*3.5. Mitochondrial Function and Content in Skeletal Muscle and Heart*

An analysis of mitochondrial respiratory enzymes in skeletal muscle homogenates (Table 1) revealed a decreased activity of citrate synthase in P5, the activity of respiratory chain complex IV, and the quantity of mitochondrial respiratory chain proteins were decreased (Figure 5A). An oxygraphy analysis of P6 skeletal muscle fibers further showed a decrease in coupled (state 3-ADP) oxidation of NADH-dependent substrates (pyruvate + malate) to 35% of the mean of the controls (Table 1). More consistent data were provided by the analysis of isolated mitochondria from skeletal muscles of P4–P6. Specific activities of respiratory chain complexes I + III (NADH: Cytochrome c reductase), complex IV (cytochrome c oxidase), and citrate synthase were decreased to 30%–50% of the mean of the controls. Both P5 and P6 had a decreased content of coenzyme Q (Table 1). A low specific content of respiratory chain enzymes, citrate synthase, and porin was further apparent in isolated muscle mitochondria of P4–P6, with the most pronounced decrease observed in P5 (Figure 5A).

**Table 1.** Activities of respiratory chain enzymes in skeletal muscle homogenates (**A**), muscle fibers (**B**), and isolated mitochondria (**C**) of proband P4–P6. For analysis samples of m. tibialis (P4, P5) or m. deltoideus sin. (P6) were used. Enzyme activities and Coenzyme Q10 content are expressed per mg protein.


Abbreviations: ADP–adenosine diphosphate, ATP–adenosine triphosphate, and NADH–reduced form of nicotinamide adenine dinucleotide.

Myocardium of two patients with desminopathy (P2, P6) (Table 2) revealed a general decrease in respiratory chain enzyme activities. An oxidation of NADH and succinate and cytochrome c oxidase respiration decreased to 20%–55% of the controls and activities of respiratory complexes I+III, II+III, and IV decreased to 15%–81%, respectively, indicating more extensive impairment in P2 heart ventricles (Table 2). The impairment of succinate respiration was the most profound with a mean of 277 pmol O2/s/mg. This was much lower than in our historical controls from donor hearts unsuitable for transplantation (653 ± 244 pmol O2/s/mg, *n* = 38) and even myocardium explanted during heart transplantation or ventricular assist device implantation (508 ± 211 pmol O2/s/mg, *n* = 91) [25]. Western blot quantification of mitochondrial proteins showed a decrease in specific content of respiratory chain complexes, also more pronounced in P2, where a very low content of complexes IV and I was associated with the upregulation of complex II (Figure 5B). Other mitochondrial proteins, as porin

(shown in Figure 5A) and adenine nucleotide translocator (not shown) were less affected. Analysis of native forms of respiratory chain complexes by BlueNative electrophoresis (Figure 5C) confirmed a marked reduction of complexes I and IV in P2 heart and further showed that it led to a pronounced decrease of high molecular weight respiratory supercomplexes consisting of complexes I, III, and IV. Similar, yet a smaller decrease of supercomplexes was observed in soleus of R349P desmin knock-in mouse [15] or heart of desmin knockout mouse [27]. The content of mitochondrial DNA (relative to nuclear DNA, D-loop/GAPDH, and 16S RNA/GAPDH) was slightly decreased in P6 heart ventricles (60%–90% of the average value of the controls) but was unchanged in P2 heart. These data indicate mild to pronounced attenuation of the energetic function of mitochondria due to a decreased content and activity of respiratory complexes and supercomplexes in failing hearts of patients with desminopathy.

**Figure 5.** Western blot detection of mitochondrial proteins in skeletal muscle and heart. Analysis of SDS-PAGE resolved proteins from (**A**) muscle homogenates (6 μg protein aliquots) and isolated muscle mitochondria (2 μg protein aliquots) demonstrated a pronounced decrease of respiratory chain complexes (CI-CV), citrate synthase (CS), and porin in skeletal muscle of P5 compared to controls (**C**), P4 and P6 were less affected. Analysis of (**B**) heart homogenates (4 μg protein aliquots) from left and right heart ventricles (LV, RV) demonstrated a marked decrease of respiratory chain enzymes in P2 and a mild decrease in P6 compared to controls (**C**). CS and porin were less affected. BlueNative electrophoresis (**C**) further showed a marked decrease of native respiratory supercomplexes consisting of CI + CIII + CIV in P2 heart ventricles (12 μg protein aliquots).


**Table 2.** Activities of respiratory chain enzymes and mtDNA content in hearts of proband 2 and 6.

Enzyme activities are expressed per mg protein, mtDNA content is expressed as 2−ΔCt value indicating the number of mtDNA copies per a haploid genome. Abbreviation: GADPH–glyceraldehyde 3-phosphate dehydrogenase.

