title: Cobra venom cardiotoxin induces perturbations of cytosolic calcium homeostasis and hypercontracture in adult rat ventricular myocytes.
abstract: The effects of Cobra venom cardiotoxin (CTX) on the cellular morphology, twitch amplitude and intracellular calcium ([Ca2+]i) of the ventricular myocytes were studied. [Ca2+]i and twitch amplitude were determined with a fluorometric ratio method using Fura-2/AM and Calcium Green-1 as calcium indicators, and a videomicroscopic technique, respectively. Addition of 0.001-1 microM CTX led to a time-dependent loss of rod shaped cells, beginning at 1 min, and remaining stable by 20 min. CTX 1 microM initially caused a transient augmentation in amplitude of the electrically induced-[Ca2+]i transient and twitch amplitude in the single cardiac myocyte. This was followed by a prolongation in duration of [Ca2+]i. Eventually, cells became inexcitable and abruptly underwent contracture, and [Ca2+]i remained elevated. In the absence of electrical stimulation, 1 microM CTX induced a Ca2+ spike followed by a sustained elevation of [Ca2+]i, an effect different from that of 40 mm KCl or 10 mm caffeine, which caused a transient elevation in [Ca2+]i. Digital imaging microscopy of Calcium Green-1 fluorescence revealed that the increase in [Ca2+]i was accompanied by changes in cell shape without leakage of fluorescence dye in the early stage after administration of the toxin. In the absence of [Ca2+]o, the initial [Ca2+]i spike was reduced, but the second phase of elevation of [Ca2+]i still occurred. In addition, experiments using Mn2+ quench technique suggested that Ca2+-influx was induced by CTX, and that both ryanodine and thapsigargin, known to deplete Ca2+ from its intracellular pool, abolished the second phase of the elevation of [Ca2+]i. The effects of cardiotoxin were abolished by 10 mM Ni2+ and 10 mM -Ca2+-o, but not by 5 microM verapamil. In conclusion, the observations indicate that CTX causes an initial increase followed by a second sustained elevation in [Ca2+]i, which is accompanied by changes in cell shape-from rod to round-and hypercontracture. The initial [Ca2+]i spikes were attributed to the extracellular Ca2+ influx, while the second [Ca2+]i elevation was related to internal Ca2+ release. The high [Ca2+]i may be responsible for hypercontracture and cell death. Further studies are needed to verify it.
authors.affiliation: Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: H X
authors.initials: HX
authors.lastname: Wang
authors.forename: S Y
authors.initials: SY
authors.lastname: Lau
authors.forename: S J
authors.initials: SJ
authors.lastname: Huang
authors.forename: C Y
authors.initials: CY
authors.lastname: Kwan
authors.forename: T M
authors.initials: TM
authors.lastname: Wong
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004179:Cobra Cardiotoxin Proteins / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D017815:Elapidae; D004558:Electric Stimulation; D006321:Heart / Q000187:drug effects* / Q000502:physiology; D006352:Heart Ventricles; D006706:Homeostasis; D007091:Image Processing, Computer-Assisted; D008345:Manganese / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D009532:Nickel / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012433:Ryanodine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D004179:Cobra Cardiotoxin Proteins; D012433:Ryanodine; D008345:Manganese; D019284:Thapsigargin; D009532:Nickel; D014700:Verapamil; D002118:Calcium