Mitochondrial transplantation for therapeutic use

James D McCully, Sidney Levitsky, Pedro J Del Nido, Douglas B Cowan, James D McCully, Sidney Levitsky, Pedro J Del Nido, Douglas B Cowan

Abstract

Mitochondria play a key role in the homeostasis of the vast majority of the body's cells. In the myocardium where mitochondria constitute 30 % of the total myocardial cell volume, temporary attenuation or obstruction of blood flow and as a result oxygen delivery to myocardial cells (ischemia) severely alters mitochondrial structure and function. These alterations in mitochondrial structure and function occur during ischemia and continue after blood flow and oxygen delivery to the myocardium is restored, and significantly decrease myocardial contractile function and myocardial cell survival. We hypothesized that the augmentation or replacement of mitochondria damaged by ischemia would provide a mechanism to enhance cellular function and cellular rescue following the restoration of blood flow. To test this hypothesis we have used a model of myocardial ischemia and reperfusion. Our studies demonstrate that the transplantation of autologous mitochondria, isolated from the patient's own body, and then directly injected into the myocardial during early reperfusion augment the function of native mitochondria damaged during ischemia and enhances myocardial post-ischemic functional recovery and cellular viability. The transplanted mitochondria act both extracellularly and intracellularly. Extracellularly, the transplanted mitochondria enhance high energy synthesis and cellular adenosine triphosphate stores and alter the myocardial proteome. Once internalized the transplanted mitochondria rescue cellular function and replace damaged mitochondrial DNA. There is no immune or auto-immune reaction and there is no pro-arrhythmia as a result of the transplanted mitochondria. Our studies and those of others demonstrate that mitochondrial transplantation can be effective in a number of cell types and diseases. These include cardiac and skeletal muscle, pulmonary and hepatic tissue and cells and in neuronal tissue. In this review we discuss the mechanisms leading to mitochondrial dysfunction and the effects on cellular function. We provide a methodology for the isolation of mitochondria to allow for clinical relevance and we discuss the methods we and others have used for the uptake and internalization of mitochondria. We foresee that mitochondrial transplantation will be a valued treatment in the armamentarium of all clinicians and surgeons for the treatment of varied ischemic disorders, mitochondrial diseases and related disorders.

Keywords: Ischemia/reperfusion injury; Mitochondria; Myocardium; Surgery.

Figures

Fig. 1
Fig. 1
Mitochondrial damage following ischemia. Representative transmission electron photomicrographs from left ventricular tissue from Control (non-ischemic) and Ischemia (30 min ischemia) hearts. In Control hearts sarcomere structure is preserved and mitochondria have electron dense intracristae matrix and normal size. In Ischemia hearts there is separation myofilaments and severely swollen and electron transparent mitochondria containing numerous mitochondrial calcium granules. Calcium granules (Ca2+), mitochondrion (M), sarcomere (S) and nucleus (N) are indicated. Scale bars (2 um are shown)
Fig. 2
Fig. 2
Myocardial mitochondrial changes occurring during ischemia. The changes to mitochondria occurring during ischemia are noted. These changes extend following the restoration of blood flow and oxygen delivery to the myocardium (reperfusion) and significantly decrease myocardial function and cell viability. Mitochondrial transplantation (the direct injection of viable exogenous mitochondria) into the ischemic myocardium, just prior to reperfusion significantly enhances myocardial function and cell viability
Fig. 3
Fig. 3
Mitochondrial isolation. Our protocol for mitochondrial isolation is illustrated. In brief a mini-thoracotomy or a sternotomy is performed and tissue is obtained from either pectoralis major or from rectus abdominous muscle. The amount of tissue is small and is shown in comparison to an American 10 cent piece. Mitochondria are isolated. Our methodology for mitochondrial isolation is shown [27]. Mitochondrial isolation can be performed in less than 30 min. Quality control parameters for mitochondrial isolation and transplantation have been established [28]
Fig. 4
Fig. 4
Experimental outline for mitochondrial transplantation studies [19, 20]
Fig. 5
Fig. 5
Transplanted mitochondria are internalized by a variety of cell types: a neonatal rat cardiomyocytes, b adult rat cardiomyocytes, c rat skeletal myoblasts, d embryonic rat neurons and e mixed embryonic rat neuronal cells. Internalized pHrodo Red-labeled rat liver mitochondria (red) following 4 h co-culture are shown in each cell type. In all images the blue stain is DAPI, alpha-actinin 2 is depicted in green. Scale bars are 10 um

