Protective effects of kaempferol against cardiac sinus node dysfunction via CaMKII deoxidization

Abstract

Kaempferol exerts cardioprotective actions through incompletely understood mechanisms. This study investigated the molecular mechanisms underlying the cardioprotective effects of kaempferol in sinus node dysfunction (SND) heart. Here, we demonstrate that angiotensin II (Ang II) infusion causes SND through oxidized calmodulin kinase II (CaMKII). In contrast to this, kaempferol protects sinus node against Ang II-induced SND. Ang II evoked apoptosis with caspase-3 activation in sinus nodal cells. However, kaempferol lowered the CaMKII oxidization and the sinus nodal cell death. To block the CaMKII oxidization, gene of p47phox, a cytosolic subunit of NADPH oxidase, was deleted using Cas9 KO plasmid. In the absence of p47phox, sinus nodal cells were highly resistance to Ang II-induced apoptosis, suggesting that oxidized-CaMKII contributed to sinus nodal cell death. In Langendorff heart from Ang II infused mice, kaempferol preserved normal impulse formation at right atrium. These data suggested that kaempferol protects sinus node via inhibition of CaMKII oxidization and may be useful for preventing SND in high risk patients.

Keywords: Angiotensin II; CaMKII; Kaempferol; Sinus node dysfunction.

Fig. 1. CaMKII oxidization and sinus node dysfunction in Ang II-infused mice. (A) Representative ECG recordings from Langendorff-perfused hearts isolated from mice infused with Ang II or saline for 3 weeks. (B) Ang II-infused mice have more sinus pauses than saline-infused mice. (C) Western blots shows ox-CaMKII and T-CaMKII from right atrial tissue obtained from infused with Ang II or saline for 3 weeks. Ang II, angiotensin II; CaMKII, calmodulin kinase II; ECG, electrocardiography; ox-CaMKII, oxidized-CaMKII; T-CaMKII, total CaMKII. Data are shown as the means±SEM of 3-6 mice per group. ^a)Significantly different (P<0.05) from saline infused control.

Fig. 2. Effect of Ang II on apoptosis with CaMKII oxidation. Cells were isolated from right atria and incubated with FBS for 1 day or 3 days. (A) Following the indicated days, protein was extracted to determine TBX3 (sinus node marker) and MLCA2 (cardiomyocyte marker) using Western blotting. Cells with dominant TBX3 expression were isolated and maintained using 20% FBS-DMEM. Subsequently, Ang II (20 µM) was added to the culture medium for 5 days. (B) Apoptotic cells were counted using TUNEL assay. Protein was extracted to measure caspase-3 activity (C) and determined ox-CaMKII and T-CaMKII using Western blotting (D). Ang II, angiotensin II; CaMKII, calmodulin kinase II; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ox-CaMKII, oxidized-CaMKII; PBS, phosphate buffered saline; T-CaMKII, total CaMKII; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Data are shown as the means±SEM of 3-6 mice per group. ^a)Significantly different (P<0.05) from PBS treated control. ^b)Significantly different (P<0.05) from right atrial cells at 1st day. Scale bar=50 µm.

Fig. 3. Knockout of p47phox prevents sinus nodal cell death and CaMKII oxidization observed with Ang II. Cas9 KO plasmid transfection of p47phox in sinus nodal cell was performed using a Lipofectamine 3000. Plated cells were exposed to the Cas9 KO plasmid or empty vector control. After this, Ang II (20 µM) was added to the culture medium for 5 days. From these cells, ox-CaMKII levels (A), apoptotic cells (B), and caspase-3 activity (C) were evaluated. Ang II, angiotensin II; CaMKII, calmodulin kinase II; ox-CaMKII, oxidized-CaMKII; PBS, phosphate buffered saline; T-CaMKII, total CaMKII; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Data are shown as the means±SEM of 3-6 mice per group. ^a)Significantly different (P<0.05) from PBS treated control. ^b)Significantly different (P<0.05) from Ang II treated control. Scale bar=50 µm.

Fig. 4. Kaempferol reduces cell death by lowering CaMKII oxidization. Sinus nodal cells were incubated with Ang II (20 µM) in the absence or presence of a kaempferol (15 mM) for 5 days. Protein was extracted to determine ox-CaMKII using Western blotting (A) and measure caspase-3 activity (C). Apoptotic cells were counted using TUNEL assay (B). Ang II, angiotensin II; CaMKII, calmodulin kinase II; ox-CaMKII, oxidized-CaMKII; T-CaMKII, total CaMKII; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Data are shown as the means±SEM of 3-6 mice per group. ^a)Significantly different (P<0.05) from Ang II treated control. Scale bar=50 µm.

Fig. 5. Kaempferol improves the recovery of Ang II-induced sinus nodal dysfunction. (A) ECG recordings from Langendorff-perfused hearts isolated from mice infused with Ang II in the absence or presence of a kaempferol for 3 weeks. (B) Together with kaempferol, Ang II infused mice decreased sinus pauses than Ang II-infused alone. (C) Western blots demonstrates ox-CaMKII and T-CaMKII from right atrial tissue obtained from infused with Ang II, Ang II+kaempferol, or saline for 3 weeks. Ang II, angiotensin II; CaMKII, calmodulin kinase II; ECG, electrocardiography; ox-CaMKII, oxidized-CaMKII; T-CaMKII, total CaMKII. Data are shown as the means±SEM of 3-6 mice per group. ^a)Significantly different (P<0.05) from Saline treated control. ^b)Significantly different (P<0.05) from Ang II treated control.

Fig. 6. Schematic mechanism for kaempferol regulation of sinus node protection. After activation of sinus nodal NADPH oxidase with Ang II, increased amounts of ROS promote CaMKII oxidization which then mediates apoptosis through caspase-3. Following the apoptosis, reducing the sinus node volume causes sinus node dysfunction. Inhibition of NADPH oxidase by kaempferol attenuates CaMKII oxidization and apoptosis of sinus nodal cells, leading to a reduction of sinus node dysfunction. Ang II, angiotensin II; CaMKII, calmodulin kinase II; ox-CaMKII, oxidized-CaMKII; ROS, reactive oxygen species.