Dyssynchrony of Ca2+ release from the sarcoplasmic reticulum as subcellular mechanism of cardiac contractile dysfunction

Journal of Molecular and Cellular Cardiology

Volumn 50, Issue 3, 2011, Pages 390-400

  Heinzel, Frank R ^b   MacQuaide, Niall ^a   Biesmans, Liesbeth ^a   Sipido, Karin ^a  

^a UNIVERSITY OF LEUVEN   (Belgium)

^b MEDICAL UNIVERSITY OF GRAZ   (Austria)

Author keywords

Calcium release; Dyssynchrony; Heart failure; Ryanodine receptor; Sarcoplasmic reticulum

Indexed keywords

CALCIUM CHANNEL L TYPE; CALCIUM ION; RYANODINE RECEPTOR; CALCIUM;

ACTION POTENTIAL; CALCIUM TRANSPORT; CHANNEL GATING; DIASTOLE; DISEASE COURSE; HEART ARRHYTHMIA; HEART CONTRACTION; HEART FAILURE; HEART MUSCLE CELL; HEART VENTRICLE HYPERTROPHY; HISTOPATHOLOGY; HUMAN; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION; PROTEIN FUNCTION; PROTEIN PROTEIN INTERACTION; REVIEW; SARCOPLASMIC RETICULUM; TRANSVERSE TUBULAR SYSTEM; ANIMAL; METABOLISM; PATHOPHYSIOLOGY; PHYSIOLOGY;

ANIMALS; ARRHYTHMIAS, CARDIAC; CALCIUM; HUMANS; MYOCARDIAL CONTRACTION; SARCOPLASMIC RETICULUM;

EID: 79551604054     PISSN: 00222828     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.yjmcc.2010.11.008     Document Type: Review

References (98)

