Adiponectin secretion from cardiomyocytes produces canonical multimers and partial co-localization with calsequestrin in junctional SR


Adiponectin (ADN) is an abundant protein in serum, secreted by adipocytes, that acts as a signal for fat metabolism. It is marked by a complex molecular structure that results from processes within the secretory pathway, producing a canonical set of multimers. ADN may also be secreted from cardiomyocytes, where a unique sarcomeric endoplasmic/sarcoplasmic reticulum (ER/SR) substructure has been characterized primarily for its Ca handling. We expressed ADN in cultured primary adult cardiomyocytes and nonmuscle (COS) cells. After 48 h of ADN expression by adenovirus treatment, roughly half of synthesized ADN was secreted from cardiomyocytes, and half was still in-transit within inner membrane compartments, similar to COS cells. Cardiomyocytes and COS cells both produced ADN in the three canonical forms: trimers, hexamers, and 18-mers. Higher rates of secretion occurred for higher-molecular weight multimers, especially 18-mers. The highest levels of ADN protein, whether in transit or secreted, were present as trimers and hexamers. In nonmuscle cell lines, ADN trafficked through ER and Golgi compartments as expected. In contrast, ADN in primary adult cardiomyocytes populated ER/SR tubules along the edges of sarcomeres that emanated from nuclear surfaces. Prominent co-localization of ADN occurred with calsequestrin, a marker of junctional SR, the Ca2+-release compartment of the cell. The early steps in ADN trafficking re-trace those recently described for newly made junctional SR proteins, involving a nuclear envelope (NE) translocation into SR tubules that are oriented along sarcolemmal transverse (T)-tubules (NEST pathway).

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Endoplasmic reticulum


Junctional sarcoplasmic reticulum




Multiplicity of infection


Plaque-forming units


High molecular weight


Medium molecular weight


Low molecular weight


Wild-type (human) adiponectin


Flag-tagged adiponectin


Native polyacrylamide gel electrophoresis


Brefeldin A

NEST pathway:

Nuclear envelope to SR along T-tubules pathway


SR tubules that traffic newly made proteins roughly aligned with the sarcomeric z-line and T-tubules


  1. 1.

    Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF (1995) A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270:26746–26749

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Hu E, Liang P, Spiegelman BM (1996) AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697–10703

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K (1996) cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). Biochem Biophys Res Commun 221:286–289.

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Fang H, Judd RL (2018) Adiponectin regulation and function. Compr Physiol 8:1031–1063.

    Article  PubMed  Google Scholar 

  5. 5.

    Holland WL, Miller RA, Wang ZV, Sun K, Barth BM, Bui HH, Davis KE, Bikman BT, Halberg N, Rutkowski JM, Wade MR, Tenorio VM, Kuo MS, Brozinick JT, Zhang BB, Birnbaum MJ, Summers SA, Scherer PE (2011) Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin. Nat Med 17:55–63.

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T (2003) Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352–40363.

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Richards AA, Stephens T, Charlton HK, Jones A, Macdonald GA, Prins JB, Whitehead JP (2006) Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications. Mol Endocrinol 20:1673–1687.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Liu M, Liu F (2014) Regulation of adiponectin multimerization, signaling and function. Best Pract Res Clin Endocrinol Metab 28:25–31.

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Schraw T, Wang ZV, Halberg N, Hawkins M, Scherer PE (2008) Plasma adiponectin complexes have distinct biochemical characteristics. Endocrinology 149:2270–2282.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Tsao TS, Tomas E, Murrey HE, Hug C, Lee DH, Ruderman NB, Heuser JE, Lodish HF (2003) Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways. J Biol Chem 278:50810–50817.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Suzuki S, Wilson-Kubalek EM, Wert D, Tsao TS, Lee DH (2007) The oligomeric structure of high molecular weight adiponectin. FEBS Lett 581:809–814.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T, Engel J, Brownlee M, Scherer PE (2003) Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications fpr metabolic regulation and bioactivity. J Biol Chem 278:9073–9085.

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Wang Y, Xu A, Knight C, Xu LY, Cooper GJ (2002) Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J Biol Chem 277:19521–19529.

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Amin RH, Mathews ST, Alli A, Leff T (2010) Endogenously produced adiponectin protects cardiomyocytes from hypertrophy by a PPARgamma-dependent autocrine mechanism. Am J Physiol Heart Circ Physiol 299:H690–H698.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Ding G, Qin Q, He N, Francis-David SC, Hou J, Liu J, Ricks E, Yang Q (2007) Adiponectin and its receptors are expressed in adult ventricular cardiomyocytes and upregulated by activation of peroxisome proliferator-activated receptor gamma. J Mol Cell Cardiol 43:73–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Shibata R, Ouchi N, Ito M, Kihara S, Shiojima I, Pimentel DR, Kumada M, Sato K, Schiekofer S, Ohashi K, Funahashi T, Colucci WS, Walsh K (2004) Adiponectin-mediated modulation of hypertrophic signals in the heart. Nat Med 10:1384–1389.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Guo B, Li Y, Jin X, Liu S, Miao C (2017) Nitric oxide/cyclic GMP pathway mediates the endothelin-1-upregulation of adiponectin expression in rat cardiomyocytes. Biomed Rep 7:267–271.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    McFarland TP, Milstein ML, Cala SE (2010) Rough endoplasmic reticulum to junctional sarcoplasmic reticulum trafficking of calsequestrin in adult cardiomyocytes. J Mol Cell Cardiol:556–564

  19. 19.

