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Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2

Journal of Muscle Research and Cell Motility volume 36, pages 205–214 (2015)Cite this article

Abstract

In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca2+ homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 ADA/ADA) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca2+ sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344–1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 ADA/ADA hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 ADA/ADA mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.

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References

  • Arnaiz-Cot JJ, Damon BJ, Zhang XH, Cleemann L, Yamaguchi N, Meissner GW et al (2013) Cardiac calcium signaling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor. J Physiol 591:4287–4299

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Baddeley D, Jayasinghe ID, Lam L, Rossberger S, Cannell MB, Soeller C (2009) Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes. Proc Natl Acad Sci U S A 106:22275–22280

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Balshaw DM, Yamaguchi N, Meissner G (2002) Modulation of intracellular calcium-release channels by calmodulin. J Membr Biol 185:1–8

    Article  CAS  PubMed  Google Scholar 

  • Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415:198–205

    Article  CAS  PubMed  Google Scholar 

  • Bers DM (2008) Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 70:23–49

    Article  CAS  PubMed  Google Scholar 

  • Bers DM, Perez-Reyes E (1999) Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res 42:339–360

    Article  CAS  PubMed  Google Scholar 

  • Bers DM, Eisner DA, Valdivia HH (2003) Sarcoplasmic reticulum Ca2 + and heart failure: roles of diastolic leak and Ca2+ transport. Circ Res 93:487–490

    Article  CAS  PubMed  Google Scholar 

  • Boncompagni S, Loy RE, Dirksen RT, Franzini-Armstrong C (2010) The I4895T mutation in the type 1 ryanodine receptor induces fiber-type specific alterations in skeletal muscle that mimic premature aging. Aging Cell 9:958–970

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Brette F, Orchard C (2007) Resurgence of cardiac t-tubule research. Physiology (Bethesda) 22:167–173

    Article  CAS  Google Scholar 

  • Chakraborty A, Pasek DA, Huang TQ, Gomez AC, Yamaguchi N, Anderson ME, Meissner G (2014) Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor. PLoS One 9:e104338

    Article  PubMed Central  PubMed  Google Scholar 

  • Chu A, Sumbilla C, Inesi G, Jay SD, Campbell KP (1990) Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle. Biochemistry 29:5899–5905

    Article  CAS  PubMed  Google Scholar 

  • Eisner DA, Choi HS, Diaz ME, O’Neill SC, Trafford AW (2008) Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87:1087–1094

    Article  Google Scholar 

  • Fabiato A (1983) Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 245:C1–C14

    CAS  PubMed  Google Scholar 

  • Fernández-Velasco M, Gómez AM, Benitah J-P, Neco P (2012) Ryanodine receptor channelopathies: the new kid in the arrhythmia neighborhood. In: Yamada PT (ed) Tachycardia. ISBN: 978-953-51-0413-1

  • Fill M, Copello JA (2002) Ryanodine receptor calcium release channels. Physiol Rev 82:893–922

    Article  CAS  PubMed  Google Scholar 

  • Franzini-Armstrong C (2010) RyRs: their disposition, frequency, and relationships with other proteins of calcium release units. In: Serysheva I (ed) Structure and function of calcium release channels. Current Topics in Membranes, vol 66. Elsevier Inc, pp 1–26

  • Franzini-Armstrong C, Jorgensen AO (1994) Structure and development of E-C coupling units in skeletal muscle. Annu Rev Physiol 56:509–534

    Article  CAS  PubMed  Google Scholar 

  • Franzini-Armstrong C, Protasi F, Ramesh V (1999) Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles. Biophys J 77:1528–1539

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gorelik J, Wright PT, Lyon AR, Harding SE (2013) Spatial control of the betaAR system in heart failure: the transverse tubule and beyond. Cardiovasc Res 98:216–224

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Guo A, Zhang C, Wei S, Chen B, Song LS (2013) Emerging mechanisms of T-tubule remodelling in heart failure. Cardiovasc Res 98:204–215

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Guo A, Zhang X, Iyer VR, Chen B, Zhang C, Kutschke WJ, Weiss RM, Franzini-Armstrong C, Song LS (2014) Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress. Proc Natl Acad Sci U S A 111:12240–12245

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hayashi T, Martone ME, Yu Z, Thor A, Doi M, Holst MJ, Ellisman MH, Hoshijima M (2009) Three-dimensional electron microscopy reveals new details of membrane systems for Ca2+ signaling in the heart. J Cell Sci 122:1005–1013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hom J, Sheu SS (2009) Morphological dynamics of mitochondria—a special emphasis on cardiac muscle cells. J Mol Cell Cardiol 46:811–820

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hom J, Yu T, Yoon Y, Porter G, Sheu SS (2010) Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes. Biochim Biophys Acta 1797:913–921

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Houser SR (2014) Role of RyR2 phosphorylation in heart failure and arrhythmias: protein kinase A-mediated hyperphosphorylation of the ryanodine receptor at serine 2808 does not alter cardiac contractility or cause heart failure and arrhythmias. Circ Res 114:1320–1327

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Huang X, Sun L, Ji S, Zhao T, Zhang W, Xu J et al (2013) Kissing and nanotunneling mediate intermitochondrial communication in the heart. Proc Natl Acad Sci U S A 110:2846–2851

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ibrahim M, Terracciano CM (2013) Reversibility of T-tubule remodelling in heart failure: mechanical load as a dynamic regulator of the T-tubules. Cardiovasc Res 98:225–232

    Article  CAS  PubMed  Google Scholar 

  • Lai FA, Liu QY, Xu L, El-Hashem A, Kramarcy NR, Sealock R et al (1992) Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle. Am J Physiol 263:C365–C372

