Cobra Venom Cardiotoxin Induces Perturbations of Cytosolic Calcium Homeostasis and Hypercontracture in Adult Rat Ventricular Myocytes

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+]iand 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 0.001–1μ CTX led to a time-dependent loss of rod shaped cells, beginning at 1 min, and remaining stable by 20 min. CTX 1μ initially caused a transient augmentation in amplitude of the electrically induced-[Ca2+]itransient 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+]iremained elevated. In the absence of electrical stimulation, 1μ CTX induced a Ca2+spike followed by a sustained elevation of [Ca2+]i, an effect different from that of 40 m KCl or 10 m caffeine, which caused a transient elevation in [Ca2+]i. Digital imaging microscopy of Calcium Green-1 fluorescence revealed that the increase in [Ca2+]iwas 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+]ispike was reduced, but the second phase of elevation of [Ca2+]istill 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 m Ni2+and 10 m [Ca2+]o, but not by 5μ 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+]ispikes were attributed to the extracellular Ca2+influx, while the second [Ca2+]ielevation was related to internal Ca2+release. The high [Ca2+]imay be responsible for hypercontracture and cell death. Further studies are needed to verify it.