The UW CTMR supports the development of exciting new muscle research through a pilot and feasibility grant program that allows investigators to obtain preliminary data needed to apply for follow-on funding and receive training in new skills. Funding up to $25,000 (direct costs) is available.

Current Awardees

Applying

Applications are now open! To apply, please see the information below or download the PDF version of the request for proposals. The due date for proposals is Monday, March 10, 2025.

If you are considering applying, reach out to CTMR Core Resource Directors & Personnel to discuss integrating the Cores into your proposal (a proposal requirement):

2025- Download a PDF of the Request for Proposals Here Cover Page Form SUBMIT your application
  • Submission Deadline for 2025 Pilot Grants Applications is March 10, 2025.
  • Target project period: May 1, 2025 – March 31, 2026 (estimated)

Two categories of funding are offered:

  • Pilot Grants will provide up to $25,000 (direct costs) per award to support projects designed to collect preliminary data for new research with the purpose of obtaining follow-on funding. Priority will be given to new and junior faculty doing innovative research. The CTMR will also support more senior faculty who are changing career objectives in order to study skeletal muscle. In both cases, this funding allows investigators rapid accumulation of evidence needed to demonstrate feasibility for novel studies and (especially for new investigators) training in new skills. Pilot Grant funding may be used for salaries and benefits for graduate students, undergraduate students, postdoctoral scholars, and research staff; materials and supplies; and CTMR core services.
  • Mini Pilot Grants will provide up to $5,000 for pilot studies to new users of CTMR core services who need a limited, specified set of data from one of the cores. Examples of the use of this grant mechanism would be (1) to collect preliminary data on muscle mechanical performance for a small set of animals or to create a device prototype using the Mechanics and Devices Core; (2) to use the Metabolism Core to obtain metabolomic data for 10-20 animals (~$100/sample), and (3) to work with the Quantitative Analysis Core to develop computational models or algorithms for a specific set of simulations. Another possible use of is funding NMR time on the 14T magnet at the University of Washington to develop protocols, or troubleshoot or run preliminary imaging studies. This magnet is run as part of a High-Resolution Imaging (HRIM) user group. Please contact Dan Raftery (draftery@uw.edu) or Mike Regnier (mregnier@uw.edu) if your proposal includes this research. Mini Pilot Grant funding may be used for materials and supplies and CTMR core services, but not salaries and benefits.

The Principal Investigator (PI) of the proposal must have a faculty-level appointment at the UW. An applicant may serve as PI on only one proposal and as an investigator (co-PI or PI) on no more than two proposals. Current CTMR senior investigators may not receive pilot award funding, but can participate in the proposed work as unfunded collaborators.

Submission

Proposals are due by 5:00 PM on Monday, March 10, 2025. Submissions after this deadline will not be considered. Please complete this form to submit your application: https://forms.office.com/r/DPvSLPEHYZ

Proposal Preparation

  • Formatting: 11-point Arial or Helvetica; 0.5” margin.
  • Sections:
    • Cover Page– Please use this form, which requests you fill in the following information:
        • Project title
        • Funding category (Pilot Grant or Mini Pilot Grant)
        • Contact PI information: Name, Position Title, UW Department, Mailing Address, Phone Number, Email Address
        • Name of Co-PI, if applicable
        • Name(s) of Co-I(s), if applicable
        • CTMR Cores that will be utilized (Mechanics & Devices, Metabolism, and/or Quantitative Analysis)
      • Human subjects research? (Yes/No). IRB approval is required before submission.
      • Vertebrate Animals (Yes/No). IACUC approval is required before submission. You will need to provide an IACUC congruence letter that includes this specific proposal title by March 31, 2025, in order for your project to be funded. Please reach out to Katie (kmitz@uw.edu) if you would like an example congruence letter. Work with your IACUC official to complete this letter. We suggest starting early as this process can take some time.
    • Project Description. The page limit for Pilot Grant applications is 4 pages, the page limit for Mini Pilot Grant applications is 2 pages
      • Specific Aims
      • Research Plan
        • Significance and potential impact
        • Innovation
        • Approach
        • Use of CTMR Cores. How will the project make use of the Cores? How might CTMR support of the project lead to follow-on funding, and/or the broader CTMR program?
        • Project timeline
      • References (not included in the page limit)
    • Human Subjects. If applicable, describe the use of human subjects. IRB approval is required before submission.
    • Vertebrate Animals. If the project involves the use of vertebrate animals, address the criteria listed in section 5.5.5 Vertebrate Animals of Instructions for Grant Applications using PHS 398. IACUC approval is required before submission and a congruence letter including your specific project title should be submitted by March 31, 2025.
    • Budget. Download the PHS 398 Detailed Budget for Initial Budget Period form and complete only the following sections, if applicable:
      • Project Period: From 05/01/2025 Through 03/31/2026 (estimated) [**NOTE: After proposals have been reviewed and selected for funding, they are sent NIH for approval. We must receive a Notice of Award from NIH before we can distribute funds. Due to uncertainty in timing, it is ideal to budget for an 9- or 10- month project.]
      • Personnel (N/A for mini pilot grants). Funding can be used to support graduate students, undergraduate students, postdoctoral scholars, and research staff. Other salary types (such as faculty salaries) are not allowed.
      • Supplies (itemize by category)
      • Other Expenses (itemize by category). Include CTMR Core services. Facilities/instrumentation expenses will be charged for data collection and analysis of data that are collected by Core staff. For project personnel, training for instrumentation use is provided at no cost, but a fee for the time of Core instrument use by trained individuals will be assessed. Contact the Core Director to determine costs (see INQUIRIES below for contact information).
  • Budget Justification
  • NIH-formatted Biosketches for the PI and any Co-PIs.

Proposals longer than the stated page limits, deviating from the stated instructions, or omitting information requested above will not be reviewed.

