NOTE: End date changed to 05/31/2023 per F. Hernandez/ARC (Ed., 6/20/22).

NOTE: Start/end dates changes to 6/1/2019-7/31/2022 (originally 3/1/2019-5/31/2022) per NSSC award documents per F. Hernandez/ARC (Ed., 12/3/2020)

Mechanotransduction is the ability to sense and regulate adaptive responses to increased or decreased loading. New paradigms have emerged from ground analogs of µG that have contributed to a leap of our understanding of mechanotransduction and muscle atrophy. Specifically, the mu-splice variant of neuronal nitric oxide synthase (nNOSµ) was discovered as causal in both muscle hypertrophy with overloading and atrophy with unloading. Our laboratory has found that reactive oxygen species (ROS) directly contribute to both muscle fiber atrophy and fiber-type shift from slow to fast. Pilot Data and cutting-edge research have identified mitochondria, the Nox2 isoform of NADPH oxidase, and upstream angiotensin II receptor 1 (AT1R) as sources of ROS during mechanical unloading. Preliminary Data show that inhibition of Nox2 translocation of nNOSµ away from the sarcolemma, muscle fiber atrophy, and fiber-type shift. However, the upstream mechanisms that regulate Nox2 during µG are poorly understood, impeding progress in space biology and novel countermeasure development. The lack of such knowledge impedes our development in understanding the mechanisms that underlie redox regulation of mechanotransduction in skeletal muscle. This grant application serves as a renewal and extension and Renewal of our research team's NNX13AE45G award, particularly stretching our horizons in understanding how Nox2 assembly is enhanced during microgravity in skeletal muscle.

New studies have identified novel inhibitors for proteins recently govern assembly of the Nox2 complex at the cell membrane acid—sphingomyelinase (ASMase) and cyclophilin A. We hypothesize that the following novel countermeasures will protect against nNOSµ translocation and the spaceflight phenotype— (a) the ASMase inhibitor etidronate (Didronel) and (b) cyclophilin A inhibitor TMN-355. We further postulate that Nox2 is causal in ROS-induced suppression of anabolic signaling. The efficacy and specificity of the above countermeasures will be confirmed with gene knockdown experiments. Texas A&M is a rich research environment for NASA research, including the Space Life Science Program. We will use the latest molecular and image analysis tools in the development of highly novel countermeasures against spaceflight sarcopenia during microgravity. Dr. Lawler and Dr. Fluckey's laboratories have continued to be supported by NASA, and are dedicated to finding targeted, antioxidant countermeasures against spaceflight sarcopenia. The ground hindlimb unloading model will be used in short and long-term experiments.

Our research will also directly translate to skeletal muscle wasting in clinical setting on Earth, an important mission of the Space Biology program. For example, hospitalization, particularly in an ICU (intensive care unit) can reduce skeletal muscle by 25%. Cast immobilization can decrease affected muscle mass by 30% as well. In addition, mechanical unloading due to disuse (e.g., bedrest) and illness (e.g., sepsis, chronic obstructive pulmonary disease, chronic heart failure) exacerbates atrophy, weakness, and impedes recovery.

NASA Task Book Space Biology (SB) 2022-2023 bullet points:

- We developed a novel Bioreactor to simulate microgravity, radiation exposure, and over/reloading of skeletal muscle cells. We intend to develop co-cultures.

Kamal KY, Othman M, Lawler JM. Proof of Concept: Developing a novel bioreactor for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain. ASGSR abstract, 2022, Houston, TX.

- We developed a new receptor activator of nuclear factor kappa-B ligand (RANKL) knockdown package with adeno-associated virus serotype 9 (AAV9) gene delivery. RANKL is elevated by microgravity, aging, and Duchenne muscular dystrophy in skeletal muscles.

Othman, Mariam A, Khaled Y. Kamal, Devon Roeming, Samhitha Ramanuja, John M. Lawler. RANKL protein knockdown applying AAV9/shRNA Systemic Drug Delivery to mitigate spaceflight-induced muscle atrophy, ASGSR Abstract 2022, Houston, TX.