1. Spragg D.D., Kass D.A. Pathobiology of left ventricular dyssynchrony and resynchronization. Prog Cardiovasc Dis 2006, 49:26-41.
2. Yu C.M., Chan Y.S., Zhang Q., Yip G.W., Chan C.K., Kum L.C., et al. Benefits of cardiac resynchronization therapy for heart failure patients with narrow QRS complexes and coexisting systolic asynchrony by echocardiography. J Am Coll Cardiol 2006, 48:2251-2257.
3. Kort A.A., Lakatta E.G. Spontaneous sarcoplasmic reticulum calcium release in rat and rabbit cardiac muscle: relation to transient and rested-state twitch tension. Circ Res 1988, 63:969-979.
4. Capogrossi M.C., Stern M.D., Spurgeon H.A., Lakatta E.G. Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+-dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium. J Gen Physiol 1988, 91:133-155.
5. Schlotthauer K., Bers D.M. Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials. Circ Res 2000, 87:774-780.
6. Houser S.R. When does spontaneous sarcoplasmic reticulum CA(2+) release cause a triggered arrythmia? Cellular versus tissue requirements. Circ Res 2000, 87:725-727.
7. Spitzer K.W., Pollard A.E., Yang L., Zaniboni M., Cordeiro J.M., Huelsing D.J. Cell-to-cell electrical interactions during early and late repolarization. J Cardiovasc Electrophysiol 2006, 17(Suppl 1):S8-S14.
8. Sato D., Xie L.H., Sovari A.A., Tran D.X., Morita N., Xie F., et al. Synchronization of chaotic early afterdepolarizations in the genesis of cardiac arrhythmias. Proc Natl Acad Sci USA 2009, 106:2983-2988.
9. Wasserstrom J.A., Shiferaw Y., Chen W., Ramakrishna S., Patel H., Kelly J.E., et al. Variability in timing of spontaneous calcium release in the intact rat heart is determined by the time course of sarcoplasmic reticulum calcium load. Circ Res 2010, 107(9):1117-1126.
10. Franzini-Armstrong C., Protasi F., Ramesh V. Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles. Biophys J 1999, 77:1528-1539.
11. Brochet D.X., Yang D., Di M.A., Lederer W.J., Franzini-Armstrong C., Cheng H. Ca2+ blinks: rapid nanoscopic store calcium signaling. Proc Natl Acad Sci USA 2005, 102:3099-3104.
12. Stern M.D., Pizarro G., Rios E. Local control model of excitation-contraction coupling in skeletal muscle. J Gen Physiol 1997, 110:415-440.
13. Bers D.M., Stiffel V.M. Ratio of ryanodine to dihydropyridine receptors in cardiac and skeletal muscle and implications for E-C coupling. Am J Physiol 1993, 264:C1587-C1593.
14. Inoue M., Bridge J.H. Variability in couplon size in rabbit ventricular myocytes. Biophys J 2005, 89:3102-3110.
15. Baddeley D., Jayasinghe I.D., Lam L., Rossberger S., Cannell M.B., Soeller C. Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes. Proc Natl Acad Sci USA 2009, 106:22275-22280.
16. Hayashi T., Martone M.E., Yu Z., Thor A., Doi M., Holst M.J., et al. Three-dimensional electron microscopy reveals new details of membrane systems for Ca2+ signaling in the heart. J Cell Sci 2009, 122:1005-1013.
17. Polakova E., Zahradnikova A., Pavelkova J., Zahradnik I., Zahradnikova A. Local calcium release activation by DHPR calcium channel openings in rat cardiac myocytes. J Physiol 2008, 586:3839-3854.
18. Bridge J.H., Torres N.S., Sobie E.A. New insights into the structure and function of couplons. J Physiol 2008, 586:3735.
19. Cheng H., Lederer W.J., Cannell M.B. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 1993, 262:740-744.
20. Lopez-Lopez J.R., Shacklock P.S., Balke C.W., Wier W.G. Local, stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy. J Physiol 1994, 480(Pt 1):21-29.
21. Lederer J.W., Gmosim S., Song L.S., Sobie E.A., Hartmann H., Lukyanenko V. Ca2+ sparks in heart muscle. J Muscle Res Cell Motil 2004, 25:602-603.
22. Xu L., Mann G., Meissner G. Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions. Circ Res 1996, 79:1100-1109.
23. Subramanian S., Viatchenko-Karpinski S., Lukyanenko V., Gyorke S., Wiesner T.F. Underlying mechanisms of symmetric calcium wave propagation in rat ventricular myocytes. Biophys J 2001, 80:1-11.
24. Gyorke S., Gyorke I., Lukyanenko V., Terentyev D., Viatchenko-Karpinski S., Wiesner T.F. Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 2002, 7:d1454-d1463.