    CAS  PubMed  Google Scholar 

  • MacLennan DH, Kranias EG (2003) Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4:566–577

    Article  CAS  PubMed  Google Scholar 

  • Marx SO, Marks AR (2013) Dysfunctional ryanodine receptors in the heart: new insights into complex cardiovascular diseases. J Mol Cell Cardiol 58:225–231

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Meissner G, Henderson JS (1987) Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem 262:3065–3073

    CAS  PubMed  Google Scholar 

  • Newham DJ, McPhail G, Mills KR, Edwards RH (1983) Ultrastructural changes after concentric and eccentric contractions of human muscle. J Neurol Sci 61:109–122

    Article  CAS  PubMed  Google Scholar 

  • Nixon GF, Mignery GA, Somlyo AV (1994) Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J Muscle Res Cell Motil 15:682–700

    Article  CAS  PubMed  Google Scholar 

  • Pieske B, Maier LS, Bers DM, Hasenfuss G (1999) Ca2 + handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. Circ Res 85:38–46

    Article  CAS  PubMed  Google Scholar 

  • Rhoads AR, Friedberg F (1997) Sequence motifs for calmodulin recognition. FASEB J 11:331–340

    CAS  PubMed  Google Scholar 

  • Schlotthauer K, Schattmann J, Bers DM, Maier LS, Schutt U, Minami K et al (1998) Frequency-dependent changes in contribution of SR Ca2+ to Ca2+ transients in failing human myocardium assessed with ryanodine. J Mol Cell Cardiol 30:1285–1294

    Article  CAS  PubMed  Google Scholar 

  • Scriven DR, Asghari P, Moore ED (2013) Microarchitecture of the dyad. Cardiovasc Res 98:169–176

    Article  CAS  PubMed  Google Scholar 

  • Sipido KR, Cheng H (2013) T-tubules and ryanodine receptor microdomains: on the road to translation. Cardiovasc Res 98:159–161

    Article  CAS  PubMed  Google Scholar 

  • Smith JS, Rousseau E, Meissner G (1989) Calmodulin modulation of single sarcoplasmic reticulum Ca2+-release channels from cardiac and skeletal muscle. Circ Res 64:352–359

    Article  CAS  PubMed  Google Scholar 

  • Soeller C, Baddeley D (2013) Super-resolution imaging of EC coupling protein distribution in the heart. J Mol Cell Cardiol 58:32–40

    Article  CAS  PubMed  Google Scholar 

  • Song LS, Sobie EA, McCulle S, Lederer WJ, Balke CW, Cheng H (2006) Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA 103:4305–4310

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tijskens P, Jones LR, Franzini-Armstrong C (2003) Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins. J Mol Cell Cardiol 35:961–974

    Article  CAS  PubMed  Google Scholar 

  • van Oort RJ, Garbino A, Wang W, Dixit SS, Landstrom AP, Gaur N, De Almeida AC, Skapura DG, Rudy Y, Burns AR, Ackerman MJ, Wehrens XH (2011) Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice. Circulation 123:979–988

    Article  PubMed Central  PubMed  Google Scholar 

  • Vatner DE, Sato N, Kiuchi K, Shannon RP, Vatner SF (1994) Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure. Circulation 90:1423–1430

    Article  CAS  PubMed  Google Scholar 

  • Wehrens XH, Lehnart SE, Marks AR (2005) Intracellular calcium release and cardiac disease. Annu Rev Physiol 67:69–98

    Article  CAS  PubMed  Google Scholar 

  • Xu L, Meissner G (2004) Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor). Biophys J 86:797–804

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yamaguchi N, Takahashi N, Xu L, Smithies O, Meissner G (2007) Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca release channel. J Clin Invest 117:1344–1353

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yamaguchi N, Chakraborty A, Huang TQ, Xu L, Gomez AC, Pasek DA, Meissner G (2013) Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1. Am J Physiol Heart Circ Physiol 305:H86–H94

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yang Y, Guo T, Oda T, Chakraborty A, Chen L, Uchinoumi H et al (2014) Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure. Circ Res 114:295–306

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhang L, Franzini-Armstrong C, Ramesh V, Jones LR (2001) Structural alterations in cardiac calcium release units resulting from overexpression of junctin. J Mol Cell Cardiol 33:233–247

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by NIH HL 48093 to CFA and Cardiac Center Grant, Children's Hospital of Philadelphia 27115-526326000 to VRI.

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Affiliations

  1. Department of Cell and Developmental Biology, Perelmann School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Manuela Lavorato, Stefano Perni & Clara Franzini-Armstrong

  2. Department of Biochem/Biophys, University of North Carolina, Chapel Hill, NC, 27599, USA

    Tai-Qin Huang & Gerhard Meissner

  3. Division of Cardiology, Children Hospital of Philadelphia, Philadelphia, PA, 19104, USA

    Venkat Ramesh Iyer

Authors
  1. Manuela Lavorato
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  2. Tai-Qin Huang
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  3. Venkat Ramesh Iyer
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  4. Stefano Perni
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  5. Gerhard Meissner
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  6. Clara Franzini-Armstrong
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Correspondence to Manuela Lavorato.

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Lavorato, M., Huang, TQ., Iyer, V.R. et al. Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2. J Muscle Res Cell Motil 36, 205–214 (2015). https://doi.org/10.1007/s10974-015-9405-5

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  • DOI: https://doi.org/10.1007/s10974-015-9405-5

Keywords

  • Calmodulin
  • Cardiac ryanodine receptor
  • Dyads
  • Z line streaming
  • Cardiac myocytes