Discussion
The main findings of this study include: (1) Reduction by IVA of
intrinsic atrial rate could be attenuated in hearts with increased
either vagal activity or late INa; (2) IVA prolonged
MAPD90, ERP and PRR in atria and decreased the APA and
Vmax in myocytes at relatively low therapeutic
concentrations (≥ 0.1 µM) and lengthened QRS and QT intervals at high
concentration range (>3 µM) in isolated hearts; (3)
Modulation of IVA on atrial MAPD90 and APD were
condition dependent, it prolonged
MAPD90/APD
in ACh-treated but shortened MAPD90/APD in ATX-II
treated hearts or cells, respectively; (4) IVA (0.03-10 μM) induced
greater incidence of atrial arrhythmias either at slow heart rate or in
the presence of ATX-II or ACh, and DADs in atrial myocytes; (5) IVA
increased the frequency, amplitude, FWHM of calcium spark, up-regulated
RyR2 and NCX1 protein expression, and down-regulated SERCA2 protein
expression, leading to intracellular Ca2+ overload.
In denervated rabbit isolated hearts,
the intrinsic sinus heart rate was reduced by IVA at therapeutic
concentration range, i.e., approximately 0.02-0.05 µM14. The results in this study conform to the findings
of previous basic and clinical investigations that IVA inhibits
If in the sinus node to slow sinus rate15. Meanwhile, IVA also lengthened the PR interval due
to the prolongation of the conduction time in the AV node at relative
high concentrations, i.e., ≥ 1 µM, presumably due to a prolongation of
the ERP in the AV node following a prolongation of the APD in the heart
(see below) and the increase in the atrial rate due to atrial
arrhythmias. Interestingly, in isolated hearts treated with low
concentration of ATX-II or ACh to increase atrial late
INa 16 or vagal activity , the
amplitude of HR reduction by IVA would be reduced, which may be result
from the slower basal HR by drugs or under pathological conditions of
the heart. Further investigation will be needed to clarify the
conditions at which the efficacy of IVA on HR would be attenuated.
The MAPD90, ERP and PRR were prolonged by IVA at
concentrations higher than the therapeutic range (i.e., 0.02-0.05 μM)14), consistent with other research in rabbit hearts17. Previous studies have shown that IVA, at
concentrations higher than the therapeutic concentration, blocks
IKr (IC50=2.8 µM) 18,
which was attributed to the prolongation of APD in this study. The overt
prolongation in the APD by drugs that inhibit IKr (class
III antiarrhythmic drugs, macrolide and quinolone antibiotics) is
undesirable, because it is associated with torsade de pointes
ventricular tachycardia in the heart 19.
These results support the hypothesis
that atrial proarrhythmic risk of IVA was increased in hearts with slow
rate, enhanced late INa and vagal activation. IVA
induced much greater incidence of atrial arrhythmias in hearts paced at
CL of 570ms than at CL of 350 ms (76.9% vs. 26.1%). In hearts with
increased late INa by ATX-II or ACh to simulate vagal
excitation, IVA modulated the MAPD90, lengthened the ERP
and PRR, and induced atrial arrhythmias in 44.4% and 61.9% of hearts
paced at a fixed CL of 350 ms, suggesting that risk of proarrhythmia by
IVA was increased under pathological conditions in the atria. This
result is consistent with the findings from clinical studies that the
risk of AF is increased by 24% in patients treated with IVA9 and that sodium channelopathies are associated with
an increased risk of atrial arrhythmias, including AFs20. Wu et al. reported that an increase in late
INa by ATX-II potentiated the proarrhythmic activity of
low-risk QT-prolonging drugs 21. The prolongation of
the MAPD caused by drugs that purely inhibit IKr is
synergistically increased in hearts treated with late
INa enhancers 21. However, drugs that
potentially inhibit late INa cause an increase (i.e.,
pentobarbital) or sometimes a shortening (such as ranolazine) of the
MAPD 16.
If is a kind of
Na+/K+ mixed current (a net inward
current) and is mainly involved in the automatic depolarization of
sinoatrial node cells in phase 4 22. IVA decreased the
amplitude and Vmax of an AP without affecting AP
duration at relatively low concentration which might be attributed to
the inhibitory effect on the INa of the atrial myocytes.
