Sequences of the authentic and alternative first exons, and the second exon of δ-SG gene. The primers used in primer-extension experiment (PE), PCR (exIF, alexIF, alexIR, exIIF, exIIR1, and exIIR2), and 5′ RACE (M1 and M2) are located by arrows. (Upper Left) The authentic first exon of δ-SG. The major transcription initiation site in the LV of normal hamsters is located at position +1 in the sequence. One of the several binding sequences for Sp1 is boxed. (Lower Left) The alternative first exon of δ-SG. (Right) The sequences for the second exon and the deduced amino acids for δ-SG. The single transmembrane domain (TMD) and one of the three potential N-glycosylation sites are indicated by a box and an asterisk, respectively.

Analysis of δ-SG transcript in the CM hamsters. (a) Multiplex RT-PCR. (Upper) The sizes of PCR products for GAPDH and δ-SG (indicated by arrows) are 452 and 150 bp, respectively. Lanes: 1, 2/1 μl; 2, 2/4 μl; 3, 2/16 μl; 4, 2/64 μl; 5, 2/256 μl of template cDNA. The forward (exIIF) and reverse (exIIR2) primers were both located in the second exons. (Lower) The densities of each band quantified by nih image software are plotted. Note that GAPDH, an internal standard, was equivalently amplified. The amount of δ-SG transcript in B (•) was estimated to be 20–40 times lower than that of G (○). The similar data were also obtained for U and T (data not shown). (b) RT-PCR targeted for the authentic first and the second exons of δ-SG. The forward (exIF) and reverse (exIIR1) primers were located in the authentic first and the second exons, respectively. Note that a 217-bp band was detected only for G. N, no template. (c) 5′ RACE. The size of the bands was 0.5 kb. N, no template. (d) Localization of the alternative first exon of δ-SG. (Left) Ethidium bromide staning. (Right) Southern blot analysis with an alternative first exon probe (AlEx 1; 274-bp PCR products amplified with a primer set of alexIF and alexIR). Lanes: 1, 13.3-kb EcoRI–EcoRI fragment of the first intron of normal δ-SG gene containing the deletion breakpoint (Fig. 3 Middle); 2, 1.1-kb Ex 2 fragment (Fig. 3 Upper); 3, AlEx 1 probe as a positive control for hybridization. Note that AlEx 1 probe did not hybridize the genomic fragments of normal hamsters corresponding to lanes 1 and 2 that cover the genomic region between the deletion breakpoint and the second exon of δ-SG of the CM hamsters.

Microscopic and macroscopic features of CM hamster hearts. G, B, U, and T denote Golden, BIO14.6, UMX7.1, and TO-2 hamsters, respectively. More than 10 animals of each subline were analyzed and representative photographs are presented. (a) Hematoxylin and eosin (H&E) staining of the LVs. Note necrosis, fibrosis, and calcification in B, U, and T. (Bar = 200 μm.) (b) Macroscopic appearances of the whole hearts. The remaining blood within a heart was cleared by a Langendorff perfusion apparatus (16). The white streaks visible in B, U, and T indicate calcification. (Bar = 1 cm.) (c) Cross-section of the LVs and right ventricles stained with H&E. (Bar = 2 mm.) (d) The electrocardiogram recording from the first lead. The amplitudes (volts) of QRS complex for G, B, U, and T were 0.55 ± 0.06 (mean ± SEM, n = 11), 0.98 ± 0.15 (n = 7), 0.45 ± 0.05 (n = 17), and 0.22 ± 0.08 (n = 3), respectively. The vertical and horizontal bars indicate 0.5 V and 1 sec, respectively.

Cloning of the genomic DNA containing the deletion breakpoint of the CM hamsters. Genomic DNAs digested with EcoRI were hybridized by the Int 0 (a), Ex 1 (b), or Ex 2 (c) probes. The sizes of hybridized bands are shown by arrows. (d) Genomic DNAs digested with EcoRI followed by EcoRV, SpeI, or BstXI were hybridized by the Int 1a probe. Note that restriction fragment length polymorphisms were detected for EcoRI/EcoRV but not for EcoRI/SpeI digestion. (e) Genomic DNAs digested with SpeI followed by EcoRI or PstI were hybridized by the Int 1b probe. Note that the 1.7-kb polymorphic band (shown by an arrow) was detected for SpeI/PstI digestion. The same results as d and e were obtained for both U and T (data not shown). (f) Amplification of polymorphic PstI–SpeI fragments of the CM hamster genomes. The size of the amplified fragment (1.6 kb; shown by an arrow) is smaller than that of the SpeI–PstI restriction fragment length polymorphism band (1.7 kb), because the GS primer used for this cloning is located 80 bp 5′ upstream from the SpeI cleavage site (Fig. 3 Bottom).

