4.3 Effects of isorhamnetin on electrical remodeling
Electrical remodeling involves cellular Ca2+-handling abnormalities that can cause arrhythmogenic post-depolarization and spontaneous ectopic beats while promoting the development of arrhythmogenic substrates that initiate and maintain re-entry (Nattel et al., 2021). CaMKII activation and enhanced reactive oxygen species (ROS) are widely known to contribute to cardiac arrhythmias (Anderson, 2015). Among ROS, the p47(phox)-mediated activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in CaMKII oxidation (Wang et al., 2018). Previous reports have demonstrated that CaMKII activation is initiated by the oxidation of methionine 281/282 under AngII infusion. Additionally, when CaMKII is oxidized, it promotes the phosphorylation of ryanodine receptor type 2 (RyR2) serine-2814, which is a key downstream molecular target for the arrhythmogenesis of AngII (Wang et al., 2018). In short, through CaMKII oxidation, a diastolic SR Ca2+ leak from the RyR2 can be observed. Furthermore, it has been reported that CaMKII activation enhances the Ca2+ sensitivity of RyR2, which lowers the threshold for spontaneous Ca2+ release and predisposes the heart to DAD-induced arrhythmias (Wehrens, 2011). In addition, CaMKII activation affects AP morphology by changing various ionic currents and ionic channel activities related to calcium, sodium, and potassium (Thompson et al., 2017; Wagner et al., 2009). On the other hand, previous reports have indicated that isorhamnetin treatment prevents ROS production by suppressing the overexpression of p47 (phox), a major component of CaMKII oxidation (Romero et al., 2009; Sanchez et al., 2007). Furthermore, a previous report demonstrated that isorhamnetin could modulate Ca2+channels (Cav1.2) and Ca2+ currents, which play an important role in AP morphology (Saponara et al., 2011). In our study, AngII induced diastolic SR Ca2+ sparks and CaMKII oxidation. However, isorhamnetin diminished the occurrence of diastolic SR Ca2+ sparks, the phosphorylation of RyR2 at serin 2814, and the oxidation of CaMKII in the atrium (Fig. 2B, Fig. 7.1E-F). In addition, although AngII promoted APD prolongation and DAD formation, isorhamnetin brought the wave morphology closer to that of the control group and diminished the occurrence of DADs (Fig. 4K). Aberrant spontaneous Ca2+ waves were observed more frequently in the AngII group than in the control and isorhamnetin groups (Fig. 3F). These spontaneous abnormal Ca2+ waves may be caused by arrhythmogenic depolarizations, such as DADs. Interestingly, isorhamnetin also normalized the elevated expression of CACNA1C, which encodes Cav1.2 (Fig. 7.1C, G). This may have contributed to the normalization of wave morphology (Fig. 4G). Collectively, these results suggest that isorhamnetin reduces abnormal diastolic intracellular Ca2+ activity and the occurrence of DADs by abrogating CaMKII oxidation and RyR2 phosphorylation, while isorhamnetin normalizes AP morphology by modulating Ca2+ channels and currents.