NOTE: End date changed to 6/30/2022 per Space Radiation element and NSSC information (Ed., 7/6/21)

NOTE: End date changed to 6/30/2021 per NSSC information (Ed., 11/6/20)

NOTE: End date changed to 6/30/2020 per NSSC information (Ed., 9/26/19)

New studies and Preliminary Data from our laboratory suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress and cardiovascular disease, increases protective heat shock proteins. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.

Spaceflight appears to elicit morphological (e.g., collagen fibrosis) and functional changes of the heart that could impede performance, lead to fatigue and orthostatic hypotension upon re-entry to a gravitational environment, and increase the risk of heart and vascular disease. In addition, disuse that occurs with microgravity may predispose the heart to arrhythmias (Moffitt et al. 2013). Radiation enhances apoptosis and loss of myocytes as well as accumulation of collagenous tissue, or “fibrosis.” The average age of a typical astronaut has increased to over 50 years of age, and progressive age increases oxidative stress in the heart (Kwak et al. 2006).

Spaceflight imposes a unique set of stressors on astronauts as a result of the loss of gravity during spaceflight, while tissues are bombarded by galactic and solar radiation. The cardiovascular system is adversely affected by the disuse and fluid shifts that occur with spaceflight. However, there is a growing concern that cardiovascular disease may be substantially elevated during spaceflight. Indeed, increasing evidence indicates that radiation exposure causes damage and fibrosis in the heart and vasculature. Weightlessness and space radiation during long-duration spaceflight, particularly in outer space between the Earth and the moon or Mars, increases inflammation and oxidative stress in the heart, vasculature, and muscles, joints, and bones. The body is exposed to X-ray and heavy ion (HZE) radiation that damages cell components such as mitochondria, nuclei, and the cell membrane through increase release of oxidants (i.e., oxidative stress). Astronaut age has increased into the 50s, and thus has the risk of damage, cell death, and fibrotic connective tissue, as published by our laboratory and other scientists. However, the contribution by which space radiation (X-Ray, HZE) contributes to secondary oxidative stress and fibrosis in the heart is poorly understood. We argue that space radiation accelerated the aging process in heart and skeletal muscle, increased fibrosis, and contributed to cell death.

New publications and pilot data from our laboratories indicate that a potential source of oxidative stress in the heart during radiation is called the renin-angiotensin system (RAS). RAS can trigger the assembly of NADPH oxidase-2 (Nox2), a cluster of proteins that produces oxidative stress. We recently found that Nox-2 is elevated in a ground spaceflight analog in skeletal muscle and heart, and contributed directly to changes in muscle cell size, shape, and infiltration of connective tissue. Antioxidant compounds and nutritional supplement choices that are based upon causal studies may have alleviated changes in the heart, vasculature, and skeletal muscle with spaceflight. For example, fish oil reduces oxidative stress, and thus increases protective heat shock proteins, and reduces cardiovascular disease. For example, a combination of fish oil and curcumin recently prevented muscle fiber atrophy and increased protective stress response proteins in a spaceflight analog. Dietary pectin ingestion reduces oxidative stress and cell death. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. We propose to determine the effects of a combination of fish oil and pectin on heavy ion-induced radiation in the heart.

The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We are conducting a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University focuses on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice were sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Effectiveness of fish oil + pectin in reducing heart damage and fibrosis is being tested. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. A protein called p53 also contributes to cell death, fibrosis of the heart, and muscle atrophy. We will thus also query archived cardiac samples irradiated at the Brookhaven National Laboratory. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involving combined X-Ray and HZE radiation, where mice with a single p53 allele deletion were irradiated.

References

Moffitt JA, Henry MK, Welliver KC, Jepson AJ, Garnett ER. (2013) Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression. Am J Physiol Regul Integr Comp Physiol. 304(5):R362-73.

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006) Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

Published Articles 1. Wesolowski LT, Simons JL, et al., Lawler JM, Kamal KY, White-Springer SH. The Impact of SRT2104 on Skeletal Muscle Mitochondrial Function, Redox Biology, and Loss of Muscle Mass in Hindlimb Unloaded Rats. Int. J. Mol. Sci. 2023, 24(13).

2. Kamal KY, Othman MA, Kim JH, Fluckey JF, Lawler JM. A novel bioreactor for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain: Proof of Concept. npj Microgravity. 2024 Jun 11;10(1):62. doi: 10.1038/s41526-023-00320-

3. Christopher E. Mason, James Green, Konstantinos I. Adamopoulos, Evan E. Afshin, Jordan J. Baechle, Mathias Basner, Susan M. Bailey, Josef Borg, Joseph Borg, Jared T. Broddrick, Marissa Burke, Andrés Caicedo, Verónica Castañeda, Subhamoy Chatterjee, George Church, Sylvain V. Costes, Rajeev I. Desai, Raja Dhir, Juan Esteban Diaz, Sofia M. Etlin, David Furman, J. Sebastian Garcia-Medina, Stefania Giacomello, Anjali Gupta, Amira Hassanin, Nadia Houerbi, Iris Irby, Peter Jirak, Christopher W. Jones, Khaled Kamal, Brian D. Kangas, JangKeun Kim, Joo-Hyun Kim, Ashley Kleinman, John M. Lawler, Jessica A. Lee, Charles L. Limoli, J. Tyson McDonald, Jakub Mieczkowski, Masafumi Muratani, Deena Najjar, Mariam A. Othman, Eliah G. Overbey, Vera Paar, Jiwoon Park, Amber M. Paul, Adrian Perdyan, Jacqueline Proszynski, Robert J. Reynolds, April E. Ronca, Kate Rubins, Lindsay A. Rutter, Krista A. Ryon, Lauren M. Sanders, Patricia Savi Glowe, Ryan T. Scott, Bader Shirah, Karolina Sienkiewicz, Keith Siew, Corey A Theriot, Braden T Tierney, Kasthuri Venkateswaran, Jeremy Wain Hirschberg, Stephen B. Walsh, Daniel A. Winer, Min Yu, Luis Zea, Jaime Mateus, Afshin Beheshti. The Second Space Age and Precision Aerospace Medicine. Nature, 2024 Aug;632(8027):995-1008. doi: 10.1038/s41586-024-07586-8.

