NOTE: Extended to 5/31/2014 per NSSC information (Ed., 12/4/13)

Our central hypothesis is that low-dose space radiation-induced DNA damage repair is inefficient in BM-derived EPC and this may lead to increased mutagenesis with subsequent long-term loss of endothelial function of BM-derived EPCs. This may then pose significant degenerative CV risk on physiologic homeostasis in the aging heart and on the regeneration and neovascularization processes in the heart under pathologic conditions such as acute myocardial infarction (AMI).

Comparisons will be made between two types of low-dose radiation -- proton versus iron and single versus fractionated exposures. In short-term in vitro studies (minutes, hours, and up to 30 days after exposure) will evaluate in ex-vivo expanded EPCs and cardiomyocytes DNA damage and repair as well as radiation-induced bystander effects (irradiated cells emit signals to "un-hit" cells), angiogenic gene expression in EPCs. In short-term in vivo studies in the heart we will evaluate acute damage (inflammation and cell death), DNA damage, and repair kinetics. In our long-term studies (3, 6, 9, and 12 months after exposure) we will evaluate oxidative stress and antioxidant defense in BM-derived EPCs, alterations in several EPC endothelial functions, number of circulating peripheral blood EPCs, and cardiomyocyte contractility. In the last part of our studies we will assess CV risks as a result of low-dose radiation plus aging and CV risks under pathological condition -- radiation plus aging plus adverse CV event (i.e., AMI). Here we will evaluate post-AMI survival, alterations in cardiac physiology (echocardiography), infarct size, inflammation, cardiac regeneration, neovascularization, and mobilization of EPCs from BM.

Our studies will address two high-priority research topics of this specific solicitation and NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

In addition, our studies will also provide novel insights into the alterations in cardiac function processes on the molecular and cardiac physiology levels that may allow for estimation of degenerative risks to cardiovascular system in the civilian population exposed to full body low-dose radiation due to accidental exposures (Chernobyl, Fukushima, etc.) and cancer patients undergoing very frequent imaging tests (i.e., full body Computer Tomography, PET Scans).

Our studies will address two high-priority research topics for NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

SUB-PROJECT 1:

Title: DELAYED CARDIOMYOCYTE RESPONSE TO TOTAL BODY HEAVY ION PARTICLE RADIATION EXPOSURE – IDENTIFICATION OF REGULATORY GENE NETWORKS. Background: Understanding the effects of low dose ionizing radiation (IR) for ions of high atomic number (Z) and energy (HZE) on cardiovascular (CV) tissue is important for characterizing the risk of human space exploration. Cardiomyocytes (CM) are the basic contractile cells within the heart, whose contractile function directly influences the pathogenesis of heart disease. Understanding of complex processes that take place in CMs after insults caused by IR, such as proton and HZE particles, is paramount to our understanding of CV system function during and after exploration-type space missions.

Methodology: Adult (8-9 months old) male C57Bl/6NT mice were exposed to single full-body IR of proton (90 cGy, 1 GeV) or 56Fe, iron particles (15 cGy, 1 GeV/nucleon); 0.15 Gy was delivered using 1 GeV/nucleon iron ions (linear energy transfer-LET ~151 keV per µm) or 0.9 Gy at 1 GeV energy protons (LET ~0.22 keV per µm). Primary left ventricular (LV) CMs were isolated using standard preparation protocol that includes cannulation of the aorta and collagenase digestion followed by a Ca2+ gradient selection, yielding >90% of Ca2+ tolerant CMs. We looked at molecular responses using transcriptome profiling in isolated LV murine CMs to 1H- and 56Fe-IR at 1, 3, 7, 14, and 28 days post-IR using Ingenuity IPA and NIH DAVID programs.

Main Findings: Unsupervised clustering analysis of gene expression segregated samples according to the IR response, and time after exposure with iron IR showing the greatest level of gene modulation. Little differential transcript modulation was seen in response to whole body proton IR. There were two notable types of upstream regulators – cytokines and transcriptional regulators. Individual transcription factors were inferred to be active at 1, 3, 7, 14, and 28 days after exposure. The activation of several developmental transcription factors, such as TBX5, GATA-4, MEF2C two weeks after 56Fe-IR, strongly suggest initiation of cardio-protective and regeneration responses in 56Fe-IR hearts. To identify time independent changes in biological pathways, we also analyzed differences between iron and proton arrays. The top 5 significant pathways identified were Parkinson’s disease, Alzheimer’s, oxidative phosphorylation, cardiac muscle contraction, and Huntington’s disease, followed by hypertrophic cardiomyopathy and dilated cardiomyopathy. These top 5 significant pathways shared the majority of transcripts that were involved in mitochondrial and oxidative phosphorylation function illustrating the inter-relationship between the same oxidative phosphorylation genes that play a role in neurodegenerative and cardiovascular disorders/diseases.

Summary: These data suggest that the molecular response to 15 cGy, 1 GeV/n of iron is unique and shows long-lasting gene expression in cardiomyocytes, up to 28 days after exposure. In addition, proteins involved in signal transduction and transcriptional activation via DNA binding play a role in the response to HZE particles. Our study may have implications for NASA’s efforts to develop more accurate heart risk estimates for astronaut safety via identification of specific to HZE IR molecular markers as well as for patients receiving conventional and particle radiotherapy.

SUB-PROJECT 2:

Title: DIFFERENT SEQUENCE OF FRACTIONATED PROTON AND SINGLE LOW DOSE IRON RADIATION INDUCE DIVERGENT BIOLOGICAL RESPONSES IN THE HEART.

Background: Astronauts will be exposed to IR composed of a spectrum of low-fluence protons (1H) and high charge and energy (HZE) nuclei iron (i.e., 56Fe). During GCR each cell in an astronaut’s body will be traversed by a 1H about every 3 days and HZE nuclei about every few months. Hence, the traversal sequence of cells with an ion in space could be random. Therefore, a scenario that a cell in human body may be hit first with several 1H particles then with HZE or an HZE then several 1H should be equally probable. The effect of cosmic IR during and after space flights on cardiovascular (CV) system is unknown.

