Discussion
The findings from the in vitro studies are congruent with previous experiments demonstrating the acute cardiotoxic effects of chloroquine (Essien and Ette, 1986; Tona et al., 1990). At the concentrations of chloroquine used, left atria were more sensitive to detrimental effects on mechanical performance than either ventricular or papillary tissue preparations. Decreases in developed isometric tension, together with increases in times to peak tension were observed, which are indicative of impaired atrial contractility. An increase in the time to peak tension by chloroquine reflects a prolongation of one or more phases of the cardiac excitation-contraction cycle, and is consistent with the ability of chloroquine to block cardiac ion channels (Essien and Ette, 1986; Tona et al., 1990; Sánchez-Chapula et al., 2001; Rodríguez-Menchaca et al., 2008). Ikhinmwin et al., (1981) demonstrated a negative inotropic response which was reversed in the presence of increased extracellular calcium aimed to promote calcium influx via unblocked L-type calcium channels. Tona et al., (1990) demonstrated chloroquine to inhibit the Treppe response in atrial guinea pig preparations, but without effect on post-extrasystolic potentiation of contractile force, suggesting that chloroquine interferes with cellular calcium influx upon which the Treppe response is dependent, but not the latter response which is dependent on intracellular calcium mobilisation. Increases in the refractoriness of cardiac tissues are indicative of potassium and /or sodium ion channel blockade. Using voltage-clamped cat ventricular myocytes Sánchez-Chapula et al., (2001) observed that chloroquine blocked several inward and outward membrane currents. The order of potency (1-10 μM range) was inward rectifying potassium current > rapid delayed rectifying potassium current > sodium current > L-type calcium current. Neither the transient outward potassium current nor the slow delayed rectifying potassium current were modified by chloroquine. Salinas and Cebada, (1993) also demonstrated that chloroquine blocks the inward rectifying potassium current in dog cardiac myocytes, but had no effects on either the transient outward, or the delayed rectifier currents. Other quinolone antimalarials also have known actions in modulating cardiac electrical activity, including blockade of human ether-a-go-go related gene (hERG) potassium and L-type calcium channels (Coker at al., 2000; Michel et al., 2002; Kim et al., 2010).
Diazepam had little effect on the function of myocardial tissue, other than at 100 μM, where it increased the effective refractory period and peak developed tension in left atrial preparations; and increased the times to peak tension in right ventricular strips. Diazepam inhibits phosphodiesterase (PDE) 4 (Collado et al., 1998), suggesting a possible mechanism for cardioprotection, although this occurs at lower concentrations (IC50 of 8.7 μM) than required to elicit responses in the present investigation. The responses of cardiac tissues to chloroquine in the presence of diazepam were no different from vehicle controls, supporting previous observations that diazepam does not attenuate the cardiac effects of chloroquine via a direct action upon the heart (Riou et al., 1989).
The in vivo experimental models of chloroquine toxicity indicated that rabbits and rats responded in a similar manner to chloroquine administration, with impaired cardiac contractility being the primary event in the sequalae of toxicity. Hypotension, bradycardia, changes in ECG intervals, arrhythmias and death followed, in a similar manner as described previously (Sofola, 1980). The provision of mechanical ventilation did not appear to influence the onset or the severity of these effects. Significant changes in cardiovascular function occurred in the absence of changes in either arterial blood gas levels or pH, suggesting that toxic manifestations due to chloroquine are not secondary to hypoxia. Chloroquine was about twice as potent in its toxic effects in rabbits than in rats, where whole blood concentrations were within the 10-20 μM range, and comparable with the concentrations used in the in vitro experiments.
The series of randomised controlled trials were designed to assess whether modulation of the GABAA receptor or other effects of diazepam might account for previous reports of reduced toxicity with chloroquine. However, diazepam, whether administered prior, during or after the administration of chloroquine or at high dose, failed to attenuate chloroquine-induced cardiotoxicity in anaesthetized rats. These results are consistent with previous studies in spontaneously breathing rats anaesthetised with thiobutobarbitone (Buckley et al., 1996), but contrast with experiments performed in conscious rats (Crouzette et al., 1983) and pentobarbitone-anesthetized, mechanically-ventilated pigs (Riou et al., 1988b). Possible explanations for these discrepancies might include the choice of species, doses of chloroquine and diazepam, and anaesthesia. Effects were similar, however, in the trial in which urethane was chosen as an anaesthetic for its lack of interaction with GABAA receptors.
Experiments aimed to differentiate any GABA mediated versus other effects of diazepam, used Ro5-4864, which has activity at the mitochondrial TSPO benzodiazepine binding site distinct from the GABAA receptors in the central nervous system; and clonazepam, which rapidly crosses the blood-brain barrier and is a potent, positive allosteric modulator of GABAAreceptors, while having low affinity towards TSPO. Mitochondrial TSPO is ubiquitously expressed in various tissues, including the heart with a putative role in regulating heart rate and contractility (Surinkaew et al., 2011). As neither diazepam nor either of these agents protected against or attenuated chloroquine toxicity, it is unlikely that any cardiovascular effects – in the context of chloroquine toxicity – can be attributed to interaction with benzodiazepine binding sites.
In view of the fact that the principal adverse effect of chloroquine is negative inotropy (Sofola, 1980), and the absence of positive inotropic effects of diazepam under basal conditions (and negative inotropy under certain conditions (Zeegers et al., 1998)), the use of a positive inotrope seems essential for the improvement in the cardiac function following chloroquine toxicity. While neither diazepam nor adrenaline alone reversed any chloroquine-induced cardiovascular changes, the improvement in cardiac contractility observed with their combined administration may indicate a beneficial interaction. Studies in rat ventricular tissues demonstrated diazepam (10 μM) to augment contractility due to isoprenaline (Martinex et al., 1995), noradrenaline (Juan-Fita et al., 2003) and dopamine (Juan-Fita et al., 2006). These effects were not mimicked by GABA nor antagonized by the selective TSPO inhibitor PK11195, or flumazenil, an antagonist of the GABAA benzodiazepine binding site. Rather, they were attributed to diazepam’s ability to inhibit PDE-4, the main isoenzyme responsible for the inotropic effect of β-adrenoceptor agonists in the rat myocardium. This offers a plausible mechanism for the observed effects in chloroquine intoxicated rats. However, there are differences between species in the expression of PDE-4, with a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents, and this will impact on the effect of enzyme inhibition (Richter et al., 2011).