Identification of the deletion breakpoint of δ-SG gene in CM hamster genomes. (Top and Middle) Partial restriction maps of normal hamster (Normal) δ-SG gene. (Bottom) The sequence around the genomic breakpoint of the three CM hamsters (CM). The breakpoint (C in the sequences) locates 6.1 kb 5′ upstream from the 5′ end of the second exon of δ-SG gene. Genomic probes (Ex 1, Ex 2, Int 0, Int 1a, and Int 1b) and the GS primer used for cloning of the polymorphic SpeI–PstI genomic fragment of the three CM hamsters are indicated by double and single arrows, respectively. E, EcoRI; Sl, SalI; P, PstI; EV, EcoRV; Sp, SpeI; B, BstXI; Sc, SacI.

Specific interactions of SGs and DGs. The predicted hamster α-, β-, γ-, and δ-SG polypeptides comprised 387, 320, 291, and 289 amino acids, respectively, and exhibited the following essentially similar structures: (i) a large extracellular domain with a putative N-glycosylation site(s), (ii) a single transmembrane domain, and (iii) a relatively short intracellular domain. The N-terminal domain of α-SG is predicted to be extracellular, due to the presence of a signal sequence. The C-terminal domains of the β-, γ-, and δ-SGs are assumed to be extracellular. α-DG is an extracellular globular glycoprotein and β-DG is another membrane protein with a topography similar to α-SG (14). Accordingly, putative extracellular domains of α-, β-, γ-, and δ-SGs and β-DG are designated as α-SG N, β-SG C, γ-SG C, δ-SG C, and β-DG N. (a) In vitro pull-down study for SGs and DGs. Specific interaction of labeled (input indicated at the bottom) and immobilized (shown in the lanes) proteins is detected as a band. For SGs, reciprocal pull-down combinations were obtained as follows. α-SG-N bound β-SG-C and δ-SG-C. β-SG-C bound α-SG-N, γ-SG-C, and δ-SG-C. γ-SG-C bound β-SG-C and δ-SG-C. δ-SG-C bound α-SG-N, β-SG-C, and γ-SG-C. Strong signals indicate high affinities between the two proteins tested (such as β-SG-C vs. δ-SG-C). None of the intracellular domains of the four SGs and β-DG bound each other (data not shown). α-DG bound α-SG-N, β-SG-C, δ-SG-C, and β-DG-N. β-DG-N bound α-SG-N, β-SG-C, γ-SG-C, and δ-SG-C. In ×0.1 indicates 10% of the input. (b) Ligand overlay assay for DGs. Specific interaction of labeled (incubated; indicated at the bottom) and electrophoresed (shown in the lanes) proteins is detected as a band. CBB, Coomassie staining. The same binding profiles as in a were confirmed.

δ-SG as a candidate causative gene common to both HCM and DCM hamsters. (a) Western blot analyses of α- and δ-SGs and α- and β-DGs for LVs. CBB; Coomassie staining. Note that reduction of α-DG was more prominent than that of β-DG in all the CM hamsters. (b) RNA blot analyses of α-, β-, γ-, and δ-SGs. The transcript sizes are 1.5 kb for α-, 4.5 and 3.0 kb for β-, 1.7 kb for γ-, and 9.5 kb for δ-SGs. Besides the 9.5-kb transcript, there were also 4.3-, 2.3-, and 1.4-kb δ-SG transcripts, none of which was detectable in any CM hamsters. Full-length cDNAs for Golden hamster α-, β-, γ-, and δ-SGs were used as probes. BF, biceps femoris.

Hypothetical model of the DAGC architecture in normal cardiac sarcolemma and its disruption caused by deficiency of δ-SG in CM hamsters. The horizontal planes indicate sarcolemma, above which is the extracellular space. α-DG directly binds extracellular matrix laminin (14) and β-DG directly binds dystrophin with the C-terminal intracellular domain (25). (Left) In normal hamsters, the four SGs bind one another at the extracellular domains and constitute the SG subcomplex, which serves as a molecular stabilizer for the DG subcomplex. (Center and Right) Deficiency of δ-SG in the CM hamsters could disrupt the SG subcomplex (Center) because, for example, α-SG does not bind to γ-SG, and eventually the whole DAGC (Right), rendering cardiomyocytes more susceptible to mechanical stress generated by contraction of cardiac muscle. Molecular weights of α-, β-, γ-, and δ-SGs and α- and β-DGs are 50, 43, 35, 35, 156, and 43 kDa, respectively (7–10, 12, 14).