As changes in mitochondria energetic function can affect a generation of reactive oxygen species, the content of antioxidative enzymes was analyzed by Western blot analysis in skeletal muscle and heart samples from patients with desminopathy (Figure 6). Both P4 and P5 muscle homogenates revealed highly increased glutathione reductase (GR) and superoxide dismutase 1 (SOD1) as well as a variable increase of catalase (CAT) and superoxide dismutase 2 (SOD2). Higher CAT was found in P5 isolated mitochondria, while the most increased SOD1 was of extra-mitochondrial origin (also apparent from the SOD1/SOD2 ratio). An increased content of GR and SOD1 was also found in heart ventricles of P6.

**Figure 6.** Western blot detection of antioxidative enzymes in skeletal muscle and heart. Both P4 and P5 muscle homogenates revealed variable increase in antioxidative enzymes glutathione reductase (GR), catalase (CAT), and superoxide dismutases 1 and 2 (SOD1, SOD2). Increased content of CAT, GR, and SOD1 was also found in heart ventricles of P6. Protein aliquots–muscle homogenate 30 μg, muscle mitochondria 15 μg, and heart homogenate 20 μg. For comparison, citrate synthase signal (CS) from Figure 5 is shown.

#### **4. Discussion**

Firstly, the prevalence of desminopathy in a large cohort of patients with an unexplained etiology of cardiomyopathy assessed with WES was 1.8%. Secondly, the presence of pathological desmin aggregates in myocardial/skeletal muscle samples of P1–P3 and decreased myocardial desmin expression in P4 suggested a pathogenicity of two novel *DES* variants and two *DES* variants previously classified as of uncertain significance. Thirdly, a pathogenicity of one variant of uncertain significance (DES-p.(K43E)) was supported also by abnormal desmin filament formation and its cytoplasmic aggregation in transfected HT1080 cells and in iPSC-derived cardiomyocytes. Fourthly, we provided further evidence for LVNC as a novel phenotype of desminopathy. Fifthly, we described secondary mitochondrial dysfunction in skeletal muscle and in myocardium, which was in case of myocardial succinate respiration more profound than in end-stage heart failure of other etiology. To the best of our knowledge, this seems to be the first comprehensive description of mitochondrial dysfunction in human myocardium affected by desminopathy. Finally, secondary mitochondrial dysfunction and/or an extensive left ventricular late gadolinium enhancement in desminopathy may imitate a primary mitochondrial disease or an inflammatory cardiomyopathy.

#### *4.1. Clinical and Histopathological Correlates of Desminopathy*

The majority of 68 pathogenic desmin variants that were reported so far are missense or small in-frame deletion variants localized in the helical rod domain [26,28]. A phenotype-genotype correlation meta-analyses revealed that pathogenic variants in the rod 2B domain of *DES* are common among patients with both skeletal and cardiac muscle phenotype, whereas head and tail domain pathogenic variants result mainly in clinically isolated cardiac phenotype [1,29,30]. In agreement with these findings, we found that two *DES* variants in head region (p.(K43E), p.(S57L)) and one novel *DES* variant (p.(A210D)) in the 1B helical domain had in probands isolated cardiac involvement. On the other hand, the novel *DES* mutation located in the 2B helical domain (p.(Q364H)) and two known *DES* variants (p.(R406W), p.(R454W)) affected both the cardiac and skeletal muscle.

Immunohistochemistry revealed pathological desmin aggregates in skeletal or cardiac myocytes in five probands from our study group. Importantly, desmin aggregates were absent in the deltoid muscle of proband 6 (*DES*-p.(R454W)), but present in her myocardial samples. Desmin aggregates were completely absent in proband 4 (*DES*-p.(Q364H)). Immunohistochemistry and electron microscopy of diagnostic muscle soleus biopsy and post-mortem myocardial and intercostal muscle samples failed to detect any pathological protein aggregates. Interestingly, Western blot analysis of the myocardial sample showed a decreased expression of desmin suggesting decreased protein synthesis. This is in agreement with the experience of pathologists that myopathological findings in genetically proven desminopathies may range from no overt pathology over subtle myopathic changes with sporadic protein aggregates to the picture of a vacuolar myopathy [31]. An absence of desmin aggregates has been recently documented both in autosomal dominant [32] and autosomal recessive [33] desminopathy.

