Third party funded individual grant
Start date : 01.01.2017
End date : 31.12.2018
Skeletal muscle is the prime source of locomotion in humans. Quality of life is vastly challenged in muscle disease and loss of muscle function. There are many conditions that lead to myopathic changes with acute or chronic course. On the other hand, muscle is an organ with extreme plasticity, and muscle exercise and training can help to overcome some of the mechanisms leading to muscle weakness by boosting muscle contractile performance and endurance, e.g. through nitrate supplementation that leads to better metabolic supply and endurance (A/Prof. Leonardo Ferreira’s lab at HHP). A model of acute acquired myopathies that is routinely used in the host’s college is inflammation-induced myopathy in sepsis (rodent models; Prof. Thomas Clanton’s lab at HHP), while another rodent model of chronic inherited myopathy is available through Duchenne Muscular Dystrophy mice (mdx) that have an additional mutation predisposing them to more complete muscle fibrosis and make them more similar to the human form of the disease (Prof. Elisabeth Barton’s at HHP). The characterization of muscle dysfunction and muscle performance enhancement mechanisms requires a focused multidisciplinary approach including physiology, biochemistry but also new strategies from medical biotechnology, bioengineering and kinesiology. Our overall goal is to develop and apply screening technologies for muscle functionality at the biomechanical and ultrastructural cytoarchitecture level developed in OF’s Institute of Medical Biotechnology (MBT) to be applied to muscle disease and performance enhancement models available at the host’s College of Health and Human Performance (HHP). Both partners complement each other very well since the technologies developed at OF’s labs are not available at University of Florida and the other argument applies to the muscle biology rodent models.
Scientific goals are related to each of the models and ongoing collaborations:
SA 1: Biomechanics analysis of skeletal limb and diaphragm muscle in response to in vitro exposure to single or combinations of inflammatory cytokines or in animals treated with LPS to induce sepsis in vivo. Muscle fibre bundles of 3-5 single fibres are dissected from EDL and diaphragm and will be mounted on a newly developed MyoRobot system for fully automated high-end cellular biomechanics and kinesiology experiments. Fibres are skinned to activate Ca2+ release and force transients with caffeine stimulation. Ca2+-sensitivity of the contractile apparatus is assessed with pCa-force recordings. Passive biomechanics related to visco-elasticity of the muscle fibres will be assessed with modern voice coil technology in an automated recording environment. Experiments are performed under control conditions and following differential incubations with IL-1, TNF-a, interferon-ß/g and LPS directly. Bundles from septic animals will also be used and blood samples analysed for pro-inflammatory cytokine profiling. SA 1 is a collaboration with Prof. Clanton.
SA 2: Biomechanics analysis of whole muscles (limb, diaphragm) from animals having undergone an oral dietary supplementation treatment with nitrate over several weeks. Nitrate is known to enhance muscle performance in elite athletes by NO-mediated effects, primarily affecting muscle metabolism and blood supply. Our hypothesis is that nitrate treatment also affects excitation-contraction coupling by reducing cellular fatigue and increasing Ca2+ sensitivity of the contractile apparatus. Whole muscles from treated mice will be subjected to a manual setup in a Radnoti-based organ bath with external field stimulation and set to optimum resting length. Then, force frequency curves will be assessed by varying the external stimulation frequency and recording tetanic force. The results will be compared to results from whole muscle recordings using our MyoRobot technology which will have to be further modified to take up whole muscles and electrical field stimulation. SA 2 is a collaboration with Prof. Ferreira.
SA 3: Morphometric multiphoton microscopy of ultrastructural remodelling of extracellular matrix collagen components and sarcomere reorganization in an mdx model with increased tissue fibrosis. This model is available at Prof. Elisabeth Barton’s lab, and our goal is to perform label-free high-end multiphoton Second Harmonic Generation microscopy and quantitative morphometry on limb and diaphragm muscles obtained from these mice. Muscle will be collected during a visit of the German team at Prof. Barton’s lab and transferred fixed to OF’s labs in Germany where a unique high-end multiphoton-multifocal imaging system is available.