4. Kamal KY, Lawler JM. Cellular and Molecular Signaling Meet the Space Environment. Int. J. Mol. Sci. 2023, 24, 5955.

5. Lawler, JM, RE Botchlett, SL Woo, H Xu, H Li, JM Hord, JD Fluckey, K. Kamal, and C Wu. Metformin-sensitive Effects of a High Fat Diet on Skeletal Muscle Morphology and Sarcolemmal Protein Signaling in Young Mice. Connective Tissue Research. Published online Mar 7, 2025. 1–15. https://doi.org/10.1080/03008207.2025.2471853

6. Lawler JM, Hord JM, Ryan P, Holly D, Janini Gomes M, Rodriguez D, Guzzoni V, Garcia- Villatoro E, Green, C, Lee Y, Little S, 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. International Journal of Molecular Science. 2021 Mar 23;22(6):3252. doi: 10.3390/ijms22063252.

7. Hord, JM, MM Garcia, JM Kuczmarski, KR Farris, V Guzzoni, Y Lee, MS Lawler, JM Lawler. Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading. Physiological Reports. 2021 Jan;9(1):e14606. doi: 10.14814/phy2.14606.

8. Wu CS, Q. Wei, DM Kim, M Balderas, G Wu, J Lawler, S Safe, S Devaraj, Z Chen, and Y. Sun. Protective effects of ghrelin on fasting-induced muscle atrophy in aging mice. Journal of Gerontology. 2020 Mar 9;75(4):621-630.

Submitted ‘Under Review’ 1. Othman M., Kim JH, Kamal KY, Lawler JM. Exercise-induced HSP72 Mitigation of Insulin Resistance in Skeletal Muscle. ‘In preparation.’ 2. Lawler JM, Kim JH, Kamal KY, Othman M. Ford JF, Turner ND, Lawler JM. Pectin and Fish Oil Supplementation Mitigate HZE Radiation-induced Damage, Immune Cell Invasion, and Fibrosis of the Mouse Heart. In Preparation, 2025.

Conferences Abstracts 1. Kim JH, Kamal KY, Othman MA, Ford JR, Turner ND, Lawler JM, editors. Fish Oil and Pectin Supplementation Attenuates Heart Damage, RANKL, and Inflammation 28 Days After Exposure to Space Radiation. Human Research Project Meeting; 2024; Galveston, TX, USA. 2. Kendra J, Golpasandi S, Othman M; et al, Micronized Biocompatible Ceramic Promotes Muscle IL-6 Release in Disuse, 2024 TACSM 3. Kim JH, Kamal KY, Othman MA, Ford JR, Turner ND, Lawler JM. Fish Oil and Pectin Supplementation Attenuates Heart Damage, RANKL, and Inflammation 28 Days After Exposure to Space Radiation. Human Research Project Meeting, Galveston, TX 2024. 4. Othman MA, Kamal KY, Kim JH, Raugh R, Weslowski LT, Simons JL, Semanchik PL, Kendra JA, Morton AB, Janini Gomes M, White-Springer-S, Lawler JM. Sirtuin-1 Agonist SRT2014 Mitigates Unloading-induced Skeletal Muscle Atrophy and Inflammation. Human Research Project Meeting, Galveston, TX 2024. 5. Lawler JM, Kim JH, Kamal KY, Othman MA, Kendra S, Ford JR, Sun Y, Raugh R, Fluckey JD. We Have Ignition: Redox Regulation of Mechanotransduction with Spaceflight and Translation into Myopathies on Earth. Human Research Project Meeting, Galveston, TX 2024. 6. Kamal KY, Othman M, et al, Lawler JM, The Sirtuin-1 Agonist SRT2104 Mitigates Unloading-Induced Elevation of Inflammatory Markers, Mitochondrial Dysfunction, and Skeletal Muscle Atrophy, 2023 ASGSR Annual meeting, 14-18 November 2023, Washington, D.C. (Oral) 7. Othman M*, Kim JH*, Kamal KY, Lawler JM, Myeloid Cell-Specific Knockout of the Ghrelin Receptor: Effect on Inflammation, RANKL, and Sarcopenia, 2023 ASGSR Annual meeting, 14-18 November 2023, Washington, D.C. (Poster) 8. Kim JH*, Kamal KY, Othman M, Ford JF, Turner ND, Lawler JM, Consumption of Fish Oil and Pectin Attenuates Heart Damage from the Risk of Space Radiation, 2023 ASGSR Annual meeting, 14-18 November 2023, Washington, D.C. (Poster) 9. Lawler JM, Kim JH, Othman M, Kamal KY, Front to the Future: Translating Spaceflight Research to Muscle Myopathies on Earth, 2023 ASGSR Annual meeting, 14-18 November 2023, Washington, D.C. (Poster) 10. Kim JH, Othman M, Kamal KY, Lawler JM, The RANKL and Nox2 Signaling in the Duchenne Muscular Dystrophy models of skeletal muscle, 2023, TACSM, February 23-24, 2023, Waco, TX. 11. Othman M, Kim JH, Kamal KY, Lawler JM, Role of Ghrelin receptor in sarcopenia: involvement of Redox regulation and RANKL, 2023, TACSM, February 23-24, 2023, Waco, TX. 12. Kamal KY, Othman M, Lawler JM, Proof of Concept: Developing a novel bioreactor for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain, 2022 ASGSR Annual meeting, 9-12 November 2022, Houston, TX. 13. Roeming D, Ramanuja S, Othman M, Kim JH, Kamal KY, Lawler JM. AAV9/shRNA Knockdown of Cyclophilin A Utilizing Systemic Drug Delivery to Mitigate the Effects of Microgravity, 2022 ASGSR Annual meeting, 9-12 November 2022, Houston, TX. 14. Othman M, Kamal KY, Roeming D, Ramanuja S, Kim JH, Lawler JM. RANKL protein knockdown using AAV9/shRNA Systemic Drug Delivery to mitigate spaceflight-induced muscle atrophy, 2022 ASGSR Annual meeting, 9-12 November 2022, Houston, TX. 15. 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, 2022 ASGSR Annual meeting, 9-12 November 2022, Houston, TX. 16. Lawler JM., Kamal KY, Spaceflight Sarcopenia: Solutions in Redȯx Biology, TACSM 22 meeting, Waco, TX, March 2022. 17. Kamal KY, Hord JM, Wu C, Talcott S., Gomes MJ, Fluckey JF, Ford JF, Turner ND, and Lawler JM. Combination Nutrition Interventions Against Spaceflight Sarcopenia. NASA HRP-IWS 2022.