Roeming D, Ramanuja S, Kamal KY, Othman M, Lawler JM. AAV9/shRNA Knockdown of RANKL Protein Expression Utilizing Systemic Drug Delivery: Proof of Concept. Student Research Week, Texas A&M University, 2022.

- We used a combination of nutritional interventions (fish oil + curcumin) to mitigate skeletal muscle oxidative stress, atrophy, and fibrosis with microgravity.

- Also, fish oil + pectin against cardiac fibrosis.

Kamal KY, Hord JM, Wu C, Talcott S, Janini Gomes M, Fluckey JF, Ford JF, Nancy D. Turner, Lawler JM. Combination Nutrition Interventions Against Spaceflight Sarcopenia. Human Research Project Meeting. NASA. Galveston, February, 2022.

- Partial loading and high energy particle (HZE) radiation resulted in significant damage and markers of fibrosis in skeletal muscle. This paper has been accepted and is in press.

Wiggs MP, Lee Y, Shimkus KL, O'Reilly CI, Lima F, Macias BR, Shirazi-Fard Y, Greene ES, Hord JM, Braby LA, Carroll CC, Lawler JM, Bloomfield SA, Fluckey JD. Combined effects of heavy ion exposure and simulated lunar gravity on skeletal muscle. Life Sci Space Res. 2023 May;37:39-49.

- Involvement of RANKL, which causes bone loss in spaceflight in sarcopenia and inflammatory cell invasion.

Lawler JM, Kamal K, Othman M, Lee J. Myeloid specific knockout of the ghrelin receptor mitigates aging-linked elevation of RANKL, Ca2+ overload, and sarcopenia. Pending ASGSR abstract submission.

Kamal KY, Lawler JM. Cellular and molecular signaling meet the space environment. International Journal of Molecular Science. 2023 Mar 22;24(6):5955. doi: 10.3390/ijms24065955.

- Assistant Research Assistant Dr. Khaled Kamal was awarded a SHINE scholarship for radiation research.

NASA SHINE (Space Health Impacts for the NASA Experience) Training Program- Virtual Space Radiation Curriculum (# NNJ22ZSA001L). Dr. Khaled Kamal received this award and participated in the first annual SHINE (Space Health Impacts for the NASA Experience) Space Radiation virtual course.

Kim J, M Othman, KY Kamal, JM Lawler. RANKL and Nox2 signaling in Duchenne Muscular Dystrophy models of skeletal muscle. Texas Affiliate – American College of Sports Medicine. Waco, 2023.

Othman M, J Kim, KY Kamal, JM Lawler. Role of Ghrelin Receptor in Sarcopenia: Involvement of Redox Signaling and RANKL. Texas Affiliate – American College of Sports Medicine. Waco, 2023.

Lawler, JM. Redox Regulation of nNOS and Skeletal Muscle Atrophy During Microgravity. Graduate Faculty of Nutrition Seminar, Texas A&M University, 2022.

Lawler JM, Kamal KY, Othman M, Roeming D, Ramanuja S. Redox Regulation of Mechanotransduction in Skeletal Muscle During Spaceflight: Translation to Duchenne Muscular Dystrophy and New Insights, ASGSR abstract, 2022 Houston, TX.

NOTE: Start/end dates changes to 6/1/2019-7/31/2022 (originally 3/1/2019-5/31/2022) per NSSC award documents per F. Hernandez/ARC (Ed., 12/3/2020)

Mechanotransduction is the ability to sense and regulate adaptive responses to increased or decreased loading. New paradigms have emerged from ground analogs of µG that have contributed to a leap of our understanding of mechanotransduction and muscle atrophy. Specifically, the mu-splice variant of neuronal nitric oxide synthase (nNOSµ) was discovered as causal in both muscle hypertrophy with overloading and atrophy with unloading. Our laboratory has found that reactive oxygen species (ROS) directly contribute to both muscle fiber atrophy and fiber-type shift from slow to fast. Pilot Data and cutting-edge research have identified mitochondria, the Nox2 isoform of NADPH oxidase, and upstream angiotensin II receptor 1 (AT1R) as sources of ROS during mechanical unloading. Preliminary Data show that inhibition of Nox2 translocation of nNOSµ away from the sarcolemma, muscle fiber atrophy, and fiber-type shift. However, the upstream mechanisms that regulate Nox2 during µG are poorly understood, impeding progress in space biology and novel countermeasure development. The lack of such knowledge impedes our development in understanding the mechanisms that underlie redox regulation of mechanotransduction in skeletal muscle. This grant application serves as a renewal and extension and Renewal of our research team's NNX13AE45G award, particularly stretching our horizons in understanding how Nox2 assembly is enhanced during microgravity in skeletal muscle.