  • Will the application substantially contribute to the applicant’s career development? Is the applicant a junior investigator who would be likely to derive greatest benefit from the relatively modest Pilot support or, if not, from a more senior investigator who is likely, if funded, to change career direction towards muscle research?
  • Does the proposal have high scientific merit? Is the proposed project designed to pursue a new finding or timely research opportunity and will it, if successful, lead to a significant scientific contribution to the understanding of muscle biology, pathology or therapeutic development?
  • Will the proposed research capitalize on the strengths of one or more CTMR cores so the CTMR can maximize the impact of the modest level of Pilot funding?
  • What are the prospects that the Pilot proposal will lead to demonstration of feasibility sufficient to support successful new external peer reviewed funding?

All selected proposals are subject to terms and conditions that will be detailed in the award notification letter, including:

  • Each publication, press release, or other document of research supported by CTMR funding must include an acknowledgment of the CTMR support and a disclaimer such as “Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number P30AR074990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”
  • All funded PIs must submit a brief pilot proposal report and an NIH “highlight” slide for inclusion in the CTMR annual progress report, as requested by the Center Director.
  • All purchases and HR appointments will be conducted in the home departments of the Pilot Grant PI.
  • All funded PIs will present their work at the annual CTMR symposium (Fall 2025, exact date TBD)

For help with determining a budget for CTMR Core Services, contact the appropriate Core Director(s):

For other inquiries, contact kmitz@uw.edu.

Current Awardees

Project Period: September 1, 2024 – March 31, 2025 (Year 6)
Joe Powers, PhD

PI: Joe Powers, PhD, Assistant Professor Laboratory Medicine & Pathology Mechanical Engineering

Project: Measuring and modeling emergent mechanical behavior of remodeled myocytes due to Filamin C deletion

The contractile properties of myocytes strongly depend on the spatial organization and interconnectivity of myofilaments, sarcomeres, and myofibrils. This multiscale structural organization and mechanical coupling in myocytes is regulated by protein networks that physically link the Z-disks of sarcomeres to each other in adjacent myofibrils, to the cytoskeleton, and to the extracellular matrix (ECM). Mechanotransmission and mechanotransduction in myocytes rely on these molecular networks to transmit mechanical forces throughout the myocyte, and to convert mechanical information into biochemical signals that regulate myocyte morphology and function. Mutations in genes encoding Z-disk-associated proteins are often found in patients with skeletal and/or cardiac myopathies, which are often associated with myofibrillar disarray and Z-disk abnormalities. However, our current understanding of the role of Z-disk proteins in regulating subcellular structure & contractile mechanics is far from complete, thus limiting our ability to engineer new therapeutic strategies for muscular disorders associated with loss-of-function Z-disk protein variants. Recently, variants of the striated muscle specific Z-disk protein filamin C (FLNC) have been found in patients with distal myopathy, myofibrillar myopathy, and all major forms of cardiomyopathies. FLNC variants typically cause myofibril disarray and contractile abnormalities, but the role of FLNC in regulating muscle structure/function are widely unknown. Thus, our overall goal is to develop a novel computational and experimental platform from which to investigate the relationship between Z-disk interconnectivity and mechanosignaling pathways underlying subcellular remodeling and contractile dysfunction in FLNC-associated myopathies, with the ultimate goal of informing new therapeutic approaches for muscular diseases associated with extra-sarcomeric protein defects.

Silvia Marchianò, PhD, PharmD & Elaheh Karbassi, PhD

PI: Silvia Marchianò, PhD, PharmD, Acting Instructor, Department of Laboratory Medicine and Pathology & Elaheh Karbassi, PhD, Acting Instructor, Department of Laboratory Medicine and Pathology

Project: Mutational screening of ribonucleotide reductase activity using human pluripotent stem cell derived cardiomyocytes.

Improving contraction represents the ultimate goal in the treatment and, potentially prevention, of heart failure. Multiple pharmacological agents, namely calcium sensitizers and myosin activators, although successful in providing inotropic benefits, demonstrated limited clinical application. The Regnier Lab has demonstrated that overexpression of ribonucleotide reductase (RNR) can increase intracellular concentrations of 2 deoxyadenosine triphosphate (dATP), which can fuel myosin to dramatically enhance force generation and cardiac contractility. The Murry Lab pioneered the use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as cell-based cardiac remuscularization therapy. The collaboration between the two labs generated compelling data demonstrating the additive effect of the two technologies for enhanced global contractility after injury: hPSC-CMs remuscularization and engineering these cells to deliver dATP to neighboring myocardium. To increase the efficacy of cell-based/gene therapy approach, we will 1) generate hPSC lines to screen de novo mutations in RNR, identified from computational modeling, that will modulate enzymatic activity and boost dATP levels and 2) test the physiological effects of RNR mutations on dATP production, contractile force and transcriptome of hPSC-CMs. We will build a donor vector that contains RNR subunits, and introduce it at the AAVS1 locus in hPSCs. From computational modeling, we will identify 3 mutations that tune RNR activity and generate mutant lines by site-specific mutagenesis. HPSC-CMs from individual mutant lines will be characterized using muscle cell force assays. This study will generate hPSC lines that can be readily used for translational studies and also provides an experimental framework to screen a library of RNR mutations in hPSC-CMs.

Matthew Childers, PhD

PI:  Matthew Childers, Research Scientist 4, Department of Bioengineering

Project: Examining the Mechanisms of the Dilated Cardiomyopathy-Associated Myosin Mutation R369Q

Understanding disease pathogenesis strengthens and streamlines the design of novel and highly effective treatments for familial cardiomyopathies. Mutations associated with these cardiomyopathies are diverse and can occur in multiple regions of several sarcomeric proteins. Further complicating research efforts, mutations in the same region of a protein can result in different disease phenotypes. Thus, at this stage, careful study of individual pathogenic mutations is required to understand their contribution to disease. Myosin, the motor protein of cardiac muscle, is a sarcomeric protein with a vast array of mutations that can contribute to different forms of inherited cardiomyopathy. Here, we will study the dilated cardiomyopathy-associated myosin mutation R369Q with the aim of developing a computational and experimental workflow to more effectively unmask the mechanisms of such mutations. We will combine insights from molecular dynamics simulations and an experimental human induced pluripotent stem cell-derived cardiomyocyte model to develop a multiscale understanding of disease pathogenesis. By integrating atomistic studies from computational simulations with functional data from reductionist assays, we will establish the validity of our platform for studying any number of mutations in sarcomeric proteins, laying the groundwork for a testing platform that can be used for discovery and testing of novel therapeutics. The support of the CTMR through this pilot grant funding and the institutional expertise in the Quantitative Analysis and Mechanics and Devices cores will provide key resources to develop these approaches and generate preliminary data for grant applications to expand the scope and success of this work.

