Coexpression of Myosin Heavy Chain 2b with Myosin Heavy Chain 1- Fact or Artefact?

In skeletal muscle, pure fibres expressing one myosin heavy chain (MyHC) isoform, and intermediate fibres, expressing two and exceptionally three MyHCs have been described. When skeletal muscle adapts its fibre type profile to changed functional demands MyHC isoform transformation follows the pathway: MyHC-1 ↔ MyHC-2a ↔ MyHC-2x/d ↔ MyHC-2b. Therefore, in hybrid fibres only successive isoforms from the proposed pathway should coexist. However, jump fibres in which MyHC-1 is co-expressed with MyHC-2x/d have been described recently. The present study describes possible coexpression of MyHC-1 with MyHC-2b in transforming as well as in normal control mouse and rat muscle fibres. The study is only descriptive and provides not sufficient proof to exclude the possible artefact resulting from unknown technical reasons. Key words: coexpression, mouse, myosin heavy chains, rat

Challenges to evidence synthesis and identification of data gaps in human biomonitoring

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The increasing number of human biomonitoring (HBM) studies undertaken in recent decades has brought to light the need to harmonise procedures along all phases of the study, including sampling, data collection and analytical methods to allow data comparability. The first steps towards harmonisation are the identification and collation of HBM methodological information of existing studies and data gaps. Systematic literature reviews and meta-analyses have been traditionally put at the top of the hierarchy of evidence, being increasingly applied to map available evidence on health risks linked to exposure to chemicals. However, these methods mainly capture peer-reviewed articles, failing to comprehensively identify other important, unpublished sources of information that are pivotal to gather a complete map of the produced evidence in the area of HBM. Within the framework of the European Human Biomonitoring Initiative (HBM4EU) initiative-a project that joins 30 countries, 29 from Europe plus Israel, the European Environment Agency and the European Commission-a comprehensive work of data triangulation has been made to identify existing HBM studies and data gaps across countries within the consortium. The use of documentary analysis together with an up-to-date platform to fulfil this need and its implications for research and practice are discussed.HBM4EU has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 733032.info:eu-repo/semantics/publishedVersio

PLASTICITY OF SKELETAL MUSCLE STUDIED BY STEREOLOGY

The present contribution provides an overview of stereological methods applied in the skeletal muscle research at the Institute of Anatomy of the Medical Faculty in Ljubljana. Interested in skeletal muscle plasticity we studied three different topics: (i) expression of myosin heavy chain isoforms in slow and fast muscles under experimental conditions, (ii) frequency of satellite cells in young and old human and rat muscles and (iii) capillary supply of rat fast and slow muscles. We analysed the expression of myosin heavy chain isoforms within slow rat soleus and fast extensor digitorum longus muscles after (i) homotopic and heterotopic transplantation of both muscles, (ii) low frequency electrical stimulation of the fast muscle and (iii) transposition of the fast nerve to the slow muscle. The models applied were able to turn the fast muscle into a completely slow muscle, but not vice versa. One of the indicators for the regenerative potential of skeletal muscles is its satellite cell pool. The estimated parameters, number of satellite cells per unit fibre length, corrected to the reference sarcomere length (Nsc/Lfib) and number of satellite cells per number of nuclei (myonuclei and satellite cell nuclei) (Nsc/Nnucl) indicated that the frequency of M-cadherin stained satellite cells declines in healthy old human and rat muscles compared to young muscles. To access differences in capillary densities among slow and fast muscles and slow and fast muscle fibres, we have introduced Slicer and Fakir methods, and tested them on predominantly slow and fast rat muscles. Discussing three different topics that require different approach, the present paper reflects the three decades of the development of stereological methods: 2D analysis by simple point counting in the 70's, the disector in the 80's and virtual spatial probes in the 90's. In all methods the interactive computer assisted approach was utilised

THE CAPILLARY PATTERN IN HUMAN MASSETER MUSCLE DURING AGEING

The effect of ageing on the capillary network in skeletal muscles has produced conflicting results in both, human and animals studies. Some of the inconsistencies are due to non-comparable and biased methods that were applied on thin transversal sections, especially in muscles with complicated morphological structures, such as in human masseter muscle. We present a new immunohistochemical method for staining capillaries and muscle fibres in 100 µm thick sections as well as novel approach to 3D visualization of capillaries and muscle fibres. Applying confocal microscopy and virtual 3D stereological grids, or tracing capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to muscle fibre per fibre length, fibre surface or fibre volume were evaluated in masseter muscle of young and old subjects by an unbiased approach. Our findings show that anatomic capillarity is well maintained in masseter muscle in old subjects; however, vascular remodelling occurs with age, which could be a response to changed muscle function and age-related muscle fibre type transformations

IMPROVING METHODOLOGICAL STRATEGIES FOR SATELLITE CELLS COUNTING IN HUMAN MUSCLE DURING AGEING

Stereological methods, based on the optical disector principle and fluorescent staining, were developed for estimating frequency of satellite cells in skeletal muscles. The parameter NL(sc, fib) (number of satellite cells per fibre length) was compared with the parameter NN(sc, nucl) (the percentage of satellite cell nuclei in all muscle nuclei), most often published in the literature, by applying unbiased sampling and counting procedures and using a confocal microscope. The methods were tested in autopsy samples of four young vs. four old human vastus lateralis muscles. Both parameters NL(sc, fib) and NN(sc, nucl) declined during ageing. However, it appears that the two parameters cannot be substituted one by the other because the number of nuclei per fibre length tends to be increased during aging. Using the introduced methods, it is more straightforward to estimate NL(sc, fib) than NN(sc, nucl)

3D Visualization and Measurement of Capillaries Supplying Metabolically Different Fiber Types in the Rat Extensor Digitorum Longus Muscle During Denervation and Reinnervation

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential. (J Histochem Cytochem 57:437–447, 2009