25. Song L.S., Sham J.S., Stern M.D., Lakatta E.G., Cheng H. Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes. J Physiol 1998, 512(Pt 3):677-691.
26. Altamirano J., Bers D.M. Voltage dependence of cardiac excitation-contraction coupling: unitary Ca2+ current amplitude and open channel probability. Circ Res 2007, 101:590-597.
27. Lakatta E.G. Beyond Bowditch: the convergence of cardiac chronotropy and inotropy. Cell Calcium 2004, 35:629-642.
28. Haddock P.S., Coetzee W.A., Cho E., Porter L., Katoh H., Bers D.M., et al. Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes. Circ Res 1999, 85:415-427.
29. Blatter L.A., Kockskamper J., Sheehan K.A., Zima A.V., Huser J., Lipsius S.L. Local calcium gradients during excitation-contraction coupling and alternans in atrial myocytes. J Physiol 2003, 546:19-31.
30. Woo S.H., Cleemann L., Morad M. Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+ release in rat atrial myocytes. Circ Res 2003, 92:e1-e11.
31. Lenaerts I., Bito V., Heinzel F.R., Driesen R.B., Holemans P., D'hooge J., et al. Ultrastructural and functional remodeling of the coupling between Ca2+ influx and sarcoplasmic reticulum Ca2+ release in right atrial myocytes from experimental persistent atrial fibrillation. Circ Res 2009, 105:876-885.
32. Dibb K.M., Clarke J.D., Horn M.A., Richards M.A., Graham H.K., Eisner D.A., et al. Characterization of an extensive transverse tubular network in sheep atrial myocytes and its depletion in heart failure. Circ Heart Fail 2009, 2:482-489.
33. Huser J., Lipsius S.L., Blatter L.A. Calcium gradients during excitation-contraction coupling in cat atrial myocytes. J Physiol 1996, 494(Pt 3):641-651.
34. Soeller C., Cannell M.B. Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ Res 1999, 84:266-275.
35. Heinzel F.R., Bito V., Volders P.G., Antoons G., Mubagwa K., Sipido K.R. Spatial and temporal inhomogeneities during Ca2+ release from the sarcoplasmic reticulum in pig ventricular myocytes. Circ Res 2002, 91:1023-1030.
36. Song L.S., Sobie E.A., McCulle S., Lederer W.J., Balke C.W., Cheng H. Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA 2006, 103:4305-4310.
37. Brillantes A.B., Ondrias K., Scott A., Kobrinsky E., Ondriasova E., Moschella M.C., et al. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 1994, 77:513-523.
38. Marx S.O., Gaburjakova J., Gaburjakova M., Henrikson C., Ondrias K., Marks A.R. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ Res 2001, 88:1151-1158.
39. Marx S.O., Reiken S., Hisamatsu Y., Jayaraman T., Burkhoff D., Rosemblit N., et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 2000, 101:365-376.
40. Loughrey C.M., Seidler T., Miller S.L., Prestle J., MacEachern K.E., Reynolds D.F., et al. Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes. J Physiol 2004, 556:919-934.
41. Louch W.E., Bito V., Heinzel F.R., Macianskiene R., Vanhaecke J., Flameng W., et al. Reduced synchrony of Ca2+ release with loss of T-tubules-a comparison to Ca2+ release in human failing cardiomyocytes. Cardiovasc Res 2004, 62:63-73.
42. Gyorke S., Terentyev D. Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc Res 2008, 77:245-255.
43. Wang W., Cleemann L., Jones L.R., Morad M. Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes. J Physiol 2000, 524(Pt 2):399-414.
44. Jones L.R., Suzuki Y.J., Wang W., Kobayashi Y.M., Ramesh V., Franzini-Armstrong C., et al. Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J Clin Invest 1998, 101:1385-1393.
45. Shacklock P.S., Wier W.G., Balke C.W. Local Ca2+ transients (Ca2+ sparks) originate at transverse tubules in rat heart cells. J Physiol 1995, 487(Pt 3):601-608.
46. Lukyanenko V., Ziman A., Lukyanenko A., Salnikov V., Lederer W.J. Functional groups of ryanodine receptors in rat ventricular cells. J Physiol 2007, 583:251-269.
47. Dolber P.C., Sommer J.R. Corbular sarcoplasmic reticulum of rabbit cardiac muscle. J Ultrastruct Res 1984, 87:190-196.
48. Jorgensen A.O., Shen A.C., Arnold W., McPherson P.S., Campbell K.P. The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle. J Cell Biol 1993, 120:969-980.
49. Sobie E.A., Guatimosim S., Gomez-Viquez L., Song L.S., Hartmann H., Saleet J.M., et al. The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice. Prog Biophys Mol Biol 2006, 90:172-185.
