Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome

Figures

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  Figure 1. In vivo evaluation of skeletal muscle morphology and functionality (n. animal used for these evaluations: 4 animals per genotype). (A) Medium forelimb strength was 0.203 ± 0.003 kg in WT mice (n. measurements = 16), 0.185 ± 0.003 in SUR2wt/AV kg mice (n. measurements = 21) and 0.191 ± 0.005 kg in SUR2AV/AV mice (n. measurements = 20), respectively. In ultrasound evaluation, (B) the mean pixel echodensity for the hind limb was 91.2 ± 4.7 in WT mice, 113.4 ± 2.4 in SUR2wt/AV mice and 128.7 ± 2 in SUR2AV/AV mice. (C) Percentage of hind limb vascularization was 36.6 ± 0.6% in WT mice, 32.3 ± 2.5% in SUR2wt/AV mice and 59.8 ± 6.2% in SUR2AV/AV mice. (D) Hind limb volume was 536.7 ± 29.2 mm3 in WT mice, 630.8 ± 79.9 mm3 in SUR2wt/AV mice and 575.8 ± 77.8 mm3 in SUR2AV/AV mice. (E) Diaphragm amplitude was 1.35 ± 0.14 mm in WT mice, 0.86 ± 0.04 mm in SUR2wt/AV mice and 0.93 ± 0.03 mm in SUR2AV/AV mice. (F) The mean pixel echodensity for the diaphragm was 84 ± 11.4 in WT mice, 99.3 ± 7.5 in SUR2wt/AV mice and 118 ± 2.8 in SUR2AV/AV mice. Data are presented as mean ± SEM. Data significantly different as evaluated with one-way ANOVA and Bonferroni test (*** p < 0.001, ** p < 0.005, * p <…

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  Figure 2. Channel subunits expression and current amplitude distribution in FDB fibers at physiological membrane voltage of −60 mV (Vm). (A) Sample gel of PCR analysis in an FDB muscle: high levels of expression of ABCC9 and KCNJ11 genes, encoding respectively for SUR2 and Kir6.2, were found in all the phenotypes. (B) In inside-out patch-clamp experiments on FDB isolated fibers, the calculated mean was −1152.8 ± 164 pA for WT, −1381.2 ± 155.5 pA for SUR2wt/AV mice and −1499.2 ± 151.7 pA for SUR2AV/AV mice (n. patches = 20, 38, 45 from 3 muscles/3 mice per genotype, respectively); * data significantly different as evaluated with one-way ANOVA and Bonferroni test for p < 0.05. (C) Sample traces of recorded KATP currents, illustrating a tendency toward increased current amplitude in FDB fibers from SUR2wt/AV and SUR2AV/AV mice with respect to WT but more marked reduction of sensitivity to 5 × 10−3 M MgATP.

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  Figure 3. MgATP- and glibenclamide-sensitivity of KATP currents in FDB fibers. (A) Sample traces of currents in zero (CTRL), 5 × 10−5 M and 5 × 10−3 M MgATP in FDB isolated fibers from WT, SUR2wt/AV and SUR2AV/AV mice. (B) Sample traces of currents in zero (CTRL), 10−7 M glibenclamide (Glib) and 5 × 10−3 M MgATP in FDB fibers from WT, SUR2wt/AV and SUR2AV/AV mice (n. patches = 30, 48, 55 from 3 muscles/3 mice per genotype, respectively). (C,D) Sensitivity to inhibition by MgATP (C) or Glib (D) in FDB patches from WT, SUR2wt/AV and SUR2AV/AV mice. Data were fitted using the Hill equation (see Table 1). Each experimental point represents the mean ± SEM of at least three patches. Data significantly different as evaluated with one-way ANOVA and Bonferroni test (*** p < 0.001, * p < 0.05).

