot of p-CaMKII and CaMKII. (C) Representative immunocytochemistry images showing co-localization of CaMKII and CaV1.1 in single WT and EK FDB fibers. (D and E) Representative line profiles of immunofluorescence for CaMKII and CaV1.1 in (D) WT and (E) EK FDB fibers. (F) Representative images for the PLA assay confirming a close association of CaMKII with CaV1.1. Scale bar = 20 μm. For negative control (right), normal rabbit IgG was used instead of CaMKII antibody. (G) Analysis of average spot density in the proximity ligation assay in fiber resting and electrically stimulated. Spots are analyzed in fibers from three mice of each genotype. (H) Effect of AIP on the amplitude of the Ca2+ transient with repetitive stimulation. (I) 4CmC-induced Ca2+ release post stimulation in the presence and absence of AIP. (J) Changes associated with the EK mutation in CaV1.1. Values are shown as mean ± SEM. *P <0.05, **P < 0.01, and ***P < 0.001.
Figure 5
Figure 5
Effect of EK mutation on growth signaling pathways. (A) Representative anti-puromycin western blot for soleus muscle homogenates from EK and WT mice treated with insulin 25 minutes after puromycin. Also shown is a protein stain as a loading control. (B) Representative anti-puromycin western blot for EDL muscle homogenates from EK and WT mice treated with insulin 25 minutes after puromycin. Also shown is a protein stain as a loading control. (C) Analysis of anti-puromycin/protein in the soleus and EDL of saline and insulin-treated EK and WT mice. (D) Western blot for protein involved in protein synthesis as %WT average for each western blot using homogenates from the soleus and EDL muscles from mice treated with saline or insulin. (E and F) Analysis of indicated phospho protein to dephosphorylated proteins (plotted as %WT average) in soleus and EDL. (G and H) Analysis of indicated phospho protein to dephosphorylated protein (plotted as %WT average) in soleus and EDL. (I) Pathways altered by the EK mutation alter protein synthesis. Values are shown as mean ± SEM. *P <0.05 and **P <0.01.
Figure 6
Figure 6
Ca V 1.1/CaMKII modulates Ras/Raf/ERK1/2 signaling to increase mTORC1 signaling in response to electrical stimulation. (A) Representative western blot for activated Ras (Ras-GTP) in Raf-RBD pull-down from WT and EK muscle. (B) Analysis of activated Ras (Ras-GTP) in Raf-RBD pull-down in muscle from WT and EK mice. (C) Electrical stimulation-induced changes in mTORC1 signaling. Representative western blots of isolated solei and EDL muscles subjected to a five-minute electrical stimulation (as described in Methods) in the presence and absence of KN-93 (5 μM). For all analyses the values for the western blots of individual bands from WT and EK muscle are each normalized to the values obtained in the absence of electrical stimulation. For statistical analyses the WT and EK are each compared to their unstimulated controls. (D) Analysis of pCaMKII/GAPDH in the soleus. (E) Analysis of pCaMKII/GAPDH in the EDL. (F) Analysis of p-Raf (a CaMKII target) in the EDL. (G) Analysis of pERK1/2/GAPDH in the soleus. (H) Analysis of pERK1/2/GAPDH in the EDL. (I) Analysis of pS6/S6 in the soleus. (J) Analysis of pS6/S6 in the EDL. (K) Analysis of p4EBP1/4EBP1 in the soleus. (L) Analysis of p4EBP1/4EBP1 in the EDL. (M) Summary of changes in signaling pathways. Values are shown as mean ± SEM. *P <0.05, **P <0.01, and ***P <0.001.
Figure 7
Figure 7
Muscle function. (A) Force frequency relationship for the soleus of eight-week-old mice. (B) Force- frequency relationship for the EDL of eight-week-old mice. (C) Fatigue plotted as % initial force in the soleus of eight-week-old mice. (D) Fatigue plotted as % initial force in the EDL of eight-week-old mice. (E) Force frequency relationship for the soleus of mice over nine months old (F) Fatigue plotted as % initial force in the EDL of mice over nine months old. (G and H) Representative images from muscle (soleus and EDL, respectively) sections pseudo colored for the different fiber types are shown. Primary antibodies against MHC I (clone BA-F8) and IIa (clone sc-71) were probed in the same sections to detect type I (red) and type IIa (green) fibers and antibody for MHC IIb (clone BF-F3) was used in different sections for the detection of type IIb fibers (red), bar is 100 μm. (I and J) Fiber type distributions in soleus and EDL, respectively. (K and L) Evaluation of average cross-sectional area (CSA) for the soleus and EDL, respectively. Fibers from seven mice of each genotype were analyzed. Number of individual fibers in soleus measured: type I: 980 (WT), 1,116 (EK); IIA: 1,898 (WT), 1,980 (EK), IIb/x: 417 (WT), 407 (EK). Number of individual fibers in EDL measured: IIA: 612 (WT), 505 (EK), IIb/x: 1,965 (WT), 2,323 (EK). Values in bar graphs are presented as mean ± SEM, n numbers are indicated. *P <0.05, **P <0.01, and ***P <0.001.

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