NOTE: End date changed to 6/30/2022 per Space Radiation element and NSSC information (Ed., 7/6/21)

NOTE: End date changed to 6/30/2021 per NSSC information (Ed., 11/6/20)

NOTE: End date changed to 6/30/2020 per NSSC information (Ed., 9/26/19)

New studies and Preliminary Data from our laboratory suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress and cardiovascular disease, increases protective heat shock proteins. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.

Spaceflight appears to elicit morphological (e.g., collagen fibrosis) and functional changes of the heart that could impede performance, lead to fatigue and orthostatic hypotension upon re-entry to a gravitational environment, and increase the risk of heart and vascular disease. In addition, disuse that occurs with microgravity may predispose the heart to arrhythmias (Moffitt et al. 2013). Radiation enhances apoptosis and loss of myocytes as well as accumulation of collagenous tissue, or “fibrosis.” The average age of a typical astronaut has increased to over 50 years of age, and progressive age increases oxidative stress in the heart (Kwak et al. 2006).

Spaceflight imposes a unique set of stressors on astronauts as a result of the loss of gravity during spaceflight, while tissues are bombarded by galactic and solar radiation. The cardiovascular system is adversely affected by the disuse and fluid shifts that occur with spaceflight. However, there is a growing concern that cardiovascular disease may be substantially elevated during spaceflight. Indeed, increasing evidence indicates that radiation exposure causes damage and fibrosis in the heart and vasculature. Weightlessness and space radiation during long-duration spaceflight, particularly in outer space between the Earth and the moon or Mars, increases inflammation and oxidative stress in the heart, vasculature, and muscles, joints, and bones. The body is exposed to X-ray and heavy ion (HZE) radiation that damages cell components such as mitochondria, nuclei, and the cell membrane through increase release of oxidants (i.e., oxidative stress). Astronaut age has increased into the 50s, and thus has the risk of damage, cell death, and fibrotic connective tissue, as published by our laboratory and other scientists. However, the contribution by which space radiation (X-Ray, HZE) contributes to secondary oxidative stress and fibrosis in the heart is poorly understood. We argue that space radiation accelerated the aging process in heart and skeletal muscle, increased fibrosis, and contributed to cell death.

New publications and pilot data from our laboratories indicate that a potential source of oxidative stress in the heart during radiation is called the renin-angiotensin system (RAS). RAS can trigger the assembly of NADPH oxidase-2 (Nox2), a cluster of proteins that produces oxidative stress. We recently found that Nox-2 is elevated in a ground spaceflight analog in skeletal muscle and heart, and contributed directly to changes in muscle cell size, shape, and infiltration of connective tissue. Antioxidant compounds and nutritional supplement choices that are based upon causal studies may have alleviated changes in the heart, vasculature, and skeletal muscle with spaceflight. For example, fish oil reduces oxidative stress, and thus increases protective heat shock proteins, and reduces cardiovascular disease. For example, a combination of fish oil and curcumin recently prevented muscle fiber atrophy and increased protective stress response proteins in a spaceflight analog. Dietary pectin ingestion reduces oxidative stress and cell death. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. We propose to determine the effects of a combination of fish oil and pectin on heavy ion-induced radiation in the heart.

The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We are conducting a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University focuses on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice were sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Effectiveness of fish oil + pectin in reducing heart damage and fibrosis is being tested. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. A protein called p53 also contributes to cell death, fibrosis of the heart, and muscle atrophy. We will thus also query archived cardiac samples irradiated at the Brookhaven National Laboratory. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involving combined X-Ray and HZE radiation, where mice with a single p53 allele deletion were irradiated.

References

Moffitt JA, Henry MK, Welliver KC, Jepson AJ, Garnett ER. (2013) Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression. Am J Physiol Regul Integr Comp Physiol. 304(5):R362-73.

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006) Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

- 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 RANKL knockdown package with AAV9 gene delivery. RANKL is elevated by microgravity, aging, 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 combination nutritional interventions (fish oil + curcumin to mitigate skeletal muscle oxidative stress, atrophy, 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.