References

    1. Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N. Importing mitochondrial proteins: machineries and mechanisms. Cell. 2009;138:628–644. doi: 10.1016/j.cell.2009.08.005.
    1. Chen L, Winger AJ, Knowlton AA. Mitochondrial dynamic changes in health and genetic diseases. Mol Biol Rep. 2014;41:7053–7062. doi: 10.1007/s11033-014-3663-y.
    1. Frolkis VV, Frolkis RA, Mkhitarian LS, Sherchuk VG, Fraifeld VE, Vakulenko LG, Syrouy I. Contractile function and Ca2+ transport system of myocardium in ageing. Gerontology. 1988;34:64–74. doi: 10.1159/000212932.
    1. Faulk EA, McCully JD, Tsukube T, Hadlow NC, Krukenkamp IB, Levitsky S. Myocardial mitochondrial calcium accumulation modulates nuclear calcium accumulation and DNA fragmentation. Annals Thorac Surg. 1995;60:338–344. doi: 10.1016/0003-4975(95)00446-R.
    1. Faulk EA, McCully JD, Hadlow NC, Tsukube T, Krukenkamp IB, Federman M, Levitsky S. Magnesium cardioplegia enhances mRNA levels and the maximal velocity of cytochrome oxidase I in the senescent myocardium during global ischemia. Circulation. 1995;92:405–412. doi: 10.1161/01.CIR.92.9.405.
    1. Rousou AJ, Ericsson M, Federman M, Levitsky S, McCully JD. Opening of mitochondrial KATP enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation and respiration. Am J Physiol Heart Circ Physiol. 2004;287:H1967–1976. doi: 10.1152/ajpheart.00338.2004.
    1. Suleiman MS, Halestrap AP, Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther. 2001;89:29–46. doi: 10.1016/S0163-7258(00)00102-9.
    1. McCully JD, Rousou AJ, Parker RA, Levitsky S. Age and gender differences in mitochondrial oxygen consumption and free matrix calcium during ischemia/reperfusion and with cardioplegia and diazoxide. Ann Thorac Surg. 2007;83:1102–1109. doi: 10.1016/j.athoracsur.2006.10.059.
    1. Ozcan C, Holmuhamedov EL, Jahangir A, Terzic A. Diazoxide protects mitochondria from anoxic injury: implication for myopreservation. J Thorac Cardiovasc Surg. 2001;121:298–306. doi: 10.1067/mtc.2001.111421.
    1. Cross RL. Turning the ATP motor. Nature. 2004;427:407–408. doi: 10.1038/427407b.
    1. Garlid KD. Opening mitochondrial KATP in the heart–what happens, and what does not happen. Basic Res Cardiol. 2000;95:275–279. doi: 10.1007/s003950070046.
    1. Tsukube T, McCully JD, Metz RM, Cook CU, Levitsky S. Amelioration of ischemic calcium overload correlates with high energy phosphates in the senescent myocardium. Am J Physiol. 1997;273:H418–427.
    1. Levitsky S, Laurikka J, Stewart RD, Campos CT, Lahey SJ, McCully JD. Mitochondrial DNA deletions in coronary artery bypass grafting patients. Eur J Cardiothorac Surg. 2003;24:777–784. doi: 10.1016/S1010-7940(03)00501-3.
    1. Van Tuyle GC, Gudikote JP, Hurt VR, Miller BB, Moore CA. Multiple, large deletions in rat mitochondrial DNA: evidence for a major hotspot. Mut Res. 1996;349:95–107. doi: 10.1016/0027-5107(95)00165-4.
    1. Gaweda-Walerych K, Zekanowski C. The impact of mitochondrial DNA and nuclear genes related to mitochondrial functioning on the risk of Parkinson’s disease. Curr Genomics. 2013;14:543–559. doi: 10.2174/1389202914666131210211033.
    1. McCully JD, Bhasin MK, Wakiyama H, Daly C, Guerrero M, Dillon S, Libermann TA, Cowan DB, Mably JD, Parker RA, Ericsson M, McGowan FX, Levitsky S. Transcriptomic and proteomic analysis of the cardioprotection afforded by cardioplegia. Physiol Genomics. 2009;38:125–137. doi: 10.1152/physiolgenomics.00033.2009.