Methodology: We evaluated the effect of low-dose fractioned and sequential IR dose regimens in 8-9 month old C57BL/6NT male mice in the following groups: Group 1- control; Group 2 - 1H 17 cGy once every 2 days for a total of 3 doses (fractionated proton); Group 3 – Fractionated 1H 17 cGy once every 2 days for a total of 3 doses followed by a single dose of 56Fe 15 cGy 2 days after the last dose of proton; Group 4 – a single dose of 56Fe 15 cGy/followed by fractionated 1H 17 cGy once every 2 days for a total of 3 doses 2 days after a single 56Fe-IR. The energy for both ions was 1 GeV. Cardiac function was assessed by echocardiography and hemodynamic measurements. To determine the effect of fractionated/sequential regimens of 1H and 56Fe-IR on recovery after an ischemic event, acute myocardial infarct (AMI) was induced by ligation of left anterior descending coronary artery 1 and 3 months post-IR.

Main Findings: In IR + Aging group, at 1 and 3 months post-IR, fractionated 1H-IR alone or fractionated 1H-IR followed by a single 56Fe-IR did not induce negative effect on post-IR mice survival and cardiac function, and cardiac fibrosis was decreased in mice of these IR groups. However, when a single 56Fe-IR was followed by a fractionated 1H-IR, there were several negative and CV degenerative developments. In this group post-IR survival was decreased by >20% at 1 and 3 months, left ventricular (LV) end diastolic pressure (LVEDP) was increased at 3 months, and LV maximum pressure change (dP/dtmax) was decreased at 1 month; both of which are indicative of negative hemodynamic developments in the heart of the surviving fraction of mice in 56Fe-IR/1H+1H+1H radiation regimen - Group 4.

In IR + Aging + AMI group, fractionated 1H-IR alone or 56Fe-IR followed by fractionated 1H-IR did not have significant negative effect on post-AMI survival and cardiac function at 1 month post-IR. However, when fractionated 1H-IR was followed by a single 56Fe-IR, there were several negative developments. In this group, at 1 month, there was a 24% decrease in post-AMI survival and a substantial increase in post-AMI cardiac fibrosis. These findings are indicative of significant negative effects for post-AMI recovery after 1H+1H+1H/56Fe-IR regimen in the surviving fraction of mice in Group 3.

Summary: Taken together, our findings in mixed ion fractionated/sequential IR groups strongly suggest dramatically different biological responses due to diverse sequence and fractionation of 1H vs. a single 56Fe-IR. These findings emphasize the necessity to determine underlying molecular mechanisms responsible for this significant mix ion fractionation and sequence-dependent divergent responses in the heart during aging and in case of a possible ischemic cardiovascular event.

SUB-PROJECT 3:

Title: PARTICLE RADIATION INDUCED LONG-LASTING CYCLICAL DECREASES IN THE NUMBER OF BONE MARROW PREGENITOR CELLS IS ASSOCIATED WITH UPREGULATION OF SEVERAL PLURIPOTENT STEM CELL MARKERS OVER TEN MONTHS POST-IR. Background: Radiation-induced decreases in the number of bone marrow (BM)-derived endothelial progenitor cell (BM-EPCs) and their lineage precursors which include Early- and Late-Multi-Potent Progenitor cells (E-MPPs and L-MPPs) could contribute to the pathogenesis of ischemic and vascular diseases. We examined the effect of full-body single dose of proton (1H) at 0.5 Gy, 1 GeV, and 0.15 Gy, 1 GeV/nucleon of iron (56Fe) ionizing radiation (IR) on survival of multipotent progenitor cell populations. The survival of E-MPPs and L-MPPs in the BM after particle IR in C57BL/6NT mice were determined at 1, 2, 4, 8, 12, 28, and 40 weeks post-IR.

Methodology: Total BM-derived mononuclear cells were triple-stained with FITC-labeled RAM34 antibody (that consists of CD34, c-kit, and Sca1 antibodies), PE-Cy7-AC133, and PE-hematopoietic lineage cocktail, then sorted by Fluorescence Associated Cell Sorting (FACS) analysis for Early-MPPs and Late-MPPs.

Main Findings: Compared to control mice, 1H-IR increased the number of both E-MPPs (665%) and L-MPPs (203%) by 1 week, whereas 56Fe-IR decreased E-MPP (74%) and L-MPPs (65%) at 1 week post-IR, suggesting an initial stimulation by 1H and “a hefty” damage by 56Fe in the BM milieu. In 56Fe-IR mice, E-MPPs were partially recovered between 4 and 12 weeks but declined again below ~55-70% of control levels between 28-40 weeks. In 1H-IR mice, E-MPPs were close to control levels up to 4 weeks, but declined >50% at 8 and 28 weeks. These long-lasting cyclical effects on the number of BM-derived E-MPPs and L-MPPs suggest the presence of prolonged non-targeted effects in BM milieu. Total RNA isolated from L-MMP cell pellets up to 10 months post-IR to determine the expression of three pluripotent stem cell markers - Sox-2, Nanog and Oct-4. Our targeted gene expression results reveal long-lasting and cyclical (2 and 10 months) upregulation of these Sox-2, Nanog and Oct-4 in Late-MPPs. Without a significant preconceived notion, the decrease in the number of Early- and L-MPPs associated with upregulation of three pluripotent stem cell factors in BM may indicate a significant depletion/loss of stem and progenitor cells in the BM and an activation of endogenous re-programing signaling. The specific mechanism(s) of this phenomenon is not known.

Summary: These long-lasting and cyclical effects of IR on the BM E-MPPs and L-MPPs after a single 1H or 56Fe IR dose suggest the presence of prolonged and non-targeted effects in BM milieu, particularly in cells that were not traversed by IR directly. The function of the surviving fraction of E-MPPs and L-MPPs and, more importantly, their impact on cardiac homeostasis, cardiac repair, and regeneration, remain unknown. These findings warrant inquiry into the mechanistic studies of the stem cell functions such as, pluripotency and self-renewal, in surviving fraction of BM progenitor cell populations. In addition, future longitudinal studies are necessary to determine whether BM progenitor cell populations may be affected in astronauts after deep space missions as well as after low dose terrestrial IR exposure, such as full body CT and PET scans.

SUB-PROJECT 4:

Title: IONIZING PARTICLE RADIATION INDUCE CYCLICAL INCREASE IN BONE MARROW-DERIVED ENDOTHELIAL PROGENITOR CELL APOPTOSIS. Background: Long-lasting radiation (IR)-induced chromosomal instability has been demonstrated in the bone-marrow (BM) after full body IR with either X-rays or neutrons. It has been shown that after space flights, the numbers of myeloid and lymphoid BM-derived stem and progenitor cells are reduced to just one-half of their normal levels. This result suggests that endothelial progenitor cells (EPCs) may be similarly reduced in the bone marrow milieu. During future exploration-type space missions, astronauts will be exposed to space IR composed of a spectrum of low-fluence protons (1H) and high charge and energy (HZE) nuclei (e.g., 56Fe) for extended time. BM-derived EPCs are critical to endothelial cell (EC) maintenance and repair. The data on the effects of low-dose ionizing space-type particle IR on survival and proliferation of BM-EPCs are limited.