Lawler JM, Holly H, Ryan P, Janini Gomes M, Brooks M-C, Jennifer Cardona J, Nancy D. Turner ND, Ford JR. Effect of Fish Oil and Pectin on Fibrosis and Inflammation in Mouse Hearts Exposed to HZE Radiation. 2018 Integrative Physiology of Exercise, San Diego, CA, 2019.

- Partial loading and 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.

- 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.

NOTE: End date changed to 6/30/2021 per NSSC information (Ed., 11/6/20)

NOTE: End date changed to 6/30/2020 per NSSC information (Ed., 9/26/19)

New studies and Preliminary Data from our laboratory suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress and cardiovascular disease, increases protective heat shock proteins. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.

Spaceflight appears to elicit morphological (e.g., collagen fibrosis) and functional changes of the heart that could impede performance, lead to fatigue and orthostatic hypotension upon re-entry to a gravitational environment, and increase the risk of heart and vascular disease. In addition, disuse that occurs with microgravity may predispose the heart to arrhythmias (Moffitt et al. 2013). Radiation enhances apoptosis and loss of myocytes as well as accumulation of collagenous tissue, or “fibrosis.” The average age of a typical astronaut has increased to over 50 years of age, and progressive age increases oxidative stress in the heart (Kwak et al. 2006).

Spaceflight imposes a unique set of stressors on astronauts as a result of the loss of gravity during spaceflight, while tissues are bombarded by galactic and solar radiation. The cardiovascular system is adversely affected by the disuse and fluid shifts that occur with spaceflight. However, there is a growing concern that cardiovascular disease may be substantially elevated during spaceflight. Indeed, increasing evidence indicates that radiation exposure causes damage and fibrosis in the heart and vasculature. Weightlessness and space radiation during long-duration spaceflight, particularly in outer space between the Earth and the moon or Mars, increases inflammation and oxidative stress in the heart, vasculature, and muscles, joints, and bones. The body is exposed to X-ray and heavy ion (HZE) radiation that damages cell components such as mitochondria, nuclei, and the cell membrane through increase release of oxidants (i.e., oxidative stress). Astronaut age has increased into the 50s, and thus has the risk of damage, cell death, and fibrotic connective tissue, as published by our laboratory and other scientists. However, the contribution by which space radiation (X-Ray, HZE) contributes to secondary oxidative stress and fibrosis in the heart is poorly understood. We argue that space radiation accelerated the aging process in heart and skeletal muscle, increased fibrosis, and contributed to cell death.

New publications and pilot data from our laboratories indicate that a potential source of oxidative stress in the heart during radiation is called the renin-angiotensin system (RAS). RAS can trigger the assembly of NADPH oxidase-2 (Nox2), a cluster of proteins that produces oxidative stress. We recently found that Nox-2 is elevated in a ground spaceflight analog in skeletal muscle and heart, and contributed directly to changes in muscle cell size, shape, and infiltration of connective tissue. Antioxidant compounds and nutritional supplement choices that are based upon causal studies may have alleviated changes in the heart, vasculature, and skeletal muscle with spaceflight. For example, fish oil reduces oxidative stress, and thus increases protective heat shock proteins, and reduces cardiovascular disease. For example, a combination of fish oil and curcumin recently prevented muscle fiber atrophy and increased protective stress response proteins in a spaceflight analog. Dietary pectin ingestion reduces oxidative stress and cell death. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. We propose to determine the effects of a combination of fish oil and pectin on heavy ion-induced radiation in the heart.

The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We are conducting a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University focuses on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice were sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Effectiveness of fish oil + pectin in reducing heart damage and fibrosis is being tested. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. A protein called p53 also contributes to cell death, fibrosis of the heart, and muscle atrophy. We will thus also query archived cardiac samples irradiated at the Brookhaven National Laboratory. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involving combined X-Ray and HZE radiation, where mice with a single p53 allele deletion were irradiated.

References

Moffitt JA, Henry MK, Welliver KC, Jepson AJ, Garnett ER. (2013) Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression. Am J Physiol Regul Integr Comp Physiol. 304(5):R362-73.

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006) Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

We have been collaborating with Dr. Nancy Turner’s laboratory at Texas A&M on two sets of radiation studies. Low LET gamma-ray exposure of mice will be conducted at Texas A&M overseen by Dr. John Ford’s laboratory in Nuclear Engineering. Acute 0.5 Gy exposures will be used. Sacrifice of the mice and extraction of the heart and skeletal muscle will occur at 12 hours, 4 weeks, and 8 weeks following radiation treatment. Astronaut age (40-42 weeks) mice were split into (n=6/group) controls, non-irradiated with pectin + fish oil, X-ray irradiation, X-ray irradiation with pectin + fish oil. Pectin (6% by weight) and fish oil (15% by weight) will be provided in the diet as previously described (Cho et al. 2012). We have completed collecting samples and are currently analyzing irradiated hearts.

In addition, the impact of HZE (28Si, 48Ti) radiation on cardiac markers of pro-oxidant and pro-fibrotic signaling is currently being examined. We postulate that intervention with fish oil and pectin will abrogate radiation-induced oxidative stress and fibrosis in the heart.

Outcome Markers for Specific Aim 1 include (1) left ventricle damage, (2) oxidative stress, (3) Nox2 subunits (gp91phox, p67phox), (4) pro-fibrotic signaling (TGF-ß, p-smad 2/3, FSP-1), fibrosis (collagen I), and (5) nuclear damage. We expect that pectin and fish oil will reduce oxidative stress and boost stress response proteins (grp94, HSP70) concomitant with protection against pro-inflammatory signaling (TGF-ß, MMP-9, FSP-1, NF-kappaB). These data would demonstrate a reduction in fibrosis linked to antioxidant and anti-inflammatory properties of fish oil combined with pectin under low LET radiation in the mouse heart. Initial results indicated increased markers of oxidative stress and fibrosis in the irradiated heart.

