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COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoPedia topics: EAGLE
|Isola Raffaella, Dott.ssa
University Citadel of Monserrato, IT
|SS 554 Bivio Sestu, 09042
|Isola 2022 Abstract Bioblast
|Dubouchaud 2018 MiPschool Tromso
|Isola 2018 MiPschool Tromso C3
|Isola 2017 MiPschool Obergurgl
MitoEAGLE Short-Term Scientific Mission
- Work Plan summary
- 1. Aim and motivation-please explain the scientific and/or other motivation for the STSM and what scientific and/or other outcomes you aim to accomplish with the STSM Rationale. AMP-activated protein kinase (AMPK) is an heterotrimeric enzyme, which is aimed at keeping the energetic state of the cell at a constant level. It is activated by an increase in AMP/ATP (or ADP/ATP) ratio or by a calcium rise (being activated by Ca2+/calmodulin-dependent protein kinase B) or by another upstream kinase, liver kinase B1 (LKB1). Its activation results in multiple outcomes, among others, increase of glucose uptake, mitochondrial fatty acid oxidation and mitochondria biogenesis. Its role has been relevant to skeletal muscle energy homeostasis and cardiac tissue. In the latter, other roles have been ascribed to AMPK, since myocardium seldom goes into an energy demanding status, such as skeletal muscles faces during, e.g., endurance training. AMPK is activated during cardiac hypertrophy and ischemia, and it seems to play an important role in preventing myocardial fibrosis (Jiang et al., 2017), hypertrophic cardiomyopathy (Li et al., 2017) and in counteracting ischemia (Qi and Young, 2015). AMPK exists in two isoforms of its catalytic subunit: AMPKÎ±1 and AMPKÎ±2, which are ubiquitous and often overlapping in their intracellular functions. A previous report (Lantier et al., 2014) showed that selective knocking down of both AMPKÎ±1 and AMPKÎ±2 in mouse skeletal muscle tissue lead to intolerance to exercise, but not in alteration of glucose uptake and muscle contractility, rather, to a defective mitochondrial OXPHOS (oxidative phosphorylation) limited to the complex I substrates. Aim. The project that will be developed during the STSM, will assess whether selective AMPK deletion in heart muscle affects mitochondrial oxidative capacity in heart mitochondria. To accomplish that, a novel model of conditional tamoxifen-inducible cardiac AMPK KO mouse will be used (Sohal et al., 2001). Heart-specific AMPK deletion will be achieved by tamoxifen administration in adult mice to avoid any developmental defect or compensatory mechanisms. The aim is to highlight whether the specific deficiency of heart AMPK develops mitochondrial respiratory capacity defects as seen in skeletal muscles, or whether other defects or none are secondary to knocking out of AMPK, due to the typical characteristic of myocardial tissue. To this end, experiments will be performed on isolated cardiac mitochondria, as well as on both wild type and KO male and female mice. Substrates against each complex of the respiratory chain and of fatty acid oxidation will be exploited. Markers of mitochondrial density, such as citrate synthase activity or succinate dehydrogenase, will be searched, too. Motivation of the study. Despite its undoubtful value on understanding the basic myocardial physiology, this study is preliminary to a wider study on the effect of anthracyclines (the known antitumoral drugs) on cardiac tissue. The hypothesis is that anthracyclins-elicited cardiotoxicity is mediated by AMPK and the AMPK- KO animal model is functional to support this idea. Thus, the set of experiments presented in this project are essential to the following ones. Motivation of the STSM. The applicant’s motivation in accomplishing this STSM is, in primis, to become familiar with the proper technique on mitochondria bioenergetics both in isolated cardiac mitochondria and in permeabilized skeletal muscle fibers, by Clark-type electrode. Oxygen consumption experiments on permeabilized fibers would be performed only for training in a separate set of experiments. Moreover, the possibility of confrontation in the interpretation of OXPHOS data will be of prominent importance for the applicant.
- Being an autodidactic in this field, she realized the value of having a reference guide in a training period on mitochondria studies. Last, being involved mainly in cardiac mitochondria studies, the present investigation would widen her knowledge on cardiac mitochondria physiology and homeostasis. An STSM mission would combine the opportunity of improving her personal scientific culture on mitochondria, together with the possibility of starting a new international collaboration that might lead to a long-term cooperation. Maybe in the future it might be planned the writing of joint funding applications.
- 2. Proposed contribution to the scientific objectives of the Action The applicant is a senior investigator who is an academic with a permanent position at the University of Cagliari, Italy. Although having a long-term experience in mitochondrial potential studies and mitochondrial ultrastructural studies, the applicant’s experience in the mitochondrial bioenergetic field is rather new. Thus, this STSM would follow the aim of CA15203 first because it would allow “the dissemination of updated knowledge and know-how among the partners”, secondly because this mission would set the basis for a long-term collaborative relationship, according to the aim of “forming a unique well-coordinated network of senior researchers and young investigators, to include well established stakeholders, and to establish a spirit of mentorship and collaboration”. This experience would increase the applicant’s quality and validity of research and, hopefully, it might lead to a joint request for European funding for a research proposal.
