‘Weak by Structure’—Limb Muscle Fibre Cytoarchitecture Remodelling During Critical Illness and Effects of Chaperone Co-Inducer BGP-15 and Dissociative Glucocorticoid VBP-15

Figures

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  Figure 1. Experimental workflow of the rat ICU model, pharmacotherapy and single muscle fibre label-free multiphoton Second Harmonic Generation (SHG) morphometry. (A), mechanical ventilation (MV) and neuromuscular blockade (NMBA) of a rat over the course of 10 d. Rats were either subjected to no drug treatment (‘none’) or treated with either Prednisolone (PRED), BGP-15 or VBP-15. (B), single fibres were manually isolated from soleus (SOL) and EDL muscles harvested at day 0 (control), 5 or 10, and subjected to SHG imaging, followed by quantitative morphometry applying custom-made image processing algorithms. The sterical order of myofibrillar motor protein arrays is reflected by two parameters: ‘Cosine Angle Sum’ (CAS) and ‘Vernier Density’ (VD), reflecting angular disarray and axial out-of-register alignment of adjacent myofibrils, respectively. CAS was analysed with a slice-wise 2D CAS and a recently developed true 3D approach. The bottom schematics visualise typical patterns in healthy, i.e., regularly organised, sarcomeres and in an irregular lattice with angular and axial distortions.

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  Figure 2. Myofibrillar lattice angular geometry of single soleus and EDL muscle fibres following 5 or 10 d MV+NMBA intervention without (none) or with PRED, VBP-15 or BGP-15 treatment. (A), middle plane images from within an XYZ SHG image stack are shown for single EDL (top) and soleus muscle fibres (bottom) at day 0 (control) and following 5 or 10 days of VBP-15 or BGP-15 treatment. As indicated for each image (each XYZ stack, respectively), the two-dimensional (CAS2D), the three-dimensional (CAS3D) Cosine Angle Sum (CAS), and the fibre diameter (d) are given. (B), statistical comparison of CAS values during the course of ICU intervention reveals a significant decline in untreated ICU rats (‘None’) in both muscles. Within each duration group (5 d and 10 d), neither treatment (BGP-15, VBP-15, PRED) could ameliorate the decline in CAS values (CAS2D and CAS3D) in EDL muscles but instead, further increased myofibrillar disorder (significantly smaller CAS values as indicated by asterisks). For soleus muscle, the different treatments did not negatively impact myofibrillar order (except for BGP-15 at day 10 in CAS3D). Instead, they significantly prevented the decline seen in the untreated rats.…

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  Figure 3. Diameter (d) and resting sarcomere length (SL) of single soleus and EDL muscle fibres following a 5 or 10 d MV+NMBA intervention without (none) or with PRED, VBP-15 or BGP-15 treatment. (A), middle plane images are shown for single EDL (top) and soleus muscle fibres (bottom) at day 0 (control) and following 5 or 10 days of applying VBP-15, BGP-15, or no treatment (‘none’). Fibre diameter and SL values are given for each fibre. (B), statistical comparison of diameters (left) and SL values (right) for EDL (top) and SOL (bottom) muscle fibres over the course of ICU condition and treatment regimens. A significant decline in fibre diameters can be seen in both muscles, worsening with duration. BGP-15 slowed down atrophy compared with no treatment in EDL fibres but was less efficient in SOL fibres, leading to even more pronounced atrophy. In contrast, VBP-15 ameliorated atrophy in those fibres after 10 days of treatment. As for sarcomere lengths, those were significantly declined already after 5 days of ICU condition in EDL fibres while not being altered in SOL fibres at day 5 but significantly increased at day 10. BGP-15 had no major effects at day 5 in EDL fibres, but induced a…

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  Figure 4. Vernier Densities (VD) in single soleus and EDL muscle fibres following a 5 or 10 d MV+NMBA intervention without (none) or with PRED, VBP-15 or BGP-15 treatment. (A), middle plane images are shown for single EDL (top) and soleus muscle fibres (bottom) at day 0 (control) and following 5 or 10 days of applying PRED, BGP-15 or no treatment (none). VD values and fibre diameters are given for each fibre. (B), statistical comparison of VD values in EDL (left) and SOL (right) muscles over the course of ICU condition and for the given treatment regimens. During the course of ICU condition, a significant increase in myofibrillar lattice misalignment (i.e., increased VD values) was seen in both muscle types, becoming significant at day 10. Within each ICU duration group, BGP-15 was able to markedly reduce VD values in EDL muscle, while acting oppositely in SOL muscle fibres. This effect was also seen by VBP-15 in SOL muscle (note: for EDL muscle, no VBP-15 samples were available). BGP-15 restored VD levels back to control fibre levels in EDL muscle at day 10. In SOL muscle, no small-molecule treatment was capable of restoring VD levels back to control levels. **: p < 0.05 vs. ‘None’ within…