Molecular and Cellular Biochemistry

, Volume 462, Issue 1–2, pp 1–10 | Cite as

Exosomes derived from cardiac parasympathetic ganglionic neurons inhibit apoptosis in hyperglycemic cardiomyoblasts

  • Reetish Singla
  • Kaley H. Garner
  • Mohtashem Samsam
  • Zixi Cheng
  • Dinender K. SinglaEmail author


Diabetic cardiomyopathy is known to involve two forms of cardiac cell death: apoptosis and necrosis. However, it remains unknown whether hyperglycemia-induced apoptosis in the H9c2 cell culture system is inhibited by parasympathetic ganglionic neurons (PGN) derived exosomes (exos). We isolated PGN and sympathetic ganglionic neurons (SGN) from the right stellate ganglion in rats, and derived exos from these sources. H9c2 cells were divided into 4 groups: (1) Control, (2) H9c2 + Glucose (100 mmol/L), (3) H9c2 + Glucose + PGN-exos, and (4) H9c2 + Glucose + SGN-exos. We determined cell proliferation and viability with an MTT assay kit, and assessed apoptotic cell death with TUNEL staining and ELISA. Data were further confirmed by analyzing the presence of pro-apoptotic proteins Caspase-3 and Bax, and anti-apoptotic protein Bcl-2. Glucose exposed H9c2 cells significantly reduced cell viability, which was improved by PGN-exos, but not by SGN-exos. Furthermore, increased apoptosis in hyperglycemia in H9c2 cells was confirmed with TUNEL staining and cell death ELISA which demonstrated significantly (p < 0.05) reduction with PGN-exos treatment, but not with SGN-exos. Moreover, high expression of pro-apoptotic proteins Caspase-3 and Bax was reduced following treatment with PGN-exos; however, SGN-exos were unable to reduce the expression. Significantly reduced anti-apoptotic protein Bcl-2 following glucose treatment was improved with PGN-exos. Therefore, our data suggest that hyperglycemia induces apoptosis in H9c2 cells and decreases cell viability, and that PGN-exos are able to inhibit apoptosis, improve cell viability, and restore levels of anti-apoptotic protein Bcl-2.


Diabetes Apoptosis Parasympathetic ganglionic neurons Sympathetic ganglionic neurons Exosomes 



This publication was derived from studies performed in partial fulfillment of Honors in the major thesis requirements for Mr. Reetish Singla at the University of Central Florida. The authors are thankful to Zahra Tavakoli Dargani for her technical assistance.


Internal resources were used to fund this project.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest to report.


