Biotechnology of Muscle Dysfunction


Background

Many chronic disorders of muscle function, either of inflammatory or degenerative origin, are accompanied by specific remodeling processes of the tissue, e.g. increased fibrosis. A functional constraint of many muscle pathologies is reflected by a reduced biomechanical performance, either affecting active force development or impaired passive compliance of the muscle. Apart from an increase in extracellular linker proteins (i.e. collagen), also ultrastructural changes of the cytoarchitecture can prevail. Such biophysical long-term changes can, in particular, be triggered by defective or aberrant mechanotransduction of cells.

Cellular mechanisms

The cellular mechanisms underlying a loss in muscle force strongly depend on the respective model investigated. Within the institute, we study interactions of single mechanisms and their temporal development in genetically modified mice carrying mutations within genes encoding for extra-sarcomeric or sarcomeric proteins.  Such animals are provided by the Institute of Neuropathology, Prof. Schröder, who also provides the medical and clinical input and expertise while our institute (MBT) provides biomedical cell technology expertise and optical technologies.

In particular, studies of contractility, of membrane excitation, calcium homeostasis, mechanotransduction, inflammatory processes and cellular remodeling of the cytoskeleton are a strong focus of our work. For this, we apply cell technologies and metrologies on the single cell level and combine them with a broad spectrum of fluorescence- and laser-microscopy into subcellular domains. As an example, the figure shows results from past studies in dystrophic mdx mice.


Dystrophie

Figure: A, confocal recordings of elementary Ca2+ release events (ECRE) and their frequency over time in single dystrophic mdx muscle cells that were mechanically challenged by hyper- and hypo-osmotic shock. Shortly after challenge, ECRE frequency markedly increases but can be resuscitated to pre-challenge values by application of blockers of mechanosensitive channels (streptomycin, Gd3+). These and further data resulted in a proposed model in which aberrant mechanosensitive channel activity relieves the inhibition of the DHPR (dihydropyridine receptor) on intracellular Ca2+ release from the SR (sarcoplasmic reticulum) via the ryanodine receptor [from Teichmann et al. 2008]. B, 3D reconstruction of Second Harmonic Generation (SHG) XYZ image stacks which were required by multiphoton microscopy in single muscle cells from aged mdx mice. The normally regular, highly ordered ultrastructure that is seen in wild-type cells is vastly abrogated in mdx cells showing massive tilting and twisting of myofibrils. This prevents an coordinated contraction if one imagines that adjacent force generator would pull in totally different directions [from Friedrich et al. 2010].

Abberant mechanotransduction

In previous studies, we were able to unravel the mechanism of aberrant mechanotransduction in dystrophic muscle. Aberrantly active mechanosensitive channels in the muscle membrane modulate the normally inhibitory interaction between the DHP receptor (dihydropyridine) and the RyR receptor (ryanodine) on the membrane of the intracellular sarcoplasmic Ca2+ store (see figure A). This link usually prevents uncontrolled Ca2+ release into the resting cell. It is disinhibited in dystrophic muscle and results in increased frequency of elementary Ca2+ release events (ECRE) that can be resolved using confocal Ca2+ fluorescence microscopy (see figure A). Aberrant ECRE response upon mechanical stress in dystrophic muscle cells can be overcome pharmacologically by established blockers of mechanosensitive channels.

Ultrastructure imaging

In another study using multiphoton microscopy of intrinsic second harmonic generation (SHG) signals, we could show that the progressive force loss in aged mdx muscle could be explained by a vast remodeling of the contractile ultrastructure within the cells. The ‚Second Harmonic Generation‘ microscopy technology has also already been applied to young animals carrying the DesR350P mutation in the desmin gene. Preliminary data also suggest an already early derangement of the sarcomeric order which will now be applied across the whole age range of animals. With the use of high-end automated image processing strategies (e.g. pattern recognition algorithms), we aim to build an image database of multiphoton patterns in disease models for later translation into a diangostic tool by medical colleagues. Such an SHG image data-base could potentially help to aid diagnoses as well as to judge progression in disorders using defined ultrastructural parameters.

Finally, a linking focus of our current research addresses the mechanisms bridging early functional and late structural changes to diseased skeletal muscle. We think inflammation should provide the answer.

Literature

  • Friedrich O, Both M, Weber C, Schürmann S, Teichmann MDH, von Wegner F, Fink RHA, Vogel M, Chamberlain JS, Garbe C (2010). Microarchitecture is severely compromised but motor protein function is preserved in dystrophic mdx skeletal muscle. Biophys J 98(4): 606-616. doi:10.1016/j.bpj.2009.11.005
  • Teichmann MDH, Wegner FV, Fink RH, Chamberlain JS, Launikonis BS, Martinac B, Friedrich O (2008). Inhibitory control over Ca2+ sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression. PLoS One 3(11): e3644. doi:10.1371/journal.pone.0003644