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First published online 30 October 2007
doi: 10.1242/jcs.009241

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Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization

Michele R. Stone^1,*,, Andrea O'Neill^1,*, Richard M. Lovering^1,*, John Strong¹, Wendy G. Resneck¹, Patrick W. Reed¹, Diana M. Toivola^2,3, Jeanine A. Ursitti⁴, M. Bishr Omary^2,3 and Robert J. Bloch^1,

¹ Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
² Department of Medicine, Palo Alto VA Medical Center, 3801 Miranda Avenue, Mail code 154J, Palo Alto, CA 94304, USA
³ Stanford University Digestive Disease Center, 300 Pasteur Drive, Stanford, CA 94305, USA
⁴ Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA

View larger version (51K): [in this window] [in a new window]   Fig. 1. Analysis of mutant mice for the presence of K19. (A) Extracts of muscle were analyzed by RT-PCR for the presence of mRNA encoding K8 and K19. K19–/– extracts contained mRNA encoding K8, at levels similar to controls, but lacked mRNA encoding K19. (B) K19 was immunoprecipitated from extracts of TA muscle from wild-type or K19–/– mice, then immunoblotted with anti-K19 antibodies. Lanes show immunoblots of: K19, purified K19; +/+, immunoprecipitate from wild-type muscle; –/–, immunoprecipitate from K19–/– muscle; IgG, immunoprecipitate from wild-type muscle probed with a non-immune control (MOPC). Equal loading and transfer was confirmed by Ponceau S staining (not shown). K19 was not detected in muscle of K19–/– mice. (C) Immunoblots of desmin and K8 in extracts of TA muscle from wild-type (+/+) and K19–/– mice. Desmin is slightly but significantly elevated in the K19–/– TA muscle compared with wild type; K8 levels are unchanged.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Measurement of specific force and susceptibility to eccentric injury. Maximal tetanic tension was measured in wild-type (WT) and K19–/– TA muscles before (black bars) and after (gray bars) injury caused by a single high-strain lengthening contraction. Data are reported as specific force (force normalized to cross-sectional area). Details are provided in Materials and Methods. Six animals were tested in each group. *P0.001, significant difference between noninjured and injured; #P0.003, significant difference between WT and K19–/–. The differences in the extent of injury between WT and K19–/– were not significant (see text).

View larger version (104K): [in this window] [in a new window]   Fig. 3. Fiber type, nuclear location and size of K19–/– myofibers. (A,B) TA muscles from wild-type (A) and K19–/– (B) mice were snap frozen, cryosectioned (20-µm cross sections) and fluorescently immunolabeled with antibodies against βI-spectrin to visualize the sarcolemma of the myofibers (shown in green), and counterstained with propidium iodide (PI) to visualize myonuclei (shown in red). Many fibers in K19–/– muscle were smaller than controls but did not have central nuclei. (C,D) Cross sections of mouse TA muscle were labeled with antibodies against βI-spectrin (red) and antibodies against the myosin heavy chain of slow-twitch muscle (MHCS; green). K19–/– muscle (D) showed 10% of fibers labeled for the slow isoform of myosin, whereas wild-type muscle showed none (C). Bars, 5 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Ultrastructure of subsarcolemmal region of control and K19–/– muscle fibers. (A-C) TA muscles of control (A) and K19–/– (B,C) mice were fixed in situ, processed for electron microscopy of longitudinal thin sections, and viewed near the fiber surfaces. The distance between the sarcolemma and the nearest Z-disks (white lines) was greater in K19–/– samples (B,C) than in controls (A). Mitochondria accumulated in the subsarcolemmal gap to moderate (B) or large (C) extents in mutant but not in control (A) muscle. Sarcomeres in the mutant appeared unchanged. (D) Distances between neighboring Z-disks in adjacent myofibrils were measured and are shown in µm. Control values were 180 nm (line). K19–/– samples showed a mean value of 240 nm (line). These differences are highly significant (see text). (E) The percent myofibers with slight (category 1), moderate (category 2) or large (category 3) accumulations of mitochondria under the sarcolemma (see Materials and Methods) were scored in TA and soleus muscle fibers of wild-type (gray bars) and K19–/– (white bars) mice. TA but not soleus muscle showed significant increases in the number of myofibers with large numbers of subsarcolemmal mitochondria. (F) Distances between the sarcolemma and the nearest Z-disks were measured and are shown in µm. Control values were 150 nm, with little variation (line). K19–/– samples showed a mean value of 1 µm, with greater variation (line). These differences are highly significant (see text).

View larger version (144K): [in this window] [in a new window]   Fig. 5. Costameres at the sarcolemma of K19–/– muscle are disrupted. Frozen, longitudinal cryosections of tibialis anterior muscles from wild-type (A-C) and K19–/– (D-I) mice were immunofluorescently labeled with pairs of antibodies to membrane skeletal proteins at the sarcolemma (βI-spectrin: A,D,G; dystrophin: B,E) and nearby structures (desmin, H). Color overlays (C,F,I) show βI-spectrin in red (C,F) or green (I), and the other proteins in the contrasting color. Regions labeled by both antibodies are shown in yellow. Insets show twofold magnifications of the boxed areas in each panel. The results show that the normally rectilinear pattern of costameres (A-C; large arrow indicates a Z-domain; small arrow indicates an M-domain; arrowhead indicates an L-domain) is disrupted in K19–/– muscle (these domains are missing in the examples shown in D-I) without, however, altering the organization of desmin in nearby myofibrils (H,I). Bars, 5 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Quantitation of costameric organization in K19–/– myofibers. Longitudinal sections tangential to the sarcolemma of myofibers were labeled with antibodies against βI-spectrin, and costameres were scored as extensively disrupted (category 1, panel 1; see also Fig. 5D,G), moderately disrupted, with only one set of costameric domains remaining (category 2, panel 2), or normal, with costameres present over Z- and M-lines and oriented longitudinally as well (category 3, panel 3; see also Fig. 5A). Costameres in the K19–/– fibers (gray bars) were significantly more disrupted than in controls (striped bars; see text).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Isolation of the dystrophin-dystroglycan complex of the K19–/– mouse. The dystrophin-dystroglycan complex was partially purified on a wheat germ lectin affinity column, eluted, and analyzed by SDS-PAGE and immunoblotting, as described in Materials and Methods. K19, K8 and -actin eluted with dystrophin (Dys) and β-dystroglycan (Bdg) from control tissue, but only -actin eluted with these proteins from K19–/– tissue.

View larger version (50K): [in this window] [in a new window]   Fig. 8. Effect of K19–/– mutation on costameres and nearby structures. (A) Organization of the costameres in the myofibrils of striated muscle. Image is adapted from Ursitti et al. (Ursitti et al., 2004). For clarity, only the Z- and M-domains of costameres and a few of the integral and peripheral proteins present at costameres are shown. Emphasis is on the alignment of integral and peripheral membrane proteins with filaments composed of -actin, desmin, and K8 and K19. (B) Changes documented by us in K19–/– TA muscle, including the disruption of costameres, and the increase in the gap between the sarcolemma and the nearest myofibrils in which mitochondria accumulate. The retention of costameres in some K19–/– myofibers might be owing to filaments composed of -actin and desmin that persist in the absence of K19. Not drawn to scale.

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