Nicotinamide Phosphoribosyltransferase (Nampt)/Nicotinamide Adenine Dinucleotide (NAD) Axis Suppresses Atrial Fibrillation by Modulating the Calcium Handling Pathway

Figure 1. Gene and protein expressions of Nampt and Sirt1 in the atrial tissues. (A) Nampt mRNA expression levels in the atrial tissues evaluated by quantitative PCR (n = 4 mice per each group). (B) Sirt1 mRNA expression levels in the atrial tissues evaluated by quantitative PCR (n = 4 mice per group). (C) Nmrk2 mRNA expression levels in the atrial tissues evaluated by quantitative PCR (n = 4 mice per group). (D) Representative western blots for Nampt and Sirt1 in the atrial tissues (n = 3 mice per group). (E) Nampt protein expression levels in the atrial tissues (n = 3 mice per group). (F) Sirt1 protein expression levels in the atrial tissues (n = 3 mice per group). (G) Representative images of Nampt immunohistochemistry in the atrial tissues. Nampt was mainly expressed in the cytoplasm of atrial cardiomyocytes. Scale bar: 50 µm. (H) Positively stained areas in the atrial tissues by immunohistochemistry. (I) Atrial NAD/NADH ratio in four studied groups (n = 4 mice per group). * p < 0.05 vs. WT+ND mice. Data are shown as mean ± SD. Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.

Figure 2. Echocardiography, histology, and adipose tissue measurements. (A) Representative M-mode echocardiographic images of a mouse heart. (B) Representative images of Masson trichrome staining of the atrial tissues. (C) Ratios of fibrotic area to total area (n = 5 mice per group). (D) calculated atrial cardiomyocytes width (n = 12 cells in 3 mice per each group). (E) Representative images of abdominal CT scans. Yellow: subcutaneous adipose tissue; pink: intestinal (visceral) adipose tissue. (F) Visceral adipose tissue weights measured by abdominal CT scans. (G) Subcutaneous adipose tissue weight measured by abdominal CT scan (n = 5 mice in WT+ND group, n = 6 in NKO+ND group, n = 6 in WT+HFD group, n = 4 in NKO+HFD group). *p < 0.05 vs. WT+ND mice. Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.

Figure 3. Electrophysiological study. (A) Typical mouse surface electrocardiogram (top) and endocardial electrogram (ECG; bottom) recordings during induced AF. A: intra-atrial ECG; V: intra-ventricular ECG. (B) scatter chart of AF inducibility. AF inducibility defined as the percentage of AF occurrence in 5 times pacing. (C) scatter chart of AF duration time. AF duration defined as the average AF duration time when AF happened. (D) AERP at the basic cycle length of 150 ms. * p < 0.05 vs. WT+ND mice; # p < 0.05 vs. WT+HFD mice (n = 10 in WT+ND group, n = 7 in NKO+ND group, n = 10 in WT+HFD group, n = 7 in NKO+HFD group). Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.

Figure 4. Calcium imaging of isolated cardiomyocytes. (A) Representative calcium images of isolated cardiomyocytes at baseline and after application of Iso obtained by confocal microscopy. (B) Representative images of 3D surface plot of calcium sparks of each groups. (C) Numbers of calcium sparks and mini waves per scan. Mini waves are defined as the calcium waves which did not transport throughout the cardiomyocyte, but only a portion of the cell. Base indicates baseline condition, and Iso indicates condition after adding isoproterenol (Iso). (D) Scatter chart of fractional fluorescence increases (F/F0). (E) Scatter chart of time to peak florescence signal. * p < 0.05 vs. WT+ND mice; † p < 0.05 vs. WT+ND+Iso mice; ‡ p < 0.05 vs. WT+HFD+Iso mice (n = 10 in WT+ND group, n = 7 in NKO+ND, n = 7 in WT+HFD group, n = 6 in NKO+HFD group). Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.

Figure 5. Western blotting of calcium handling pathway-related molecules. (A) Representative western blots for proteins with key roles in the calcium handling pathway. β-Actin was assessed as an internal control. (B) Ratios of oxidized CaMKII to total CaMKII. (C) Ratios of phosphorylated RyR2 to total RyR2. * p < 0.05 vs. WT+ND mice (n = 3 per each groups). Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.

Figure 6. Effect of NR on HFD-induced AF. (A) Representative images of western blots for Nampt. (B) Nampt protein expression levels in the atrial tissues (n = 3 mice per each group). (C) Scatter chart of NAD/NADH ratio (n = 4 mice per each group). (D) Scatter chart of AF inducibility. (E) Scatter chart of AF duration. (F) Scatter chart of AERP. * p < 0.05 vs. ND mice; † p < 0.05 vs. HFD mice (n = 6 in ND group, n = 5 in HFD group, n = 7 in HFD+NR group). Statistical comparisons between multiple groups: one-way ANOVA followed by a post-hoc Bonferroni test.