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.