3.6 Computational simulations of HCQ and AZM on human
ventricular action potentials
To translate the experimental data describing the multi-ion channel
blocking effects of HCQ and/or AZM to human ventricular excitation, the
ORd model was implemented to reconstruct the impacts of HCQ alone and
combined with AZM, on human ventricular action potentials including
APD90 and maximal upstroke velocities.
Figure 8A displays predicted action potentials under control conditions
and in the presence of HCQ, and HCQ and AZM combined. It was shown that
with the 10 µM HCQ alone, the APD was prolonged by 100 ms as compared to
the control condition. With the combined action of HCQ and AZM, a
synergistic action of the two drugs on APD prolongation was seen, which
was manifested in a greater increase of APD90 of ~300 ms
(Figure 8B). Such an APD prolongation can be attributable to the
integrated action of reduced repolarisation potassium channel currents
of IKr and IK1, and reduced depolarising
currents of INa, ICaL and
INaCa. A small reduction in action potential maximum
upstroke velocities was also observed by both HCQ and HCQ+AZM due to the
effects on INa.
DISCUSSION
The main findings of this study are: (1) Reported therapeutic levels of
HCQ blocked multiple human cardiac ion channels, particular hERG
(mediating IKr) and Kir2.1 (IK1,) and,
to a lesser degree, hNav1.5 (INa). AZM potentiated the
effects of HCQ on hERG and Kir2.1. (2) Computational molecular docking
studies correspondingly predicted HCQ binding to hERG. Accordingly, (3)
at the level of intact hearts, HCQ increased QT intervals as well as
ventricular AP and Ca2+ transient durations and their
heterogeneities. However, (4) HCQ further reduced heart rates, A-V and
ventricular conduction as well as increasing conduction heterogeneities.
All these effects were accentuated by combining HCQ challenge with AZM.
Furthermore, (5) The HCQ+AZM combination, but not HCQ alone, then went
on to increase susceptibilities to ventricular arrhythmic events
consistent with significant clinical cardiac safety risks. (6) Humanin-silico modelling of ion channel effects of HCQ and AZM
recapitulated the altered APDs but not the reduced conduction. This
mechanistic electrophysiological data complements ongoing multiple
randomized trials evaluating clinical efficacy and cardiac safety
profiles of these drugs as a treatment for Covid-19. The findings
directly align with recent FDA guidelines concerning their use.
These successive lines of investigation were combined to reach an
integrated view of HCQ and AZM action. This began with the approach
recommended by the CiPA guidelines (Strauss et al., 2018) in which we
examined effects of HCQ and AZM on human cardiac ion channels
responsible for the main inward (hNav1.5 (INa) Cav1.2
(ICa,L)) and outward (Kv4.3 (Ito), hERG
(IKr), KCNQ1/E1 (IKs) and Kir2.1
(IK1)) currents in the human ventricular myocardium. HCQ
and AZM were studied both alone and in combination through a wide
concentration range encompassing and extending beyond reported
therapeutic levels. The latter typically fell around 1.15-4.5 µM(Durcan,
Clarke, Magder & Petri, 2015) and 0.5-0.7 µM(Miller, Fiechtner,
Carpenter, Brown, Stroshane & Stecher, 1987) for HCQ in SLE and
rheumatoid arthritis patients on a daily 400 mg dose. In contrast, its
dose used in Covid-19, 1.2 g/day, would give substantially higher
therapeutic concentrations of ~10 µM(Borba et al.,
2020). Blood AZM levels following successive daily 500 mg and two 250 mg
doses were reported at 0.22 μM(Lai, Ho, Jain & Walters, 2011). HCQ both
alone and combined with AZM synergistically inhibited
IKr and IK1 with IC50s
at the high end of reported therapeutic levels, with some minor
inhibition of INa in this range.
Consistent with these experimental findings, computational molecular
docking simulations utilizing a recent hERG homotetramer structure (PDB
ID: 5va2) demonstrate potential HCQ binding. This is predominantly with
Y652 and F656 from one or more hERG subunits. Additional residues
involved in interactions with HCQ frequently included F557, L622, T623,
S624, and S660. In contrast, the model predicted approximately two-fold
weaker AZM-hERG binding.
Others have also explored the electrophysiological effects of HCQ or AZM
on some cardiac ion channels but did so at three times the clinically
relevant concentration and above (Capel et al., 2015; Lai, Ho, Jain &
Walters, 2011). Furthermore, the present study systematically evaluates
HCQ and AZM in combination for the first time. Furthermore, the only
report on electrophysiological effects of HCQ specifically studied
sinoatrial cells, demonstrating significant reductions in
hyperpolarization-activated currents (If), with reduced
ICaL and IKr observed at 3 µM (Capel et
al., 2015). Nevertheless, this HCQ action on If explains
its bradycardic effects observed here, further illustrating its broad
cardiac electrophysiological actions. Such a bradycardic medication in
the presence of a hypotension accompanying sepsis or SARS could
potentially exacerbate a septic shock.
