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Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

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Abstract

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca2+-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.

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Abbreviations

CS:

Canton-special wild-type

CSK:

Cytoskeleton buffer

CyO :

Curly of Oster, multiply inverted second chromosome balancer

DHPR:

Dihydropyridine receptor

FITC:

Fluorescein isothiocyanate

FR:

Frequency

HL3:

Hemolymph like solution 3

MESA:

Maximum entropy spectral analysis

PBS:

Phosphate buffered saline

PIPES:

1,4 Piperazine bis (2-ethanosulfonic acid)

PMSF:

Phenyl methyl sulfonyl fluoride

RI:

Rhythmicity index

SERCA:

Sarco-endoplasmic reticulum calcium ATPase

SR:

Sarcoplasmic reticulum

UAS:

Upstream Activator Sequence

WT:

Wild type

References

  • Asahi M, McKenna E, Kurzydlowski K, Tada M, MacLennan DH (2000) Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP. J Biol Chem 275:15034–15038

    Article  PubMed  CAS  Google Scholar 

  • Ashburner M (1989) Drosophila, a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor

    Google Scholar 

  • Bers D (2002) Calcium and cardiac rhythms: physiological and pathological. Circ Res 90:14–17

    PubMed  CAS  Google Scholar 

  • Bodmer R, Wessels RJ, Johnson E, Dowse H (2004) Heart development and function. Comprehensive molecular insect science. In: Gilbert LI, Iatrou K, Gill S (eds) V 2. Elsevier, London

  • Bour BA, O’Brien MA, Lockwood WL, Goldstein ES, Bodmer R, Taghert PH, Abmayr SM, Nguyen HT (1995) Drosophila MEF2, a transcription factor that is essential for myogenesis. Genes Dev 9:730–741

    Article  PubMed  CAS  Google Scholar 

  • Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415

    PubMed  CAS  Google Scholar 

  • Chatfield C (1980) The analysis of time series. Chapman and Hall, London

    Google Scholar 

  • Chien KR (1999) Stress pathways and heart failure. Cell 98:555–558

    Article  PubMed  CAS  Google Scholar 

  • Clarke DM, Loo TW, Inesi G, MacLennan DH (1989) Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature 339:476–478

    Article  PubMed  CAS  Google Scholar 

  • Curtis NJ, Ringo JM, Dowse HB (1999) Morphology of the pupal heart, adult heart, and associated tissues in the fruit fly, Drosophila melanogaster. J Morphol 240:225–235

    Article  PubMed  CAS  Google Scholar 

  • Dowse H, Ringo J (1989) The search for hidden periodicities in biological time series revisited. J Theor Biol 139:487–515

    Google Scholar 

  • Dowse H, Ringo J, Power J, Johnson E, Kinney K, White L (1995) A congenital heart defect in Drosophila caused by an action-potential mutation. J Neurogenet 10:153–168

    Article  PubMed  CAS  Google Scholar 

  • Drago GA, Colyer J, Lederer WJ (1998) Immunofluorescence localization of SERCA2a and the phosphorylated forms of phospholamban in intact rat cardiac ventricular myocyte. Ann N Y Acad Sci 853:273–279

    Article  PubMed  CAS  Google Scholar 

  • Dulcis D, Levine RB (2003) Innervation of the heart of the adult fruit fly, Drosophila melanogaster. J Comp Neurol 465:560–578

    Article  PubMed  Google Scholar 

  • Einot I, Gabriel K (1975) A study of the power of several methods of multiple comparisons. J Am Stat Assoc 70:574–583

    Article  Google Scholar 

  • Felder E, Protasi F, Hirsch R, Franzini-Armstrong C, Allen PD (2002) Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle. Biophys J 82:3144–3149

    Article  PubMed  CAS  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

    PubMed  CAS  Google Scholar 

  • Franzini-Armstrong C (1999) The sarcoplasmic reticulum and the control of muscle contraction. Faseb J 13(Suppl 2):S266–S270

    PubMed  CAS  Google Scholar 

  • Fyrberg C, Ketchum A, Ball E, Fyrberg E (1998) Characterization of lethal Drosophila melanogaster alpha-actinin mutants. Biochem Genet 36:299–310

    Article  PubMed  CAS  Google Scholar 

  • Gajewski K, Kim Y, Lee YM, Olson EN, Schulz RA (1997) D-mef2 is a target for Tinman activation during Drosophila heart development. Embo J 16:515–522

    Article  PubMed  CAS  Google Scholar 

  • Gu GG, Singh S (1995) Pharmacological analysis of heartbeat in Drosophila. J Neurobiol 28:269–280

    Article  PubMed  CAS  Google Scholar 

  • Hagiwara N, Irisawa H, Kameyama M (1988) Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 395:233–253

    PubMed  CAS  Google Scholar 

  • Jensen H (1977) Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (insecta: diptera). Cell Tissue Res 180:293–302

