A Novel DES L115F Mutation Identified by Whole Exome Sequencing is Associated with Inherited Cardiac Conduction Disease

Figures

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  Figure 1. Mutation identification. (A) Pedigree of the studied family. The pedigree shows the affection statuses, individual identifiers, and genotypes at DES c.C343T. The phenotype of sample III:1 was uncertain. The samples (marked by arrows) II:1, II:2, and III:3 (proband) were exome sequenced. (B) Sequencing result showing the heterozygous DES c.C343T (p.L115F) mutation. WT, wild-type allele; MT, mutant allele. (C) Schematic of desmin protein. * L115F mutation in the 1A region.

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  Figure 2. Structural models of DES. (A) Model of the wild-type (WT) DES (residues 109−141) was built using the Coot program (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/). (B) The L115F DES mutant model. L115 and F115 are marked with arrows.

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  Figure 3. Expression of desmin in cultured cells. (A) HL-1 atrial cardiomyocytes, HeLa, and HaCaT cells transfected with wild-type (WT) or p.L115F desmin in fluorescent expression vectors, showing cytoplasmic aggregates with p.L115F independent of the transfected cell type. (B) SW-13 cells transfected with WT or p.L115F desmin in fluorescent expression vectors, showing p.L115F desmin unable to develop an extended filamentous network as WT. (C) SW-13 cells cotransfected with the same amount of WT and p.L115F desmin expression vectors, showing that WT/p.L115F desmin was unable to develop an extended filamentous network as WT/WT. Scale bar = 25 µm.

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  Figure 4. Confocal microscopy of ventricular myocardium from wild-type (WT) (A) and desL114F/L114F mice (E). (B–D) Zoomed region of the white square in (A). (F–H) Zoomed region of the white square in (E). Representative confocal images showed numerous desmin-positive but connexin 43-negative (marked by white square in Figure F–H) irregularly amorphous aggregates among the myofibrils, and altered connexin 43 distribution (open arrow) in the desL114F/L114F mice. The arrows indicate colocalization of desmin and connexin 43 at intercalated discs. Green signals indicate desmin; red signals indicate connexin 43 gap junctions; yellow signals indicate the colocalization of desmin and connexin 43; and blue signals indicate nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 25 µm.

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  Figure 5. Confocal microscopy of ventricular Purkinje fibers from wild-type (A) and desL114F/L114F mice (E). (B–D) Zoomed region of the white square in (A). (F–H) Zoomed region of the white square in (E). Representative confocal images showed numerous desmin-positive but connexin 40-negative (arrows in Figure F–H) irregularly amorphous aggregates in the Purkinje fibers from desL114F/L114F mice, but not from wild-type mice. Anti-connexin 40 immunostaining aided in identifying cardiac conduction cells (Purkinje fibers). The open arrows indicate colocalization of connexin 40 and desmin at intercalated disc. Green signals indicate desmin; red signals indicate connexin 40 gap junctions; and yellow signals indicate the colocalization of desmin and connexin 40. Scale bar = 25 µm.

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  Figure 6. Transmission electron microscopy. A–D, 7-week-old desL114F/L114F homozygote; E, 12-week-old wild type; Electron microscopy of ventricular myocardium from 7-week-old des L114F/L114F homozygote revealed numerous highly electron-dense, amorphous, irregularly lobulated aggregates (marked by stars) in the intermyofibrillar space (A) and enriched at the levels of peri-nucleus (B) and intercalated discs (C), associated with disintegrated and thinning of myofibrils (B,D), as well as aggregates of swollen and disorganized mitochondria (arrows). N, nucleus; *, intercalated disc. (A,B,D,E) scale bar = 2 µm; (C) scale bar = 1 µm.

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  Figure 7. Transmission electron microscopy of the soleus muscle from wild-type (WT) and desL114F/L114F mice. Electron microscopy of the soleus muscle from 7-week-old desL114F/L114F mice revealed numerous electron-dense aggregates (marked by stars) in the perinuclear region with sarcomeres disrupted and discontinued at Z-discs (arrows). Upper panel, scale bar = 5 µm; lower panel, scale bar = 2 µm.

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  Figure 8. Founder (lane marked by the arrow) containing the knock-in (KI) allele was confirmed by sub-cloning and direct sequencing analysis.