We are currently testing the ability of fish oil + pectin on inflammatory cell invasion in the heart exposed to HZE radiation. CD45 antigen was used as a marker for all leukocytes. Antibodies for CD68+ detected monocytes and macrophage. CD11c+ was our marker for all inflammatory macrophages, with iNOS a tag for M1 macrophages, while co-localization with CD206 for M2 macrophages. CD8+ antigen was used as a tag for T-cells.

Data shows a trend towards higher infection of cardiac myocytes after radiation than controls. A smaller trend was noted for CD45+ cells invading cells with pectin + fish oil. Similar results were found for CD68-positive staining. These data are consistent with the hypothesis that radiation-induced damage and pathology in the heart is linked to chronic inflammation. Secondly, a nutritional intervention, fish oil + curcumin partially mitigated the effects of radiation on cardiac inflammation. Therefore, nutraceutical and potential antioxidant approaches may attenuate cardiac damage, inflammation, and fibrosis in astronauts, thus improving long-term health outcomes against cardiovascular disease.

Reference

Cho, Y., N.D. Turner, L.A. Davidson, R.S. Chapkin, R.J. Carroll, and J.R. Lupton. 2012. A chemoprotective fish oil/pectin diet enhances apoptosis via Bcl-2 promoter methylation in rat azoxymethane-induced carcinomas. Experimental Biology & Medicine 237:1387-1393.

NOTE: End date changed to 6/30/2020 per NSSC information (Ed., 9/26/19)

New studies and Preliminary Data from our laboratory suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress and cardiovascular disease, increases protective heat shock proteins. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.

Spaceflight appears to elicit morphological (e.g., collagen fibrosis) and functional changes of the heart that could impede performance, lead to fatigue and orthostatic hypotension upon re-entry to a gravitational environment, and increase the risk of heart and vascular disease. In addition, disuse that occurs with microgravity may predispose the heart to arrhythmias (Moffitt et al. 2013). Radiation enhances apoptosis and loss of myocytes as well as accumulation of collagenous tissue, or “fibrosis.” The average age of a typical astronaut has increased to over 50 years of age, and progressive age increases oxidative stress in the heart (Kwak et al. 2006).

Spaceflight imposes a unique set of stressors on astronauts as a result of the loss of gravity during spaceflight, while tissues are bombarded by galactic and solar radiation. The cardiovascular system is adversely affected by the disuse and fluid shifts that occur with spaceflight. However, there is a growing concern that cardiovascular disease may be substantially elevated during spaceflight. Indeed, increasing evidence indicates that radiation exposure causes damage and fibrosis in the heart and vasculature. Weightlessness and space radiation during long-duration spaceflight, particularly in outer space between the Earth and the moon or Mars, increases inflammation and oxidative stress in the heart, vasculature, and muscles, joints, and bones. The body is exposed to X-ray and heavy ion (HZE) radiation that damages cell components such as mitochondria, nuclei, and the cell membrane through increase release of oxidants (i.e., oxidative stress). Astronaut age has increased into the 50s, and thus has the risk of damage, cell death, and fibrotic connective tissue, as published by our laboratory and other scientists. However, the contribution by which space radiation (X-Ray, HZE) contributes to secondary oxidative stress and fibrosis in the heart is poorly understood. We argue that space radiation accelerated the aging process in heart and skeletal muscle, increased fibrosis, and contributed to cell death.

New publications and pilot data from our laboratories indicate that a potential source of oxidative stress in the heart during radiation is called the renin-angiotensin system (RAS). RAS can trigger the assembly of NADPH oxidase-2 (Nox2), a cluster of proteins that produces oxidative stress. We recently found that Nox-2 is elevated in a ground spaceflight analog in skeletal muscle and heart, and contributed directly to changes in muscle cell size, shape, and infiltration of connective tissue. Antioxidant compounds and nutritional supplement choices that are based upon causal studies may have alleviated changes in the heart, vasculature, and skeletal muscle with spaceflight. For example, fish oil reduces oxidative stress, and thus increases protective heat shock proteins, and reduces cardiovascular disease. For example, a combination of fish oil and curcumin recently prevented muscle fiber atrophy and increased protective stress response proteins in a spaceflight analog. Dietary pectin ingestion reduces oxidative stress and cell death. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. We propose to determine the effects of a combination of fish oil and pectin on heavy ion-induced radiation in the heart.

The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We are conducting a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University focuses on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice were sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Effectiveness of fish oil + pectin in reducing heart damage and fibrosis is being tested. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. A protein called p53 also contributes to cell death, fibrosis of the heart, and muscle atrophy. We will thus also query archived cardiac samples irradiated at the Brookhaven National Laboratory. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involving combined X-Ray and HZE radiation, where mice with a single p53 allele deletion were irradiated.

References

Moffitt JA, Henry MK, Welliver KC, Jepson AJ, Garnett ER. (2013) Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression. Am J Physiol Regul Integr Comp Physiol. 304(5):R362-73.

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006) Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

[Ed. note June 2021: compiled from PI's annual report from June 2020; received November 2020]

New studies and Preliminary Data from our laboratory suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress and cardiovascular disease, increases protective heat shock proteins. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.

Spaceflight appears to elicit morphological (e.g., collagen fibrosis) and functional changes of the heart that could impede performance, lead to fatigue and orthostatic hypotension upon re-entry to a gravitational environment, and increase the risk of heart and vascular disease. In addition, disuse that occurs with microgravity may predispose the heart to arrhythmias (Moffitt et al. 2013). Radiation enhances apoptosis and loss of myocytes as well as accumulation of collagenous tissue, or “fibrosis.” The average age of a typical astronaut has increased to over 50 years of age, and progressive age increases oxidative stress in the heart (Kwak et al. 2006).

