Q&A Report: Redox Regulation of Skeletal Muscle: A Journey from Liverpool to the International Space Station
The answers to these questions have been provided by:
Are the inhibitors actually inhibitors of ROS generation of rather scavenging them away?
It depends what you use, we prefer to use inhibitors of the specific enzymes that generate the species such as inhibitors of NADPH Oxidase 2. It would be possible to use antioxidants which would have a much more general and less predictable effect.
Is there any effect of ageing on the switching of myosin heavy chain as well? i mean on conversion of slow to fast or fast to slow myosin heavy chain?
There is a general switch to an overall slower fiber type composition, meaning that the proportion of myosin changes but not known to be due to any direct conversion.
What are your preferred methods for exploring mitochondrial fusion and fission?
There are several potential approaches. Western blotting approaches appear to be relatively insensitive and provide a snap-shot, whereas the process is very dynamic. We are currently focusing on imaging using EM or confocal with the potential for the use of live imaging.
Did you measure the cytokines concentration during contraction, or (immediately?) after contraction?
Cytokines released into the media over 24 hours post-contractions were measured upon return to earth after freezing on the ISS.
What role do you think cytokines are playing in the mitochondria?
We think that mitochondria signaling is core to cytokine production by the muscle rather than direct actions of cytokines on the mitochondria.
Are you seeing any similarities between changes in muscle physiology in long covid and ME, when compared to aging and space travel?
We do see some changes in patterns of cytokine/chemokines in patients with ME/CFS. Of course both show a muscle weakness.
How do you link mitochondria health to muscle atrophy? Do you think it's predominantly driven by E3 ligases in space? If so, did you measure and/or look into downstream analysis to determine more specific mechanisms?
Proteomic and genomic analyses from the space flown muscle constructs are ongoing and this hasn’t focussed on E3 ligases at this stage. These bioinformatics approaches are producing some exciting information regarding downstream mechanisms and we look forward to presenting these data soon.
Do you have a sense of how compartmental hydrogen peroxide concentrations are altered in dystrophin deficient mdx skeletal muscle?
We are not aware of anyone having loaded muscle fibers from mdx mice with HyPer probes localized to specific compartments. HyPer7 constructs with localization sequences for mitochondria, ER, nuclei and cytosol are widely available. We have previously used HyPer probes in an in vivo model using an AAV vector, but mainly use transduced isolated FDB fibers ex vivo.
Are you able to measure OXPHOS in your muscle constructs in the cassettes in space?
It is not possible with the current hardware. However, we have established a method for measuring mitochondrial complex function from frozen muscle, including constructs, upon return.
Did you see a decline in mitophagy related markers in muscle under microgravity?
There are some changes in various components and pathways related to mitochondria but we are still analyzing the data in detail.
Are there fiber type specific changes? What kind of model or tools can be used to study specific muscle fibers (oxidative vs. glycolytic fibers) ?
The muscle constructs remain relatively immature and so this would require a different model such as isolated muscle fibers from rodents.
Is there any changes you found in Calpain 1 and 2 activities during the Microgravity conditions ?
Unfortunately, we can’t answer this question yet. Proteomic and genomic analyses from the space flown muscle constructs are ongoing. These bioinformatics approaches are producing some exciting information and we look forward to presenting these data soon.
How do mitochondria regulate inflammatory marker (such as Nfkb) expression?
We and others propose that changes in mitochondria during ageing or in microgravity, particularly increased H2O2 generation leads to constitutively activated NFkB in muscle. This is supported by the data in our lab, including that from Lightfoot et al 2015.
What methods would you recommend for measuring reactive oxygen species (ROS) generation in vivo, say for benchmarking changes with aging in a comparative training study, particularly approaches that are reliable yet relatively low-cost?
We think it is important to carefully define what you are trying to measure. It is not possible to measure the whole spectrum of Reactive Oxygen Species (ie ROS) in vivo with any current methods. Many of the widely used approaches are scientifically poor. If you had access to muscle tissue then it would be possible to analyze specific changes (such as peroxiredoxin oxidation).
Have you tried to incubate the fibers with cytokines chronically and then test contraction and response?
Yes, this has been studied extensively in our laboratory and others showing that treatment of muscle cells or mice in vivo with cytokines such as TNF-a causes a reduction in force generation.