Overview

The Mechanics & Devices Core provides state-of-the-art instrumentation, training, and resources to investigators studying the structure and functional properties of skeletal muscle during development and dysfunction with diseases.

Intact muscle and engineered muscle tissue: contractile properties
Sub-cellular contractile organelle (myofibril) contractile kinetics
The in vitro motility assay
Stopped flow kinetics: muscle protein chemo-mechanical analysis

The Mechanics & Devices Core provides state-of-the-art instrumentation, training, and resources to investigators studying the structure and functional properties of skeletal muscle during development and dysfunction with diseases. The Core can assess contractile function at the protein, filament, cell, tissue and organ levels, and provide assays for measuring the developing properties of early stage muscle from animal and human sources such as inducible pluripotent stem cell derived muscle cells. The Core trains investigators individually and with workshops, and works with them to develop novel research assays and platforms for drug and small molecule testing, and investigating engineered cells and tissues and gene therapies that are being developed for clinical application and commercialization.

Services

  • Provide detailed analysis of muscle contractile properties at multiple levels of organization. State-of-the-art customized apparatus are used to measure the contractile properties of muscle at multiple levels of organization. This includes isolated contractile proteins, contractile organelles (myofibrils), single cells and whole muscle. Measurements can be made from either skeletal or cardiac muscle, sourced from human biopsies, animal models or stem cell sources. Each level offers unique advantages to understand molecular mechanisms of contractile function and dysfunction. These assays can be coupled with protein biochemical and proteomic analysis using multiple state-of-the art techniques, and with structural analysis.
  • Develop new assays and protocols for analysis of muscle development, pathologies, and therapeutics. Smaller sample volumes can be analyzed through the use of engineered platforms that recapitulate the mechanical and biochemical cues of the native environment. Solutions for new assays and protocols can be developed through the Core’s expertise in 3D printing, microfabrication, and nanofabrication.
  • Test pharmaceutical, small molecules, gene therapies for treatment of muscle diseases. Development and testing of these approaches using the Core resources, often in collaboration with other Translational Research Centers.
  • Provide high resolution (nano-meter scale) dynamic measurements of muscle (sarcomere) structure with low-angle x-ray diffraction. UW CTMR partners with the Biophysics Collaborative Access Team (BioCAT) at the Advanced Photon Source (APS) of Argonne National Laboratory (ANL). We provide expertise, personnel and training to perform experiments at BioCAT for static and time-resolved information on muscle protein and sarcomere structure in human and animal models of disease. These experiments can provide detailed information for understanding muscle disease processes and translation of therapies.

Primary Researchers:

Dr. Michael Regnier, Core Director
Dr. Nathan Sniadecki

Overview

Marcinek Lab

Metabolism and mitochondrial function are at the center of muscle health and degeneration. Mitochondrial dysfunction is a key component of many skeletal muscle degenerative disorders, including muscular dystrophy, disuse atrophy, and sarcopenia. New capabilities, in the form of metabolite profiling, provide advanced methods to investigate muscle metabolism. Metabolomics approaches, which focus on the quantitative analysis of large numbers of metabolites in complex bio-specimens, are providing a wealth of new data. Metabolites (<1000 Da), represent the end products of gene, transcripts and protein function, and provide an instantaneous snapshot of biological status. To date, numerous pathological aspects of muscle metabolism including myopathy disorders, endocrine disorders and consequences of aging, have been investigated using metabolomics methods. The conditions such as mitochondrial oxidative stress, apoptosis and cellular stress responses have all been implicated in skeletal muscle atrophy and degeneration. However, despite the growing interest and research focus, the mechanistic links between skeletal muscle dysfunction and mitochondrial function remain unclear.

With a focus of bridging the knowledge gap between mechanistic understanding and skeletomuscular diseases, the Metabolism Core offers novel tools and resources for investigation of cellular and subcellular functions under normal and a wide variety of muscular disease conditions. Additionally, the Metabolism Core offers Training and Pilot Studies to encourage new users and enable current collaborators to access the state-of-the-art resources that are available. The Core develops unique scientific tools that expand access to a variety of complementary methods for measuring metabolism and in vivo mitochondrial function to the broader scientific community. Access to these tools will allow diverse groups to move beyond traditional biochemical characterization of the role of mitochondria in skeletal muscle disease and therapies. Our increased access to diverse models of skeletal muscle disease will also provide insight into common disease mechanisms by providing a comparative datasets to identify whether there are general mechanisms that lead to successful adaptation or failure of energy homeostasis and disease. The integration of in vivo measurements of mitochondrial function and energetics with ex vivo analysis of oxidative phosphorylation and metabolism via metabolomics are ideally suited for the investigation of skeletomuscular diseases.