Spaceflight imposes a unique set of stressors on astronauts as a result of the loss of gravity during spaceflight, while tissues are bombarded by galactic and solar radiation. The cardiovascular system is adversely affected by the disuse and fluid shifts that occur with spaceflight. However, there is a growing concern that cardiovascular disease may be substantially elevated during spaceflight. Indeed, increasing evidence indicates that radiation exposure causes damage and fibrosis in the heart and vasculature. Weightlessness and space radiation during long-duration spaceflight, particularly in outer space between the Earth and the moon or Mars, increases inflammation and oxidative stress in the heart, vasculature, and muscles, joints, and bones. The body is exposed to X-ray and heavy ion (HZE) radiation that damages cell components such as mitochondria, nuclei, and the cell membrane through increase release of oxidants (i.e., oxidative stress). Astronaut age has increased into the 50s, and thus has the risk of damage, cell death, and fibrotic connective tissue, as published by our laboratory and other scientists. However, the contribution by which space radiation (X-Ray, HZE) contributes to secondary oxidative stress and fibrosis in the heart is poorly understood. We argue that space radiation accelerated the aging process in heart and skeletal muscle, increased fibrosis, and contributed to cell death.

New publications and pilot data from our laboratories indicate that a potential source of oxidative stress in the heart during radiation is called the renin-angiotensin system (RAS). RAS can trigger the assembly of NADPH oxidase-2 (Nox2), a cluster of proteins that produces oxidative stress. We recently found that Nox-2 is elevated in a ground spaceflight analog in skeletal muscle and heart, and contributed directly to changes in muscle cell size, shape, and infiltration of connective tissue. Antioxidant compounds and nutritional supplement choices that are based upon causal studies may have alleviated changes in the heart, vasculature, and skeletal muscle with spaceflight. For example, fish oil reduces oxidative stress, and thus increases protective heat shock proteins, and reduces cardiovascular disease. For example, a combination of fish oil and curcumin recently prevented muscle fiber atrophy and increased protective stress response proteins in a spaceflight analog. Dietary pectin ingestion reduces oxidative stress and cell death. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. We propose to determine the effects of a combination of fish oil and pectin on heavy ion-induced radiation in the heart.

The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We are conducting a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University focuses on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice were sacrificed and tissues extracted 12 hrs, 4 wks, or 8 wks after radiation exposure. Effectiveness of fish oil + pectin in reducing heart damage and fibrosis is being tested. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. A protein called p53 also contributes to cell death, fibrosis of the heart, and muscle atrophy. We will thus also query archived cardiac samples irradiated at the Brookhaven National Laboratory. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involving combined X-Ray and HZE radiation, where mice with a single p53 allele deletion were irradiated.

References

Moffitt JA, Henry MK, Welliver KC, Jepson AJ, Garnett ER. (2013) Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression. Am J Physiol Regul Integr Comp Physiol. 304(5):R362-73.

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006) Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

Effect of Exercise on Nox2-induced Signaling and Fibrosis in the Aging Heart: We have conducted a series of studies that have investigated the ability of exercise training to ameliorate oxidative stress and cardiac remodeling in the astronaut age hearts. NADPH oxidase-2 (Nox2) activity and subunit protein levels were elevated with aging in the heart. Indeed, we found that 12 weeks of exercise training significantly attenuated the age-related increase in Nox2 activity. Exercise training also attenuated age-associated increases in Nox2 subunit localization and protein abundance for gp91phox and p47phox. These data clearly indicate that exercise can ameliorate Nox2 and oxidative stress levels in the astronaut age heart.

Given Nox2 contributes to fibrosis of the heart, and exercise training reduces Nox2, we tested the hypothesis that exercise training reduces connective tissue and collagen I levels in the aging heart. Per our hypothesis, exercise training significantly ameliorated fibrotic tissue in the aging heart. Collagen was markedly elevated with age, and suppressed with daily exercise. Similarly, alpha-smooth muscle actin (alpha-SMA) staining, a marker of myofibroblasts commonly seen in fibrotic tissue, was significantly enhanced by aging. However, exercise training provided a significant inhibitory effect on alpha-SMA. Transforming growth factor-ß (TGF-ß) is a cytokine usually involved in activation of myofibroblasts and collagen production. As predicted, exercise training not only decreased TGF-ß localization in the aging heart, but also significantly reduced TGF-ß abundance when compared with the age-matched sedentary group. Furthermore, smad2/3 phosphorylation (Ser423/425) increased 2.4-fold with aging and was attenuated by regular exercise.

Upregulation of Nox2 in the aging heart has been linked to renin-angiotensin II signaling (RAS). We tested the hypothesis that 12 weeks of endurance treadmill training would significantly reduce abundance of angiotensin II receptor I levels (AT1R). Indeed, while old rats expressed higher AT1R protein levels in the heart, exercise training provided substantial protection against age-induced upregulation of AT1R. These data suggest that exercise-induced protection against fibrosis and remodeling of the aging heart is accomplished by downregulation a pathway involving AT1R, Nox2, and TGF-ß.

The Role of Nox2 in Fibrosis in the mdx Mouse Heart: Similar to aging, Duchenne muscular dystrophy (DMD) causes profound and progressive fibrosis of the heart, impaired function, and the risk of heart failure. Nox2 is upregulated in mdx mice, a model for DMD (Whitehead et al. 2010). We have observed that apocynin, a Nox2 inhibitor and antioxidant, significantly reduces fibrosis in the mdx mouse heart. In addition, apocynin also significantly reduced TGF-ß levels in the dystrophic heart. We also determined that Nox2 effects were linked to oxidative stress by utilizing EUK-134, a mimetic of the antioxidant enzymes superoxide dismutase and catalase. Indeed, EUK-134 reduces muscle damage, inflammation and weakness in the diaphragm muscle of mdx mice (Kim and Lawler 2012).