IVA mainly affected the AP duration and triggering activity, i.e., DAD,
of atrial cells pretreated with either ACh or ATX-II. When IVA was
applied to ATX-II-/ACh-treated cells, APD30,
APD50 and APD90 were either shortened or
prolonged, indicating that IVA could also affect IK1 and
IKACh under certain conditions without affecting RMP,
APA and Vmax at low therapeutic concentration range23. Finally, IVA increased DADs but not EADs in both
in the absence and presence of either ACh or ATX-II in atrial myocytes.
DAD is related to intracellular calcium overload and abnormal
Ca2+ handling associated with the increase of
Na+/Ca2+ exchange24, 25.
IVA mainly enhanced the frequency, amplitude and FWHM (spatial
characteristics) with little effects on the FDHM (temporal properties)
of Ca2+ sparks. Ca2+ sparks are
local Ca2+ release events from the sarcoplasmic
reticulum (SR), with one spark representing the flux of
Ca2+ through a single SR release channel or RyR26. Spontaneous Ca2+ sparks are
thought to play a major role in SR Ca2+ leakage, and
the frequency, amplitude and FWHM of these sparks are highly dependent
on the Ca2+ concentration in the SR
([Ca2+]SR)27. In rabbit ventricular cells, a higher
[Ca2+]SR (>600 μM)
led to increased calcium spark amplitude and width,
Ca2+ sparks became a significant pathway of SR
Ca2+ leakage, and Ca2+ sparks
disappeared at ~300 µΜ
[Ca2+]SR, the
Ca2+ spark termination threshold 28.
Increased Ca2+signaling instability occurs in AF 29, 30 and
contributes to atrial arrhythmia and the maintenance of AF24, especially in patients with cardiovascular
diseases, including heart failure and ischemic heart disease, etc. These
mechanisms may be attributed to the change in the Ca2+release flux as the Ca2+ gradient across the SR
membrane or to luminal Ca2+-dependent RyR regulation31. Diastolic
Ca2+ sparks are spontaneous bouts of localized
inter-RyR Ca2+-induced Ca2+ release
(CICR) that are likely triggered by a rare stochastic opening of a
single RyR channel. A spark occurs if the RyR Ca2+flux amplitude mediated by that rare channel opening is sufficient to
drive inter-RyR CICR. The results in this study indicated that IVA
increased the Ca2+ release and
Ca2+-based arrhythmogenic substrate may contribute to
the initiation of AF caused by IVA.
Drug-induced AF of IVA application may be atrial DADs-related and
activation of Ca2+ sparks, which contribute to the AF
trigger. The differences in calcium instability between cells from atria
and pulmonary sleeve need to be determined because intracellular
Ca2+ was reported to be reduced in pulmonary veins.
Predominant resource of increased intracellular calcium is yet to be
fully determined in this study and is worth of further investigation.
When [Ca2+]i increases due to
spontaneous Ca2+ release events, a
Ca2+-based membrane current is activated during
diastole. This arrhythmogenic transient inward current
(Iti), which is carried by the sarcolemmal NCX, is
responsible for DAD generation. Enhancement of late INais one of the causes to increase
[Ca2+]i because it increase
[Na+]i and then
[Ca2+]i through NCX to facilitate
DAD formation and therefore was used in this project to augment the
proarrhythmic risk of IVA. When DADs are sufficiently large, they can
trigger extrasystole. Additionally, Ca2+-activated
Iti can occur during repolarization and then contribute
to triggered activities, which trigger extrasystoles and atrial
tachyarrhythmias.
Phosphorylation-mediated RyR2 sensitization is implicated in unstable
Ca2+ signaling, i.e., increased Ca2+spark frequency and diastolic Ca2+ leak in the genesis
of AF. Ca2+-based arrhythmic events caused by unstable
[Ca2+]i signaling are mediated by
intracellular Ca2+ waves and
Ca2+-activated inward currents. Ca2+overload (i.e., increased
[Ca2+]SR) increases the
sensitivity of the RyR2s to activation by cytosolic
[Ca2+]i 32. This
higher [Ca2+]i sensitivity leads
to an increased probability of spontaneous Ca2+ sparks
and Ca2+ waves.
Drug induced AF, including both
cardiovascular and non-cardiovascular agents, may have diverse
mechanisms 33. Adequate understanding of these
mechanisms underlying the increased risk of drug induced AF is critical
for the prevention and management of this kind of AF. The results in
this study indicated that
intracellular
Ca2+ overload associated triggered activities and the
reduction of HR by IVA may have synergistic effects to increase the risk
of IVA induced AF in the heart. Further study will be needed to
determine how does the IVA to increase the calcium release from the
sarcoplasm reticulum and the characteristics of IVA induced AF under
different pathophysiological conditions.