    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hendricks GL IIIrd, Hadley JA, Krzysik-Walker SM, Prabhu KS, Vasilatos-Younken R, Ramachandran R (2009) Unique profile of chicken adiponectin, a predominantly heavy molecular weight multimer, and relationship to visceral adiposity. Endocrinology 150:3092–3100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Lippincott-Schwartz J, Yuan LC, Bonifacino JS, Klausner RD (1989) Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56:801–813

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116:1071–1080

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Milstein ML, Houle TD, Cala SE (2009) Calsequestrin isoforms localize to different ER subcompartments: evidence for polymer and heteropolymer-dependent localization. Exp Cell Res 315:523–534.

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Gomez-Navarro N, Miller E (2016) Protein sorting at the ER-Golgi interface. J Cell Biol 215:769–778.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Lippincott-Schwartz J, Roberts TH, Hirschberg K (2000) Secretory protein trafficking and organelle dynamics in living cells. Annu Rev Cell Dev Biol 16:557–589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Xie L, Boyle D, Sanford D, Scherer PE, Pessin JE, Mora S (2006) Intracellular trafficking and secretion of adiponectin is dependent on GGA-coated vesicles. J Biol Chem 281:7253–7259.

    Article  CAS  PubMed  Google Scholar 

  28. 28.

    Sleiman NH, McFarland TP, Jones LR, Cala SE (2015) Transitions of protein traffic from cardiac ER to junctional SR. J Mol Cell Cardiol 29:35–45

    Google Scholar 

  29. 29.

    Jorgensen AO, Shen AC, Daly P, MacLennan DH (1982) Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle. J Cell Biol 93:883–892

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Sommer JR, Jennings RB (1986) Ultrastructure of cardiac muscle. In: Fozzard HA (ed) The heart and cardiovascular system. Raven Press, New York, pp 61–100

    Google Scholar 

  31. 31.

    Cala SE, Jones LR (1983) Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl- sepharose. J Biol Chem 258:11932–11936

    CAS  PubMed  Google Scholar 

  32. 32.

    Mao X, Kikani CK, Riojas RA, Langlais P, Wang L, Ramos FJ, Fang Q, Christ-Roberts CY, Hong JY, Kim RY, Liu F, Dong LQ (2006) APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nat Cell Biol 8:516–523.

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T (2003) Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762–769.

    Article  CAS  PubMed  Google Scholar 

  34. 34.

    van Andel M, Heijboer AC, Drent ML (2018) Adiponectin and Its Isoforms in Pathophysiology. Adv Clin Chem 85:115–147.

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Wang ZV, Scherer PE (2016) Adiponectin, the past two decades. J Mol Cell Biol 8:93–100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Nilsson I, Kelleher DJ, Miao Y, Shao Y, Kreibich G, Gilmore R, von Heijne G, Johnson AE (2003) Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol 161:715–725.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Nikonov AV, Hauri HP, Lauring B, Kreibich G (2007) Climp-63-mediated binding of microtubules to the ER affects the lateral mobility of translocon complexes. J Cell Sci 120:2248–2258.

    Article  CAS  PubMed  Google Scholar 

  38. 38.

    Kaisto T, Metsikko K (2003) Distribution of the endoplasmic reticulum and its relationship with the sarcoplasmic reticulum in skeletal myofibers. Exp Cell Res 289:47–57

    Article  CAS  PubMed  Google Scholar 

  39. 39.

    Zhang L, Kelley J, Schmeisser G, Kobayashi YM, Jones LR (1997) Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem 272:23389–23397

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    Scriven DR, Dan P, Moore ED (2000) Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J 79:2682–2691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Sommer JR, Waugh RA (1976) The ultrastructure of the mammalian cardiac muscle cell–with special emphasis on the tubular membrane systems. A review. Am J Pathol 82:192–232

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Bers DM, Shannon TR (2013) Calcium movements inside the sarcoplasmic reticulum of cardiac myocytes. J Mol Cell Cardiol 58:59–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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This work was supported by an Incubator Grant 1-77311 from the Office of Vice President for Research, Wayne State University.

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Correspondence to Steven Cala.

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Solarewicz, J., Manly, A., Kokoszka, S. et al. Adiponectin secretion from cardiomyocytes produces canonical multimers and partial co-localization with calsequestrin in junctional SR. Mol Cell Biochem 457, 201–214 (2019).

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  • Adiponectin
  • Calsequestrin
  • Trafficking
  • Cardiomyocyte
  • Junctional SR
  • NEST