DHPR alpha1S subunit controls skeletal muscle mass and morphogenesis

Abstract

The alpha1S subunit has a dual function in skeletal muscle: it forms the L-type Ca(2+) channel in T-tubules and is the voltage sensor of excitation-contraction coupling at the level of triads. It has been proposed that L-type Ca(2+) channels might also be voltage-gated sensors linked to transcriptional activity controlling differentiation. By using the U7-exon skipping strategy, we have achieved long-lasting downregulation of alpha1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of alpha1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy-related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of alpha1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.

Figure 1

Generation of a local knockdown of α1S subunit. (A) Schema of exonic chaining of pre-mRNA, exons 15, 16 and 17 are represented by blue boxes. Sequences of exon acceptor site SA (in blue) and exon splicing enhancer ESE (in black) of exon 16 were targeted by antisense sequences introduced in U7smOPT cassettes. (B) qRT–PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non-skipped α1S form: 18±6% (^**P⩽0.001, n=19) and 10±7% (^**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1-(U7-ESE) and AAV1-(U7-SA):ΔDHPR or AAV1-(U7-Ctrl): c as a control, respectively. (C) Six months post injection, lysates from TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were immuno-blotted for α1S or α-actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TA^ΔDHPR normalized to TA^Ctrl for each mice, ^**P⩽0.001, n=4. (D) Longitudinal cryo-sections from TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were stained with anti-α1S subunit (red) and anti-laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.

Figure 2

Consequence of α1S subunit knockdown. (A) Six months post injection, TAs from eight mice were dissected and weighed. (B) The internal diameters (shortest diameter) from all fibres throughout the total muscle section were recorded and analysed. Muscles from five different animals were examined. The bar graph presents mean±s.e.m. of the number of myofibres by fibre diameter class for TA^ΔDHPR (black) and TA^Ctrl (grey). (C) Transversal sections of TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were stained with haematoxylin and eosin, bars represent 100 μm. (D) To quantify fibrosis, transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software (marked in blue), data were normalized with total surface of each muscle (orange line surrounding the sections), bars represent 500 μm. (E) Quantification is presented in bar graph and showed 4.1±0.1 fold increase of fibrosis in TA^ΔDHPR (ΔDHPR) compared to TA^Ctrl (c) (^**P<0.001, n=4).

Figure 3

nNOS membrane dissociation, FoxO3A accumulation in myonuclei and upregulation of autophagy-related genes as consequences of α1S subunit loss. (A) mRNA from TA^ΔDHPR and TA^Ctrl tissues were extracted and nNOS expression was quantified by qRT–PCR. Results are expressed as mean±s.e.m., ^**P<0.001, n=4. (B) Transversal cryo-sections of TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were stained with anti-nNOS (red), anti-laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (C) The tissues from extracts were analysed by western blot with FoxO3a-P antibody and normalized with α-actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a-P determined by densitometry and nomalized to the α-actin expression for each muscle, ^*P⩽0.005, n=3. (D) Longitudinal cryo-sections of TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were stained with anti-FoxO3a (red), anti-laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (E) mRNA from TA^ΔDHPR and TA^Ctrl tissues were extracted and regulation of autophagy genes expression was followed by qRT–PCR. Bnip, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TA^ΔDHPR compared with the controlateral TA^Ctrl, ^**P<0.001, n=4.

Figure 4

Formation of autophagosomes in TA^ΔDHPR. (A) Ultrathin sections were imaged by electron microscopy, M, mitochondria; C, collagen fibres; T, T-tubule. Arrows show double-membrane vesicules called auphagosomes. (B) Transversal cryo-sections were stained with anti-LC3b (red), anti-dystrophin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Upper panel: TA^Ctrl (Ctrl) and lower panel: TA^ΔDHPR (ΔDHPR). Bars represent 20 μm. (C) Myofibres isolated from control (Ctrl) or 6 months post-injected FDB (ΔDHPR) muscles were processed for immuno-fluorescent labelling for P62 (green) and LC3b (red) and imaged by confocal microscopy. Scale bars, 10 μm.

Figure 5

Impact of the α1S subunit knockdown in maintenance of muscle ultrastructure. (A) Ultrathin sections of TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm. (B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno-fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.

Figure 6

α1S subunit knockdown and RyR1 expression. (A) Lysates from TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were immuno-blotted for RyR1 or α-actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of RyR1 in TA^ΔDHPR normalized to TA^Ctrl for each mice, ^*P⩽0.005, n=4. (B) Longitudinal cryo-sections of TA^ΔDHPR (ΔDHPR) and TA^Ctrl (c) were stained with anti-α1S subunit (red), anti-RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.

Figure 7

Localization of α 1S subunit. Whole skeletal muscle fibres enzymatically isolated from Ctrl (A–D) or ΔDHPR (E–H) FDB muscles 6 months post injection were processed for immuno-fluorescent labelling for α1 S subunit (red) and laminin (green). Scale bars, 10 μm; Arrows indicate α1 S subunit expression on sarcolemma; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections (Supplementary Movies S5A and S5B). (D, H) Present projections of confocal Z-series (step between each frame is 1 μm) along XZ and YZ planes as indicated.