Wentao Zhu, PhD

PI:  Wentao Zhu, PhD, Acting Assistant Professor, Department of Anesthesiology & Pain Medicine

Project: A Comprehensive Metabolomics Investigation into the Molecular Mechanisms of ZFYVE1 in a Mouse Model of Heart Failure

Understanding the molecular mechanisms that underlie cardiac function and dysfunction is crucial given the persistent threat of cardiovascular diseases worldwide. Patients with heart failure and acute coronary syndromes often experience elevated levels of free fatty acids (FFAs), exacerbating myocardial dysfunction and arrhythmias.[1] Additionally, the accumulation of lipid droplets (LDs) in cardiac cells may contribute to cardiomyopathy and heart failure.[2] Recent research has identified ZFYVE1 as a novel NTPase involved in LD and FFAs metabolism in Hep3B and U2OS cells.[3] Specifically, ZFYVE1 knockdown results in increased cellular FFA’s and the formation of smaller LD’s, particularly during periods of starvation. Meanwhile, in our unpublished preliminary data, we observed decreased levels of ZFYVE1 mRNA expression and protein in the hearts of mice experiencing heart failure, with ZFYVE1 knockout mouse hearts exhibiting more severe heart failure functions under stress. However, the molecular mechanism and relation of ZFYVE1 with cardiovascular disease remains relatively unexplored. In this pilot study, we hypothesize that the metabolic profile of ZFYVE1 knockout mouse hearts and their mitochondria will show distinct alterations compared to wild type, under both normal conditions and surgically induced heart failure. We aim to comprehensively investigate the metabolic changes associated with ZFYVE1 knockout in mouse hearts by integrating targeted and untargeted metabolomics approaches and identifying key unknown metabolites associated with ZFYVE1 knockout. Results from this study could advance our understanding of ZFYVE1 regulatory pathways and offer mechanistic insights into the pathological processes driving cardiovascular diseases, thereby presenting potential avenues for novel therapeutic interventions.

Shabnam Salami, MD, MSc, FAHA

PI:  Shabnam Salami, MD, MSc, FAHA, Acting Instructor, Department of Anesthesiology & Pain Medicine

Project: Mechanistic Insights into DOX-induced Accelerated Aging and Mitigation Strategies

This research investigates the molecular mechanisms underlying sarcopenia following chemotherapy, focusing on the impact of doxorubicin (DOX) on muscle metabolism and function. Sarcopenia, characterized by muscle mass decline, poses significant health risks, particularly in cancer patients undergoing chemotherapy. Leveraging advanced techniques such as isotope tracing and untargeted metabolomics, this study aims to elucidate how DOX-induced cellular senescence and metabolic reprogramming contribute to muscle loss. Additionally, the project seeks to explore the potential of anti-aging drugs like rapamycin, spermidine, and alpha ketoglutarate (AKG) in mitigating these effects by modulating autophagy, inflammation, and DNA methylation pathways. Through a comprehensive approach involving in vitro cell culture models, mouse models, and metabolomic analyses, this research aims to uncover novel therapeutic strategies to prevent and treat sarcopenia in cancer patients receiving chemotherapy. By understanding the intricate interplay between chemotherapy-induced cellular senescence, metabolic alterations, and muscle loss, this study aims to improve the quality of life and long-term health outcomes of cancer survivors. The findings from this research may inform the development of targeted interventions aimed at preserving muscle mass and function in vulnerable patient populations, ultimately enhancing their overall well-being and survivorship.

Joel Chamberlain, PhD
Seung Wook Oh, PhD

PI:  Joel Chamberlain, PhD, Research Associate Professor, Division of Medical Genetics & Seung Wook Oh, PhD, Chief Operating Officer, Kinea Bio, Inc.

Project: A novel non-viral delivery of RNA therapeutics to treat myotonic dystrophy type 1 (DM1)

The most common adult-onset muscular dystrophy, myotonic dystrophy type 1 (DM1), is caused by the expansion of an unstable CTG-repeat in the myotonic dystrophy protein kinase gene DMPK. Expression of non coding nucleotide repeats of DMPK mRNA leads to toxic DMPK mRNA in muscle cells triggering wide-ranging clinical features, like muscle hyperexcitability, progressive skeletal muscle weakness and degeneration, and cardiac defects. There is no effective treatment for this devastating disorder. We propose to test natural cell derived nanovesicles to modulate toxic DMPK mRNA with improved safety compared to risk-prone viral mediated gene therapy. We will demonstrate the feasibility of a nanovesicle-based approach in a two-fold experimental approach. In Aim 1, we will first assess the delivery efficiency of nanovesicles to muscle cells (C2C12) in comparison with other previously characterized cell types. Then, we will test the functional relevance by subjecting nanovesicles loaded with selected (N=5-10) siRNA constructs that reduce toxic DMPK mRNA in human induced pluripotent stem cells (iPSC) derived from DM1 patients. In Aim 2, we propose to evaluate the effect of nanovesicles on muscle cell viability and contractility using a myocardial cell model in collaboration with the Mechanics & Devices Core at the UW Center for Translational Muscle Research (CTMR). By demonstrating a highly efficient modulation of target genes, along with proven safety, this UW-CTMR pilot grant project will provide critical proof-of-concept data for successful resubmission of the SBIR grant and lay a crucial foundation toward advancing this novel nanovesicle-based technology as a viable therapy for DM1 patients.