New studies have identified novel inhibitors for proteins recently govern assembly of the Nox2 complex at the cell membrane acid—sphingomyelinase (ASMase) and cyclophilin A. We hypothesize that the following novel countermeasures will protect against nNOSµ translocation and the spaceflight phenotype— (a) the ASMase inhibitor etidronate (Didronel) and (b) cyclophilin A inhibitor TMN-355. We further postulate that Nox2 is causal in ROS-induced suppression of anabolic signaling. The efficacy and specificity of the above countermeasures will be confirmed with gene knockdown experiments. Texas A&M is a rich research environment for NASA research, including the Space Life Science Program. We will use the latest molecular and image analysis tools in the development of highly novel countermeasures against spaceflight sarcopenia during microgravity. Dr. Lawler and Dr. Fluckey's laboratories have continued to be supported by NASA, and are dedicated to finding targeted, antioxidant countermeasures against spaceflight sarcopenia. The ground hindlimb unloading model will be used in short and long-term experiments.

Our research will also directly translate to skeletal muscle wasting in clinical setting on Earth, an important mission of the Space Biology program. For example, hospitalization, particularly in an ICU (intensive care unit) can reduce skeletal muscle by 25%. Cast immobilization can decrease affected muscle mass by 30% as well. In addition, mechanical unloading due to disuse (e.g., bedrest) and illness (e.g., sepsis, chronic obstructive pulmonary disease, chronic heart failure) exacerbates atrophy, weakness, and impedes recovery.

• We found that microgravity depresses protective proteins, i.e., heat shock protein 70 (HSP70), sirtuin-1 (SIRT1), nuclear factor erythroid 2–related factor 2 (Nrf2), and Manganese – specific superoxide dismutase (MnSOD) were depressed with 7 days of unloading, allowing excessive oxidative stress that led to muscle atrophy (Lawler 2021, 2022).

• Mitigation of elevated assembly of the NADPH oxidase-2 complex by using the peptide inhibition of gp91ds-tat significantly protected against unloading-induced reduction of HSP70 in skeletal muscle.

• Nox2 was found to be causal in skeletal muscle atrophy associated with microgravity.

• Nox2 peptide inhibition protected against loss of nNOS at the muscle cell membrane.

• Angiotensin II receptor 1 (AT1R) inhibition protected against Nox2 elevation, oxidative stress, and loss of sarcolemmal nNOS (Hord 2021).

• Demonstrated development and proof of concept of a new Bioreactor to simulate overloading and microgravity in skeletal muscle cells.

• Translation to Duchenne muscular dystrophy (DMD): Nox2 was elevated in both Duchenne muscular dystrophy and spaceflight, leading to skeletal muscle myopathy.

• Mice with DMD had significant muscle damage and elevated levels of pro-inflammatory proteins RANKL, Cyclophilin A, and Acid Sphingomyelinase compared with normal, healthy mice.

• Inhibition of Nox2 protected against upregulation of RANKL in skeletal muscle of dystrophic mice.

• Stress protective proteins SIRT1, HSP70, Nrf2, and MnSOD were depressed in mice with Duchenne muscular dystrophy, similar to spaceflight -- thus contributing to oxidative stress and myopathy.

• Our research team also found elevated levels of mRNA transcripts (gene expression) for Cyclophilin A and Acid Sphingomyelinase in dystrophic muscle -- golden retriever muscular dystrophy (GRMD) muscle.

• We demonstrated proof of concept of systemic AAV9 delivery to skeletal muscle and heart. This new technology development and application holds great promise for SAFE gene therapy against inflammation, oxidase stress, and impaired stress proteins.

