Role Of Ryanodine Receptor In Triggered Ventricular Arrhythmia

Objective Ryanodine receptor is a key protein that influences intracellular calcium concentration. In abnormal state it can cause imbalance of calcium homeostasis and lead to triggered ventricular arrhythmia. A main downstream of catecholamine phosphorylation signaling transduction pathway, Calmodulin Kinaseâ…¡(CaMKâ…¡), can phosphorylate the ryanodine receptor and regulate its function. In this study we will develop two pathological heart models, rabbits of catecholaminergic polymorphic ventricular tachycardia (CPVT) and rabbits with left ventricular hypertrophy (LVH), and observe the effect of KN-93 (a blocker of the CaMKâ…¡) and ryanodine (a blocker of the ryanodine receptor) perfusion on the frequency of ventricular arrhythmia in both models, respectively. With the analysis of western blot result, we will investigste the role of the ryanodine receptor in triggered ventricular arrhythmia and the benefit of ryanodine receptor blockage as a new means of treatment.Methods The research includes three parts as the following:(1) To observe the effect of KN-93 and ryanodine perfusion on the frequency of ventricular arrhythmia in left ventricular wedge preparations of rabbit CPVT models: Japanese rabbits were randomized into four groups:control group, model group, KN-93 group and ryanodine group. Arterially perfused left ventricular wedge preparations were made, and transmembrane action potentials (APs) of epicardium and endocardium and transmural ECG were simultaneously recorded. The control group was perfused with Tyrode's solution, while the model group with isoproterenol+caffeine to mimic CPVT. The KN-93 group was pre-perfused with KN-93 and then with KN-93+isoproterenol+ caffeine. And the ryanodine group was pre-perfused with ryanodine and then with ryanodine+isoproterenol+caffeine. The frequency of triggered APs and ventricular tachycardia of each group was recorded under high-frequency stimulation.(2) To observe the effect of KN-93 and ryanodine perfusion on the frequency of ventricular arrhythmia in left ventricular wedge preparations of rabbits with LVH:Japanese rabbits were randomized into four groups:sham group, LVH group, KN-93 group and ryanodine group. Rabbits in the LVH, KN-93 and ryanodine groups were used to establish a LVH model by the coarctation of the abdominal aorta, while those in the sham group did not undergo the coarctation. After eight weeks, action potentials (APs) were recorded simultaneously in the endocardium and epicardium, and a transmural electrocardiogram was also recorded in the left ventricular wedge preparations. The KN-93 group was pre-perfused with KN-93 and the ryanodine group was pre-perfused with Ryanodine, then the frequency of triggered APs and ventricular tachycardia was recorded under perfusion of isoprenaline and high-frequency stimulation in each group. And the total ryanodine receptor protein and phosphorylated level at Ser2815 were detected by western blot.(3) To observe the effect of KN-93 and ryanodine perfusion on the frequency of delayed afterdepolarization (DAD) and triggered activity in single left ventricular myocyte of rabbits with LVH:Japanese Rabbits were used to establish a LVH model by the coarctation of the abdominal aorta, while those in the sham group did not undergo the coarctation. After eight weeks, single left ventricular myocyte was isolated by enzymatic dissociation method and then action potential (AP) was recorded using whole cell patch clamp technique. Perfused with Tyrode's solution containing isoprenaline, the frequency of DAD and triggered activity was recorded under high-frequency stimulation stimulation. And the effect of KN-93 and ryanodine perfusion on the frequency of DAD and triggered activity was observed.Results (1) The frequency (animals/group) of triggered APs were 0/10 in the control group,10/10 in the model group,4/10 in the KN-93 group, and 2/10 in the ryanodine group. The frequencies of polymorphic ventricular tachycardia or ventricular fibrillation were 0/10, 9/10,2/10, and 1/10, respectively. The frequencies of triggered ventricular arrhythmias in the KN-93 and ryanodine groups were much lower than those in the model group (P< 0.05).(2) The frequency (animals/group) of triggered APs were 0/10 in the sham group, 10/10 in the model group,4/10 in the KN-93 group and 2/10 in the ryanodine group. The frequencies of ventricular tachycardia or fibrillation were 0/10,9/10,3/10 and 1/10, respectively. The frequencies in the KN-93 and ryanodine groups were much lower than those in the LVH group (P< 0.05). The total ryanodine receptor protein and phosphorylated level at Ser2815 were much higher in the LVH group than those in the sham group (P< 0.05), while the phosphorylated level at Ser2815 was much lower in the KN-93 group than that in the LVH group (P<0.05).(3) The frequency (animals/group) of DAD was 0/20 in the sham group,17/20 in the LVH group,7/20 in the KN-93 group, and 4/20 in the ryanodine group. The frequencies of triggered activity were 0/20,12/20,4/20, and 2/20, respectively. The frequencies of DAD and triggered activity in the KN-93 and ryanodine groups were much lower than those in the LVH group<P<0.05). Conclusion Either down-regulating the phosphorylated level of the ryanodine receptor at Ser2815 or blocking the receptor directly can effectively reduce the occurrence of triggered ventricular arrhythmia in the two pathological heart models. Ryanodine receptor plays an important role in the development of triggered ventricular arrhythmia and may be used as a new means of treatment.