Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

Abstract

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.

Figure 1.

Generation of Syt VII −/− mice by gene targeting. (A) Schematic representation of the Syt VII genomic locus, the targeting vector, and the targeted locus. Exons 4–6 are shown as boxes. Restriction enzyme sites (N, NotI; K, KpnI; S, SalI; R, EcoRI; B, BamHI; Nd, NdeI; X, XhoI; H, HindIII) and the 5′ and 3′ external probes are indicated. (B) Southern blot analysis of ES cells showing one of the injected clones (+/−) and two wild-type clones (+/+). (C) Southern blot analysis of tail DNA derived from Syt VII wild-type (+/+), homozygous (−/−), and heterozygous (+/−) animals analyzed with 5′ and 3′ external probes. The sizes of the wild-type and mutant alleles are indicated. (D) Northern blot analysis of RNA from wild-type (+/+) and homozygous (−/−) MEFs (bottom). The concentration of the RNA samples was assessed by ethidium bromide staining of the 28S and 18S RNA (top). (E) Immunoblot analysis of extracts prepared from wild-type (+/+) and homozygous (−/−) bone marrow macrophages (top) and quadriceps muscle (bottom) probed with affinity-purified antibodies against the Syt VII membrane proximal domain. (F) RT-PCR analysis of cDNA from MEFs, bone marrow macrophages (mø), and quadriceps muscle from wild-type (+/+) and Syt VII–deficient (−/−) mice, using primers directed to the C₂A (forward, 5′-CCAGACGCCACACGATGAGTC-3′; and reverse, 5′-CCTTCCAGAAGGTCTGCATCTGG-3′) or membrane proximal spacer (forward, 5′-CCCGCGATGTCCTGCTGGTCT-3′; and reverse, 5′-GTATTCACAGCCTTCCCTCCTGC-3′) domains of Syt VII. Amplification of β-actin cDNA was included as a control.

Figure 2.

Decreased susceptibility to T. cruzi invasion and defective lysosome-mediated membrane repair. (A) Immunofluorescence of wild-type (WT) and Syt VII–deficient (KO) MEFs with antibodies to Lamp-1 and to the spacer domain of Syt VII, indicating that both wild-type and mutated forms of Syt VII are targeted normally to lysosomes. Yellow reflects regions of overlay between anti–Lamp-1 (green) and anti–Syt VII spacer domain (red). (B) Invasion of WT and KO MEFs by T. cruzi. MEFs were exposed to trypomastigotes for the indicated periods of time, washed, and processed for detection of intracellular parasites using an inside/outside immunofluorescence assay. The data represent the average of triplicates ± SD. (C) Total β-hexosaminidase and LDH activity in fibroblast-collagen matrices prepared from WT or KO mice. Enzymatic activity was assayed in detergent extracts. The data represent the average of triplicate matrices ± SD. (D) Release of lysosomal β-hexosaminidase during contraction and wounding of WT or KO MEF-collagen matrices. Matrices were released from the dish at t = 0 and the supernatant was removed and replaced at 2-min intervals for determination of β-hexosaminidase activity. The data represent the average of triplicate matrices ± SD. (E) Release of cytosolic LDH during contraction and wounding of WT or KO MEF-collagen matrices. Matrices were released from the dish at t = 0 and the supernatant was removed and replaced at 2-min intervals, for determination of LDH activity. The data represent the average of triplicate matrices ± SD.

Figure 3.

Inflammation, injury, and fibrosis in skin and skeletal muscle. (A) Histological sections of quadriceps muscle, footpad, and ear of 14-wk-old male wild-type (WT) and Syt VII–deficient (KO) mice. Increased accumulation of connective tissue is evident in the sections of KO mice. Skeletal muscle sections are stained with toluidine blue, footpad sections with H&E, and skin sections with Masson's trichrome, which stains collagen fibers in blue. Bars, 100 μm. (B) Toluidine blue–stained sections of quadriceps muscle from male WT and KO mice at 4, 8, and 44 wk old. Infiltration of inflammatory cells is initially detected (black arrows), followed by tissue injury, fiber invasion by phagocytic cells (white arrow), and collagen deposition (black arrowheads). Bars, 25 μm.

Figure 4.

Inflammatory cells in skeletal muscle. (A) Electron micrographs of sections from the quadriceps muscle of 8-wk-old male wild-type (WT) and Syt VII–deficient (KO) mice. In contrast to WT muscle (top left), in mutant animals, the collagen accumulation and infiltration of inflammatory cells is evident. Arrows point to bundles of collagen fibers, large arrowheads to eosinophils, and small arrowheads to macrophages; n indicates muscle fiber nuclei; c, capillaries; and m, mast cell. (B) Detection of inflammatory cells in the skeletal muscle of Syt VII −/− mice with specific antibodies. Frozen sections of quadriceps muscle of 8-wk-old male WT or KO mice were incubated with biotinylated mAbs to CD3 (T cells), CD11b (macrophages and NK cells), or Ly6G (neutrophils and eosinophils), followed by streptavidin/peroxidase-conjugated secondary antibodies. The first two panels (stained for CD3 and CD11b, respectively) are serial sections, indicating that the same muscle fibers are simultaneously surrounded by T cells and macrophages.

Figure 5.

Myositis symptoms and antinuclear antibody production. (A) Total collagen content over time in ear, liver, and skeletal muscle from male wild-type (WT) and Syt VII–deficient mice (KO). Tissues were weighed, homogenized, hydrolyzed, and assayed for hydroxyproline content. Each column represents the average ± SD of values obtained from four mice. Asterisk indicates significant differences between the data obtained for KO and WT mice (t test, P < 0.03). (B) Serum creatine kinase (CK) levels in male WT and KO mice over time. Each column represents the average ± SD of values obtained from four mice. Asterisk indicates significant differences between the data obtained for KO and WT mice (t test, P < 0.05). (C) Forelimb grip strength over time in male WT and KO mice. Each column represents the average ± SD of measurements made in four to nine mice. Asterisk indicates significant differences between the data obtained for KO and WT mice (t test, P < 0.03). (D) Generation of antinuclear antibodies in Syt VII −/− mice. Fixed and permeabilized wild-type MEFs were incubated with a 1:100 dilution of serum from 8-, 19-, or 44-wk-old WT or KO mice, followed by FITC-conjugated anti–mouse IgG secondary antibodies.