Previous Awardees

Project Period: May 1, 2023 – February 29, 2024 (Year 5)
Shiri Levy, PhD

PI: Shiri Levy, PhD, Acting Instructor, Department of Biochemistry

Project: Epigenetic reprogramming of muscle stem cells in sarcopenia using AI-design computer protein binder

25-45% of older adults in the U.S suffer from Sarcopenia. Sarcopenia is linked to the aging process, and it involves the reduction of muscle mass and strength, which can negatively impact motor function. This deterioration in muscle quality can speed up the overall decline observed in older adults, making them more vulnerable to serious health conditions. Moreover, sarcopenia can diminish a person’s quality of life, making it more difficult for them to maintain their independence. In sarcopenia, the loss of muscle mass and function is associated with a decline in the number and function of satellite cells. This reduction in satellite cell activity is believed to be one of the key mechanisms that contribute to the progression of muscle wasting and weakness in older adults including a reduction in myofibers. Artificial intelligence (AI) computer-designed protein is a rapidly advancing field with the potential to revolutionize various industries and sectors, including healthcare and biotechnology. In this context, the design of AI-driven epigenetic protein binder has tremendous potential to be highly efficient in promoting skeletal muscle differentiation from satellite cells by activating MyoD and MyoG. Unlike cellular therapies that introduce immunogenicity and toxicity (even in autologous transplantation) or chemical drugs which lead to adverse side effects in patients, targeted epigenetic gene editing therapies are safe as they target specific genes and do not involve making changes to the underlying blueprint of the DNA sequence. Therefore, as a new junior faculty doing innovative research, I propose epigenetically remodeling the niche of satellite muscle stem cells for regenerative therapy in sarcopenia towards new therapeutic in the clinic.

Logan Murphy, PhD

PI: Logan Murphy, PhD, Acting Instructor, Department of Physiology & Biophysics

Project: Sex-Dependent Recovery from Cervical Spinal Cord Injury with Electrical Spinal Stimulation Therapy: Role of Muscle Responses

Motor deficits severely impact the quality of life of people with spinal cord injury (SCI), yet current treatments produce limited improvements in movement abilities. We have developed a novel application of electrical stimulation, termed targeted, activity-dependent spinal stimulation (TADSS), to induce neural plasticity and improve recovery of forelimb motor function in a rat model of cervical contusion SCI. Results indicate that several weeks of TADSS combined with task retraining leads to substantial improvements in movement ability that persists for months after the therapy ends. However, this functional benefit is primarily in female rats. Male rats recover much less, and recovery is highly variable from animal to animal and even from day-to-day for a single animal.
The dramatic sex difference in the effects of TADSS has huge potential clinical implications and therefore we would like to understand the underlying physiological mechanisms. One possible factor is a sex-dependent change in muscle properties that affects males’ ability to execute successful movements. SCI causes extensive changes to muscle contractile and metabolic function. Treatments that restore motor ability also reverse some of these effects. We hypothesize that differences in muscle properties in male and female rats after TADSS contribute to the sex-dependent recovery. The objective of the proposed project is to utilize two of the Center for Translational Muscle Research Cores to initiate studies to determine if muscles in male and female rats respond differently to the therapy. An investigation of muscle properties represents a new direction in our research program.

Matthew Walker, PhD

PI:  Matthew Walker, PhD, Research Assistant Professor, Mitochondria and Metabolism Center & Department of Anesthesiology and Pain Medicine

Project: Computational approaches to study redox regulation of mitochondrial RNase P:
Implications for skeletal muscle mitochondrial biogenesis in heart failure

Given that there is significant skeletal muscle dysfunction in heart failure (HF), additional strategies to treat muscle abnormalities in HF are urgently needed. Drastic shifts in mitochondrial biogenesis are well-documented in skeletal muscle from HF patients often resulting in mitochondrial dysfunction. Mitochondrial dysfunction is a hallmark of HF that directly contributes to exercise intolerance and muscle wasting. My research suggests a significant defect in mitochondrial DNA post-transcriptional processing in HF that is sensitive to the cellular redox (NAD+/NADH). This prompted me to investigate the functions of the mitochondrial RNase P complex. The structure of human mitochondrial RNase P has been determined previously by single-particle cryoEM at an overall resolution of 3.0 Å. The base of the three-subunit complex is formed by the NAD+-dependent Mrpp2 tetramer. Mrpp2 dehydrogenase active sites are located near the adaptor region of Mrpp1 and anticodon recognition site for tRNAs. To date, it is unknown if NAD+-induced conformational changes to the active site of Mrpp2 affect binding to Mrpp1 or to the tRNA substrates. Thus, I hypothesize that the imbalanced redox state in HF impairs Mrpp2 interactions with Mrpp1 resulting in impaired mitochondrial biogenesis. To test this hypothesis, we will employ molecular dynamics simulations to the study structure-function relationship of Mrpp2 when bound to its coenzyme NAD+ that might enhance or inhibit mitochondrial RNase P. To validate the simulations, we will aim to test whether increasing NAD+ level in skeletal muscle from HF mice can improve RNase P activity and thus skeletal muscle mitochondrial biogenesis in HF.

Matthew Campbell, PhD

PI:  Matthew Campbell, PhD, Acting Instructor, Department of Radiology

Project: Acute mitochondrial functional response to contraction in human skeletal myotube

Decreased skeletal muscle mass, specific force, increased overall fatty infiltration in the skeletal muscle, frailty and depressed energy maintenance are all associated with increased oxidative stress and the decline in mitochondrial function with age. We are partnering with the Study of Muscle, Mobility and Aging (R01 AG059416) to obtain primary human myotubes from well phenotyped older adults to develop a cell model of skeletal muscle aging. Mitochondrial response to exercise has been shown to be partially mediated through redox sensitive signaling control following muscle contraction. We have previously developed protocols to test mitochondrial function following high-intensity interval training (HIIT) and low-intensity steady state (LISS) muscle contraction in vivo. Following HIIT, young skeletal muscle mitochondria increased fatty acid oxidation compared to non-stimulated control muscle; however, aged muscle mitochondria decreased fatty acid oxidation. In contrast, following LISS, young skeletal muscle decreased fatty acid oxidation, whereas aged muscle increased fatty acid oxidation. We also found that HIIT inhibits oxidation of glutamate in both stimulated and non-stimulated aged muscle, suggesting HIIT stimulates circulation of an exerkine capable of altering whole-body metabolism. We will adapt these protocols for in vitro use to test the hypothesis that age inhibits mitochondrial response to acute exercise and exerkine signaling. We will test this hypothesis with two specific aims 1) we will examine mitochondrial substrate utilization response to LISS and HIIT in vitro and 2) we will characterize mitochondrial response to exerkine signaling following myotube contraction in vitro.

