OLeandrin Produces Changes in Intracellular Calcium Levels in Isolated Cardiomyocytes: A Real-Time Fluorescence Imaging Study Comparing Adult to Neonatal Cardiomyocytes

Abstract

Oleanders are common, hardy shrubs that grow throughout the southern United States. They contain cardiotonic steroids formed from cardenolides and bufadienolides, making the plant poisonous to both animals and humans. Aliquots of both commercially available oleander and fresh oleander extracts were prepared. Fresh, rod-like, calcium-tolerant adult rat cardiomyocytes and cultured neonatal cardiomyocytes were isolated and treated with 0-4 ng/ml of both preparations, challenged with verapamil and ouabain, and real-time spectrophotometric calcium transients and images were acquired. A number of effects were observed with the adult cells: (1) intracellular calcium levels were increased in a concentration-dependent manner: (2) reduced calcium transient heights and eventual cessation of beating resulted; and (3) increased sparking intensity led to subsequent beating and eventual calcium overload. In the spontaneously beating cultured neonatal myocytes increased intramyocytic calcium levels were also seen, with retention of this calcium rise leading to overload and, as in the adult myocytes, cessation of beating. These observations demonstrate that oleander extract is markedly potent with respect to the elevation of calcium concentrations in cardiomyocytes, and that the inability of the cardiomyocytes to release the accumulated calcium possibly indicates a role for oleandrin in inhibition of ryanodine receptor calcium release channels, calcium uptake via Na+,K+-ATPase inhibition [EC 3.6.1.3], and/or dysfunction of sarcolemmal calcium release channels.

Do Binucleate Cardiomyocytes Have A Role in Myocardial Repair? Insights Using Isolated Rodent Myocytes and Cell Culture

“…Cardiomyocytes were cultured as previously described [ 8 , 18 ]. No additions were made to any of the cultures, other than medium, to induce growth.…”

“…Neonatal cardiac myocytes were isolated as previously reported [ 8 , 18 ], sacrificing 2–3 day-old male rats (Harlan Sprague–Dawley, Harlan Co., IN, USA). The hearts were removed and harvested ventricles were digested with collagenase/pancreatin, separated on a Percoll (Pharmacia, Piscataway, NJ, USA) gradient and cultured on laminin coated cover slips in 35 mm petri dishes.…”

New drugs vs. old concepts: A fresh look at antiarrhythmics

“…Cardiotonic steroids inhibit Na + /K + ATPase leading to the accumulation of diastolic intracellular Na + , which increases Ca 2+ influx mediated by NCX during the AP and limits diastolic Ca 2+ extrusion, which in turn results in SR Ca 2+ store overfilling, the subsequent activation of a transient inward Na + current mediated by NCX (I ti ) and the triggering of DADs (Ferrier et al, 1973;Rosen et al, 1973;Lederer & Tsien, 1976;Levi et al, 1997). Recently, Na + -dependent SR Ca 2+ overload was shown to induce arrhythmogenic events both in a mouse model with a human CPVT mutation (Sedej et al, 2010) and with oleandrin, a cardiotonic steroid obtained from oleander extract (Poindexter et al, 2007). It should be noted that ouabain-induced abnormalities in Ca 2+ handling can be partially rescued by the RyR2 stabilizer JTV-519 (Sedej et al, 2010).…”

“Seeing is Believing”: Perspectives of Applying Imaging Technology in Discovery Toxicology

“…This can be further facilitated by fluorescent imaging dyes such as fluo-4 staining of the cardiomyocytes [46,47]. But the real advantages of cellular imaging as it applies to the cardiac myocytes are in the realm of investigating the mechanisms of observed cardiac phenotypes.…”