Mechanotransduction is the ability to sense and regulate adaptive responses to increased or decreased loading. New paradigms have emerged from ground analogs of µG that have contributed to a leap of our understanding of mechanotransduction and muscle atrophy. Specifically, the mu-splice variant of neuronal nitric oxide synthase (nNOSµ) was discovered as causal in both muscle hypertrophy with overloading and atrophy with unloading. Our laboratory has found that reactive oxygen species (ROS) directly contribute to both muscle fiber atrophy and fiber-type shift from slow to fast. Pilot Data and cutting-edge research have identified mitochondria, the Nox2 isoform of NADPH oxidase, and upstream angiotensin II receptor 1 (AT1R) as sources of ROS during mechanical unloading. Preliminary Data show that inhibition of Nox2 translocation of nNOSµ away from the sarcolemma, muscle fiber atrophy, and fiber-type shift. However, the upstream mechanisms that regulate Nox2 during µG are poorly understood, impeding progress in space biology and novel countermeasure development. The lack of such knowledge impedes our development in understanding the mechanisms that underlie redox regulation of mechanotransduction in skeletal muscle. This grant application serves as a renewal and extension and Renewal of our research team's NNX13AE45G award, particularly stretching our horizons in understanding how Nox2 assembly is enhanced during microgravity in skeletal muscle.

New studies have identified novel inhibitors for proteins recently govern assembly of the Nox2 complex at the cell membrane acid—sphingomyelinase (ASMase) and cyclophilin A. We hypothesize that the following novel countermeasures will protect against nNOSµ translocation and the spaceflight phenotype— (a) the ASMase inhibitor etidronate (Didronel) and (b) cyclophilin A inhibitor TMN-355. We further postulate that Nox2 is causal in ROS-induced suppression of anabolic signaling. The efficacy and specificity of the above countermeasures will be confirmed with gene knockdown experiments. Texas A&M is a rich research environment for NASA research, including the Space Life Science Program. We will use the latest molecular and image analysis tools in the development of highly novel countermeasures against spaceflight sarcopenia during microgravity. Dr. Lawler and Dr. Fluckey's laboratories have continued to be supported by NASA, and are dedicated to finding targeted, antioxidant countermeasures against spaceflight sarcopenia. The ground hindlimb unloading model will be used in short and long-term experiments.

Our research will also directly translate to skeletal muscle wasting in clinical setting on Earth, an important mission of the Space Biology program. For example, hospitalization, particularly in an ICU (intensive care unit) can reduce skeletal muscle by 25%. Cast immobilization can decrease affected muscle mass by 30% as well. In addition, mechanical unloading due to disuse (e.g., bedrest) and illness (e.g., sepsis, chronic obstructive pulmonary disease, chronic heart failure) exacerbates atrophy, weakness, and impedes recovery.

Lawler JM, Hord JM, Ryan P, Holly D, Janini Gomes M, Rodriguez D, Guzzoni V, Garcia-Villatoro E, Green C, Lee Y, Little S, Garcia M, Hill L, Brooks MC, Lawler MS, Keys N, Mohajeri A, Kamal KY. "Nox2 inhibition regulates stress response and mitigates skeletal muscle fiber atrophy during simulated microgravity." Int J Mol Sci. 2021 Mar;22(6):3252. https://doi.org/10.3390/ijms22063252

Hord JM, Garcia MM, Farris KR, Guzzoni V, Lee Y, Lawler MS, Lawler JM. "Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading." Physiol Rep. 2021 Jan;9(1):e14606. https://pubmed.ncbi.nlm.nih.gov/33400850

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity:

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight, bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of then angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded + 7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox+ staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Nox2 Signaling and Muscle Fiber Remodeling are Attenuated by Losartan Administration during Skeletal Muscle Unloading:

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into 4 groups: ambulatory control (CON), ambulatory + losartan (40 mg/kg/day) (CONL), 7-days of tail-traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading-induced loss of muscle fiber cross-sectional area (CSA) and fiber-type shift. Losartan mitigated unloading-induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

ABSTRACTS

Lawler JM, Hord JM, P. Ryan, D. Holly, Guzzoni, V, Janini Gomes M, D. Rodriguez, Garcia-Villatoro E Green C, Lee Y. Little S, Garcia M, Hill L, Brooks M-C, Lawler MS, Keys N, Mohajeri, A, Kamal, K. "Effect of Nox-2 Inhibition on Skeletal Muscle Atrophy and Stress Response Signaling During Mechanical Unloading" American Society for Gravitational and Space Biology & Radiation meeting. Virtual Meeting, 2020.

