A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization

Abstract

A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyopathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.

Figure 1.

A mutation at the tail domain of desmin(I451M) expressed in the myocardium of transgenic mice (Tgdes^I451M) has an apparent molecular mass 2–4 kDa lower than expected. A) Immunoblot analysis for desmin, performed using cardiac protein extracts from mice WT (lane 1), des^−/− (lane 2), homozygote des^−/−/aTgdes^I451M (lane 3), homozygote des^−/−/bTgdes^I451M (lane 4), and heterozygote des^WT/bTgdes^I451M for the mutation (lane 5). The α-desmin polyclonal antibody (Sigma) was used. B) For loading control, same amounts of protein extracts (as calculated by Bradford) used for Western blot were analyzed in the same SDS-PAGE, blotted to PVDF membrane, and stained with Brilliant Blue R (numbering of lanes same as in A). C) Western blot analysis of the soluble fraction (supernatant of 3000 g), insoluble fraction (0.6 M KCl pellet), and total cardiac protein extracts reveals that the major part of the mutant desmin(I451M) is present in the insoluble fraction (numbering of lanes same as in A).

Figure 2.

Mutant desmin(I451M) has an intact carboxyl terminus. Mass spectroscopy was performed on the mutant desmin(I451M) protein, isolated as a band from an SDS-PAGE of the corresponding cardiac extracts. The peptides identified in the mouse desmin sequence are boxed. No peptides were found to cover the first 58 aa sequence of the amino terminus. The mutant amino acid (I/M) is highlighted.

Figure 3.

The I451M mutation at the desmin carboxyl terminus promotes cleavage at the amino terminus. Immunoblot analysis of protein extracts from cardiac tissue of WT (lane 1), des^−/− (lane 2), des^−/−/Tgdes^aMHCdes (lane 3), and des^−/−/Tgdes^I451M animals (lane 4), performed using either an antibody (α-desN15) against the first 15 aa of desmin (A), an antibody (H-76; Santa Cruz) against desmin aa 15–90 (B), or the preimmune serum of α-desN15 antibody (C). Note that in A, lane 4, only the cleaved small amino terminus fragment is detected (★) and in B, lane 4, the remaining part of the desmin molecule is detected. (Sample 4 is overloaded, compared to other samples).

Figure 4.

The mutant (I451M) desmin is more acidic compared to WT desmin. Protein extracts from cardiac tissue of des^+/–/bTgdes^I451M animals, analyzed by two-dimensional gel and probed with anti-desmin polyclonal antibody.

Figure 5.

Mutant desmin(I451M) forms filaments in primary cardiomyocytes. Primary cardiomyocytes isolated from WT and des^−/− animals were transfected with WT desmin or mutant desminI451M cDNAs under the control of CMV promoter and stained for desmin (red) and α-actinin (green). In WT cardiomyocytes transfected either with WT (A) or mutant (I451M) desmin (B) and in des^−/− cardiomyocytes transfected with WT desmin (C), extended filaments formed and are more abundant in the periphery of the cardiomyocytes. In des^−/− cardiomyocytes transfected with mutant (I451M) desmin (D), abundant desmin staining is observed around the nucleus, as well as in the periphery. In A1, B1, C1, and D1, only the red channel (desmin) is shown for better resolution.

Figure 6.

The mutant (I451M) desmin loses its ability to associate with the Z discs, although its association with the intercalated disc is retained. Immunofluorescence staining for desmin of frozen cardiac tissue sections from 8-month old WT (A) and des^−/−/Tgdes^I451M mice (B) using the DE-U-10 anti-desmin monoclonal antibody. Image derived after deconvolution processing.

Figure 7.

The demin mutation (I451M) leads to altered desmoplakin (DP) appearance at the intercalated discs of the heart. Intercalated discs in transgenic animals expressing only the mutant desmin (des^−/−Tgdes^I451M) (B) show increased width of DP-covered area compared to WT animals (A). Desmin-null (des^−/−) animals (C) also have altered intercalated discs compared to WT animals, but to a lesser extent compared to transgenic animals. Frozen cardiac tissue sections of 14-month-old mice were stained for DP. Confocal images of a Z stack of 5 optical sections, 1.5 μm total thickness.

Figure 8.

The Z discs are formed properly in transgenic mice expressing only the mutant (I451M) desmin (des^−/−/Tgdes^I451M), as indicated by double immunofluorescence staining with α-sarcomeric actinin. Mutant desmin(I451M) is localized only at the intercalated discs. Frozen sections of cardiac tissue from 8-month-old mice were stained with the anti-desmin polyclonal antibody (green) and with anti-α-actinin monoclonal antibody (red).

Figure 9.

The I451M mutation in desmin does not lead to any detectable histological myocardial abnormalities. Hematoxylin-eosin staining of cardiac tissue sections from 10-month-old des^+/–/Tgdes^I451M (C) and des^−/−/Tgdes^I451M animals (D) shows a pattern similar to WT (A), with no obvious fibrosis or other lesions characteristic of the des^−/− myocardium (B).