KN-93

Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice and the pulmonary vein (PV) is the most important AF trigger. Neuregulin 1β (NRG1β), which is elevated in patients with paroxysmal AF, may activate signalling pathways that mediate cellular adaptations and subsequent stress in the myocardium. The objectives of this study were to study the effects of NRG1β on the PVs and explore the underlying mechanisms.

A conventional microelectrode, a whole‐cell patch clamp, Western blotting and immunofluorescent confocal microscopy were used to investigate electrical activity, calcium (Ca2+) regulation, protein expression, ionic currents, reactive oxygen species and cytosolic sodium ([Na+]i) in isolated rabbit PV tissue and single cardiomyocytes with or without NRG1β (10 nM) incubation for 4 h.

NRG1β‐treated PVs had faster beating rates and a higher incidence of triggered activity than control PVs. The increased PV spontaneous beating rate induced by NRG1β could be mitigated by ranolazine (a late Na+ current inhibitor, 10 μM), KN93 (1 μM) and AIP (1 μΜ) (CaMKII inhibitors) and AKTi (AKT‐1/2 inhibitor, 10 μM). NRG1β‐treated PV cardiomyocytes demonstrated larger late Na+ and Na+‐Ca2+ exchanger current than control PV cardiomyocytes. AIP decreased late Na+ current in NRG1β‐treated PV cardiomyocytes. Furthermore, NRG1β‐treated PV cardiomyocytes had smaller intracellular Ca2+ transients and reduced sarcoplasmic reticulum Ca2+ contents, but higher levels of [Na+]i, oxidative stress and RyR‐dependent SR Ca2+ leak than control PV cardiomyocytes. The increased RyR‐dependent SR Ca2+ leak by NRG1β could be alleviated by KN93. Additionally, NRG1β–treated PV cardiomyocytes exhibited upregulated AKT, pAKT, ERK, pERK, CaMKII and pCaMKII, while SERCA2a and PLB were downregulated. AKTi can downregulate oxi‐CaMKII and CaMKII in NRG1β–treated PV cardiomyocytes.

By modulating electrophysiological characteristics, Ca2+ homeostasis, and enhancing oxidative stress through AKT/CaMKII signalling, NRG1β increased PV arrhythmogenesis with increasing RyR‐dependent SR Ca2+ leak of PV cardiomyocytes.

To explore the mechanism by which moxibustion promotes synaptic plasticity of hippocampal neurons and improves learning and memory in mice with Alzheimer's disease (AD) based on the calmodulin-dependent protein kinase Ⅱ (CaMKⅡ)/ryanodine receptor (RyR)3 pathway.

Forty APP/PS1 mice were randomly divided into the model (n=15), moxibustion (n=25) groups, and 15 C57BL/6J mice served as the blank control group. The mice in the moxibustion group were given moxibustion at the "Baihui"(GV20) and "Yongquan"(KI1) acupoints, 30 minutes each time, once a day. A course of treatment lasted for 5 days, and there were a total of 4 courses of treatment. Ten mice from the moxibustion group were randomly selected as the moxibustion+inhibitor group, and were intraperitoneally injected with the CaMKⅡ protein inhibitor KN-93 (2 mg/100 g) one day before sample collection on the basis of moxibustion. After the treatment, the shuttle box test was used to evaluate the learning and memory ability of the mice. Transmission electron microscopy was used to detect the changes in the ultrastructure of synapses in the CA1 region of the hippocampus. Western blot was used to detect the expression levels of β-amyloid protein (Aβ) and CaMKⅡ protein in the hippocampus of the mice. Immunofluorescence staining was used to detect the positive expression of CaMKⅡ in the hippocampus and the co-expression of RyR3/PSD95.

Compared with the blank control group, the number of active avoidance responses, the protein expression and positive expression of CaMKⅡ in the hippocampus, and the percentage of the co-expression area of RyR3/PSD95 of the mice in the model group were decreased (P<0.01, P<0.05), and the number of synaptic vesicles in the CA1 region of the hippocampus was decreased;while the expression of Aβ protein in the hippocampus was increased (P<0.01). Compared with the model group, the number of active avoidance responses, the protein expression and positive expression of CaMKⅡ in the hippocampus, and the co-expression area percentage of RyR3/PSD95 of the mice in the moxibustion group were increased significantly (P<0.01), and the number of synaptic vesicles in the CA1 region of the hippocampus was increased;while the expression of Aβ protein was decreased (P<0.05). Compared with the moxibustion group, the number of active avoidance, the positive expression of CaMKⅡ and the co-expression area percentage of RyR3/PSD95 in the moxibustion+inhibitor group were decreased (P<0.01), and the number of synaptic vesicles in the CA1 region of the hippocampus was decreased.

Moxibustion can improve the learning and memory ability of AD model mice, and its mechanism may be related to up-regulating the expression of CaMKⅡ, activating the endoplasmic reticulum RyR3 channel, inducing the release of Ca²⁺, and then promoting neuronal synaptic plasticity.