#### *4.2. Novel Cardiac Phenotypes of Desminopathy*

In addition to known cardiac phenotypes of desminopathy like DCM, RCM HCM, and ACM [1,28,29], we observed LVNC as a relatively novel phenotype. So far, *DES* variants have been associated with LVNC just in a few individuals [34–37]. The first report from Arbustini et al. [34] described a family with a segregation of *DES* variant p.(G84S) with non-obstructive hypertrophic cardiomyopathy and one case of LVNC. Another group [35] reported one sporadic case of LVNC in a child with a *DES* variant p.(L398P). An occurrence of two cases of LVNC in one family has been recently associated with an in-frame mutation of desmin p.(Q113\_L115del) affecting the α–helical rod domain [36] with a formation of typical desmin-immunoreactive aggregates. We expanded the available evidence by a description of another familial occurrence of two cases of LVNC associated with the desmin variant p.(Q364H) with a decreased myocardial expression of desmin and absent desmin aggregates in myocardial/skeletal biopsy.

Recently, a novel phenotype of desminopathy describing left ventricular arrhythmogenic cardiomyopathy was reported to have a significant amount of subepicardial fibrosis [32]. A similar phenotype had our P2, which mimicked inflammatory cardiomyopathy by an extensive late gadolinium enhancement in the left ventricle and persistent elevation of cardiac troponins. However, ventricular ectopy and an inversion of T waves in inferior and precordial leads were absent. Taken together, the presence of desminopathy should be considered also in unexplained cases of LVNC and non-ischemic left ventricular systolic dysfunction with an extensive subepicardial or intramural fibrosis.

#### *4.3. Mitochondrial Dysfunction in Desminopathy*

Desminopathy may also imitate a mitochondrial disease, as was shown by Mc Cormic et al. [16]. The presence of SDH positive/COX negative muscle fibers, decreased activities of mitochondrial respiratory chain enzymes, and reduced mitochondrial DNA content in skeletal muscle biopsy lead to the suspicion of mitochondrial disease. We observed similar findings in our patient (P6) with an absence of desmin aggregates and signs of mitochondrial dysfunction in deltoid muscle biopsy. The correct diagnosis in our case provided genetic testing and immunostaining of myocardial samples.

Studies in desmin null mice and patients with recessive desmin-null muscular dystrophy revealed abnormalities in nuclear and mitochondrial localization and morphology, as well as impaired mitochondrial respiratory capacity [13,14]. Secondary mitochondrial dysfunction was also confirmed by Schröder et al. [38] and Vincent et al. [39] in skeletal muscle biopsies of heterozygous patients with desminopathy. Furthermore, Vincent et al. [39] reported a deficiency of respiratory chain complex I and IV compared to age matched controls and a low mitochondrial mass compared to controls. Our morphological and functional data from skeletal muscle samples are in agreement with the above mentioned studies and further evidence [40,41]. We observed a variable mitochondrial dysfunction characterized by a decreased expression of mitochondrial respiratory chain components and other mitochondrial proteins, as well as decreased enzyme activities, suggesting secondary changes in mitochondrial energetic function. The upregulation of several anti-oxidative enzymes, in particular that of superoxide dismutase 1 in homogenates, but not in isolated mitochondria, indicated increased antioxidative defense outside mitochondria.

Novel findings provided our analysis of myocardial energetic function in the explanted failing hearts of two probands with desminopathy. In one case harboring p.(R454W) desmin tail mutation we found a mild decrease in the content and activities of respiratory chain complexes while in the other case with p.(S57L) mutation in the desmin head region was present a very pronounced decrease in mitochondria proteins and an alteration of the bioenergetics function. Interestingly, changes in respiratory chain enzymes thus also caused a downregulation of respiratory supercomplexes that are expected to modulate the catalytic function as well as reactive oxygen species production by the respiratory chain [42]. This was associated with a decrease in several marker proteins of different mitochondrial compartments suggesting a complex mitochondrial dysfunction. The most pronounced was the impairment of myocardial succinate respiration, which was in our patients with desminopathy more profound than in end-stage heart failure of other etiologies.

#### *4.4. Study Limitations*

There are several limitations to our study. First, the small study group size reflects the rare occurrence of desminopathy and may limit the general applicability of the study results. Secondly, the small size of affected families limited the segregation studies. However, WES enabled us to exclude the presence of other pathogenic variants in cardiomyopathy- and skeletal myopathy-related genes, including genes coding mitochondrial proteins. Thirdly, an assessment of the mitochondrial function in tissues was possible only in a subgroup of patients with a clinical indication to biopsy or undergoing cardiac surgery. Finally, decreased desmin expression in P4 with a missense variant of *DES* might be

related to replacement fibrosis of the myocardium or epigenetic factors. Unfortunately, we cannot provide data supporting any of these hypotheses.
