Abstract
Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases, including those of skeletal muscle. We show that mouse skeletal muscle can produce high quality X-ray diffraction patterns establishing the mouse intact skeletal muscle X-ray preparation as a potentially powerful tool to test structural hypotheses in health and disease. A notable feature of the mouse model system is the presence of residual myosin layer line intensities in contracting mouse muscle patterns. This provides an additional tool, along with the I1,1/I1,0 intensity ratio, for estimating the proportions of active versus relaxed myosin heads under a given set of conditions that can be used to characterize a given physiological condition or mutant muscle type. We also show that analysis of the myosin layer line intensity distribution, including derivation of the myosin head radius, Rm, may be used to study the role of the super-relaxed state in myosin regulation. When the myosin inhibitor blebbistatin is used to inhibit force production, there is a shift towards a highly quasi-helically ordered configuration that is distinct from the normal resting state, indicating there are more than one helically ordered configuration for resting crossbridges.
Highlights
Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of actively contracting muscle from living and skinned muscle preparations in concert with mechanical data, such as muscle force and length changes, in real physiological time
There has been a shift in focus towards attempting to understand the molecular basis of muscle disease states, and how this can inform novel therapeutic strategies for these often-times untreatable diseases. This new direction for the field has led to a burgeoning of functional and biochemical work using mouse models of inherited myopathies but, so far, with little emphasis on understanding how disease mutations in sarcomeric proteins alter their structural dynamics leading to the functional phenotype
One physiological parameter that can be used to explain deficiencies in force production is the fraction of myosin heads that are attached to actin, and available to generate force
Summary
Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of actively contracting muscle from living and skinned muscle preparations in concert with mechanical data, such as muscle force and length changes, in real physiological time. There are very few reports [15,16], of X-ray diffraction of murine skeletal muscle in the literature to indicate how useful a preparation this can be for biophysical studies of the relationship of mutations of sarcomeric proteins to disease phenotype One such phenotype is the maximum activated force per unit area, often deficient in disease. X-ray diffraction has the potential to provide such information but, hitherto, these inferences have been indirect, with considerable variability between estimates arrived at by different means [17,18,19]
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