Mohajeri A, Kamal Khaled, and Lawler JM. "The Evaluation of Nox2 Role in Microgravity–Induced Skeletal Muscle Atrophy," International Journal of Exercise Science: Conference Proceedings: Vol. 2: Iss. 13, Article 76. 2021.

Mohajeri A., Kamal K., & Lawler J. Peptidyl inhibition of Nox2 enhances stress response & mitigates muscle fiber atrophy with simulated microgravity. Experimental Biology, virtual event, April 27–30, 2021.

Kamal K., Mohajeri A., & Lawler J. Stress response proteins & Nox2 signaling in the gastrocnemius muscle of dystrophic mice. Experimental Biology, virtual event), April 27–30, 2021.

Mohajeri A., Kamal K., & Lawler J. The evaluation of Nox2 role in microgravity–induced skeletal muscle atrophy. Texas chapter of ACSM, virtual event, February 25–26, 2021.

Mechanotransduction is the ability to sense and regulate adaptive responses to increased or decreased loading. New paradigms have emerged from ground analogs of µG that have contributed to a leap of our understanding of mechanotransduction and muscle atrophy. Specifically, the mu-splice variant of neuronal nitric oxide synthase (nNOSµ) was discovered as causal in both muscle hypertrophy with overloading and atrophy with unloading. Our laboratory has found that reactive oxygen species (ROS) directly contribute to both muscle fiber atrophy and fiber-type shift from slow to fast. Pilot Data and cutting-edge research have identified mitochondria, the Nox2 isoform of NADPH oxidase, and upstream angiotensin II receptor 1 (AT1R) as sources of ROS during mechanical unloading. Preliminary Data show that inhibition of Nox2 translocation of nNOSµ away from the sarcolemma, muscle fiber atrophy, and fiber-type shift. However, the upstream mechanisms that regulate Nox2 during µG are poorly understood, impeding progress in space biology and novel countermeasure development. The lack of such knowledge impedes our development in understanding the mechanisms that underlie redox regulation of mechanotransduction in skeletal muscle. This grant application serves as a renewal and extension and Renewal of our research team's NNX13AE45G award, particularly stretching our horizons in understanding how Nox2 assembly is enhanced during microgravity in skeletal muscle.

New studies have identified novel inhibitors for proteins recently govern assembly of the Nox2 complex at the cell membrane acid—sphingomyelinase (ASMase) and cyclophilin A. We hypothesize that the following novel countermeasures will protect against nNOSµ translocation and the spaceflight phenotype— (a) the ASMase inhibitor etidronate (Didronel) and (b) cyclophilin A inhibitor TMN-355. We further postulate that Nox2 is causal in ROS-induced suppression of anabolic signaling. The efficacy and specificity of the above countermeasures will be confirmed with gene knockdown experiments. Texas A&M is a rich research environment for NASA research, including the Space Life Science Program. We will use the latest molecular and image analysis tools in the development of highly novel countermeasures against spaceflight sarcopenia during microgravity. Dr. Lawler and Dr. Fluckey's laboratories have continued to be supported by NASA, and are dedicated to finding targeted, antioxidant countermeasures against spaceflight sarcopenia. The ground hindlimb unloading model will be used in short and long-term experiments.

Our research will also directly translate to skeletal muscle wasting in clinical setting on Earth, an important mission of the Space Biology program. For example, hospitalization, particularly in an ICU (intensive care unit) can reduce skeletal muscle by 25%. Cast immobilization can decrease affected muscle mass by 30% as well. In addition, mechanical unloading due to disuse (e.g., bedrest) and illness (e.g., sepsis, chronic obstructive pulmonary disease, chronic heart failure) exacerbates atrophy, weakness, and impedes recovery.