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  Figure 4. Sample representative histological analysis with hematoxylin–eosin (H.E.) reaction on extensor digitorum longus (EDL) muscles of (A) WT, (B) SUR2AV/AV and SUR2wt/AV mice. (B) EDL muscle of homozygous CS mice showed homogeneous myofibers population and normal nuclei, except for pink colored materials in the endomysia and perimysium, suggestive of edema of non-inflammatory origin (arrow) and no atrophy (H.E. 10×, Bar 50 μm). Heterozygous (C) and WT (A) EDL muscles sections were normal.

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  Figure 5. Sample representative histological analysis with hematoxylin–eosin (H.E.) reaction on soleus (SOL) muscles of (A) WT, (B,D) SUR2AV/AV and (C) SUR2wt/AV mice. (B) SOL muscles from SUR2AV/AV mice showed marked atrophy (H.E. 10×, Bar 50 μm) vs. (A) WT and (C) SUR2wt/AV EDL muscles. (D) SOL muscle from SUR2AV/AV mice showed myofibers with small diameter (arrow 1) and inflammatory cells (arrow 2) and rare area of degeneration and necrosis with some atrophy (H.E. 20×, Bar 50 μm).

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  Figure 6. Sample representative histological analysis with hematoxylin–eosin (H.E.) reaction on tibialis anterioris (TA) muscles of (A) WT, (B,D,E) SUR2AV/AV and (C) SUR2wt/AV mice. (B) TA muscle from SUR2AV/AV mice showed heterogenous population of myofibers with reduced area (arrows) (10×, Bar 50 μm), (D) no longer polygonal in shape but elongated, rounded and angular, with hyper-eosinophil sarcoplasm (arrow) (H.E. 40×, Bar 50 μm). (E) Hyperchromatic nuclei can be grouped together, and present fragmented myo-fibers separated by an accumulation of pale pink interstitial material of inflammatory origin in TA muscle from SUR2AV/AV mice (H.E. 40×, Bar 50 μm).

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  Figure 7. Cross sectional area of tibialis anterior (TA) and soleus (SOL) muscles of CS mice. The CSA of TA muscle was significantly reduced in CS mice, and it was 1937.5 ± 121 μm2 in heterozygous (F = 2.982, * p < 0.05) (n muscle/mice = 3/3) and 1647.8 ± 131 μm2 in homozygous CS mice (F = 3.321, * p < 0.05) (n muscle/mice = 3/3) vs. 2769.6 ± 221 μm2 in WT mice (n muscle/mice = 3/3). The CSA of SOL muscle was significantly reduced in homozygous mice, and it was 1142.7 ± 99 μm2 (F = 1.9821, * p < 0.05) (N mice = 2) vs. 1465 ± 91 μm2 in WT (n muscle/mice = 3/3). CSA was calculated on 53 fibers per mouse.

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  Figure 8. Atrogin-1 and MuRF1 gene expression in soleus (SOL), tibialis anterior (TA) and gastrocnemius (GA) of WT, SUR2wt/AV and SUR2AV/AV mice. The figure shows quantification of transcriptional level with qPCR for (A) atrogin-1 and (B) MuRF1 normalized by β-actin gene. In each graph, bars represent mean ± SEM for 3 muscles per each genotype. * Data significantly different with respect to the WT (p < 0.05).

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  Figure 9. Evaluation of ex vivo survival of FDB isolated fibers from WT, SUR2wt/AV and SUR2AV/AV mice. Fiber survival was reduced at 3 h in SUR2AV/AV fibers and progressively more so in SUR2wt/AV and SUR2AV/AV at 24 h. Short-time incubation with Glib did not significantly affect fiber viability, whereas 24 h of fibers treatment with Glib further reduced fiber viability with respect to untreated fibers (n. of fibers = 100–150 for each experimental condition; fibers were collected from ≥ 3 FDB from ≥3 mice per genotype). Values of percentage of fiber survival are presented as individual data points for each repetition. Data significantly different as evaluated with one-way ANOVA and Bonferroni test (*** p < 0.001, * p < 0.05).