50. Cannell M.B., Crossman D.J., Soeller C. Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure. J Muscle Res Cell Motil 2006, 27:297-306.
51. Li Y., Kranias E.G., Mignery G.A., Bers D.M. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes. Circ Res 2002, 90:309-316.
52. Song L.S., Wang S.Q., Xiao R.P., Spurgeon H., Lakatta E.G., Cheng H. beta-Adrenergic stimulation synchronizes intracellular Ca(2+) release during excitation-contraction coupling in cardiac myocytes. Circ Res 2001, 88:794-801.
53. Zhou P., Zhao Y.T., Guo Y.B., Xu S.M., Bai S.H., Lakatta E.G., et al. Beta-adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca2+ channel. Proc Natl Acad Sci USA 2009, 106:18028-18033.
54. Guo T., Zhang T., Mestril R., Bers D.M. Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res 2006, 99:398-406.
55. Curran J., Hinton M.J., Rios E., Bers D.M., Shannon T.R. Beta-adrenergic enhancement of sarcoplasmic reticulum calcium leak in cardiac myocytes is mediated by calcium/calmodulin-dependent protein kinase. Circ Res 2007, 100:391-398.
56. Ogrodnik J., Niggli E. Increased Ca(2+) leak and spatiotemporal coherence of Ca(2+) release in cardiomyocytes during beta-adrenergic stimulation. J Physiol 2010, 588:225-242.
57. Brette F., Rodriguez P., Komukai K., Colyer J., Orchard C.H. beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules. J Mol Cell Cardiol 2004, 36:265-275.
58. Goldhaber J.I., Lamp S.T., Walter D.O., Garfinkel A., Fukumoto G.H., Weiss J.N. Local regulation of the threshold for calcium sparks in rat ventricular myocytes: role of sodium-calcium exchange. J Physiol 1999, 520(Pt 2):431-438.
59. Litwin S.E., Li J., Bridge J.H. Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J 1998, 75:359-371.
60. Sobie E.A., Cannell M.B., Bridge J.H. Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes. Biophys J 2008, 94:L54-L56.
61. Viatchenko-Karpinski S., Terentyev D., Jenkins L.A., Lutherer L.O., Gyorke S. Synergistic interactions between Ca2+ entries through L-type Ca2+ channels and Na+-Ca2+ exchanger in normal and failing rat heart. J Physiol 2005, 567:493-504.
62. Bers D.M. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol 2004, 37:417-429.
63. Trafford A.W., Sibbring G.C., Diaz M.E., Eisner D.A. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium 2000, 28:269-276.
64. Janczewski A.M., Spurgeon H.A., Stern M.D., Lakatta E.G. Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells. Am J Physiol 1995, 268:H916-H920.
65. Cheng H., Lederer M.R., Lederer W.J., Cannell M.B. Calcium sparks and [Ca2+]i waves in cardiac myocytes. Am J Physiol 1996, 270:C148-C159.
66. MacQuaide N., Dempster J., Smith G.L. Assessment of sarcoplasmic reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular cardiomyocytes. Biophys J 2009, 96:2744-2754.
67. Shannon T.R., Guo T., Bers D.M. Ca2+ scraps: local depletions of free [Ca2+] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2+ reserve. Circ Res 2003, 93:40-45.
68. Hasenfuss G., Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol 2002, 34:951-969.
69. Sipido K.R., Eisner D. Something old, something new: changing views on the cellular mechanisms of heart failure. Cardiovasc Res 2005, 68:167-174.
70. Litwin S.E., Zhang D., Bridge J.H. Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts. Circ Res 2000, 87:1040-1047.
71. Kaprielian R., Wickenden A.D., Kassiri Z., Parker T.G., Liu P.P., Backx P.H. Relationship between K+channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction. J Physiol 1999, 517(Pt 1):229-245.
72. Janse M.J. Electrophysiological changes in heart failure and their relationship to arrhythmogenesis. Cardiovasc Res 2004, 61:208-217.
73. Sah R., Ramirez R.J., Backx P.H. Modulation of Ca(2+) release in cardiac myocytes by changes in repolarization rate: role of phase-1 action potential repolarization in excitation-contraction coupling. Circ Res 2002, 90:165-173.
74. Nerbonne J.M., Kass R.S. Molecular physiology of cardiac repolarization. Physiol Rev 2005, 85:1205-1253.
75. Sah R., Ramirez R.J., Oudit G.Y., Gidrewicz D., Trivieri M.G., Zobel C., et al. Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (I(to)). J Physiol 2003, 546:5-18.
