4.4 Effects of isorhamnetin on structural remodeling.
The findings of our study also suggest that the inhibitory effect of isorhamnetin on structural remodeling may be another factor involved in its proarrhythmic effects. JNK, a major member of the MAPK family, is activated in response to stress. It has been reported that JNK activation leads to cell proliferation, apoptosis, ROS production, cytokine production, fibrosis, and hypertrophy. Moreover, JNK promotes diastolic SR Ca2+leakage from the RyR2 channel through CaMKII activation (Nattel et al., 2020). In addition, ERK is a Thr/Ser kinase that leads to cell proliferation, cell differentiation, cell cycle regulation, cell apoptosis, and tissue formation when activated. In addition, ERK activation is associated with CaMKII activation (Lu et al., 2009). Furthermore, the importance of TRPCs in the Ca2+signaling mechanism of cardiomyocytes has recently been discussed (Tse, 2016). TRPCs are transmembrane non-selective cation channels that play important roles in various biological functions, including neuronal cell survival, immune cell maturation, cardiac fibrosis, and hypertrophy. TRPCs have also been associated with arrhythmic remodeling stimuli by modulating Ca2+ entry via cardiac fibroblast responses (Rose et al., 2012). Among these, the role of TRPC3/6 arrhythmias, including AF, has been discussed (Harada et al., 2012; Nikolova-Krstevski et al., 2017). TRPC3 regulates cardiac fibroblast function via Ca2+-dependent ERK phosphorylation (Harada et al., 2012). In contrast, TRPC6 contributes to the regulation of myofibroblast formation caused by endothelin-1 via JNK signaling (Nishida et al., 2007). Although some reports have shown that isorhamnetin inhibits cardiac remodeling by suppressing the JNK and ERK pathways, there are no reports indicating the evaluation of Ca2+ channels and molecules related to the suppression of ERK and JNK and their upstream or downstream pathways (Sun et al., 2012; Sun et al., 2013). Our study showed that isorhamnetin reversed JNK and ERK activation and TRPC3/6 protein expression induced by AngII (Fig. 7.1H-I, Fig. 7.2B-C). These results suggest that isorhamnetin diminishes CaMKII activation and normalizes Ca2+ entry through Ca2+channels to suppress the expression of these proteins. Moreover, similar to the results shown previously for ventricles, isorhamnetin morphologically inhibited atrial fibrosis and hypertrophy and reversed the related gene expression (Fig. 5A-D, Fig. 6A-D). Isorhamnetin also suppressed the AngII-induced upregulation of NF-κB, which is the most fundamental indicator of inflammation and crosstalk with MAPK (Fig. 7.2D). These results emphasize the assertion that isorhamnetin strongly diminishes AngII-induced fibrosis and the enlargement of the atrium via normalized TRPC-mediated MAPK expression.