We have completed seven sub-projects within the scope of our main project. Each of the summaries below represent progress for the particular sub-project.
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.