    Article  PubMed  CAS  Google Scholar 

  • Johnson E, Ringo J, Bray N, Dowse H (1998) Genetic and pharmacological identification of ion channels central to the Drosophila cardiac pacemaker. J Neurogenet 12:1–24

    Article  PubMed  Google Scholar 

  • Johnson E, Ringo J, Dowse H (1997) Modulation of Drosophila heartbeat by neurotransmitters. J Comp Physiol [B] 167:89–97

    CAS  Google Scholar 

  • Johnson E, Ringo J, Dowse H (2000) Native and heterologous neuropeptides are cardioactive in Drosophila melanogaster. J Insect Physiol 46:1229–1236

    Article  PubMed  CAS  Google Scholar 

  • Johnson E, Ringo J, Dowse H (2001) Dynamin, encoded by shibire, is central to cardiac function. J Exp Zool 289:81–89

    Article  PubMed  CAS  Google Scholar 

  • Johnson E, Sherry T, Ringo J, Dowse H (2002) Modulation of the cardiac pacemaker of Drosophila: cellular mechanisms. J Comp Physiol [B] 172:227–236

    CAS  Google Scholar 

  • Lakatta EG, Maltsev VA, Bogdanov KY, Stern MD, Vinogradova TM (2002) Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance. Circ Res 92:e45–e50

    Article  CAS  Google Scholar 

  • Levine JD, Funes P, Dowse HB, Hall JC (2002) Signal analysis of behavioral and molecular cycles. BMC Neurosci 3:1

    Article  PubMed  Google Scholar 

  • MacLennan DH (2000) Ca2+ signaling and muscle disease. Eur J Biochem 267:5291–5297

    Article  PubMed  CAS  Google Scholar 

  • Magyar A, Bakos E, Varadi A (1995) Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca(2+)-ATPase gene. Biochem J 310(Pt 3):757–763

    PubMed  CAS  Google Scholar 

  • Maruyama K, MacLennan DH (1988) Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. Proc Natl Acad Sci USA 85:3314–3318

    Article  PubMed  CAS  Google Scholar 

  • Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J Jr, Kranias EG, Giles WR, Chien KR (1999) Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99:313–322

    Article  PubMed  CAS  Google Scholar 

  • Morgan JP (1991) Abnormal intracellular modulation of calcium as a major cause of cardiac contractile dysfunction. N Engl J Med 325:625–632

    Article  PubMed  CAS  Google Scholar 

  • Papaefthmiou C, Theophilidis G (2001) An in vitro method for recording the electrical activity of the isolated heart of the adult Drosophila melanogaster. In Vitro Cell Dev Biol Anim 37:445–449

    Article  PubMed  CAS  Google Scholar 

  • Park M, Wu X, Golden K, Axelrod JD, Bodmer R (1996) The wingless signaling pathway is directly involved in Drosophila heart development. Dev Biol 177:104–116

    Article  PubMed  CAS  Google Scholar 

  • Periasamy M, Huke S (2001) SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility. J Mol Cell Cardiol 33:1053–1063

    Article  PubMed  CAS  Google Scholar 

  • Peterson BZ, Lee JS, Mulle JG, Wang Y, de Leon M, Yue DT (2000) Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels. Biophys J 78:1906–1920

    PubMed  CAS  Google Scholar 

  • Pogwizd SM, Shclotthauer K, Li L, Weilong Y, Bers DM (2001) Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium–calcium exchange, inward rectifier potassium current, and residual β-adrenergic responsiveness. Circ Res 88:1159–1167

    Article  PubMed  CAS  Google Scholar 

  • Qi M, Shannon TR, Euler DE, Bers DM, Samarel AM (1997) Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy. Am J Physiol 272:H2416–2424

    PubMed  CAS  Google Scholar 

  • Ranganayakulu G, Schulz RA, Olson EN (1996) Wingless signaling induces nautilus expression in the ventral mesoderm of the Drosophila embryo. Dev Biol 176:143–148

    Article  PubMed  CAS  Google Scholar 

  • Ray V, Dowse H (2005) Mutations in and deletions of the Ca2+ channel-encoding gene cacophony, which affect courtship song in Drosophila, have novel effects on heartbeating. J Neurogenet 19:39–56

    Article  PubMed  CAS  Google Scholar 

  • Rigg L, Heath B, Cui Y, Terrar D (2000) Localisation and functional significance of ryanodine receptors during β-adrenoceptor stimulation in the guinea-pig sino-atrial node. Cardiovasc Res 48:254–264

    Article  PubMed  CAS  Google Scholar 

  • Robinow S, White K (1988) The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev Biol 126:294–303

    Article  PubMed  CAS  Google Scholar 

  • Saide JD, Chin-Bow S, Hogan-Sheldon J, Busquets-Turner L, Vigoreaux JO, Valgeirsdottir K, Pardue ML (1989) Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J Cell Biol 109:2157–2167