Effect of Microgravity (Hindlimb Unloading) on Nox2 in the Heart: Oxidative stress is increased with spaceflight and ground analogs for µG in heart and skeletal muscle. We found that Nox2 is upregulated in the heart with the hindlimb unloading µG model in the heart. The specific Nox2 inhibitory peptide gp91ds-tat mitigated oxidative stress in the heart.

Oxidative Stress Is Causal in Age-Related Fibrosis and Apoptosis of the Aging Heart: Our laboratory consistently observed reduction in oxidative stress and cardiac remodeling (e.g., fibrosis, apoptosis) of the aging rat heart (Kwak et al. 2006, 2011, 2015). In addition to downregulation of an AT1R – Nox2 pathway, oxidative stress may also be suppressed with exercise by upregulating antioxidant enzymes and other stress response proteins. Indeed, we found that while aging reduced MnSOD activity in the left ventricle, exercise training increase MnSOD activity in both the old and young hearts. We found that overexpression of MnSOD partially protected against oxidative stress and elevation of collagen I of old mice. Furthermore, the transgenic MnSOD mouse also significantly reduced TGF-ß in the old heart. Overexpression of MnSOD also reduced apoptosis in the aging heart, as indicated by TUNEL+ staining (Kwak 2015). The old transgenic mouse displayed lower TUNEL+ staining vs. old wild-type mice. These data are supportive of the notion that oxidative stress drives apoptosis and remodeling in the aging heart.

Alterations in Redox State in the Heart with Radiation:

Pilot data reveal that redox balance, as assessed by the ratio of reduced to oxidized glutathione (GSH/GSSG) is significant higher with fish oil and pectin in the diet than controls, when exposed to gamma radiation. These data suggest that fish oil and pectin enhance antioxidant protection when tissues are irradiated. Increased antioxidant protection was evident both in the early and delayed responses to radiation.

Fish Oil & Curcumin Intervention in Ground-Based Microgravity: Dietary Fish Oil (FO) supplementation reduces unloading-induced changes in muscle morphology reduced oxidative stress. Similarly, curcumin, a polyphenol that is found in turmeric, inhibits inflammatory signaling and mitigates skeletal muscle atrophy.

We proposed that a treatment combining 5% fish oil and 1% curcumin (FOC) in the diet would be synergistic in reducing unloading-induced skeletal muscle atrophy because they target independent pathways. Hindlimb unloading (HU) causes muscle atrophy. We hypothesized that FOC would alleviate the translocation (or untethering) of membrane-associated proteins (ex. nNOS) away from the membrane. To test our hypothesis, C57BI/6 mice were divided in three groups: control group=CON, hindlimb unloading group=HU fed with control diet; fish oil/curcumin+HU group = FOC+HU (n=6) fed specialized diets 10 days prior to HU; continued specialized diets during 7 days of HU period.

Soleus muscle fiber cross sectional area (CSA) and membrane-associated proteins (nNOS, dysferlin, caveolin-3) expression/localization were quantified. We found a marked increase in fiber CSA and soleus mass in the FOC+HU group compared with HU, which suggests that FOC prevents the decrease of CSA during HU. We found no difference in dysferlin localization between groups. We also found that nNOS and caveolin-3 membrane expression were higher in the FOC group comparing with the HU group.

We hypothesized that fish oil, rich in omega-3-fatty acids, combined with polyphenol curcumin protect anabolic (Akt pathway) signaling and heat shock proteins in the rat soleus muscle, concomitant with protection of morphology, is a synergistic countermeasure. FOC mitigated the unloading-induced decrease in CSA and prevented the fiber-type shift normally. FOC also rescued anabolic signaling (Akt phosphorylation, p70S6K phosphorylation) and increased the abundance of HSP70. Therefore, we concluded that the combination of fish oil and curcumin prevents muscle atrophy in concert with the ability to boost heat shock proteins and anabolic signaling in an unloaded state.

HZE Irradiating in the Heart: In a pilot study, C57BL/6 mice were irradiated with a dosage of 0.50 Gy using a 60Co gamma source. Hearts were harvested and frozen in isopentane (-160°C) cooled in liquid nitrogen 8 weeks after radiation exposure. Hematoxylin and eosin (H&E) stains revealed that radiation exposure appeared to increased extracellular matrix space, in a heterogeneous pattern. There were more nuclei visualized in the extracellular matrix space as well. Follow-up experiments with TGF-ß, a cytokine activator of fibroblast, myo-fibroblasts, and fibrosis.

Intervention with Fish Oil, Pectin: TGF-ß positive staining was elevated in the irradiated hearts. These data emphasize the importance of finding effective and safe countermeasures to mitigate radiation-induced damage and fibrosis in the heart of astronauts to address inflight and post-flight cardiovascular risk. We expect Nox2 inhibition to reduce radiation-induced damage. We have been collaborating with Dr. Nancy Turner’s laboratory at Texas A&M on two sets of radiation studies. Low LET (linear energy transfer) gamma-ray exposure of mice will be conducted at Texas A&M overseen by Dr. John Ford’s laboratory in Nuclear Engineering. Acute 0.5 Gy exposures will be used. Sacrifice of the mice and extraction of the heart and skeletal muscle will occur at 12 hours, 4 weeks, and 8 weeks following radiation treatment. Astronaut age (40-42 weeks) mice were split into controls, non-irradiated with pectin + fish oil, X-ray irradiation, X-ray irradiation with pectin + fish oil, Pectin (6% by weight) and fish oil (15% by weight). We have completed collecting samples and are currently analyzing irradiated hearts. In addition, the impact of HZE (28Si, 48Ti) radiation on cardiac markers of pro-oxidant and pro-fibrotic signaling is currently being examined. We postulate that intervention with fish oil and pectin will abrogate radiation-induced oxidative stress and fibrosis in the heart.