Methodology: We studied the effects of low dose proton and 56Fe (iron) IR on BM-EPCs in mice and cultured BM-derived EPCs. Adult (8-9 months old) male C57Bl/6NT mice were exposed to a single full-body IR of proton (90 cGy, 1 GeV) or 56Fe particles (15 cGy, 1 GeV/nucleon). Mononuclear fraction of BM cells (MNCs) were isolated from full-body IR mice at 2, 5, 24 hours and 7, 14, and 28 days. The ex-vivo expanded BM-EPCs were obtained by culturing the BM-MNCs for 72 hours in EPC selective culture media. After 72 hours in culture we assessed apoptosis and proliferation of BM-derived EPCs. In the second part of the study, BM-EPCs were obtained from non-irradiated (non-IR) mice and subjected to IR. After 2, 5, and 24 hours, IR conditioned medium (IR-CM) was collected and transferred to non-IR BM-EPCs to test the non-targeted effects in the naïve non-irradiated BM-EPCs. Twenty four hours after incubation with non-IR, control and IR-CM media, the formation and decay of DNA double strand breaks were assessed by p-H2AX staining. The production and accumulation of inflammatory cytokines was measured by enzyme-linked immunesorbent assay (ELISA).

Main Findings: Our results showed that there was a cyclical (early 2-5h and delayed 28 days) increase in BM-EPC apoptosis and decreased proliferation after a single, full body, low dose 1H- or 56Fe-IR in mice. In both 1H- and 56Fe IR-CM treated naïve BM-EPCs, the number of p-H2AX foci was significantly increased between 2-24 hours after co-culture, which was associated with 2-15-fold increases in the concentration of inflammatory cytokines such as TNF-alpha, IL-1 alpha, IL-1 beta, MCP-1, and MIP-1 alpha, in IR conditioned media.

Summary: Our data indicate that early, within hours, increase in BM-EPC apoptosis may be the effect of direct IR exposure, whereas late increase in apoptosis and decrease in proliferation could be a result of non-targeted effects in the cells that were not traversed by IR directly. Further, identifying the role of specific cytokines responsible for IR-induced non-targeted effects in BM milieu may allow development of mitigating factors to reduced long-term effects of ionizing particle IR.

NOTE: Extended to 5/31/2014 per NSSC information (Ed., 12/4/13)

Our central hypothesis is that low-dose space radiation-induced DNA damage repair is inefficient in BM-derived EPC and this may lead to increased mutagenesis with subsequent long-term loss of endothelial function of BM-derived EPCs. This may then pose significant degenerative CV risk on physiologic homeostasis in the aging heart and on the regeneration and neovascularization processes in the heart under pathologic conditions such as acute myocardial infarction (AMI).

Comparisons will be made between two types of low-dose radiation - proton versus iron and single versus fractionated exposures. In short-term in vitro studies (minutes, hours, and up to 30 days after exposure) will evaluate in ex-vivo expanded EPCs and cardiomyocytes DNA damage and repair as well as radiation-induced bystander effects (irradiated cells emit signals to "un-hit" cells), angiogenic gene expression in EPCs. In short-term in vivo studies in the heart we will evaluate acute damage (inflammation and cell death), DNA damage, and repair kinetics. In our long-term studies (3, 6, 9, and 12 months after exposure) we will evaluate oxidative stress and antioxidant defense in BM-derived EPCs, alterations in several EPC endothelial functions, number of circulating peripheral blood EPCs, and cardiomyocyte contractility. In the last part of our studies we will assess CV risks as a result of low-dose radiation plus aging and CV risks under pathological condition -- radiation plus aging plus adverse CV event (i.e., AMI). Here we will evaluate post-AMI survival, alterations in cardiac physiology (echocardiography), infarct size, inflammation, cardiac regeneration, neovascularization, and mobilization of EPCs from BM.

Our studies will address two high-priority research topics of this specific solicitation and NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

In addition, our studies will also provide novel insights into the alterations in cardiac function processes on the molecular and cardiac physiology levels that may allow for estimation of degenerative risks to cardiovascular system in the civilian population exposed to full body low-dose radiation due to accidental exposures (Chernobyl, Fukushima, etc.) and cancer patients undergoing very frequent imaging tests (i.e., full body Computer Tomography, PET Scans, etc.).

Our studies will address two high-priority research topics for NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

SUB-PROJECT 1. CARDIAC HISTOLOGY (UP TO 3 MONTHS AFTER A SINGLE 1H- AND 56FE-IR). We evaluated the effect of a single low-dose full body exposure to 50 cGy, 1 GeV 1H, and 15 cGy, 1 GeV/n 56Fe IR on the formation and decay of p-H2AX foci, inflammation (CD68 staining), and oxidative DNA damage (8-oxo-deoxy-dGuanosine ELISA) in the hearts of 8-9 months old (at the time of initial IR) C57BL/6NT mice over 28 days post-IR. CD68 staining and 8-oxo-de-oxy dGuanosine ELISA was also performed at 2 and 3 months.

Summary for SUB-PROJECT 1:

• 56Fe IR-induced mean p-H2AX foci are HIGHER IN NON ECS, including CM vs. heart ECs, and the decay of 56Fe IR-induced p-H2AX foci is SLOWER IN CM vs. ECs.

• 56Fe IR-induced inflammatory responses are cyclical and longer-lived in the heart.

• 56Fe-IR-induced oxidative damage responses are longer lasting in the heart.

SUB-PROJECT 2. CARDIOMYOCYTES MICROARRAYS (DAYS 1, 3, 7, 14, AND 28 POST-IRs). Isolation of Left Ventricular (LV) Cardiomyocytes. Primary mouse LV cardiomyocytes (CM) were isolated to examine the temporal response following in vivo exposure to 15 cGy, 1 GeV/n 56Fe ions, or 50 cGy, 1GeV 1H. Early time points were collected 1 or 3 days after IR along with non-IR controls. Three additional isolations were made at 7, 14, or 28 days after IRs.

