Project Summary Mitochondrial dysfunction in skeletal muscle has devastating consequences including muscle wasting, exercise intolerance, and insulin resistance. We have discovered that a novel, highly conserved protein is critical for maintenance of mitochondrial function and cellular energy homeostasis in yeast and mammalian cells. We have designated this protein Lifespan-associated Mitochondrial Stress-responsive 1 (Lms1). Our data from yeast support a model whereby Lms1 recruits components of the ubiquitin proteasome system to mitochondria to extract damaged proteins and present them to the proteasome for degradation. The purpose of this research is to determine the function and mechanism of Lms1 action in skeletal muscle using cultured muscle cells and Lms1 knockout mice. For Specific Aims 1 and 2, which span the K99 and R00 phases, the candidate will investigate the role of Lms1 in cultured muscle cells. Studies in Aim 1 will determine whether mammalian Lms1 recruits the ubiquitin proteasome system to mitochondria as part of a mitochondrial protein quality control system. Studies in Aim 2 will determine the nature of mitochondrial defects observed with Lms1 depletion in muscle cells. Completion of the sub- aims proposed during the K99 phase will provide the candidate with training in aspects of cellular and molecular biology necessary to independently complete the R00 phase. For Specific Aims 3 and 4, the candidate will determine the role of Lms1 at the mammalian organismal level. For Aim 3 (K99 phase), the candidate will examine an Lms1 knockout mouse for mitochondrial dysfunction in heart muscle and begin studies in skeletal muscle. For Aim 4 (R00 phase), the candidate will examine an Lms1 skeletal muscle- specific knockout mouse for mitochondrial dysfunction and consequences including exercise intolerance, muscle wasting, and insulin resistance. Experiments proposed in Aim 3 will provide the candidate with the training in mitochondrial physiology necessary to independently complete Aim 4 during the R00 phase. Collectively, these experiments seek to establish a mechanistic basis for Lms1 action in cultured muscle cells and to extend these findings to mice where they will be tested for physiologic relevance. These studies will provide novel insight into the regulation of mitochondria in skeletal muscle.

Public Health Relevance Mitochondrial dysfunction in skeletal muscle has devastating consequences including muscle wasting, exercise intolerance, and insulin resistance. We have discovered that a novel protein termed Lms1 is important for maintaining mitochondrial function. These studies will determine the role of Lms1 in maintaining mitochondrial function in skeletal muscle. __SpecificAimsTextDelimiter__ SPECIFIC AIMS Mitochondrial dysfunction in skeletal muscle has devastating consequences including muscle wasting, exercise intolerance, and insulin resistance. Using yeast, C. elegans, and cultured mammalian cells, we have discovered that a novel, highly conserved protein is critical for maintenance of mitochondrial function and cellular energy homeostasis. We have designated this protein Lifespan-associated Mitochondrial Stress-responsive 1 (Lms1). In yeast, Lms1 translocates to mitochondria upon application of mitochondrial stress, Lms1 physically associates with Cdc48 and Npl4, which are components of the ubiquitin proteasome system, and LMS1 deletion causes mitochondrial accumulation of polyubiquitinated proteins and respiratory failure. We propose a model wherein Lms1 recruits Cdc48 and Npl4 to mitochondria to extract damaged proteins for degradation by the proteasome, thereby linking mitochondria to the proteasome as part of a mitochondrial protein quality control system. Lms1 depletion in cultured mammalian cells decreases cellular ATP content and mitochondrial membrane potential and initial experiments with an Lms1 knockout (Lms1-/-) mouse suggest the mitochondrial phenotype observed in model systems extends to the mammalian organismal level. Herein I propose to determine the function and mechanism of Lms1 action in skeletal muscle. Specific AIM #1: To determine the mechanism of Lms1 action in cultured muscle cells. In yeast, Lms1 translocates to mitochondria under mitochondrial stress. In yeast, Lms1 physically associates with Cdc48 and Npl4 and appears to recruit them to mitochondria to extract damaged and polyubiquitinated proteins as part of a mitochondrial protein quality control system. Using cultured muscle cells we will: 1) Determine whether mitochondrial stress causes Lms1 to translocate to mitochondria; 2) Determine whether Lms1 physically associates with p97 (Cdc48 in yeast) and Npl4 and comprehensively identify the components of the Lms1 protein complex; 3) Confirm candidate Lms1 interacting proteins and begin to study their roles in mitochondrial function; 4) Determine whether Lms1 depletion causes accumulation of mitochondrial polyubiquitinated proteins. Specific AIM #2: To determine the nature of the mitochondrial defects caused by Lms1 depletion in cultured muscle cells. Lms1 knockdown in C2C12 cells causes decreased ATP content and mitochondrial membrane potential. The goals of this aim are to determine whether Lms1 depletion in cultured muscle cells results in: 1) Decreased mitochondrial respiratory capacity and/or coupling; 2) Physical damage to mitochondria including protein carbonylation and lipid peroxidation; 3) Increased mitochondrial H2O2 emission, 4) Decreased activities of electron transport chain respiratory complexes; 5) Systematic changes in the composition of the mitochondrial proteome. Specific AIM #3: To determine the consequences of Lms1 deletion in mice. We have generated a germline Lms1 knockout mouse (Lms1-/-). These mice display decreased spontaneous activity and elevated oxygen consumption to activity ratios, characteristics consistent with known models of mitochondrial dysfunction. We will: 1) perform a general observational characterization of the Lms1-/- mouse; and 2) test the hypothesis that heart muscle from Lms1-/- mice has decreased respiratory and ATP generating capacity. Specific AIM #4: To determine the consequences of selective Lms1 deletion in mouse skeletal muscle. Unequivocally determining the specific consequences of Lms1 deletion in skeletal muscle will require selective deletion. We will generate Lms1 skeletal muscle specific knockout mice (Lms1SM-/-) and determine whether: 1) In vivo, Lms1SM-/- mice have decreased exercise tolerance, skeletal muscle strength, and insulin sensitivity; 2) Ex vivo, skeletal muscle from Lms1SM-/- mice has reduced respiratory capacity, increased H2O2 emission, and decreased insulin-stimulated glucose uptake; 3) Mitochondria in Lms1SM-/- mouse skeletal muscle accumulate damaged and polyubiquitinated proteins and exhibit gross morphological defects detectable by electron microscopy.