Project Period: April 1, 2022 – February 28, 2023 (Year 4)
Ronald Y. Kwon, Ph.D.

PI: Ronald Y. Kwon, Ph.D., Associate Professor, Department of Orthapaedics and Sports Medicine

Project: Genetic Determinants of Osteosarcopenia Risk

Osteoporosis and sarcopenia commonly occur in the same individual, a condition
termed osteosarcopenia. There is an urgent scientific need to determine the mechanisms underlying shared genetic influence on bone and muscle, as this is needed to understand how genetic factors contribute to osteosarcopenia pathophysiology and could identify clinical targets that act on bone and muscle simultaneously. Our broad objective is to leverage rapid-throughput biology
in zebrafish to identify genes that underlie genetic risk for osteosarcopenia. Genome- wide association studies have shown that the CPED1-WNT16 locus harbors genetic variants co-associated with bone and lean mass; while WNT16 is a critical regulator of bone mass, our understanding of its function fails to explain pleiotropy at this locus. Recently, our lab discovered that wnt16 exerts pleiotropic influence on muscle and bone in zebrafish. However, what remains lacking is if wnt16 regulation of muscle occurs exclusively through regulating muscle size and shape, or whether it also regulates strength and structure. Without this knowledge, how WNT16 contributes to osteosarcopenia pathophysiology cannot be fully determined. Utilizing the CTMR
Mechanics and Devices Core, we will characterize contractile properties during growth (SA1) and test whether wnt16 is necessary for muscle structure and contractile properties (SA2). We expect that this project will help identify how WNT16 contributes to genetic risk for osteosarcopenia, as well as the broader utility of zebrafish to identify genes that underlie genetic influence of osteosarcopenia-related traits. Finally, this project will accelerate a change in the PI’s career direction toward muscle research, supporting the CTMR mission.

Julie Mathieu, Ph.D.

PI: Julie Mathieu, Ph.D., Assistant Professor, Comparative Medicine

Project: ANT1 function in induced pluripotent stem cell-derived cardiomyocytes

Heart failure affects at least 65 million people worldwide and represent a global public
health burden. Cardiomyopathy is a leading cause of heart failure affecting individuals of all ages and races. Animal studies have provided important insights on the pathomechanisms of cardiomyopathies. However they do not always recapitulate the disease accurately, highlighting the need for new human in vitro models. Patients with mutation in ANT1, the cardiac and skeletal muscle isoform of the mitochondrial Adenine Nucleotide Translocase, develop cardiomyopathy and mitochondrial myopathy of limb muscles. To recapitulate the cardiac pathology of ANT1 mutation on
the cellular level in vitro, we used the CRISPR/Cas9 system in human induced pluripotent stem cells (iPSC) to generate mutations in the SLC25A4 gene that encodes for ANT1. Using a monolayer directed differentiation protocol we generated human iPSC-derived cardiomyocytes (iPSC-CM) from the ANT1 mutant and control lines. We found that the loss of ANT1 did not hinder the ability to generate beating cardiomyocytes. Here we propose to study the functional consequences of ANT1
mutation on mitochondrial bioenergetic, ROS production, oxidative stress and mitophagy. Towards that end we will generate a new mitophagy reporter line in iPSC by inserting the mt-mKeima fluorescence reporter in the AAVS1 safe harbor site. We will also determine whether the ANT1 KO iPSC-CM line recapitulates the cardiac phenotypes observed in ANT1 mutant patients. Our results will then be used to design a phenotypic screen to identify compounds that can prevent cardiac dysfunction in human ANT1 mutant cardiomyocytes.

David J. Perkel, Ph.D.
Bing W Brunton, Ph.D.

PI:  David J Perkel, Ph.D., Professor, Departments of Biology and Otolaryngology; Bing W Brunton, Ph.D., Associate Professor, Department of Biology

Project: Measuring muscle performance in a vertebrate model of enhanced bipedal balance

Bipedal organisms like humans and birds have exceptional balancing abilities. Here, we establish perching birds as a model for studying motor contributions to upright, bipedal balance control. This CTMR Pilot award will help us develop the EMG instrumentation required to measure muscle activity in freely-behaving perching birds. Specifically, with the help of the Mechanics and Devices Core we will use the Pilot Award to build an EMG amplifier and wireless data transmitter that can be worn by the birds, preventing entanglement in wires. We will also learn methods for efficient EMG data processing with the help of the Quantitative Analysis Core. This project represents a change in research direction for the two PIs as well as a new training opportunity for the two postdoctoral Co-Is and an engineering undergraduate student.

Travis Tune, Ph.D.
Matt Childers Ph.D.

PI:  Travis Tune, Ph.D., Research Scientist, Department of Biology; Matt Childers Ph.D., Postdoctoral Fellow, Department of Bioengineering

Project: Measuring muscle performance in a vertebrate model of enhanced bipedal balance

Mutations in contractile proteins lead to diseases of the musculature. However, there is a complex relationship between amino acid mutation and the resulting mechanical consequences. For example, two mutations in a similar region of a single protein can lead to drastically distinct disease phenotypes. Advances in DNA sequencing technologies have identified an ever-increasing number of mutations in contractile proteins that are linked with disease. Unfortunately, mechanistic understanding of the relationship between these mutations and diseases severely lags mutation identification. Current computational models have demonstrated a capacity to model the effects of mutations at both the single molecule level (via molecular dynamics simulations) and at the contractile organelle level (via a spatially explicit model of the half sarcomere). However, system level behaviors that arise from intricate connections among proteins within the contractile machinery can amplify the effects of mutations in unexpected ways. These system-level ’emergent properties’ demonstrate a need for development of methods and models that can investigate phenomena across biological scales. Here, we propose to bridge two computational resources within the CTMR by developing new models and methods that can both parameterize spatially explicit half-sarcomere models using molecular simulations and inform boundary conditions on molecular simulations using the spatially explicit half-sarcomere model.

Lisa Maves, Ph.D.