    1. McCully JD, Wakiyama H, Hsieh Y-J, Jones M, Levitsky S. Differential contribution of necrosis and apoptosis in myocardial ischemia/reperfusion injury. Am J Physiol Heart Circ Physiol. 2004;286:H1923–1935. doi: 10.1152/ajpheart.00935.2003.
    1. McCully JD, Toyoda Y, Wakiyama H, Rousou AJ, Parker RA, Levitsky S. Age and gender related differences in ischemia/reperfusion injury and cardioprotection: effects of diazoxide. Ann Thorac Surg. 2006;82:117–123. doi: 10.1016/j.athoracsur.2006.03.002.
    1. Masuzawa A, Black KM, Pacak CA, Ericsson M, Barnett RJ, Drumm C, Seth P, Bloch DB, Levitsky S, Cowan DB, McCully JD. Transplantation of autologously-derived mitochondria protects the heart from ischemia-reperfusion injury. Am J Phys Heart Circ Physiol. 2013;304:H966–982. doi: 10.1152/ajpheart.00883.2012.
    1. McCully JD, Cowan DB, Pacak CA, Levitsky S. Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Phys Heart Circ Phys. 2009;296:94–105.
    1. Claude A. Fractionation of mammalian liver cells by differential centrifugation II. Experimental procedures and results. J Exp Med. 1946;84:61–89. doi: 10.1084/jem.84.1.61.
    1. Pallotti F, Lenaz G. Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 2007;80:3–44. doi: 10.1016/S0091-679X(06)80001-4.
    1. Fernández-Vizarra E, Ferrín G, Pérez-Martos A, Fernández-Silva P, Zeviani M, Enríquez JA. Isolation of mitochondria for biogenetical studies: an update. Mitochondrion. 2010;10:253–262. doi: 10.1016/j.mito.2009.12.148.
    1. Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc. 2007;2:287–295. doi: 10.1038/nprot.2006.478.
    1. Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc. 2009;4:1582–1590. doi: 10.1038/nprot.2009.151.
    1. Gostimskaya I, Galkin A. Preparation of highly coupled rat heart mitochondria. J Vis Exp. 2010;10:2202.
    1. Preble JM, Pacak CA, Kondo H, McKay AA, Cowan DB, McCully JD. Rapid isolation and purification of mitochondria for transplantation. J Vis Exp. 2014;91:e51682.
    1. Preble JM, Kondo H, Levitsky S, James D, McCully JD. Quality control parameters for mitochondria transplant in cardiac tissue. JSM Biochem Mol Biol. 2014;2(1):1008.
    1. Hoppel CL, Tandler B, Fujioka H, Riva A. Dynamic organization of mitochondria in human heart and in myocardial disease. Int J Biochem Cell Biol. 2009;41(10):1949–1956. doi: 10.1016/j.biocel.2009.05.004.
    1. Hollander JM, Thapa D, Shepherd DL. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies. Am J Physiol Heart Circ Physiol. 2014;307(1):H1–14. doi: 10.1152/ajpheart.00747.2013.
    1. Bolling SF, Bies LE, Bove EL. Effect of ATP synthesis promoters on postischemic myocardial recovery. J Surg Res. 1990;49:205–211. doi: 10.1016/0022-4804(90)90120-Q.
    1. Soncul H, Ersoz A, Gokgoz L, Karasu C, Ayrancioglu K, Sinci V, Altan M. Cardioplegia with adenosine and adenosine triphosphate in the isolated guinea pig heart. Jpn Heart J. 1992;33:843–850. doi: 10.1536/ihj.33.843.
    1. Stark G, Domanowits H, Sterz F, Stark U, Bachernegg M, Kickenweiz E, Decrinis M, Laggner AN, Tritthart HA. Action of ATP on ventricular automaticity. J Cardiovasc Pharmacol. 1994;24:740–744. doi: 10.1097/00005344-199424050-00008.
    1. Pacak AP, Preble JM, Kondo H, Seibel P, Levitsky S, del Nido PJ, Cowan DB, Mccully JD. Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function. Biol Open. 2015;4:622–626. doi: 10.1242/bio.201511478.
    1. Margulis L. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof. Symp Soc Exp Biol. 1975;29:21–38.
    1. Huang X, Sun L, Ji S, Zhao T, Zhang W, Xu J, Zhang J, Wang Y, Wang X, Franzini-Armstrong C, Zheng M, Cheng H. Kissing and nanotunneling mediate intermitochondrial communication in the heart. Proc Natl Acad Sci USA. 2013;110:2846–2851. doi: 10.1073/pnas.1300741110.