Summary for SUB-PROJECT 2:

• At 50cGy, 1 GeV/n dose of 1H-IR, gene expression in CM is NOT affected over 28 days.

• At 15 cGy, 1 GeV/n dose of 56Fe-IR, cardiomyocytes exhibit a time dependent change in gene expression that results in an overall increase in the activity of inflammatory, free radical scavenging, and CV development and function genes 7 and 28 days post-IR.

• Two weeks after a single 56Fe-IR, the activation of several developmental transcription factors (TBX5, GATA4, MEF2C) that are required for the maintenance of cardiac homeostasis strongly suggest ACTIVATION OF CARDIO-PROTECTIVE RESPONSES IN 56FE-IR HEARTS, ONLY.

SUB-PROJECT 3. LONGITUDINAL STUDIES, RADIATION + AGING, 1 AND 3 MONTHS POST-IR. We evaluated the effect of a single, low-dose, full body 50 cGy, 1GeV 1H, and 15 cGy, 1GeV/n 56Fe IR in the hearts of 8-9 months old C57BL/6NT mice over 1 and 3 months post-IR. The survival between control, 1H, and 56Fe up to 3 months post-IR was 95-98% (N=100/treatment group), and no statistical difference was observed between control and either of the IR groups up to 3 months. IR-induced alterations in cardiac function were assessed by echocardiography (ECHO) and hemodynamic (HEMO) measurements and activation of signaling pathways by protein analyses.

Summary for SUB-PROJECT 3:

• Low dose, full body, single 56Fe and 1H-IR induced effects on myocardium are of long duration 56Fe-NEGATIVE, 1H-POSITIVE.

• 56FE-IR, BUT NOT 1H-IR, affects CV homeostasis under normal aging conditions.

• The significant NEGATIVE effects on systolic and diastolic heart functions post-56Fe-IR are associated with altered Ca2+ and cardiac contractility signaling, increased cardiac hypertrophy signaling, and decreased p38 MAPK signaling.

• These 56Fe-IR-mediated NEGATIVE CV developments are indicative of INCREASED RISK for cardiac dysfunction during aging, and also AN INCREASED RISK FOR IMPAIRED RESPONSES TO AN ISCHEMIC EVENT, such as heart attack.

SUB-PROJECT 4. LONGITUDINAL RADIATION + AGING + ADVERSE CV EVENT (Acute Myocardial Infarct). We evaluated the effect of a single, low-dose, full body 50 cGy, 1GeV 1H, and 15 cGy, 1GeV/n 56Fe IR in the hearts of 8-9 months old C57BL/6NT mice over 3 months in Radiation + Aging + AMI. IR-induced alterations in cardiac function were assessed by echocardiography (ECHO) and hemodynamic measurements (HEMO). Acute myocardial infarct (AMI) was induced by ligation of the left anterior descending (LAD) coronary artery 1 and 3 months post-IR, and mice were monitored over 28 days post-AMI. Post-surgical mortality (1-2 days following the surgery) was 14±1.5% at 1 month, and 11±6% for 3 months, respectively (p=NS, between all three groups). In remaining mice post-AMI survival after LAD ligation was not significantly different between control, 1H, and 56Fe groups at 1 and 3 months post-IR and up to 28 days post-AMI (100%, 100% and 88±13% survived AMI surgery, respectively, p=NS).

Summary for SUB-PROJECT 4: • A single, low dose, full body 1H-IR 3 months prior to AMI is BENEFICIAL, whereas 56Fe is DELETERIOUS for recovery after AMI.

• Low dose HZE particle, 56Fe-IR, has long-lasting NEGATIVE effect on degenerative CV risks in case of adverse CV event (e.g., heart attack).

• Multiple signaling pathways that regulate survival, proliferation, and angiogenesis ARE INHIBITED IN 56FE-IR and ACTIVATED IN 1H-IR HEARTS 3 MONTHS POST-AMI.

SUB-PROJECT 5. SURVIVAL OF BONE MARROW PROGENITOR CELL POPULATIONS UP TO 10 MONTHS AFTER PROTON AND IRON IR. After a full body, single 50 cGy 1H- and 15 cGy 56Fe-IR (both ions at 1 GeV/n) mice (C57BL/6NT) were sacrificed at 1, 2, 4, 8, 12, 28, and 40 weeks post-IR. BM cells were subjected to density gradient centrifugation to isolate mononuclear cells (MNCs). MNCs were triple-stained with FITC-labeled RAM34 (consists of CD34, c-kit, and Sca1), PE-Cy7-labeled AC133, and PE-labeled hematopoietic Linminus cocktail then sorted for Early-Multi-Potent Progenitor cells (E-MPP) and Late-MPP (L-MMP).

Summary for SUB-PROJECT 5:

• Despite an initial 1H-IR-induced increase in the number of BM multipotent progenitor (MPP) cell populations, both 1H-IR and 56Fe-IR have profound and long-lasting (28-40 weeks) NEGATIVE effects, >50% DECREASES in the number of early- and late-MPPs.

• The function of the surviving fraction of E-MPPs and L-MPPs and implications for the cardiac homeostasis, as well as cardiac repair and regeneration, remains unknown.

FRACTIONATED/SEQUENTIAL 1H AND 56Fe IR STUDIES. During GCR each cell in an astronaut’s body is being traversed by a 1H about every 3 days and HZE nuclei about every few months. Hence, the traversal sequence of cells with an ion in space is random and 99% of the space IR environment consists of 1H and 2He63. Therefore, a scenario that a cell in human body may be hit first with several 1H particles then with HZE or an HZE then several 1H should be equally probable. We present here the first set of preliminary data for fractionated/sequential mix ion IR regimens in the hearts of 8-9 months old C57BL/6NT mice over 1 and 3 months.

SUB-PROJECT 6. FRACTIONATED/SEQUENATIAL RADIATION + AGING (1 AND 3 MOUSE-MONTHS AGING). We evaluated the effect of low-dose fractioned and sequential IR dose regimens in the following groups: Group 1- Control; Group 2 - 1H 17 cGy x 3 every 2 days; Group 3 - 1H 17 cGy x 3 every 2 days/56Fe 15 cGy; Group 4 - 56Fe 15 cGy/1H 17 cGy x 3 every 2 days at the energy of 1 GeV/n, for both ions.

