Redox Balance Differentially Affects Biomechanics in Permeabilized Single Muscle Fibres—Active and Passive Force Assessments with the Myorobot

Figures

• 

  Figure 1. Sequence of recordings performed in one single muscle fibre. Each recording investigated five conditions: Control (CTRL), after 60 s of DTT exposure (1 mM), in 100 µM H2O2, in 500 µM H2O2 and in 1000 µM H2O2. For each condition, the single fibre was tested for active force generation in pCa-force curves and passive material properties in stress–strain recordings. While CTRL experiments represent the fibre’s native redox state, a 60 s DTT exposure (1 mM) shifts the redox balance towards a more reduced environment, whereas submersing the fibre in solutions with increasing H2O2 concentration produces a shift towards a more oxidizing environment. The vertical arrows indicate pCa solution exchanges. Axis are equally scaled for each respective recording protocol.

• 

  Figure 2. Maximum active forces decline with increasing H2O2 concentration in each single recording sequence. (A) example recording of a pCa-force curve at increasing Ca2+-concentrations (decreasing pCa). (B) maximum force (at pCa = 4.92) declined significantly for fibres exposed to 500 and 1000 µM H2O2, while a 60 s DTT incubation and exposure to low levels of H2O2 (100 µM) had no significant effect in relation to control levels. DTT seemed to present with forces similar to CTRL levels but was significantly larger when compared to forces produced in the presence of high concentrations of H2O2 (≥500 µM H2O2). (C) for each pCa level, the relative force was plotted and described by the mean reconstructed fit curve. While the inflection points (pCa50 value) match closely, we denote a gradual decrease in the curve’s steepness. (D) this decreasing steepness is reflected in a progressively declining Hill parameter, revealing a single significance between CTRL fibres and fibres exposed to 500 µM H2O2. Significance indicated as follows: * = p < 0.05; *** = p < 0.001.

• 

  Figure 3. Passive restoration stress increases for larger H2O2 concentrations. (A) average stress–strain curve for all CTRL fibres and all fibres exposed to 1000 µM H2O2. Maximum restoration stress is obtained at 40% strain. (B) while DTT and exposure to 100 µM H2O2 had no marked effect on maximum restoration stress, high levels of H2O2 (1000 µM) present with a significant increase over CTRL, DTT and 100 µM H2O2 in fibres. Particularly, 1000 µM H2O2 evoked twice the restoration stress in comparison to CTRL fibres. (C) axial stiffness ranged between 0.05 and 0.25 N/m, with CTRL fibres displaying the least increase from 0.05 N/m to 0.1 N/m, only. All other conditions revealed a marked gradual decrease in compliance and thus, a substantial increase in stiffness at higher strains (≥30%). Significance indicated as follows: * = p < 0.05.