    1. Islam MN, Das SR, Emin MT, Wei M, Sun L, Westphalen K, Rowlands DJ, Quadri SK, Bhattacharya S, Bhattacharya J. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med. 2012;18:759–765. doi: 10.1038/nm.2736.
    1. Lou E, Fujisawa S, Morozov A, Barlas A, Romin Y, Dogan Y, Gholami S, Moreira AL, Manova-Todorova K, Moore MA. Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One. 2012;7:e33093. doi: 10.1371/journal.pone.0033093.
    1. Spees JL, Olson SD, Whitney MJ, Prockop DJ. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci USA. 2006;103:1283–1288. doi: 10.1073/pnas.0510511103.
    1. Roche S, D’Ippolito G, Gomez LA, Bouckenooghe T, Lehmann S, Montero-Menei CN, Schiller PC. Comparative analysis of protein expression of three stem cell populations: models of cytokine delivery system in vivo. Int J Pharm. 2012
    1. Lorita J, Soley M, Ramirez I. Epidermal growth factor protects the heart against low-flow ischemia-induced injury. J Phys Biochem. 2010;66:55–62. doi: 10.1007/s13105-010-0009-7.
    1. Somers P, Cornelissen R, Thierens H, Van Nooten G. An optimized growth factor cocktail for ovine mesenchymal stem cells. Growth Factors. 2012;30:37–48. doi: 10.3109/08977194.2011.634411.
    1. Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic Z, McCarthy PM, Penn MS. Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor. Stem Cells. 2007;25:245–251. doi: 10.1634/stemcells.2006-0293.
    1. Yau TM, Tomita S, Weisel RD, Jia ZQ, Tumiati LC, Mickle DA, Li RK. Beneficial effect of autologous cell transplantation on infarcted heart function: comparison between bone marrow stromal cells and heart cells. Ann Thorac Surg. 2003;75:169–176. doi: 10.1016/S0003-4975(02)04290-X.
    1. Hamamoto H, Gorman JH, 3rd, Ryan LP, Hinmon R, Martens TP, Schuster MD, Plappert T, Kiupel M, John-Sutton MG, Itescu S, Gorman RC. Allogeneic mesenchymal precursor cell therapy to limit remodeling after myocardial infarction: the effect of cell dosage. Ann Thorac Surg. 2009;87:794–801. doi: 10.1016/j.athoracsur.2008.11.057.
    1. Kukat A, Kukat C, Brocher J, Schäfer I, Krohne G, Trounce IA, Villani G, Seibel P. Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses. Nucleic Acids Res. 2008;36:e44. doi: 10.1093/nar/gkn124.
    1. King MP, Attardi G. Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA. Cell. 1988;52:811–819. doi: 10.1016/0092-8674(88)90423-0.
    1. Elliott RL, Jiang XP, Head JF. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity. Breast Cancer Res Treat. 2012;136:347–354. doi: 10.1007/s10549-012-2283-2.
    1. Chang JC, Liu KH, Li YC, Kou SJ, Wei YH, Chuang CS, Hsieh M, Liu CS. Functional recovery of human cells harbouring the mitochondrial DNA mutation MERRF A8344G via peptide-mediated mitochondrial delivery. Neurosignals. 2013;21:160–173. doi: 10.1159/000341981.
    1. Chang JC, Wu SL, Liu KH, Chen YH, Chuang CS, Cheng FC, Su HL, Wei YH, Kuo SJ, Liu CS. Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson’s disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine–induced neurotoxicity. Transl Res. 2016;170:40–56. doi: 10.1016/j.trsl.2015.12.003.
    1. Lin HC, Liu SY, Lai HS, Lai IR. Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats. Shock. 2013;39:304–310. doi: 10.1097/SHK.0b013e318283035f.
    1. Kitani T, Kami D, Matoba S, Gojo S. Internalization of isolated functional mitochondria: involvement of macropinocytosis. J Cell Mol Med. 2014;18:1694–1703. doi: 10.1111/jcmm.12316.

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