Summary for SUB-PROJECT 6:

• 1H+1H+1H-IR – at the dose of 17 cGy x 3 and the energy of 1 GeV/n and 1H+1H+1H/56Fe-IR - at the dose of 1H 17 cGy x 3 / 56Fe 15 cGy x 1 and an energy of 1 GeV/n for both ions, does NOT reveal negative effects on the heart function.

• 56Fe/1H+1H+1H-IR – at the dose of 56Fe 15 cGy x 1 / 1H 17 cGy x 3 and an energy of 1 GeV/n for both ions, revealed significant INCREASE IN POST-IR MORTALITY, INCREASED CARDIAC FIBROSIS, REDUCED RELAXATION, AND CONTRACTILE FUNCTIONS in the heart of the surviving fraction of mice.

SUB-PROJECT 7. FRACTIONATED/SEQUENATIAL RADIATION + AGING + ACUTE MYOCARDAIL INFARCT (1 AND 3 MOUSE-MONTHS AGING). We evaluated the effect of low-dose fractioned and sequential IR dose regimens on post-AMI recovery after fractionated-sequential 1H and single 56Fe-IR regimens in the following groups: Group 1- Control; Group 2 - 1H 17 cGy x 3 every 2 days; Group 3 - 1H 17 cGy x 3 every 2 days/56Fe 15 cGy; Group 4 - 56Fe 15 cGy/1H 17 cGy x 3 every 2 days at the energy of 1 GeV/n, for both ions.

Summary for SUB-PROJECT 7: Fractionated 1H-IR alone or 56Fe/1H+1H+1H-IR regimens DO NOT have significant negative effect on post-AMI survival and cardiac function at 1 month post-IR. However, when fractionated 1H-IR was followed by a single 56Fe-IR, there are several NEGATIVE developments: (a) there is a 24% DECREASE in post-AMI survival at 1 month; (b) LV EDP in DECREASED at 1 month (altered relaxation function); (c) post-AMI cardiac fibrosis is INCREASED. These findings are indicative of significant negative effects for post-AMI recovery of 1H+1H+1H/56Fe-IR regimen in the surviving fraction of mice in this group.

MAJOR CONCLUSIONS, FRACTIONATED/SEQUENTIAL 1H AND 56FE IR: Our preliminary findings in mix ion IR studies strongly suggest dramatically different biological responses due to diverse sequence and fractionation of 1H vs. single 56Fe-IR.

• In IR + AGING group - 56Fe/1H+1H+1H-IR regimen revealed SIGNIFICANT NEGATIVE effects on the heart during aging, whereas 1H+1H+1H/56Fe-IR and 1H+1H+1H-IR had NO NEGATIVE effect, at least up to 3 months post-IR.

• In IR + AGING + AMI group, in contrary to IR + AGING group, 1H+1H+1H/56Fe-IR regimen presented SIGNIFICANT DEGENERATIVE CV RISK for the recovery of the heart after AMI, whereas both, 56Fe/1H+1H+1H-IR and 1H+1H+1H-IR had NO NEGATIVE effect on AMI recovery at least up to 1 month post-IR.

Our central hypothesis is that low-dose space radiation-induced DNA damage repair is inefficient in BM-derived EPC and this may lead to increased mutagenesis with subsequent long-term loss of endothelial function of BM-derived EPCs. This may then pose significant degenerative CV risk on physiologic homeostasis in the aging heart and on the regeneration and neovascularization processes in the heart under pathologic conditions such as acute myocardial infarction (AMI).

Comparisons will be made between two types of low-dose radiation - proton versus iron and single versus fractionated exposures. In short-term in vitro studies (minutes, hours, and up to 30 days after exposure) will evaluate in ex-vivo expanded EPCs and cardiomyocytes DNA damage and repair as well as radiation-induced bystander effects (irradiated cells emit signals to "un-hit" cells), angiogenic gene expression in EPCs. In short-term in vivo studies in the heart we will evaluate acute damage (inflammation and cell death), DNA damage, and repair kinetics. In our long-term studies (3, 6, 9, and 12 months after exposure) we will evaluate oxidative stress and antioxidant defense in BM-derived EPCs, alterations in several EPC endothelial functions, number of circulating peripheral blood EPCs, and cardiomyocyte contractility. In the last part of our studies we will assess CV risks as a result of low-dose radiation plus aging and CV risks under pathological condition -- radiation plus aging plus adverse CV event (i.e., AMI). Here we will evaluate post-AMI survival, alterations in cardiac physiology (echocardiography), infarct size, inflammation, cardiac regeneration, neovascularization, and mobilization of EPCs from BM.

Our studies will address two high-priority research topics of this specific solicitation and NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

In addition, our studies will also provide novel insights into the alterations in cardiac function processes on the molecular and cardiac physiology levels that may allow for estimation of degenerative risks to cardiovascular system in the civilian population exposed to full body low-dose radiation due to accidental exposures (Chernobyl, Fukushima, etc.) and cancer patients undergoing very frequent imaging tests (i.e., full body Computer Tomography, PET Scans, etc.).

Our studies will address two high-priority research topics for NASA research interests for degenerative risks to the heart - (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

SUB-PROJECT 1: Bioequivalent Low Dose Full Body Proton and Iron Irrradiation Mediate Comparable DNA Damage, Apoptosis, and Proliferation Responses in the Heart and BM-derived EPC.

Radiation-induced chromosomal instability was demonstrated in the bone marrow (BM) for up to 24 months after full body irradiation with either X-rays or neutrons, indicating that chromosomal instability can be initiated and maintained in vivo. However, there is a significant gap in studies to date assessing full body radiation-induced survival and function of BM-derived endothelial progenitor cells (EPCs) and effects of space radiation on the heart. It was shown for myeloid and lymphoid BM-derived stem and progenitor cells that after space flights the numbers of these cells are reduced to just one-half of their normal levels, suggesting that EPCs may be similarly reduced in the normal EPC population. Neither data on BM-derived EPCs survival and proliferation during and after space flights, nor DNA damage responses of EPCs or heart tissue to space radiation, are currently available. A growing body of evidence indicates that in the heart and other organ-tissues vascular homeostasis does not exclusively rely on proliferation of local endothelial cells (ECs) but also involves BM–derived EPCs. Decrease in the total number of BM-derived EPC or their dysfunction could contribute to the pathogenesis of ischemic and/or peripheral vascular diseases, as well as for maintenance of normal vascular homeostasis in the heart.