  1. 1.
    Rajchgot T, Thomas SC, Wang JC, Ahmadi M, Balood M, Crosson T, Dias JP, Couture R, Claing A, Talbot S (2019) Neurons and microglia; a sickly-sweet duo in diabetic pain neuropathy. Front Neurosci 13:25. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Rehman K, Akash MSH (2017) Mechanism of generation of oxidative stress and pathophysiology of type 2 diabetes mellitus: how are they interlinked? J Cell Biochem 118:3577–3585. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Faria A, Persaud SJ (2017) Cardiac oxidative stress in diabetes: mechanisms and therapeutic potential. Pharmacol Ther 172:50–62. CrossRefPubMedGoogle Scholar
  4. 4.
    Doll S, Dressen M, Geyer PE, Itzhak DN, Braun C, Doppler SA, Meier F, Deutsch MA, Lahm H, Lange R, Krane M, Mann M (2017) Region and cell-type resolved quantitative proteomic map of the human heart. Nat Commun 8:1469. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gu H, Epstein PN, Li L, Wurster RD, Cheng ZJ (2008) Functional changes in baroreceptor afferent, central and efferent components of the baroreflex circuitry in type 1 diabetic mice (OVE26). Neuroscience 152:741–752. CrossRefPubMedGoogle Scholar
  6. 6.
    Gu H, Zhang ZH, Epstein PN, Li L, Harden SW, Wurster RD, Cheng ZJ (2009) Impaired baroreflex control of renal sympathetic nerve activity in type 1 diabetic mice (Ove26). Neuroscience 161:78–85. CrossRefPubMedGoogle Scholar
  7. 7.
    Lin M, Chen QH, Wurster RD, Hatcher JT, Liu YQ, Li L, Harden SW, Cheng ZJ (2010) Maternal diabetes increases small conductance Ca2+ -activated K+ (SK) currents that alter action potential properties and excitability of cardiac motoneurons in the nucleus ambiguus. J Neurophysiol 104:2125–2138. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lin M, Hatcher JT, Chen QH, Wurster RD, Li L, Cheng ZJ (2011) Maternal diabetes increases large conductance Ca2+-activated K+ outward currents that alter action potential properties but do not contribute to attenuated excitability of parasympathetic cardiac motoneurons in the nucleus ambiguus of neonatal mice. Am J Physiol Regul Integr Comp Physiol 300:R1070–R1078. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Parati G, Esler M (2012) The human sympathetic nervous system: its relevance in hypertension and heart failure. Eur Heart J 33:1058–1066. CrossRefPubMedGoogle Scholar
  10. 10.
    Barile L, Milano G, Vassalli G (2017) Beneficial effects of exosomes secreted by cardiac-derived progenitor cells and other cell types in myocardial ischemia. Stem Cell Investig 4:93. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhu L, Kalimuthu S, Gangadaran P, Oh JM, Lee HW, Baek SH, Jeong SY, Lee SW, Lee J, Ahn BC (2017) Exosomes derived from natural killer cells exert therapeutic effect in melanoma. Theranostics 7:2732–2745. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Guay C, Kruit JK, Rome S, Menoud V, Mulder NL, Jurdzinski A, Mancarella F, Sebastiani G, Donda A, Gonzalez BJ, Jandus C, Bouzakri K, Pinget M, Boitard C, Romero P, Dotta F, Regazzi R (2019) Lymphocyte-derived exosomal microRNAs promote pancreatic beta cell death and may contribute to type 1 diabetes development. Cell Metab 29(348–361):e6. CrossRefGoogle Scholar
  13. 13.
    Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, Timmers L, van Rijen HV, Doevendans PA, Pasterkamp G, Lim SK, de Kleijn DP (2013) Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res 10:301–312. CrossRefPubMedGoogle Scholar
  14. 14.
    Jiang ZZ, Liu YM, Niu X, Yin JY, Hu B, Guo SC, Fan Y, Wang Y, Wang NS (2016) Exosomes secreted by human urine-derived stem cells could prevent kidney complications from type I diabetes in rats. Stem Cell Res Ther 7:24. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, Mackie AR, Vaughan E, Garikipati VN, Benedict C, Ramirez V, Lambers E, Ito A, Gao E, Misener S, Luongo T, Elrod J, Qin G, Houser SR, Koch WJ, Kishore R (2015) Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circ Res 117:52–64. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Katakowski M, Buller B, Zheng X, Lu Y, Rogers T, Osobamiro O, Shu W, Jiang F, Chopp M (2013) Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett 335:201–204. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ono M, Kosaka N, Tominaga N, Yoshioka Y, Takeshita F, Takahashi RU, Yoshida M, Tsuda H, Tamura K, Ochiya T (2014) Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci Signal 7:63. CrossRefGoogle Scholar
  18. 18.
    Sahoo S, Losordo DW (2014) Exosomes and cardiac repair after myocardial infarction. Circ Res 114:333–344. CrossRefPubMedGoogle Scholar
  19. 19.
    Lin M, Ai J, Harden SW, Huang C, Li L, Wurster RD, Cheng ZJ (2010) Impairment of baroreflex control of heart rate and structural changes of cardiac ganglia in conscious streptozotocin (STZ)-induced diabetic mice. Auton Neurosci 155:39–48. CrossRefPubMedGoogle Scholar