Facilities and Resources Available

  • Mass spectrometry and NMR based metabolomics – Northwest Metabolomics Research Center (NW-MRC): provides over 20 metabolite assays on 6 mass spectrometry platforms and high field NMR, including targeted and global metabolite profiling.
  • Avance III 18.8T (800 MHz) Bruker NMR with cryoprobe which provides absolute concentrations of up over 70 metabolites including coenzyme A, acetyl coenzyme A, redox coenzymes, energy coenzymes and antioxidants.
  • Avance III console and Bruker 14T (600 MHz) vertical wide-bore MRI/MRS system used for mouse imaging and in vivo spectroscopy.
  • Avance III console and Bruker 4.7T horizontal wide-bore (35 cm) MRS system for use in human limb larger rodent MRS available through the Translational Center for Metabolic Imaging.Acton backlit thermoelectrically cooled optical spectrometers (2) for in vivo optical spectroscopy.
  • Acton backlit thermoelectrically cooled optical spectrometers (2) for in vivo optical spectroscopy.
  • Custom built MR coil and probe incorporating fiber optic bundles for simultaneous measurement of magnetic resonance spectroscopy (MRS) and optical spectroscopy (OS) for rodent spectroscopy 14T Avance MRS system.
  • Custom-built dual-tuned 1H/31P coils that permit GARP broadband decoupling for human limb MRS.
  • Oxygraph 2K (O2K) high resolution respirometer with 4 chambers equipped for simultaneous respiration and fluorometry for measuring H2O2 production, membrane potential, and calcium uptake.
  • Seahorse Extracellular Flux Analyzer.

Personnel

Dr. Daniel Raftery, Core Director, is responsible for managing the scientific operations of the Metabolism Core. Dr. Raftery directs the research activities of the metabolomics work, including the targeted and global MS and NMR spectroscopy, initial data processing, and assists with the interpretation of the metabolomics results. Dr. Raftery is also responsible for coordinating with other cores for projects involving multiple CTMR resources.

Dr. David Marcinek, Core Associate Director, is responsible for overseeing all aspects of in vivo and ex vivo mitochondrial analysis, including scheduling and prioritizing MR instrument usage, data quality control, and training of new scientists in the analysis of mitochondrial function.

Dr. Hannele Ruohola-Baker has extensive experience in mitochondrial metabolism and the use of the Seahorse analyzer that is offered as a service to Metabolism Core users. She is also very experienced with cell biology and metabolism.

Dr. Nagana Gowda is the key initial contact for users of the Core, and works with the other Core staff members to integrate the measurements and data across the different platforms and capabilities offered within the Core. Dr. Gowda has extensive experience in metabolomics and its application to a wide set of biological studies including biomarker discovery and cellular metabolism studies.

Dr. Karin Fischer has extensive experience with metabolism measurements using the Seahorse analyzer, and is responsible for all such measurements on the instrument, and assists users with sample preparation and data interpretation.

Overview

The Quanititative Analysis Core provides computational modeling and statistical analysis tools and services to investigators in the CTMR. This core provides computational models at the level of single molecules (molecular dynamics) and at the level of the sarcomere. We also seek to meld mulit-scale computational tools and visualization software to be used in multi-scale research on metabolism (energy supply) and contractile mechanics (energy demand/use), how these change in developing muscle and how they are altered in skeletal muscle disease. This core is also seeking to build tools for machine learning algorithms applied to both experimental data and computational simulations.

Molecular dynamics models focus on protein simulation models and computational tools for atomic-molecular level analysis of muscle proteins to gain a molecular framework for interpretation of experiments and for future experimental design.

Sarcomere scale models focus on Monte-Carlo simulations of force generation and energy utilization during muscle activation with a focus on the mechanochemistry in the myofilament lattice. Contraction and metabolic models are valuable for mechanistic interpretations of experimental data, informing experimental design and providing predictive power in translational muscle research.