SUMMARY of sub- project 1:

(A) our ex-vivo BM-derived EPC data suggest that early increase in BM-derived EPC apoptosis may be a direct effect of single low dose proton or Iron radiation, whereas later increase in apoptosis and decrease in proliferation could be a result of delayed non-targeted effects.

(B) our in vivo heart data reveal that the decay of proton or Iron IR-induced p-H2AX foci are significantly slower in cardiac non-EC (may include cardiomyocytes and BM-derived inflammatory cells) vs. EC; and IR-induced inflammatory response are bimodal and long-lived in heart, suggesting a possibility of inflammatory signaling in bystander-type effects and kinetics of H2AX foci induction and decay in the heart tissue.

SUB-PROGECT 2: TNFR2/p75 Signaling Induces Delayed Radiobiological Bystander Responses in BM-derived EPCs: Implications for Development of Countermeasures.

Tumor necrosis factor alpha (TNF) binds two receptors TNFR1/p55 and TNFR2/p75 and activate several signaling cascades. Ionizing radiation (IR) increases tissue levels of TNF. TNF signaling regulates numerous cytokines and chemokines that known to mediate radiation-induced non-targeted effects (NTE), a phenomenon where cells that are not directly “hit” by IR exhibit IR effects as a result of signals received from nearby or distant IR cells. Little is known about the role of p55 or p75 in regulating NTE in bone marrow (BM)-derived endothelial progenitor cells (EPCs). It is well-known that aging is a risk factor for coronary and peripheral artery disease. We have previously reported significant age-associated decrease in the expression of p55 and p75 in human and mouse EPCs. We hypothesized that inhibition of TNF signaling either via p55 or p75 may alter TNF-mediated inflammatory response increasing tissue levels of cytokines/chemokines that may induce NTE.

SUMMARY of Sub-project 2:

(A) Our finding indicate that altered TNF signaling inhibits early non-targeted effects (NTEs) (hours) in BM-derived EPCs. Compared to WT, delayed (5 days) NTE were increased in naïve gamma irradiation conditioned growth media-treated TNF receptor 1 (p55) and 2 (p75) EPCs, suggesting significant role of TNF-TNFR1 and TNFR2 signaling in mediating delayed NTEs, possibly, via activation of NFkB and other stress response transcription factors.

(B) Based on gene expression profiles, prediction of transcription factor activation or inhibition our data revealed a time-dependent pattern of activation of stress response transcription factors at 1hr their disappearance at 5hrs, return at day 1, disappearance at day 3 and return back again at day 5. This “oscillating” pattern of stress response, DNA damage repair and cell cycle arrest transcription factors activation with subsequent increase in gene expression in pathways downstream of DNA damage repair, cell cycle and inflammatory cytokine genes indicates a significant role of TNF-TNFR1 and TNFR2 signaling in DNA damage responses in BM-derived EPCs. We conclude that TNF ligand-receptor axis regulates NTEs in naïve EPCs and suggest that restoring TNF signaling balance could represent a mitigating measure for prevention of delayed NTEs in tissues adjacent or distant from primary irradiation target.

SUB-PROJECT 3: Transcriptional Profiling of Cardiac Cells Reveal an Immense Complexity of Gene Expression Over One Month after a Full Body 0.15 Gy 56Fe but Not 0.9 Gy Proton Radiation.

Cardiomyocytes are the basic contractile cells within the heart, whose contractile function directly influences the pathogenesis of heart disease and the development of myocardial failure. Understanding of complex processes that take place in cardiomyocytes after insults caused by exposure to ionizing radiation, such as proton and high charge and high energy (HZE) particles, is paramount to our understanding of cardiovascular system function during and after exploration-type space missions. We performed full genome transcriptional microarray analyses using primary, ventricular cardiac muscle cells (cardiomyocytes-CM) from control and full body proton or iron irradiated mice. We isolated CM using standard preparation protocol that includes cannulation of the aorta and collagenase digestion followed by a Ca2+ gradient selection.

SUMMARY of sub-project 3:

(A) The analysis of proton-irradiated samples at False Discovery Rate (FDR) of p<0.05 did not reveal any changes in RNA transcription. When, FDR for the same samples was decreased (p<0.1), there were only 21 genes that exhibited an appreciable increase/decrease in RNA transcription, thus eliminating the need for further bioinformatics analysis of proton irradiated samples.

(B) Very stringent analysis of Iron IR samples after correction for FDR (p<0.05) and 2 or more fold change in RNA transcription using Genome Studio, IPA, Gather software revealed 51 genes with a greater than or less than 2-fold-change in RNA transcription with biphasic time course in comparison to controls. This changes included up-regulation at day 7, complete down-regulation by day 14 and a second significant up-regulation by day 28 post IR.

(C) Less stringent analysis unadjusted p<0.05, FDR p<0.2 and ±1.5-fold change revealed 878 gene list with a clear down-regulation of gene expression on days 1 and 3 compared to control, while an increase was observed on days 7 and 28 relative to control. There was a striking relationship to inflammatory genes seen just by looking at the top 10 up-regulated genes on day 28 ranging 8.0-fold to 15-fold increases.

(D) Functional pathways analysis – Ingenuity software: Functional analysis of the 878 gene list showed a time dependent switch from decreased free radical scavenging on day 3 to an increased activity on day 7. The most striking biological functions related from this data are the time dependent increase in immune/inflammatory categories on days 7 and 28.

(E) Prediction of transcription factor activation/inhibition analysis – Ingenuity software: In line with the above function analysis, prediction of the activation or inhibition of transcription factors based on downstream gene expression in the 878 gene list again shows factors related to immunity and inflammation. Our findings indicate an immense complexity of RNA transcription, regulation of biological pathways, included, but not limited to inflammation, DNA damage/repair, free radical scavenging and immune trafficking post 56Fe irradiation, but NOT bio-equivalent dose of proton.

SUB-PROJECT 4: Full Body Single Dose 0.5 Gy Proton is Beneficial Whereas Single 0.15Gy 56Fe Dose is Deleterious for Acute Myocardial Infarct recovery up to 3 month post-IR.