PI:  Lisa Maves, Ph.D., Associate Professor, Pediatrics

Project: Quantitative Modeling to Determine Muscle Myosin Gene Orthology

The human genome encodes 8 conventional class II myosin heavy chain genes that are expressed in striated muscle tissues (Berg et al., 2001): MYH6 (cardiac-a), MYH7 (cardiac-b), and six genes that cluster on chromosome 17, MYH1, MYH2, MYH3, MYH4, MYH8, and MYH13. These eight myosins have highly similar sequences and share a common 3D domain structure. Small changes in critical regions of the sequence differentiate these genes and endow their associated proteins with mechanical properties that are optimized for the specific types of muscle at specific stages of development. For example, a ~15 amino acid stretch known as loop 1 varies between species and between myosin isoforms within a single species and determines, in part, ADP release rates and ATP binding rates. Likewise, the expression of a particular MYH gene largely determines a muscle fiber’s contractile and metabolic properties. Our research seeks to take advantage of the zebrafish model to address the functions of myosin genes in cardiac and skeletal muscle development and disease. The zebrafish genome, however, contains 14 striated muscle myosin genes (Ruzicka et al., 2019): myh6, 4 myh7 genes, 6 fast myh genes that cluster on chromosome 5, and 3 slow myh genes that cluster on chromosome 24. The orthology between the zebrafish myh genes and the human MYH genes is not resolved. This presents a critical barrier to using zebrafish as a model for human myopathies. This project aims to address the orthology and function of zebrafish myh genes.

Project Period: April 1, 2021-February 28, 2022 (Year 3)
Lindsey Anderson, PhD
Lindsey Anderson, Ph.D.

PI: Lindsey Anderson, Ph.D., Acting Instructor, Division of Gerontology and Geriatric Medicine

Project: Metabolomics approach to characterize the effects of androgen deprivation therapy on skeletal muscle in prostate cancer patients

Prostate cancer (PCa) is the most common cancer among men. Androgen deprivation therapy (ADT), which minimizes endogenous androgen (i.e. testosterone) production, is the standard treatment for advanced/metastatic PCa with~400,000 men on ADT in the U.S. ADT induces a decrease in muscle mass/performance(sarcopenia), which leads to poor quality of life (QOL) and increased fatigue and mortality. Human omics analyses have reported metabolic perturbations in skeletal muscle and the circulation from non-hormone-dependent cancer patient cohorts, but this has not been assessed in the ADT setting. To date, mechanisms underlying ADT-induced sarcopenia remain unknown and constitute a significant barrier to therapeutic development.

Objective/Hypothesis: To establish the role of androgen-dependent molecular pathways leading to ADT-induced sarcopenia in men with PCa. We hypothesize that ADT will induce metabolic perturbations in skeletal muscle and plasma which will be associated with sarcopenia.

Specific Aims: To determine ADT-induced changes in 1) Muscle mass/performance; 2) Muscle and plasma targeted metabolomics; and 3) Patient-reported fatigue and QOL.

Design: Men with PCa undergoing ADT. Muscle biopsies and blood samples will be performed before and 6-months after ADT; muscle mass/performance, fatigue, and QOL will be assessed at baseline and at 3-and 6-months.

Impact: These efforts will have a major impact on 1) reducing PCa-associated morbidity by identifying mechanisms causing ADT-induced sarcopenia for targets of future interventional clinical trials, 2) Dr. Anderson’s establishment as muscle researcher, 3) Dr. Dash’s transition towards muscle research, and 4) fostering collaborations across oncology, geriatrics, and metabolomics for pursuing novel muscle research endeavors

Fausto Carnevale Neto, Ph.D.

PI: Fausto Carnevale Neto, Ph.D., Acting Instructor, Department of Anesthesiology & Pain Medicine

Project: The role of mitochondrial metabolism on quiescent muscle stem cell proliferation and myogenesis

Skeletal muscle is inherently regenerative following acute injury. Muscle tissue homeostasis and regeneration depends on muscle stem cells. In skeletal muscle, stem cells reside in a quiescent state, but little is known about the molecular mechanisms that control whether to remain quiescent, proliferate, or differentiate into myoblasts. In this pilot study, we aim to investigate how metabolism modulates muscle stem cell (SC) homeostasis and regenerative turnover. We hypothesize that the molecular mechanisms regulating reversible SC quiescence can be explored by global metabolite profiling of homogeneous populations of myoblast as well as their isolated mitochondria. Two specific aims will help us reach this goal. In aim 1, we will examine the metabolic mechanisms governing stem cell self-renewal and differentiation by manipulating culture conditions that regulate cell cycles of homogeneous populations of quiescent muscle cells. We will isolate mitochondria to inspect the influence of subcellular metabolism on muscle stem cell fate. In aim 2, we will compare the diverse metabolic phenotypes at cellular and subcellular (isolated mitochondria) levels. Results from this study promise to expand our knowledge about mitochondrial metabolite diversity and function in the regulation and maintenance of muscle stem cells.

G. A. Nagana Gowda, Ph.D.

PI: G. A. Nagana Gowda, Ph.D.,  Research Associate Professor, Department of Anesthesiology & Pain Medicine

Project: Probing Energetics of Muscle Metabolism Using 31P NMR Metabolomics

Metabolites of cellular energetics power life, health, and aging. As we age, levels of these metabolites decline substantially, leaving us at a greater risk for a multitude of muscular as well as other diseases. Hence, interest in investigation of these metabolites is increasing significantly. The ability to quantitate metabolites of energy in blood and relate those results to muscle metabolism would represent a significant advance. Using 1H-NMR spectroscopy, we recently developed methods to analyze several such metabolites in blood, tissue, cells and isolated mitochondria [1-4]. A drawback of the 1H-NMR method is that analysis is restricted to a small number of metabolites (~7) since 1H-NMR spectra are overwhelmed by the interference from numerous other classes of metabolites. Unfortunately, highly sensitive mass spectrometry methods are unsuitable for a number of fundamental reasons. To alleviate this challenge, we propose to develop 31P-NMR metabolomics method to profile a wider range of metabolites (>30) of cellular energetics including NAD+, NADH, NADP+, NADPH, ATP, ADP and AMP, and the associated glycolysis and pentose phosphate pathway metabolites. The new method will be applied to probe changes in muscle metabolism using skeletal muscle tissue and blood, that are linked to aging. Outcomes of this study will fill an important knowledge gap, which is the metabolic link between muscle metabolism and blood metabolite levels, and offers a new tool for basic research and translational studies. A successful outcome will also enable the establishment of a new analytical capability at the Metabolism Core of the CTMR.