  20. 20.
    Tavakoli Dargani Z, Singla R, Johnson T, Kukreja R, Singla DK (2018) Exosomes derived from embryonic stem cells inhibit doxorubicin and inflammation-induced pyroptosis in muscle cells. Can J Physiol Pharmacol 96:304–307. CrossRefPubMedGoogle Scholar
  21. 21.
    Singla RD, Wang J, Singla DK (2017) Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells. Mol Cell Biochem 425:77–84. CrossRefPubMedGoogle Scholar
  22. 22.
    Anversa P, Cheng W, Liu Y, Leri A, Redaelli G, Kajstura J (1998) Apoptosis and myocardial infarction. Basic Res Cardiol 93(Suppl 3):8–12CrossRefGoogle Scholar
  23. 23.
    Liu R, Li X, Zhang X (2018) Dexmedetomidine protects high-glucose induced apoptosis in human retinal pigment epithelial cells through inhibition on p75(NTR). Biomed Pharmacother 106:466–471. CrossRefPubMedGoogle Scholar
  24. 24.
    Chen M, Zheng H, Wei T, Wang D, Xia H, Zhao L, Ji J, Gao H (2016) High glucose-induced PC12 cell death by increasing glutamate production and decreasing methyl group metabolism. Biomed Res Int 2016:4125731. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Serrano-Oviedo L, Ortega-Muelas M, Garcia-Cano J, Valero ML, Cimas FJ, Pascual-Serra R, Fernandez-Aroca DM, Roche O, Ruiz-Hidalgo MJ, Belandia B, Gimenez-Bachs JM, Salinas AS, Sanchez-Prieto R (2018) Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion. PLoS ONE 13:e0200878. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Singla DK, McDonald DE (2007) Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells. Am J Physiol Heart Circ Physiol 293:H1590–H1595. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Glass C, Singla DK (2011) MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart. Am J Physiol Heart Circ Physiol 301:H2038–H2049. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gessi S, Merighi S, Bencivenni S, Battistello E, Vincenzi F, Setti S, Cadossi M, Borea PA, Cadossi R, Varani K (2019) Pulsed electromagnetic field and relief of hypoxia-induced neuronal cell death: The signaling pathway. J Cell Physiol. CrossRefPubMedGoogle Scholar
  29. 29.
    Fu C, Wang L, Tian G, Zhang C, Zhao Y, Xu H, Su M, Wang Y (2019) Enhanced anticancer effect of oncostatin M combined with salinomycin in CD133(+) HepG2 liver cancer cells. Oncol Lett 17:1798–1806. CrossRefPubMedGoogle Scholar
  30. 30.
    Shen W, Zhao Y, Chen H, Zhang T, Wu S, Liu P (2019) M3, a natural lignan xyloside, exhibits potent anticancer activity in HCT116 cells. Oncol Lett 17:2117–2122. CrossRefPubMedGoogle Scholar
  31. 31.
    Singla DK, Singla RD, McDonald DE (2008) Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway. Am J Physiol Heart Circ Physiol 295:H907–H913. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Merino H, Singla DK (2018) Secreted frizzled-related protein-2 inhibits doxorubicin-induced apoptosis mediated through the Akt-mTOR pathway in soleus muscle. Oxid Med Cell Longev 2018:6043064. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Majtnerova P, Rousar T (2018) An overview of apoptosis assays detecting DNA fragmentation. Mol Biol Rep 45:1469–1478. CrossRefPubMedGoogle Scholar
  34. 34.
    Crowley LC (2016) Marfell BJ and Waterhouse NJ (2016) Detection of DNA Fragmentation in Apoptotic Cells by TUNEL. Cold Spring Harb Protoc. CrossRefPubMedGoogle Scholar
  35. 35.
    Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ (2002) Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated Caspase-3 activation pathway. Diabetes 51:1938–1948CrossRefGoogle Scholar
  36. 36.
    Chowdhry MF, Vohra HA, Galinanes M (2007) Diabetes increases apoptosis and necrosis in both ischemic and nonischemic human myocardium: role of caspases and poly-adenosine diphosphate-ribose polymerase. J Thorac Cardiovasc Surg 134:124–131. CrossRefPubMedGoogle Scholar
  37. 37.
    Cai L, Wang Y, Zhou G, Chen T, Song Y, Li X, Kang YJ (2006) Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol 48:1688–1697. CrossRefPubMedGoogle Scholar
  38. 38.
    Fadini GP, Ferraro F, Quaini F, Asahara T, Madeddu P (2014) Concise review: diabetes, the bone marrow niche, and impaired vascular regeneration. Stem Cells Transl Med 3:949–957. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Tattersall RB (1994) The quest for normoglycaemia: a historical perspective. Diabet Med 11:618–635CrossRefGoogle Scholar
  40. 40.
    de Miguel V, Arias A, Paissan A, de Arenaza DP, Pietrani M, Jurado A, Jaen A, Fainstein Day P (2014) Catecholamine-induced myocarditis in pheochromocytoma. Circulation 129:1348–1349. CrossRefPubMedGoogle Scholar
  41. 41.
    Hare JM, Keaney JF Jr, Balligand JL, Loscalzo J, Smith TW, Colucci WS (1995) Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs. J Clin Invest 95:360–366. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Quan KJ, Van Hare GF, Biblo LA, Mackall JA, Carlson MD (2001) Endocardial stimulation of efferent parasympathetic nerves to the atrioventricular node in humans: optimal stimulation sites and the effects of digoxin. J Interv Card Electrophysiol 5:145–152CrossRefGoogle Scholar