76. Nabauer M., Kaab S. Potassium channel down-regulation in heart failure. Cardiovasc Res 1998, 37:324-334.
77. Harris D.M., Mills G.D., Chen X., Kubo H., Berretta R.M., Votaw V.S., et al. Alterations in early action potential repolarization causes localized failure of sarcoplasmic reticulum Ca2+ release. Circ Res 2005, 96:543-550.
78. Ohler A., Weisser-Thomas J., Piacentino V., Houser S.R., Tomaselli G.F., O'Rourke B. Two-photon laser scanning microscopy of the transverse-axial tubule system in ventricular cardiomyocytes from failing and non-failing human hearts. Cardiol Res Pract 2009, 2009:802373.
79. Hasenfuss G. Animal models of human cardiovascular disease, heart failure and hypertrophy. Cardiovasc Res 1998, 39:60-76.
80. Schroder F., Handrock R., Beuckelmann D.J., Hirt S., Hullin R., Priebe L., et al. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation 1998, 98:969-976.
81. Chen X., Piacentino V., Furukawa S., Goldman B., Margulies K.B., Houser S.R. L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices. Circ Res 2002, 91:517-524.
82. Hullin R., Matthes J., Bodi I., Rubio M., D'Souza K., Friedrich K.I., Rottlander D., Hoppe U.C., Mohacsi P., Schmitteckert E., Gilsbach R., Bunemann M., Hein L., Schwartz A., Herzig S. Increased expression of the auxiliary beta(2)-subunit of ventricular L-type Ca(2)+channels leads to single-channel activity characteristic of heart failure. PLoS ONE 2007, 2:e292.
83. Meyer M., Schillinger W., Pieske B., Holubarsch C., Heilmann C., Posival H., et al. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation 1995, 92:778-784.
84. Currie S., Quinn F.R., Sayeed R.A., Duncan A.M., Kettlewell S., Smith G.L. Selective down-regulation of sub-endocardial ryanodine receptor expression in a rabbit model of left ventricular dysfunction. J Mol Cell Cardiol 2005, 39:309-317.
85. Shao C.H., Rozanski G.J., Patel K.P., Bidasee K.R. Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats. J Mol Cell Cardiol 2007, 42:234-246.
86. Yaras N., Ugur M., Ozdemir S., Gurdal H., Purali N., Lacampagne A., et al. Effects of diabetes on ryanodine receptor Ca release channel (RyR2) and Ca2+ homeostasis in rat heart. Diabetes 2005, 54:3082-3088.
87. Gomez A.M., Valdivia H.H., Cheng H., Lederer M.R., Santana L.F., Cannell M.B., et al. Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science 1997, 276:800-806.
88. Xu M., Zhou P., Xu S.M., Liu Y., Feng X., Bai S.H., et al. Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy. PLoS Biol 2007, 5:e21.
89. Maron B.J., Ferrans V.J., Roberts W.C. Ultrastructural features of degenerated cardiac muscle cells in patients with cardiac hypertrophy. Am J Pathol 1975, 79:387-434.
90. Schaper J., Froede R., Hein St., Buck A., Hashizume H., Speiser B., et al. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 1991, 83:504-514.
91. Kaprielian R.R., Stevenson S., Rothery S.M., Cullen M.J., Severs N.J. Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium. Circulation 2000, 101:2586-2594.
92. He J., Conklin M.W., Foell J.D., Wolff M.R., Haworth R.A., Coronado R., et al. Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure. Cardiovasc Res 2001, 49:298-307.
93. Lyon A.R., MacLeod K.T., Zhang Y., Garcia E., Kanda G.K., Lab M.J., et al. Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart. Proc Natl Acad Sci USA 2009, 106:6854-6859.
94. Hobai I.A., O'Rourke B. Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure. Circulation 2001, 103:1577-1584.
95. Louch W.E., Mork H.K., Sexton J., Stromme T.A., Laake P., Sjaastad I., et al. T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction. J Physiol 2006, 574:519-533.
96. Mork H.K., Sjaastad I., Sande J.B., Periasamy M., Sejersted O.M., Louch W.E. Increased cardiomyocyte function and Ca2+ transients in mice during early congestive heart failure. J Mol Cell Cardiol 2007, 43:177-186.
97. Heinzel F.R., Bito V., Biesmans L., Wu M., Detre E., Von W.F., et al. Remodeling of T-tubules and reduced synchrony of Ca2+ release in myocytes from chronically ischemic myocardium. Circ Res 2008, 102:338-346.
98. Sipido K.R., Wier W.G. Flux of Ca2+ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol 1991, 435:605-630.