At the whole heart level, our optical mapping studies demonstrate that
HCQ correspondingly prolongs ventricular APD as well as CaD, to extents
further exacerbated by combination with AZM. This fulfils expectations
from its inhibition of IKr and IK1 and
translates to the observed electrocardiographic QT prolongations. It is
interesting to note that even prior to the Covid-19 pandemic, series of
case reports have led to both HCQ and AZM being listed as definite
causes of prolonged QT interval and risk of torsade de pointes for
patients with long QT syndrome on crediblemeds.org (Roden,
Harrington, Poppas & Russo, 2020).
Prolonged APD predisposes to early afterdepolarisations (EADs) during
the plateau phase of the ventricular action potential, classically
attributed to ICaL reactivation (January & Riddle,
1989). EADs are also more common under conditions of intracellular
Ca2+ overload, including increased sympathetic drive:
ICaL can be enhanced by high intracellular
Ca2+ levels and by L-type Ca2+channel phosphorylation by CaMKII(Herring, Kalla & Paterson, 2019).
EADs may potentially trigger ectopic beats and initiate arrhythmic
events given suitable substrate.
In addition to these recovery phenomena, we also demonstrate for the
first time that HCQ reduces ventricular conduction on multi-electrode
arrays and optical mapping recording, and compromise atrioventricular
and intraventricular conduction, reflected in prolonged PR intervals and
QRS durations, to extents accentuated by further addition of AZM. Such
findings are consistent with Na channel block (Reiz & Nath, 1986) of
the kind exemplified clinically by the CAST trials (Echt et al., 1991)
and/or compromised gap junction function(King, Huang & Fraser, 2013).
Together, the combination demonstrated here of prolonged and increased
dispersions of repolarisation and calcium handling combined with slowed
conduction would offer a suitable substrate for sustained re-entrant
arrhythmia. Thus, the combined exposure to HCQ and AZM, but not HCQ
alone provoked alternans, ventricular tachycardic episodes, and
re-entrant excitation following progressively accelerated cardiac
pacing.
Finally, a validated human ventricular myocyte mathematical model
integrated the observed ion channel properties with the effects on
action potential and intracellular calcium handling characteristics in
intact hearts. It separated whole heart electrophysiological features
that were explicable in terms of the single channel properties and those
attributable to altered calcium homeostasis. The modelling reproduced
effects of surface ion channel function and their interactions forming a
‘membrane clock’ underlying the measurable electrical activity (Fig.
8C)(Lei & Huang, 2019). It reproduced the action potential prolongation
and consequent prolonged QT intervals with HCQ and the synergistic
effects of combining HCQ with AZM, attributing these to their combined
effects on IKr and IK1. These latter
events have been described as feeding forward into a
‘Ca2+ clock’ driving excitation-contraction coupling
but whose events exert feedback effects modifying action potential
conduction, recovery, and post-recovery stability, exemplified by
Ca2+ feedback effects on Na+ channel
and gap junction function (Fig. 8C), as well as longer-term effects on
channel expression(Huang, 2017; Lei, Wu, Terrar & Huang, 2018). The
modelling predicted the increased calcium transient magnitudes and
durations particularly with HCQ+AZM (Fig 8C), but only small changes in
maximum upstroke velocity attributable to Na channel blockade. The
observed conduction slowing nevertheless could arise from a
Ca2+-dependent gap junction uncoupling.
These results together provide an integrated basis for the
electrophysiological and pharmacological effects of HCQ, AZM and their
combination. They thereby underpin recent FDA guidelines cautioning
against combined HCQ/AZM administration for treatment of Covid-19 on
grounds of cardiac safety. Admittedly, physiological though not
pro-arrhythmic effects occurred with HCQ administered alone at the
~10 µM concentrations associated with Covid-19 therapy
and not at the lower ~1.0 µM HCQ levels associated with
therapy for rheumatoid arthritis and SLE. HCQ alone at therapeutic doses
thus did not increase incidences of experimental ventricular arrhythmias
in healthy hearts. Nevertheless, safety threshold doses could be reduced
by accompanying cardiac injury arising from viral toxicity, sepsis,
hypoxia-related myocyte injury, and immune-mediated cytokine storm in
Covid-19. Furthermore, AZM potentiated the HCQ toxicity, now producing
arrhythmic outcomes. The present findings thus strongly indicate a
desirability for monitoring electrocardiographic, including QT, changes
and cardiac injury during 4-aminoquinoline-macrolide therapy for
Covid-19 patients.