Modification Of RyR2 And The Roles In Chronic Vagus Nerve Stimulation And S107 Attenuating Heart Failure

Calcium is a common second messenger that regulates many processes in cells (e.g., contraction, secretion, synaptic transmission, fertilization, nuclear pore regulation, transcription). Although cells are typically bathed in solutions containing a relatively high Ca2+ concentration (i.e., in the millimolar range), the cytoplasm of most cells contains much lower resting Ca2+ (?)concentrations (i.e., in the 100 nM range). Thus Ca2+ (?)entry across the surface membrane can substantially elevate cytosolic Ca2+ (?)levels providing the Ca2+ (?)trigger signals for a large number of physiological processes.The primary intracellular Ca2+ storage/release organelle in most cells is the endoplasmic reticulum (ER). In striated muscles, it is the sarcoplasmic reticulum (SR). SR contains specialized Ca2+ release channels. There are some special Ca2+ release channels:the ryanodine receptors (RyRs). Family of RyRs appears to contain three different isoforms:RyR1 is expressed in skeletal muscle; RyR2 is expressed in cardiac muscle and brain; RyR3 is expressed many tissues. The RyR channels are modulated by numerous factors, including a number of physiological agents (e.g., Ca2+, ATP, and Mg2+), various cellular processes (e.g., phosphorylation, oxidation, etc.), and several pharmacological agents (e.g., ryanodine, caffeine, and ruthenium red), and some associated proteins (FK506-binding protein, calmodulin).RyR2 is a tetramer comprised of four RyR2 polypeptides and FKBP12.6s. FKBP12.6s are regulatory subunits that stabilize RyR2 channel function and facilitate coupled gating between neighboring channels. A huge calcium release from RyR2 rapidly is the important process in excitation-contraction coupling in cardiac muscle. Abnormal Ca2+ transient always is followed by cardiac contraction failure; and HF eventually develops when such abnormal persists for a prolonged period. Now it is known that the binding of FKBP 12.6 to RyR2 are affected by phosphorylation and S-nitrosylation of RyR2 and modulate RyR2 activity. Recent many experimental proofs showed that phosphorylation and S-nitrosylation of RyR2 and dissociation of FKBP 12.6 contributed the development of heart failure.There is not an effective method to cure heart failure in present. But two potential directions to cure HF were developing now. One is chronic vigus nervus stimulation (VNS), the other is benzothiazepine derivative drugs. In this paper, we focused that the role of RyR2 played in heart failure and compared the two potential strategies, chronic vigus nervus stimulation (VNS) and benzothiazepine derivative drug S107.The binding of S107 to RyR2 using heavy SR of dog were studied in vitro firstly, and found that the very slow binding speed between S107 and RyR2. It takes about 1 hour to reach rate balance. And there are two classed binding sites of S107 in RyR2:the higher affinity binding sites yielded Bmax1～120pmol/mg protein with Kd1～40μM and the lower affinity binding sites yielded Bmax2～900pmol/mg protein with Kd2-300μM. Both Bmax values exceeded the Bmax of the SR (12pmol/mg protein) that was used in the assay. Since there are 4 RyR1 subunits per [3H]ryanodine binding site; the data indicated that there were 2.5 higher and 18.5 lower S107 binding sites per RyR1 subunit respectively and the affinity is extra low in both sites compared to ryanodine.The regulations of S107 and FKBP12.6 to RyR2 activity were studied in vitro under different condition too. Despite of reduced, oxidized condition and S-nitrosylation, FKBP12.6 significantly inhibit RyR2 activity by maximal 50%; but S107 was no effect to RyR2 activity in reduced and oxidized condition. In other words, S107 can not help FKBP12.7 binding to RyR2. Moreover GSSG and nitrosylation can result in significantly FKBP12.6 release from RyR2. It means that the increase of RyR2 activity resulted in GSSG and NOC-12 is contributed by nitrosylation, oxidization of RyR2 and dissociation of FKBP12.6.It was remarkably noticed that chronic vigus nervus stimulation attenuates heart failure (HF) recently. In this study, beneficial effect of chronic vigus nervus stimulation and S107 on canine model was studied with with a high-rate ventricular pacing induced HF. Result showd that both of vigus nervus stimulation and S107 relieved the loss of cardiac function; and there were oxidized damage and supernitrosylation of RyR2 in HF heart:GSH/GSSG ratio and free SH of RyR2 were less than control. VNS can significantly relieve oxidized damage of HF heart, but S107 was no effect on that.Moreover, supernitrosylated RyR2, FKBP12.6 loss and higher activity of RyR2 were found in HF heart. And VNS can reverse supernitrosylated RyR2, FKBP12.6 amount and RyR2 activity to normal level; but S107 only can reverse FKBP12.6 amount and RyR2 activity. It suggested that VNS improve HF in systemic level, but S107 improve HF by enhancing the FKBP12.6 binding affinity. RyR2 activity altered a lot in the four groups, but SERCA 2a activity was not altered in HF heart; and VNS and S107 were no effect on SERCA 2a activity too. So we hyprothesized that abnormal cardiac function attributed the depressed and slow Ca2+ transient characteristic of HF to Ca release by RyR2 superactivity, more than to Ca2+ uptake by a depressed SERCA2a.