Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle α-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased γ-filamin, myotilin, desmin and α-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganizatio

Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres.

Dominant mutations in TPM3, encoding α-tropomyosin(slow), cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosin(slow) was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosin(slow) likely impacts actin–tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosin(slow) (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition

Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

In skeletal muscle fibers, tropomodulin 1 (Tmod1) can be compensated for, structurally but not functionally, by Tmod3 and -4

Conformational Dynamics of Actin: Effectors and Implications for Biological Function

Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function

Short-term forecast of the power on a HV/MV transformer with a high share of renewable sources

Električno energijo s trenutno, širše dostopno tehnologijo, ne moremo skladiščiti v velikih količinah , zato je za optimalno obratovanje elektroenergetskega sistema potrebna usklajenost med porabo in proizvodnjo v sistemu. Do pred nekaj let je elektroenergetski sistem obratoval po uveljavljenem načinu proizvodnje električne energije v centraliziranih proizvodnih enotah ter distribucije energije do končnih odjemalcev. Danes vključevanje razpršenih virov proizvodnje , vse večja razširjenost elektromobilnosti, zahteve po učinkovitejši rabi energije in aktivnejši vlogi odjemalcev, ki so pripravljeni svojo porabo premikati izven okvirjev svojih standardnih vzorcev, pa postavljajo pred načrtovalce elektroenergetskega sistema kopico novih izzivov. Količina in dinamika oddane električne energije razpršenih virov v omrežje sta v veliki meri odvisna od vremenskih dejavnikov. Vse večji delež razpršenih virov, predvsem obnovljivih virov električne energije, ne prinaša velikih izzivov le na področju upravljanja elektroenergetskega sistema, temveč ima zelo izrazit vpliv tudi na dogajanje na elektroenergetskem trgu. Zato je zelo pomembno poznavanje napovedi porabe in proizvodnje, še posebej razpršenih virov, saj je ključno za stabilno obratovanje elektroenergetskega sistema, za prilagoditev proizvodnje konvencionalnih virov porabi in za trgovanje z električno energijo. V magistrskem delu je predmet našega zanimanja analiza in kratkoročna napoved pretoka (delovne) moči skozi RTP Breg. Za omrežje RTP Breg je značilno, da ima instalirano veliko število razpršenih virov. Njihov vpliv, predvsem sončnih elektrarn, katerim pripada skoraj 87 % skupne moči razpršenih virov, je najbolj opazen na dnevnem diagramu pretoka moči skozi RTP Breg. Elektrodistribucijsko podjetje Elektro Maribor nam je za potrebe analize in izračuna napovedi posredovalo zgodovinske podatke o pretoku moči skozi RTP Breg in o proizvodnji sončnih elektrarn v tem omrežju. Napovedi se lotevamo z uporabo ARIMA (avtoregresijski integrirani modeli drsečih povprečij) modela, oziroma njegove kombinacije. V magistrskem delu opišemo časovne vrste in njihove komponente, nato sledi še opis ključnih pojmov, ki so potrebni v analizi časovnih vrst. Nadalje je opisana metodologija ARIMA modela, ki spada v skupino statističnih metod. Razložen je postopek izdelave in izbire najprimernejšega ARIMA modela za kratkoročno napovedovanje pretoka moči za en dan vnaprej. Na koncu je podana primerjava treh metod napovedi pretoka moči skozi RTP Breg. Prva metoda temelji na združitvi napovedi proizvodnje in porabe električne energije, druga temelji na podlagi zgodovinskih podatkov pretoka moči, tretja pa poleg zgodovinskih podatkov pretoka moči vključuje še zunanjo pojasnjevalno spremenljivko sončnega obsevanja.With currently widely available technology, we cannot store electricity in large quantities, therefore it requires consistency between consumption and generation of electricity for optimal operation of the electric power system. Until couple of years ago, the power system operated according to the established models of electricity generation in centralized power plants and distributing electricity to consumers. Nowadays, integration of distributed energy resources (DER), increasing prevalence of electromobility, expectation of more efficient use of energy and a more active role for consumers, who are prepared to change their habits of consumption, all bring new challenges for power system planners The amount and dynamics of the electricity generated by DER in the network depend to a large extent on the weather factors. An increasing proportion of DER, especially renewable energy sources, does not only bring major challenges in the field of power system management, but it also affects the electricity market. Therefore it is very important to make forecasts of consumption and generation of electric energy, especially for DER, since it’s crucial for the stable operation of power system and for adaptation of generation of conventional sources to consumption and also for trading with electrical energy. In the master’s thesis, the subject of interest is the analysis and short-term forecast of (active) power flow through substation Breg. It is characteristic for the network of substation’s Breg, that it has installed a large number of DER. Their influence, especially solar power plants, which have almost 87 % of total power of DER, is the most noticeable on the daily diagram of power flow. Elektro Maribor d.d. gave us historical data of the power flow through substation Breg and the generation of solar power plants in that network. We tackle the forecasts with the use of ARIMA (autoregressive Integrated Moving Average) model and its combinations respectively. In the master’s thesis we describe time series and their components, followed by a description of key concepts, which are needed in the analysis of time series. Further it describes the methodology of the ARIMA model, which belongs to the group of statistical methods. The building and selection process of the most appropriate ARIMA model for short-term forecasting for one day ahead is explained. At the end, a comparison of all three methods of forecasting power flow is given. The first method is based on a combination of forecasts of production and generation of electrical energy, the second method is based only on historical data of power flow, the third method uses exogenous time series of global irradiance in addition to historical data of the power flow