Project Period: November 16, 2020 – February 28, 2021 (Year 2)
Mary Beth Brown
Mary Beth Brown, Ph.D.

PI: Mary Beth Brown, Ph.D., Associate Professor, Department of Rehabilitation Medicine

Project: Metabolomics to reveal exercise adaptations and impact of gene therapy in a novel rat model of Duchene muscular dystrophy

Duchene Muscular Dystrophy (DMD) is a severe muscle wasting disease caused by deficiency of the protein dystrophin. Due to well-characterized benefit of exercise for skeletal and cardiac muscle health, as well as for positive effects on multiple organ systems, exercise in DMD patients has been investigated for decades, but with often contradictory results. Hampering progress toward the development of clear exercise recommendations are inadequate animal models, poorly controlled/monitored exercise stimuli, and only surface-level investigation of exercise adaptations. We are currently conducting a study funded by a Research Royalty Fund (RRF) award which will be the first to investigate exercise responses in a new rat model of DMD, including for a subset of gene-therapy treated rats. There is an urgent medical need for this work as gene therapy for DMD is currently in clinical trials and a data-driven exercise regimen for boys post gene transfer must be established. Our first RRF cohort finishes a 6-week exercise training program in December 2020, where 2 types of exercise are examined- voluntary wheel running or a prescribed treadmill protocol, vs. no training. These data will support an R01 application in February 2021. While a thorough, serial assessment of physiological responses is already underway for the RRF, as well as planned biochemical/histological assays, we would like to collect proteomic data to better reveal pathways of exercise adaptations and gene therapy responses. Therefore, we are requesting CTMR mini pilot funding to support use of the Metabolism Core for skeletal and cardiac muscle samples.

Jennifer Davis
Jennifer Davis, Ph.D.
David Mack
David Mack, Ph.D.

PIs: Jennifer Davis, Ph.D, Associate Professor, Departments of Lab Medicine & Pathology and Bioengineering; David Mack, Ph.D. Associate Professor, Department of Rehabilitation Medicine

Project: The role of MBNL1-Transcriptome Reprogramming In IPS-Skeletal Muscle Differentiation

The ability to model human skeletal muscle development and disease in vitro with human induced pluripotent stem cells (iPSCs) can help answer longstanding questions regarding the generalizability of developmental and disease paradigms established in animal models. Due to limitations that include low efficiency, culture heterogeneity, and immature phenotypes iPSC-derived skeletal muscle models are lacking. Hence, this proposal seeks to examine the role of transcript reprogramming in the direct differentiation of iPSCs into skeletal muscle. To reprogram the transcriptome the expression of a myogenic RNA binding protein, MBNL1, will be perturbed by engineering 2 new iPSC lines with conditional MBNL1 loss of function and Doxycycline-regulated gain of function (Aim 1). In Aim 2 we will examine the hypothesis: MBNL1 expression is both necessary and sufficient to promote the direct differentiation of IPSCs into skeletal muscle. It is anticipated that MBNL1 overexpression will accelerate the rate of IPSC differentiation into a skeletal muscle and improve both efficiency and maturity of the differentiation. Here CTMR mechanics and metabolism cores will be used to assess the MBNL1-dependent functional state of iPSC-derived skeletal muscle as MBNL1 directly regulates both sarcomeric and metabolic genes. We anticipate the generation of new iPSC lines and implementation of transcriptome-level modulation to promote muscle differentiation will have a high overall impact for the field.

Farid Moussavi-Harami
Farid Moussavi-Harami, M.D.

PI: Farid Moussavi-Harami, M.D., Assistant Professor, Division of Cardiology

Project: Metabolomic analysis of skeletal muscle in mouse dilated cardiomyopathy

Heart failure (HF), the inability of the heart to keep up with its workload, is an emerging epidemic, affecting over five million Americans. Exercise intolerance represents a major determinant of morbidity in heart failure. HF results in underlying intrinsic structural, biochemical and metabolic skeletal muscle defects. However, the exact mechanisms are unknown. The muscle wasting in heart failure is independent of alteration in blood flow and may be attributed to decreased anabolism, increased catabolism or both. In addition to loss of fibers, there is also switch from slow twitch type I fiber to fast twitch type II fibers. The only effective treatment for skeletal muscle dysfunction in heart failure is aerobic exercise training. There are no specific pharmacologic treatments for the myopathy seen in heart failure. There is recent data in rodent models that dilated cardiomyopathy induced HF results in decreased muscle strength, inflammation and impaired regeneration after acute muscle injury. In this mini grant proposal, we propose to use metabolomics to assess changes in skeletal muscle of young and old mice with genetic dilated cardiomyopathy compared to control mice. In future studies, we will do histological and functional contractile assessment as well.

Yuliang Wang
Yuliang Wang, Ph.D.

PI: Yuliang Wang, Ph.D., Research Assistant Professor, Paul G. Allen School of Computer Science & Engineering

Project: Identify metabolic interventions to mature iPSC-SkM by network-based multi-omic data integration

The overall goal of this pilot grant is to identify metabolic strategies to drive maturity and enhance function of induced pluripotent stem cell-derived skeletal muscle in vitro (iPSC-SkM). It is widely accepted in the field that most iPSC-derived tissues naturally arrest at the embryonic or neonatal stages of development. We hypothesize that the addition or removal of certain metabolites and/or small molecule enzyme activators/inhibitors will improve iPSC-SkM maturation and thereby make this platform more powerful for juvenile and adult onset muscle disease modeling and drug discovery. There is a major knowledge gap on in vitro strategies that can improve the maturity and function of iPSC-SkM, and a methodological gap of computational tools to systematically guide the discovery of such maturation protocols.