Previous epidemiologic data in radiotherapy patients (breast, head and neck cancer), non-occupational exposure (Life Span Study), occupational exposure (radiologists/technologists, radon-exposed miner, nuclear workers) for radiation-induced circulatory diseases demonstrate that cardiovascular (CV) morbidity may occur within months or years, and CV mortality may occur within decades, after initial radiation exposure. The effect of cosmic radiation during and after space flights on CV system is unknown. The majority of space flight-associated risks identified for the CV system to date were determined shortly after International Space Station (ISS) flight missions that include: serious cardiac dysrhythmias, compromised orthostatic CV response, and manifestation of previously asymptomatic CV disease. These symptoms were determined to be a consequence of adaptation to microgravity, and are not risk factors causatively related to space radiation that could be ameliorated by post-mission exercise program. During the future Moon and Mars missions astronauts will be exposed to higher total doses of space radiation (~0.4-0.5Gy from galactic cosmic rays (GCR), especially during Mars mission that is currently estimated to be 30 to 36 months. During an exploration-class space mission to Mars, astronauts will not have access to comprehensive health care services for periods of at least 2 years. Since the majority of experienced astronauts are middle-aged (average age is 46, and the range is 33 to 58 years), they are at risk for developing serious cardiovascular events, which may be life-threatening for the astronauts and mission-threatening for NASA. In this sub-project we hypothesized that: (1) low-dose space radiation-induced biological responses may be long-lasting and are radiation type-dependent; and (2) radiation may increase CV risks of physiologic homeostasis in the aging heart (Irradiation+Aging) and affect processes of cardiac repair and regeneration due to acute myocardial infarct (Irradiation+Aging+Acute Myocardial Infarct).

In Irradiation+Aging group – no significant difference was observed between non irradiated control and proton or 56Fe irradiated mice 1 and 3 months post-IR in cardiac function and remodeling as assessed by Echocardiography (ECHO) and Hemodynamic (HEMO) heart measurements. There was a small but statistically significant (p<0.04) improvement of % Ejection Fraction (amount of the blood pumped out of the heart) in proton-IR vs. control mice.

In IR+Aging+Acute Myocardial Infarct (AMI) group – there was no difference in post-AMI mortality in any of the three groups. Four weeks after AMI, HEMO and ECHO revealed that proton-AMI mice had better cardiac functional recovery compared to control-AMI and 56Fe-AMI mice; whereas % Ejection Fraction, an independent predictive factor for increased mortality after AMI, was most decreased in 56Fe-AMI mice among the AMI groups, suggesting that 56Fe-AMI hearts may have developed cardiac De-compensation and heart failure. Masson’s trichrome staining of mid-myocardial A special staining for fibrosis (scar tissue) revealed that AMI led to small transmural (full thickness of LV) infarct in control-AMI mice, large transmural infarct in 56Fe-AMI mice and small superficial infarct in proton AMI mice, suggesting that low dose proton IR may improve, whereas 56Fe IR is deleterious for post-AMI recovery.

SUMMARY for sub-project 4: Our results reveal that low dose full body single proton or 56Fe IR-induced effects on myocardium are of long duration but they do not affect CV homeostasis under normal conditions. Further, we found that a single proton IR 3 months prior to adverse CV event (Acute Myocardial Infarct) is beneficial, whereas 56Fe deleterious for AMI recovery, strongly suggesting that low dose HZE particle radiation have long-lasting negative effect on degenerative CV risks in case of adverse CV event (e.g., AMI). Therefore, despite of possible “healthy worker factor” for astronauts our findings necessitate further extensive studies of underlying molecular mechanisms of HZE particle radiation in the heart and circulatory system that should include new studies with one or more combination of ions including simulated SPE or GCR cosmic rays, as well as combination of radiation with other confounding factors such as microgravity, history of second hand smoke exposure, etc.

Our central hypothesis is that low-dose space radiation-induced DNA damage repair is inefficient in BM-derived EPC and this may lead to increased mutagenesis with subsequent long-term loss of endothelial function of BM-derived EPCs. This may then pose significant degenerative CV risk on physiologic homeostasis in the aging heart and on the regeneration and neovascularization processes in the heart under pathologic conditions such as acute myocardial infarction (AMI).

Comparisons will be made between two types of low-dose radiation - proton versus iron and single versus fractionated exposures. In short-term in vitro studies (minutes, hours, and up to 30 days after exposure) will evaluate in ex-vivo expanded EPCs and cardiomyocytes DNA damage and repair as well as radiation-induced bystander effects (irradiated cells emit signals to "un-hit" cells), angiogenic gene expression in EPCs. In short-term in vivo studies in the heart we will evaluate acute damage (inflammation and cell death), DNA damage, and repair kinetics. In our long-term studies (3, 6, 9, and 12 months after exposure) we will evaluate oxidative stress and antioxidant defense in BM-derived EPCs, alterations in several EPC endothelial functions, number of circulating peripheral blood EPCs, and cardiomyocyte contractility. In the last part of our studies we will assess CV risks as a result of low-dose radiation plus aging and CV risks under pathological condition -- radiation plus aging plus adverse CV event (i.e., AMI). Here we will evaluate post-AMI survival, alterations in cardiac physiology (echocardiography), infarct size, inflammation, cardiac regeneration, neovascularization, and mobilization of EPCs from BM.

Our studies will address two high-priority research topics of this specific solicitation and NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.

In addition, our studies will also provide novel insights into the alterations in cardiac function processes on the molecular and cardiac physiology levels that may allow for estimation of degenerative risks to cardiovascular system in the civilian population exposed to full body low-dose radiation due to accidental exposures (Chernobyl, Fukushima, etc.) and cancer patients undergoing very frequent imaging tests (i.e., full body Computer Tomography, PET Scans, etc.).

Our studies will address two high-priority research topics for NASA research interests for degenerative risks to the heart - (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age and age plus adverse CV event.

(1) There is a different time course of changes in skeletal muscle (used as an initial model system) Ca2+ homeostasis in response to a single dose of proton and 56Fe irradiation. Specifically, our results indicate that both proton and 56Fe irradiations resulted in detectable increase in [Ca2+]i as well as in reduction of action potential evoked Ca2+ release from the sarcoplasmic reticulum (a cellular compartment). The time course of the observed changes was dependent on the type of radiation, that is - 56Fe radiation produced an increase in [Ca2+]i at the 24hr time point which then declined back to near normal levels, whereas Proton irradiation did not have an effect at the 24hr or 48hr time point, but resulted in a robust increase in [Ca2+]i by 72 hrs. We conclude that ionizing radiation may affect the functional state of skeletal muscle without a presence of obvious histological changes.