- 3. Techniques â€“ Please detail what techniques or equipment you may learn to use, if applicable.
- The techniques that will be learned during this STSM will be primarily the study of mitochondrial oxidative phosphorylation (OXHPOS) by means of Clark-type electrode oxygraphy (Hansatech). As the applicant has the same oxygraph at the home-laboratory, this fact would be of particularly of interest for her, since technical differences in other types of oxygraphs make not such valuable a scientific mission in another laboratory. The applicant in the past had a post-doc who worked with her and was trained abroad for OXPHOS assessment, but she reported wrong settings which spoiled the collected data. This period is, indeed, necessary for the applicant to learn the best practice for mitochondrial oxygen consumption estimation and for calculating respiratory capacity and coupling control.
- OXPHOS will be studied on isolated cardiac mitochondria prepared by differential centrifugation and skeletal muscle permeabilized fibers prepared according to Kuznetszov et al. (Kuznetszov et al, 2008). Working on permeabilized skeletal muscle fibers is a brand-new approach for the applicant, who will acquire a new technique that will allow her to broaden her field of research. The protocols followed will include: testing of complex I activity (by glutamate plus malate), testing of complex II activity (by adding succinate in the presence of rotenone), testing complex III activity (by adding duroquinol in the presence of rotenone), testing complex IV activity (by adding TMPD plus ascorbate in the presence of rotenone), testing OXPHOS driven by lipid substrates by adding Palmitoyl-CoA plus carnitine and malate or Palmitoyl-carnitine plus malate. These substrates will be tested in phosphorylating state (saturating ADP concentration) and non-phosphorylating condition (i.e. in the presence of oligomycine) and under various ADP concentrations (from low to high, in order to determine mitochondrial affinity for ADP). Moreover, maximal ETC capacity will be tested by adding FCCP as an uncoupler. Additionally, ROS production will be assessed on isolated mitochondria by measuring the release of H2O2 using Amplex Red as a probe, in the same conditions of substrates. The heart selective double KO mice represent an animal model the applicant had never used. Handling and using it will broaden the applicant experimental experience.
- 4. Planning â€“ Please detail the steps you will take to achieve your proposed aim. January 8-12th Arrival, becoming familiar with facilities, animals, protocols, bibliography, first experimental trial January 15-19th First set of experiments 3 days with 2 male mice per day. Other days recovery and partial elaboration of data January 22-26th Second set of experiments one day with 2 male mice and two days with 2 female mice January 29th-February 2nd two days with 2 female mice. Data elaboration February 5-9th Data elaboration, bibliography reading February 12-16th first report on data and discussion with host. Deciding whether some experiment should be repeated. February 19-23rd Assessment of citrate synthase and succinate dehydrogenase on frozen samples. February 26th-March 2nd Preparation of second report and trials on skeletal muscle permeabilized fibers (or repeat some exp.) March 5th-9th Discussion of the second report with host and trials on permeabilized fibers (or repeat some exp.) March 12-16th Preparation of scientific report and of short paper (or new data elaboration) March 19-23rd Preparation of scientific report and short paper (or and a new presentation to the host) Bibliography Jiang S, Li T, Yang Z, Yi W, Di S, Sun Y, Wang D, Yang Y. AMPK orchestrates an elaborate cascade protecting tissue from fibrosis and aging. Ageing Res Rev. 2017 38:18-27. Li T, Jiang S, Yang Z, Ma Z, Yi W, Wang D, Yang Y. Targeting the energy guardian AMPK: another avenue for treating cardiomyopathy? Cell Mol Life Sci. 2017 74(8):1413-1429 Qi D, Young LH. AMPK: energy sensor and survival mechanism in the ischemic heart. Trends Endocrinol Metab. 2015 Aug;26(8):422-9. Lantier L, Fentz J, Mounier R, Leclerc J, Treebak JT, Pehmoller C, Sanz N, Sakakibara I, Saint-Amand E, Rimbaud S, Maire P, Marette A, Ventura-Clapier R, Ferry A, Wojtaszewsk JFPi, Foretz M, Viollet B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J 2014 28: 3211â€“3224. Sohal D S, Nghiem M, Crackower M A, Witt S A, Kimball T R, Tymitz K M, Penninger J M, and Molkentin J D. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible cre protein. 2001 Circulation research 89, 20-25 Kuznetsov A V, Veksler V, Gellerich F N, Saks V, Margreiter R, and Kunz WS. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. 2008 Nature protocols 3: 965-976.
- Work Plan summary