Metabolism plays an important role in cell fate decisions. In particular, we and others have shown that metabolic manipulations can significantly improve the maturity of iPSC-derived cardiomyocytes (iPSC-CM). However, whether metabolic manipulations are similarly effective in iPSC-SkM maturation remain unclear. Therefore, using a published single cell RNA-seq dataset 1, we will identify metabolites and enzymes important for human skeletal muscle maturation in vivo, but missed in iPSC-SkM differentiation in vitro (Aim 1). Then, we will test several of our most confident predictions on iPSC-SkM at week 8, and perform metabolomics and lipidomics before and after the metabolic interventions (Aim 2).

Huiliang Zhang
Huiliang Zhang, Ph.D.

PI: Huiliang Zhang, Ph.D., Acting Instructor, Department of Laboratory Medicine & Pathology

Project: Mitochondrial function rejuvenation to restore aged heart muscle dysfunction

Aging is the greatest risk factor for cardiovascular diseases and results in progressive cardiac hypertrophy and declining cardiac function. The latter includes diastolic dysfunction, which can lead to heart failure with preserved ejection fraction (HFpEF). Unfortunately, there are no effective therapies for HFpEF or cardiac aging. Cardiomyocytes maintain a precisely coupled homeostasis of mitochondrial bioenergetics and excitation-contraction. In heart failure and the aged heart, however, mitochondrial function is compromised, impairing this homeostasis. Therefore, there is a consensus that mitochondrial function is a promising therapeutic target for cardiac dysfunction.

My recent work has shown that brief treatment of SS-31 peptide prevents mitochondrial proton leak and rejuvenates the mitochondrial function in aged cardiomyocytes. Moreover, we found that 8-week SS-31 in vivo treatment can reverse diastolic dysfunction in aged mice. Our preliminary data showed that SS-31 treatment reverses post translational modifications of key contractile proteins. Also, SS-31 in vivo treatment normalized the Ca2+ transient and Ca2+ spark in aged cardiomyocytes. Thus, we hypothesize that SS-31 restores cardiac dysfunction through the improvement of contractile machinery function and Ca2+ handling.

To test this hypothesis, I will 1) determine whether and how SS-31 improves the contractile machinery; and 2) determine the mechanisms of SS-31 improvement of Ca2+ handling in aged cardiomyocytes.

Understanding the mechanistic basis of cardiac contractility decline in aging and how this can be reversed by SS-31 treatment is critical to translate findings to the treatment of cardiac impairment in the elderly.

Project Period: April 1, 2020 – February 28, 2021 (Year 1)
Matthew Campbell
Matthew Campbell, Ph.D.

PI: Matthew Campbell, Ph.D., Acting Instructor, Department of Radiology

Project: Substrate metabolism linked redox-sensitive signaling mechanisms in mitochondria following exercise altered with age

The overall goal of this proposal is to identify redox-sensitive mechanisms in mitochondria that contribute to aging pathology. This project will utilize mouse models of aging to study how steady-state and high intensity exercise affects skeletal muscle mitochondria’s ability to utilize fatty acids as a source of energy. We will measure how exercise activates oxidant production through NADPH oxidase and what downstream redox-sensitive signals are specifically activated that may underlie the ability of mitochondria to shift toward or away from fatty acid utilization in skeletal muscle.

Nagana Gowda, Ph.D.

PI: Nagana Gowda, Ph.D., Research Associate Professor, Department of Anesthesiology & Pain Medicine

Project: Real time measurement of mitochondrial energetics in live muscle cells and isolate mitochondria

This pilot study proposes to develop nuclear magnetic resonance (NMR) spectroscopy methods for measurement of cellular and mitochondrial energetics under live conditions in real time. The investigations will build on the recent ex vivo methodological developments in our laboratory that enabled one-step measurement of many metabolites including coenzymes/cofactors, which are fundamental to mitochondrial metabolism and energetics. Development of novel methods for metabolites analysis in live cells and mitochondria in real time offers unique benefits and opportunities for the investigation of cellular and mitochondrial metabolism under pathological conditions of muscular diseases.

Farid Moussavi-Harami
Farid Moussavi-Harami, M.D.

PI: Farid Moussavi-Harami, M.D., Assistant Professor, Division of Cardiology

Project: Computational and experimental methods to evaluate the role of sarcomeric variant incorporation in striated muscle

Striated muscle activation and relaxation occurs through an intricate network of intra- and inter-filament interactions in the sarcomere. Even small disruptions in this intricate network results in altered force generation capacity and ultimately development of disease. Despite advances in our understanding of myopathy pathogenesis, there are still challenges to predicting the exact functional consequences of genetic variations in the sarcomeric genes. To address this issue, we propose to improve computational tools for studying pathogenesis of myopathies caused by sarcomeric variants in skeletal and cardiac muscle and look at sarcomeric variant incorporation using this tool. We will use a multi-filament computational model of the half sarcomere with the ability to predict how altered sarcomere activation/deactivation changes myocyte force generation and twitch kinetics. We will first start by adding Ca transients to this model to allow for twitch generation, which we have recently shown to be predictive of development of cardiomyopathies. We will demonstrate the capabilities of the model by determining the effects of increasing sarcomeric variant incorporation on striated activation and relaxation using distal arthrogryposis and hypertrophic cardiomyopathy as test cases. We will validate our results using recombinant proteins, cells and tissue.

Alec Smith
Alec Smith, Ph.D.

PI: Alec Smith, Ph.D., Acting Instructor, Department of Physiology & Biophysics

Project: Investigating the role of innervation in the development of hiPSC-derived skeletal muscle

Myotubes generated from human induced pluripotent stem cell (hiPSC) sources typically adopt embryonic or neonatal phenotypes and do not recapitulate later stages of myogenesis. This represents a significant limitation when seeking to use such cells for studies of muscle development and biology. Innervation is known to play a critical role in driving secondary myogenesis, but it remains unclear whether direct synaptic contact is necessary to achieve this. Furthermore, the specific mechanisms that underpin nerve-dependent myogenic development remain poorly characterized. Therefore, we propose to study the functional and transcriptomic impact of innervation on hiPSC-derived skeletal muscle by comparing muscle cultures maintained in isolation to those in co-culture with hiPSC-derived motor neurons.