Outcome Markers for Specific Aim 1 include (1) left ventricle damage, (2) oxidative stress, (3) Nox2 subunits (gp91phox, p67phox), (4) pro-fibrotic signaling (TGF-ß, p-smad 2/3, FSP-1), fibrosis (collagen I), and (5) nuclear damage. We expect that pectin and fish oil will reduce oxidative stress and boost stress response proteins (grp94, HSP70) concomitant with protection against pro-inflammatory signaling (TGF-ß, MMP-9, FSP-1, NF-kappaB). These data would demonstrate a reduction in fibrosis linked to antioxidant and anti-inflammatory properties of fish oil combined with pectin under low LET radiation in the mouse heart. Initial results demonstrate that HZE radiation increases oxidative stress, MP-9 levels, TGF-ß, and invasion of inflammatory cells (Macrophages, monocytes). Fish oil + pectin is having a small positive effect.

Collaboration with Aging -- HZE radiation Project with Dr. Melinda Sheffield-Moore: We are commencing a new tissue sharing project with Dr. Melinda Sheffield-Moore. C57/BL6 mice were irradiated at 1 Gy, 0.5 Gy, and 0.25 Gy with HZE. Mice were allowed a latent period of 60 days and 2 years post-radiation. We will be testing hearts from irradiated or control mice for the following outcomes: 1) oxidative stress, 2) Nox2 subunits, 3) pro-inflammatory signaling, 4) invasion of inflammatory cells, 5) profibrotic signaling.

We expect these data to reveal the long-term effects of exposure to HZE radiation, particularly important in understanding the increased risk factors for cardiac fibrosis and cardiovascular disease following long-term spaceflight, especially in deep space.

References

Whitehead NP, Yeung EW, Froehner SC, Allen DG. (2010). Skeletal muscle NADPH oxidase is increased and triggers stretch-induced damage in the mdx mouse. PLoS One 5, e15354

Kim JH, Lawler JM. (2012). Amplification of pro-inflammatory phenotype, damage, and weakness by oxidative stress in the diaphragm of mdx mice. Free Radic Biol Med. 52: 1597-1606

Kwak, H.-B., W. Song,, and J.M. Lawler. (2006). Exercise-training ameliorates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the aging rat heart. FASEB J.

Kwak, H.B., J.-H. Kim, K. Joshi, A. Yeh, D.A. Martinez, and J.M. Lawler. (2011). Exercise training reduces fibrosis and metalloproteinase dysregulation in the aging rat heart. FASEB Journal. 25: 1106-1117

Kwak, H.-B., Y. Lee, and J.-H. Kim, H. Van Remmen, A.G. Richardson and JM Lawler. (2015). MnSOD overexpression reduces fibrosis and pro-apoptotic signaling in the aging mouse heart. J Gerontology: Biological Sciences. 70: 533-44

Cho, Y., N.D. Turner, L.A. Davidson, R.S. Chapkin, R.J. Carroll, and J.R. Lupton. (2012). A chemoprotective fish oil/pectin diet enhances apoptosis via Bcl-2 promoter methylation in rat azoxymethane-induced carcinomas. Experimental Biology & Medicine 237:1387-1393

New studies and Preliminary Data from our laboratory point suggest that the renin-angiotensin signaling (RAS) are significant sources of oxidative stress, and thus pro-fibrotic signaling in the heart. Upregulation of RAS in the aging heart upregulates the Nox2 isoform of NADPH oxidase. We have also recently found that Nox2 also contributes to oxidative stress and atrophy during ground-based spaceflight of skeletal muscle. Thus secondary and amplified oxidative stress may damage nuclei and stimulate pro-fibrotic signaling, including TGF-ß, smad2/3 phosphorylation, and collagen I accumulation. The current RFA research emphasis in Space Biology Tissue Sharing provides an opportunity to promote sharing of samples with ongoing and archived studies. We will propose a series of studies with X-Ray, HZE, and X-Ray + HZE radiation. Collaboration with Dr. Nancy Turner’s laboratory at Texas A&M University will focus on two sets of radiation studies. The first cohort of studies will use X-Ray radiation (0.5 Gy) to induce damage and oxidative stress. Mouse (astronaut age) heart samples will be taken 12 hours, or 4 or 8 weeks after exposure. In the second set of experiments, mice will be exposed to 28Si and 48Ti (0.5 Gy). Mice will be sacrificed and tissues extracted 12 hrs, 4 wks or 8 wks after radiation exposure. Efficacy of an intervention of fish oil + pectin in reducing cardiac fibrotic signaling will be tested. Fish oil reduces oxidative stress, increases protective heat shock proteins, and cardiovascular disease. Our Preliminary Data reveal that fish oil + curcumin also reduces muscle atrophy. Dietary pectin ingestion reduces oxidative stress and apoptosis. Pectin and fish oil have also reduced radiation-induced tissue fibrosis in the kidney and liver, respectively. However, the effects on the irradiated heart are unknown. p53 contributes to apoptosis, cardiac fibrosis, and muscle atrophy. We will also query archived cardiac samples irradiated at the Brookhaven National Laboratory involved in combined X-Ray and 56Fe radiation, where mice with a single p53 allele deletion and wild-types were irradiated.