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).