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Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.

Aims We investigated N471D WASH complex subunit strumpellin (Washc5) knock‐in and Washc5 knock‐out mice as models for hereditary spastic paraplegia type 8 (SPG8). Methods We generated heterozygous and homozygous N471D Washc5 knock‐in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock‐out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. Results Homozygous N471D Washc5 knock‐in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock‐in mice. WASHC5‐related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock‐out mice showing normal WASHC5 levels could not be bred to homozygosity. Conclusions While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts5μg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector

Data-independent acquisition (DIA) has recently developed as a powerful tool to enhance the quantification of peptides and proteins within a variety of sample types, by overcoming the stochastic nature of classical data-dependent approaches, as well as by enabling the identification of all peptides detected in a mass spectrometric event. Here, we describe a workflow for the establishment of a sample-fitting DIA method using Spectronaut Pulsar X (Biognosys, Switzerland).

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

To counteract habitat fragmentation, the connectivity of a landscape should be enhanced. Corridors are thought to facilitate movement between disconnected patches of habitat, and linear strips of habitat connecting isolated patches are a popular type of corridor. On the other hand, the creation of new corridors can lead to fragmentation of the surrounding habitat. For example, heathland corridors connect patches of heathland and alternatively hedgerows connect patches of woodland. Nevertheless, these corridors themselves also break up previously connected patches of their surrounding habitat and in so doing fragment another type of habitat (heathland corridors fragment woodlands and woodland strips or hedgerows fragment heath-lands). To overcome this challenge we propose the use of semi-open habitats (a mixture of heathland and woodland vegetation) as conservation corridors to enable dispersal of both stenotopic heathland and woodland species. We used two semi-open corridors with a mosaic of heathland and woody vegetation to investigate the efficiency of semi-open corridors for species dispersal and to assess whether these corridors might be a suitable approach for nature conservation. We conducted a mark-recapture study on three stenotopic flightless carabid beetles of heathlands and woodlands and took an inventory of all the carabid species in two semi-open corridors. Both methodological approaches showed simultaneous immigration of woodland and heathland species in the semi-open corridor. Detrended correspondence analysis showed a clear separation of the given habitats and affirmed that semi-open corridors are a good strategy for connecting woodlands and heathlands. The best means of creating and preserving semi-open corridors is probably through extensive grazing. To counteract habitat fragmentation, the connectivity of a landscape should be enhanced. Corridors are thought to facilitate movement between disconnected patches of habitat, and linear strips of habitat connecting isolated patches are a popular type of corridor. On the other hand, the creation of new corridors can lead to fragmentation of the surrounding habitat. For example, heathland corridors connect patches of heathland and alternatively hedgerows connect patches of woodland. Nevertheless, these corridors themselves also break up previously connected patches of their surrounding habitat and in so doing fragment another type of habitat (heathland corridors fragment woodlands and woodland strips or hedgerows fragment heathlands). To overcome this challenge we propose the use of semi-open habitats (a mixture of heathland and woodland vegetation) as conservation corridors to enable dispersal of both stenotopic heathland and woodland species. We used two semi-open corridors with a mosaic of heathland and woody vegetation to investigate the efficiency of semi-open corridors for species dispersal and to assess whether these corridors might be a suitable approach for nature conservation. We conducted a mark-recapture study on three stenotopic flightless carabid beetles of heathlands and woodlands and took an inventory of all the carabid species in two semi-open corridors. Both methodological approaches showed simultaneous immigration of woodland and heathland species in the semi-open corridor. Detrended correspondence analysis showed a clear separation of the given habitats and affirmed that semi-open corridors are a good strategy for connecting woodlands and heathlands. The best means of creating and preserving semi-open corridors is probably through extensive grazing.