A metastable subproteome underlies inclusion formation in muscle proteinopathies

Abstract: Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue

Homozygous expression of the myofibrillar myopathy-associated p.W2710X filamin C variant reveals major pathomechanisms of sarcomeric lesion formation

Filamin C (FLNc) is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familial and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from FLNc and abolishes the dimerization property of Ig-like domain 24. We previously characterized “knock-in” mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: in comparison to wildtype animals (i) the expression level of FLNc protein is drastically reduced; (ii) mutant FLNc is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack Bcl2-associated athanogene 3 (BAG3) protein; (iv) the expression of heat shock protein beta-7 (HSPB7) is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective FLNc dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative, more amino-terminally located dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that FLNc is important for mechanosensing in the context of Z-disc stabilization and maintenance

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis

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

Sensitivity of Q10 and Q6 chest measurements to restraint and test parameters

Upcoming test procedures and regulations consider the use of Q-dummies. Especially Q6 and Q10 will be introduced to assess the safety of child occupants in vehicle rear seats. Therefore detailed knowledge of these dummies is important to improve safety. As recent studies have shown, chest deflection measurements of both dummies are influenced by parameters like belt geometry. This could lead to a non optimized design of child restraint systems (CRS) and belt systems. The objective of this study is to obtain a more detailed understanding of the sensitivity of chest measurements to restraint parameters and to investigate the possibilities of chest acceleration as an alternative for the assessment of chest injury risks. A study of frontal impact sled tests was performed with Q6 and Q10 in a generic rear seat environment on a bench. Belt parameters like modified belt attachment locations were varied. For the Q6 dummy, different positioning settings of the CRS (booster with backrest) and of the dummy itself were investigated. The Q10 dummy was seated on a booster cushion. Here the position of the upper belt anchorage point was varied. To simulate the influence of vehicle rotation in the ODB crash configuration, the bench was pre-rotated on the sled in additional tests with the Q10. This configuration was tested with and without pretensioner and load limiter. Chest deflection in Q6 showed a high sensitivity to changes in positioning of the CRS and the dummy itself. A more slouched position of the CRS or dummy resulted in a reduction of measured chest deflection, whereas chest acceleration increased for a more slouched position of the CRS. Chest deflection in Q10 is sensitive to belt geometry as already shown in other studies. In a more outboard position of the shoulder belt anchorage the measured chest deflection is higher. Chest acceleration shows the opposite tendency, which is highest for the rearmost location of the upper belt anchorage. On a pre-rotated bench the highest chest deflection within this test series was observed without load limiter/pretensioner and an outboard belt position. By optimizing the belt location and the use of pretensioner/load limier the chest deflection was significantly reduced. For the Q6 a criterion based on chest acceleration as well as deflection measured at two locations might be the most reliable approach, which requires further research with an additional upper deflection sensor. In the Q10 the measured chest deflection does not always correctly reflect the severity of chest loading. The deflection is depending on initial belt position and restraint parameters as well as test conditions, which result in different directions of belt migration. A3ms chest acceleration might be a better indicator for severity of chest loading independent of different conditions like belt geometries. However, in some cases the benefit of an optimized restraint system could only be shown by deflection. These findings suggest that further research is needed to identify a chest injury assessment method, which could be based on deflection as well as acceleration or other parameters related to belt to occupant interaction

Chronic Hyperglycaemia Inhibits Tricarboxylic Acid Cycle in Rat Cardiomyoblasts Overexpressing Glucose Transporter Type 4

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

Biofidelity of the WorldSID small female Revision1 dummy

In the EC FP6 Integrated Project Advanced Protection Systems, APROSYS, the first WorldSID small female prototype was developed and evaluated by BASt, FTSS, INRETS, TRL and UPM-INSIA during 2006 and 2007. Results were presented at the ESV 2007 conference (Been et al., 2007). With the prototype dummy scoring a biofidelity rating higher than 6.7 out of 10 according to ISO/TR9790, the results were very promising. Also opportunities for further development were identified by the evaluation group. A revised prototype, Revision1, was subsequently developed in the 2007-2008 period to address comments from the evaluation group. The Revision1 dummy includes changes in the half arms and the suit (anthropometry and arm biomechanics), the thorax and abdomen ribs and sternum (rib durability), the abdomen/lumbar area and the lower legs (mass distribution). Also a two-dimensional chest deflection measurement system was developed to measure deflection in both lateral and anterior-posterior direction to improve oblique thorax loading sensitivity. Two Revision1 prototype dummies have now been evaluated by FTSS, TRL, UPM-INSIA and BASt. The updated prototype dummies were subjected to an extensive matrix of biomechanical tests, such as full body pendulum tests and lateral sled impact tests as specified by Wayne State University, Heidelberg University and Medical College of Wisconsin. The results indicated a significant improvement of dummy biofidelity. The overall dummy biofidelity in the ISO rating system has significantly improved from 6.7 to 7.6 on a scale between 0-10. The small female WorldSID has now obtained the same biofidelity rating as the WorldSID mid size male dummy. Also repeatability improved with respect to the prototype. In conclusion the recommended updates were all executed and all successfully contributed in achieving improved performance of the dummy

Effects of 12 Weeks of Hypertrophy Resistance Exercise Training Combined with Collagen Peptide Supplementation on the Skeletal Muscle Proteome in Recreationally Active Men

Evidence has shown that protein supplementation following resistance exercise training (RET) helps to further enhance muscle mass and strength. Studies have demonstrated that collagen peptides containing mostly non-essential amino acids increase fat-free mass (FFM) and strength in sarcopenic men. The aim of this study was to investigate whether collagen peptide supplementation in combination with RET influences the protein composition of skeletal muscle. Twenty-five young men (age: 24.2 ± 2.6 years, body mass (BM): 79.6 ± 5.6 kg, height: 185.0 ± 5.0 cm, fat mass (FM): 11.5% ± 3.4%) completed body composition and strength measurements and vastus lateralis biopsies were taken before and after a 12-week training intervention. In a double-blind, randomized design, subjects consumed either 15 g of specific collagen peptides (COL) or a non-caloric placebo (PLA) every day within 60 min after their training session. A full-body hypertrophy workout was completed three times per week and included four exercises using barbells. Muscle proteome analysis was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). BM and FFM increased significantly in COL compared with PLA, whereas no differences in FM were detected between the two groups. Both groups improved in strength levels, with a slightly higher increase in COL compared with PLA. In COL, 221 higher abundant proteins were identified. In contrast, only 44 proteins were of higher abundance in PLA. In contrast to PLA, the upregulated proteins in COL were mostly associated with the protein metabolism of the contractile fibers. In conclusion, the use of RET in combination with collagen peptide supplementation results in a more pronounced increase in BM, FFM, and muscle strength than RET alone. More proteins were upregulated in the COL intervention most of which were associated with contractile fibers

The proteome of neuromelanin granules in dementia with Lewy bodies

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson’s disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SN$_{Surr.}$) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB

Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set