    Article  PubMed  CAS  Google Scholar 

  • Sullivan K, Scott K, Zuker CS, Rubin GM (2000) The ryanodine receptor is essential for larval development in Drosophila melanogaster. Proc Natl Acad Sci USA 97:5492–5497

    Article  Google Scholar 

  • Sanyal S, Consoulas C, Kuromi H, Basole A, Mukai L, Kidokoro Y, Krishnan KS, Ramaswami M (2005) Analysis of conditional paralytic mutants in Drosophila SERCA reveals novel mechanisms for regulating membrane excitability. Genetics. DOI 10.1534/genetics.104.031930

  • Schwinger RH, Munch G, Bolck B, Karczewski P, Krause EG, Erdmann E (1999) Reduced Ca(2+)-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation. J Mol Cell Cardiol 31:479–491

    Article  PubMed  Google Scholar 

  • Stewart BA, Atwood HL, Renger JJ, Wang J, Wu CF (1994) Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J Comp Physiol [A] 175:179–191

    Article  CAS  Google Scholar 

  • Terrar D, Rigg L (2000) What determines the initiation of the heartbeat? J Physiol 524:316

    Article  PubMed  CAS  Google Scholar 

  • Toyofuku T, Curotto Kurzydlowski K, Narayanan N, MacLennan DH (1994) Identification of Ser38 as the site in cardiac sarcoplasmic reticulum Ca(2+)-ATPase that is phosphorylated by Ca2+/calmodulin-dependent protein kinase. J Biol Chem 269:26492–26496

    PubMed  CAS  Google Scholar 

  • Toyofuku T, Kurzydlowski K, Tada M, MacLennan DH (1993) Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban. J Biol Chem 268:2809–2815

    PubMed  CAS  Google Scholar 

  • Trafford AW, Diaz ME, O’Neill SC, Eisner DA (2002) Integrative analysis of calcium signalling in cardiac muscle. Front Biosci 7:d843–d852

    Article  PubMed  CAS  Google Scholar 

  • van Straaten M, Goulding D, Kolmerer B, Labeit S, Clayton J, Leonard K, Bullard B (1999) Association of kettin with actin in the Z-disc of insect flight muscle. J Mol Biol 285:1549–1562

    Article  PubMed  Google Scholar 

  • Vigoreaux JO, Saide JD, Pardue ML (1991) Structurally different Drosophila striated muscles utilize distinct variants of Z-band-associated proteins. J Muscle Res Cell Motil 12:340–354

    Article  PubMed  CAS  Google Scholar 

  • Wu X, Golden K, Bodmer R (1995) Heart development in Drosophila requires the segment polarity gene wingless. Dev Biol 169:619–628

    Article  PubMed  CAS  Google Scholar 

  • Yoshihara M, Ensminger AW, Littleton JT (2001) Neurobiology and the Drosophila genome. Funct Integr Genomics 1:235–240

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Chen D, Wheatly MG (2000) Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/endoplasmic reticulum Ca(2+)-ATPase. J Exp Biol 203(Pt 22):3411–3423

    PubMed  CAS  Google Scholar 

  • Zhong L, Inesi G (1998) Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition. J Biol Chem 273:12994–12998

    Article  PubMed  CAS  Google Scholar 

  • Zwaal RR, Van Baelen K, Groenen JT, van Geel A, Rottiers V, Kaletta T, Dode L, Raeymaekers L, Wuytack F, Bogaert T (2001) The sarco-endoplasmic reticulum Ca2+ ATPase is required for development and muscle function in Caenorhabditis elegans. J Biol Chem 276:43557–43563

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Carol Gregorio and Davide Dulcis for useful discussions; Patty Jansma and Carl Boswell for expert assistance with confocal microscopy on instruments belonging to the ARL Division of Neurobiology and the MCB department respectively; Christos Consoulas for help with extracellular recordings. The work was funded by grants to MR from the NIH (NS34889, KO2-NS02001 and DA13338) and awards from the McKnight and Alfred P Sloan Foundations. SS is supported in part by NIH grant T32 CA09213. All experiments were performed in the USA and were conducted in accordance with all relevant current laws and statutes thereof.

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Authors and Affiliations

  1. Molecular and Cellular Biology Department and ARL Division of Neurobiology, University of Arizona, 1007 E. Lowell Street, Life Sciences South, AZ, Tucson, USA

    Subhabrata Sanyal & Mani Ramaswami

  2. Department of Biological Sciences, University of Maine, ME, Orono, USA

    Tricia Jennings & Harold Dowse

  3. Department of Mathematics and Statistics, University of Maine, ME, Orono, USA

    Harold Dowse

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  1. Subhabrata Sanyal
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  2. Tricia Jennings
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  3. Harold Dowse
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  4. Mani Ramaswami
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Correspondence to Subhabrata Sanyal.

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Communicated by H.V. Carey

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Sanyal, S., Jennings, T., Dowse, H. et al. Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila . J Comp Physiol B 176, 253–263 (2006). https://doi.org/10.1007/s00360-005-0046-7

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