During the past year, our research team has made significant progress on mechanosensing through a pathway involving proteins normally affected by angiotensin II, an enzyme involved in regulating blood pressure, inflammation, fibrosis, and recently COVID-19 pathology. In one of our studies we discovered that proteins involved in this pathway sense changes in mechanical loading in skeletal muscle, independent of angiotensin II. Simulated spaceflight triggered this pathway to produce oxidative stress and cause migration of an enzyme that produces nitric oxide. By using specific chemical inhibitors to short circuit this chain of events, we were able to substantially mitigate skeletal muscle atrophy and migration of slow to fast-twitch fibers.

The unloading of spaceflight, casting, and bedrest causes a significant amount of skeletal muscle atrophy and weakness. Chronic high-fat diet in young children and adults causes insufficient skeletal muscle mass and strength, related to a pre-diabetic state. We tested whether a calorie-rich high-fat diet in mice that had just reached puberty would cause atrophy, disruption of the ability of muscles to take up glucose, and disruption in the ability to produce nitric oxide, important in skeletal muscle atrophy and hypertrophy. Contrary to our hypothesis, we found that the capacity of skeletal muscles to take up glucose and produce nitric oxide was maintained or enhanced with a high fat diet, despite markers of insulin resistance, and was linked to profound skeletal muscle hypertrophy. Our data indicate that pubescent mammals can store protein and fat in skeletal muscle when given a calorie dense diet, possibly in preparation for their “growth spurt.” Our exciting findings may shed more light as to the underlying biological and biochemical reasons that people are susceptible to Type II diabetes as middle-age adults.

Mechanotransduction is the ability to sense and regulate adaptive responses to increased or decreased loading. New paradigms have emerged from ground analogs of µG that have contributed to a leap of our understanding of mechanotransduction and muscle atrophy. Specifically, the mu-splice variant of neuronal nitric oxide synthase (nNOSµ) was discovered as causal in both muscle hypertrophy with overloading and atrophy with unloading. Our laboratory has found that reactive oxygen species (ROS) directly contribute to both muscle fiber atrophy and fiber-type shift from slow to fast. Pilot Data and cutting-edge research have identified mitochondria, the Nox2 isoform of NADPH oxidase, and upstream angiotensin II receptor 1 (AT1R) as sources of ROS during mechanical unloading. Preliminary Data show that inhibition of Nox2 translocation of nNOSµ away from the sarcolemma, muscle fiber atrophy, and fiber-type shift. However, the upstream mechanisms that regulate Nox2 during µG are poorly understood, impeding progress in space biology and novel countermeasure development. The lack of such knowledge impedes our development in understanding the mechanisms that underlie redox regulation of mechanotransduction in skeletal muscle. This grant application serves as a renewal and extension and Renewal of our research team's NNX13AE45G award, particularly stretching our horizons in understanding how Nox2 assembly is enhanced during microgravity in skeletal muscle.

New studies have identified novel inhibitors for proteins recently govern assembly of the Nox2 complex at the cell membrane acid—sphingomyelinase (ASMase) and cyclophilin A. We hypothesize that the following novel countermeasures will protect against nNOSµ translocation and the spaceflight phenotype— (a) the ASMase inhibitor etidronate (Didronel) and (b) cyclophilin A inhibitor TMN-355. We further postulate that Nox2 is causal in ROS-induced suppression of anabolic signaling. The efficacy and specificity of the above countermeasures will be confirmed with gene knockdown experiments. Texas A&M is a rich research environment for NASA research, including the Space Life Science Program. We will use the latest molecular and image analysis tools in the development of highly novel countermeasures against spaceflight sarcopenia during microgravity. Dr. Lawler and Dr. Fluckey's laboratories have continued to be supported by NASA, and are dedicated to finding targeted, antioxidant countermeasures against spaceflight sarcopenia. The ground hindlimb unloading model will be used in short and long-term experiments.

Our research will also directly translate to skeletal muscle wasting in clinical setting on Earth, an important mission of the Space Biology program. For example, hospitalization, particularly in an ICU (intensive care unit) can reduce skeletal muscle by 25%. Cast immobilization can decrease affected muscle mass by 30% as well. In addition, mechanical unloading due to disuse (e.g., bedrest) and illness (e.g., sepsis, chronic obstructive pulmonary disease, chronic heart failure) exacerbates atrophy, weakness, and impedes recovery.