(2) Full body 0.15 Gy Iron irradiation affects survival and proliferation of bone marrow (BM)-derived endothelial progenitor cells (EPCs). Specifically, our results reveale that 2, 5, and 24 hrs after full-body irradiation there was 2-6-fold increase in EPC cell death (apoptosis) with the peak 6-fold increase in EPC death at 5hrs. The cells death was decreased by 14 days. However, by day 28 there was a second significant 4-fold increase in EPC cell death, indicating that there is a bimodal (early 5 hrs and delayed 28 days) increase in BM-derived EPC cell death after a single 0.15 Gy Iron radiation. Proliferation analysis of BM-derived EPCs revealed no changes in the rate of proliferation up to 7 days post-Iron irradiation. However, there was ~45 increase in the rate of EPC proliferation on day 14, but the rate of EPC proliferation was decreased twice on day 28. Taken together these data suggest that early increase in BM-derived EPC cell death may be a direct effect of radiation, whereas later increase in cell death and decrease in proliferation could be a result of non-targeted effects. We conclude single low dose of Iron irradiation may have long-lasting effect on survival and proliferation of BM-derived EPCs and may induce delayed non-targeted effects.

(3) Tumor necrosis factor (TNF)-TNF receptor 1 (R1)/p55 or TNFR2/p75 receptor-ligand (TNF protein) interactions inhibit early and increase delayed radiobiological bystander responses in BM-derived EPCs. Specifically, we found that in wild type (WT) naive (non-irradiated) EPC the peak of detectable mean DNA damage foci (double strand breaks-DSB in the absence of a direct radiation hit) were at 24 hrs, whereas in both TNFR1 and TNFR2 knockout (KO) DSBs were the lowest at 24 hrs. This finding indicates that ligand (TNF) - receptor (p55 or p75) interactions inhibit early (within a day) radiobiological bystander responses in BM-derived EPCs in medium transfer experiments. Interestingly, compared to WT EPCs, delayed (5 days) bystander responses in naive EPCs were amplified in p55KO and p75KO cells, suggesting significant role for TNF protein and its receptors interactions in mediating delayed bystander responses. ELISA analysis of protein levels of 8 angiogenic and 8 inflammatory genes in conditioned cell growth medium (collected from irradiated EPCs) 5 days post-radiation showed 2-16-fold increases at day 3-5 in cumulative levels of following proteins - TNF, IFNr, IL6, EGF, MIP-1, GM-SCF, Rantes , p IL1, IL1, G –CSF, MCP-1, SCF in p55KO and p75KO vs WT. Each of these proteins alone or in combination may be a causative factor in inducing DSB in the absence of the actual ionizing radiation. We conclude that radiobiological bystander effects may be regulated (decreased or increased) through modification of TNF signaling via TNFR1/p55 or TNFR2/75, suggesting this strategy may be used to develop mitigating agent(s) for prevention of delayed non-targeted effects.

(4) Low dose gamma radiation-induced early responses in the heart and BM-derived EPCs. Specifically, we evaluated the effect of a full-body single dose 1 Gy gamma-irradiation [low linear energy transfer (LET) type of radiation] on the formation of double strand breaks (DSB) in BM-derived EPCs and in the heart in C57/Bl6J mice. Our studies revealed that within 24 hrs the decay of DSB foci was slow in mouse EPCs, which may be indicative of inefficient or delayed DNA DSB repair. There was an increase in the percent of BM-derived EPCs with DSB foci and increase in DSB per cell over 7 days post-radiation, indicating a possibility of significant radiobiological bystander responses in EPCs. In medium transfer experiments (collection of cell medium from irradiated cells and treatment of naïve non-irradiated cells with this medium after filtration) BM-derived EPCs exhibit significant bystander responses in vitro. We found significant decrease in DSB in irradiated mouse heart resident endothelial cells (EC) and non-EC cells, indicating considerable DNA DSB repair, however with slower than usual repair kinetics reported for other primary cells, i.e., fibroblasts, leukocytes. We also found that radiation-induced increase in cytoplasmic [Ca2+]i concentration was sustained for longer periods of time. This was accompanied by a partial loss of mitochondrial membrane potential, which most likely resulted from mitochondrial calcium overload and subsequent activation of the permeability transition (PT) pore. Moreover, full body 1 Gy gamma-radiation led to a substantial loss of mitochondrial membrane potential for at least seven days, suggesting that if sustained for longer periods of time this may alter mitochondrial membrane integrity, affecting energy production in the cells, therefore diminishing muscle contractile function. We conclude that longitudinal studies using low-dose proton and heavy ion (HZE) radiation studies are warranted to determine space radiation-induced long-term Excess Relative Risks (ERR) for cardiovascular diseases.

Our central hypothesis is that low-dose space radiation-induced DNA damage repair is inefficient in BM-derived EPC and this may lead to increased mutagenesis with subsequent long-term loss of endothelial function of BM-derived EPCs. This may then pose significant degenerative CV risk on physiologic homeostasis in the aging heart and on the regeneration and neovascularization processes in the heart under pathologic conditions such as acute myocardial infarction (AMI).

Comparisons will be made between two types of low-dose radiation - proton versus iron and single versus fractionated exposures. In short-term in vitro studies (minutes, hours, and up to 30 days after exposure) will evaluate in ex-vivo expanded EPCs and cardiomyocytes DNA damage and repair as well as radiation-induced bystander effects (irradiated cells emit signals to "un-hit" cells), angiogenic gene expression in EPCs. In short-term in vivo studies in the heart we will evaluate acute damage (inflammation and cell death), DNA damage, and repair kinetics. In our long-term studies (3, 6, 9, and 12 months after exposure) we will evaluate oxidative stress and antioxidant defense in BM-derived EPCs, alterations in several EPC endothelial functions, number of circulating peripheral blood EPCs, and cardiomyocyte contractility. In the last part of our studies we will assess CV risks as a result of low-dose radiation plus aging and CV risks under pathological condition -- radiation plus aging plus adverse CV event (i.e., AMI). Here we will evaluate post-AMI survival, alterations in cardiac physiology (echocardiography), infarct size, inflammation, cardiac regeneration, neovascularization, and mobilization of EPCs from BM.

Our studies will address two high-priority research topics of this specific solicitation and NASA research interests for degenerative risks to the heart -- (1) development of murine models to estimate risks for degenerative heart diseases; (2) determine the progression rates and latency periods for space radiation-related degenerative CV risks as a function of radiation type (proton vs. heavy ion), exposure frequency (single vs. fractionated), age, and age plus adverse CV event.