  43. 43.
    Li L, Huang C, Ai J, Yan B, Gu H, Ma Z, Li AY, Xinyan S, Harden SW, Hatcher JT, Wurster RD, Cheng ZJ (2010) Structural remodeling of vagal afferent innervation of aortic arch and nucleus ambiguus (NA) projections to cardiac ganglia in a transgenic mouse model of type 1 diabetes (OVE26). J Comput Neurol 518:2771–2793. CrossRefGoogle Scholar
  44. 44.
    Wang N, Chen C, Yang D, Liao Q, Luo H, Wang X, Zhou F, Yang X, Yang J, Zeng C, Wang WE (2017) Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis. Biochim Biophys Acta Mol Basis Dis 1863:2085–2092. CrossRefPubMedGoogle Scholar
  45. 45.
    He JG, Li HR, Han JX, Li BB, Yan D, Li HY, Wang P, Luo Y (2018) GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes. Sci Rep 8:9047. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Xiao J, Pan Y, Li XH, Yang XY, Feng YL, Tan HH, Jiang L, Feng J, Yu XY (2016) Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4. Cell Death Dis. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Goldspink DF, Burniston JG, Ellison GM, Clark WA, Tan LB (2004) Catecholamine-induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways? Exp Physiol 89:407–416. CrossRefPubMedGoogle Scholar
  48. 48.
    Pu J, Zhu S, Zhou D, Zhao L, Yin M, Wang Z, Hong J (2019) Propofol alleviates apoptosis induced by chronic high glucose exposure via regulation of HIF-1alpha in H9c2 cells. Oxid Med Cell Longev 2019:4824035. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Wang X, Pan J, Liu D, Zhang M, Li X, Tian J, Liu M, Jin T, An F (2019) Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway. J Cell Mol Med. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Trotta MC, Maisto R, Alessio N, Hermenean A, D’Amico M, Di Filippo C (2018) The melanocortin MC5R as a new target for treatment of high glucose-induced hypertrophy of the cardiac H9c2 cells. Front Physiol 9:1475. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Yang YY, Sun XT, Li ZX, Chen WY, Wang X, Liang ML, Shi H, Yang ZS, Zeng WT (2018) Protective effect of angiotensin-(1–7) against hyperglycaemia-induced injury in H9c2 cardiomyoblast cells via the PI3KAkt signaling pathway. Int J Mol Med 41:1283–1292. CrossRefPubMedGoogle Scholar
  52. 52.
    Chai W, Chen J, Wang H, Shen J, Ma L, Ma X (2000) The effects of glucose, insulin and oxidized low density lipoprotein on apoptosis in vascular endothelial cells. Chin Med J (Engl) 113:903–906Google Scholar
  53. 53.
    Tavakoli Dargani Z, Singla DK (2019) Embryonic stem cell-derived exosomes inhibit doxorubicin induced TLR4-NLRP3 mediated cell death—pyroptosis. Am J Physiol Heart Circ Physiol. CrossRefPubMedGoogle Scholar
  54. 54.
    Zhang Y, Hu YW, Zheng L, Wang Q (2017) Characteristics and roles of exosomes in cardiovascular disease. DNA Cell Biol 36:202–211. CrossRefPubMedGoogle Scholar
  55. 55.
    Watanabe K, Jose PJ, Rankin SM (2002) Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages. J Immunol 168:1911–1918. CrossRefPubMedGoogle Scholar
  56. 56.
    Kondo M, Murakawa Y, Harashima N, Kobayashi S, Yamaguchi S, Harada M (2009) Roles of proinflammatory cytokines and the Fas/Fas ligand interaction in the pathogenesis of inflammatory myopathies. Immunology 128:e589–e599. CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Chen JJ, Sun Y, Nabel GJ (1998) Regulation of the proinflammatory effects of Fas ligand (CD95L). Science 282:1714–1717. CrossRefPubMedGoogle Scholar
  58. 58.
    Behzadi P, Behzadi E, Ranjbar R (2016) IL-12 family cytokines: general characteristics, pathogenic microorganisms, receptors, and signalling pathways. Acta Microbiol Immunol Hung 63:1–25. CrossRefPubMedGoogle Scholar
  59. 59.
    Xu H, Li J, Zhao Y, Liu D (2017) TNFalpha-induced downregulation of microRNA-186 contributes to apoptosis in rat primary cardiomyocytes. Immunobiology 222:778–784. CrossRefPubMedGoogle Scholar
  60. 60.
    Jarrah AA, Schwarskopf M, Wang ER, LaRocca T, Dhume A, Zhang S, Hadri L, Hajjar RJ, Schecter AD, Tarzami ST (2018) SDF-1 induces TNF-mediated apoptosis in cardiac myocytes. Apoptosis 23:79–91. CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Rehman K, Akash MSH, Liaqat A, Kamal S, Qadir MI, Rasul A (2017) Role of interleukin-6 in development of insulin resistance and type 2 diabetes mellitus. Crit Rev Eukaryot Gene Expr 27:229–236. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Reetish Singla
    • 1
  • Kaley H. Garner
    • 1
  • Mohtashem Samsam
    • 1
  • Zixi Cheng
    • 1
  • Dinender K. Singla
    • 1
    Email author
  1. 1.Burnett School of Biomedical SciencesUniversity of Central Florida College of MedicineOrlandoUSA

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