Paradoxical SERCA2a Dysregulation Contributes to Atrial Fibrillation in a Model of Diet-Induced Obesity

Abstract

Obesity is a major risk factor for atrial fibrillation (AF), the most common serious cardiac arrhythmia, but the molecular mechanisms underlying obesity-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation to investigate how obesity promotes AF. Surface electrocardiography revealed that obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, this combination facilitated delayed afterdepolarizations in atrial myocytes, implicating altered Ca²⁺ dynamics. Interestingly, obesity did not affect the expression of key atrial Ca²⁺-handling proteins, including the cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). However, obesity increases the proportion of inhibitory phospholamban (PLN) monomers and decreases PLN phosphorylation, suggesting reduced SERCA2a activity. Paradoxically, Ca²⁺ reuptake in atrial myocytes from obese mice was similar to that achieved by potent small-molecule SERCA2a activators. We found that adrenergic stimulation increased Ca²⁺ transient amplitude without altering Ca²⁺ reuptake in myocytes from obese mice. Transcriptomic analysis revealed that a high-fat diet upregulated neuronatin, a protein involved in obesity that enhances SERCA2-mediated Ca²⁺ reuptake in neurons. We propose that obesity enables SERCA2a activation independently of PLN regulation, while adrenergic stimulation triggers arrhythmogenic Ca²⁺-induced Ca²⁺ release, promoting AF. In conclusion, this study demonstrates that obesity causes a paradoxical dysregulation of SERCA2a in atrial myocytes, with increased activity despite higher levels of inhibitory PLN monomers and reduced PLN phosphorylation. These findings offer new insights into the cellular mechanisms of obesity-induced AF and suggest potential therapeutic targets.

Keywords: atrial fibrillation; calcium dynamics; cardiac sarcoplasmic reticulum Ca2+-ATPase SERCA; neuronatin; obesity.

Figure 1

Changes in body weight and composition induced by a high-fat diet. We monitored (A) body mass, (B) fat mass, and (C) lean mass of mice fed regular and high-fat diets for eight weeks (N = 15 mice per group).

Figure 2

Evaluation of metabolic and inflammatory derangements induced by diet-induced obesity in mice. (A) Glucose tolerance test of mice fed a regular high-fat diet for 8 weeks; N = 4 mice fed a regular diet, N = 5 mice fed a high-fat diet. (B) Insulin tolerance test of mice fed a regular and high-fat diet for 8 weeks; N = 3 mice fed a regular diet, N = 4 mice fed a high-fat diet. (C) ELISA-based quantification of tumor necrosis factor α (TNFα), interleukin 6 (IL-6), and galectin-3 (Gal-3) in the gonadal white adipose tissue of mice fed regular and high-fat diets; N = 4 mice for each group. (D) Masson’s trichrome staining of atria of mice fed regular and high-fat diets for 8 weeks or 16 weeks; for comparison, we show fibrosis in rat uterus as a positive control. (E) Western blot analysis of atria of mice fed either regular or high-fat diets for 8 weeks with or without acute isoproterenol (ISO) treatment. Western blot analysis of COL1A1 and α-SMA was normalized against GAPDH in all atrial tissue samples. N = 8 mice per group. RD, regular diet; HFD, high-fat diet. * p < 0.05, ** p < 0.01, two-tailed t-test.

Figure 3

Diet-induced obesity and acute sympathetic activation synergize to induce atrial fibrillation in mice. (A) Representative recordings of lead II surface ECG with simultaneous ventricular and atrioventricular junction intracardiac electrograms. We show an event of AF induced by obesity in mice after the heart was paced, AF stopped spontaneously and was followed by a normal sinus rhythm (SR). Expanded signals show that the AF event spontaneously stops, followed by a normal SR (red box) in a mouse fed a high-fat diet. (B) The number of mice within each treatment group that exhibited AF (black area) or remained in sinus rhythm (white area). (C) The number of AF conversions; mice were given 10 tachypacing attempts to convert into AF. (D) Mice that were fed a high-fat diet and acutely administered isoproterenol (ISO) exhibited significantly longer AF episodes compared to mice fed a regular diet and treated acutely with ISO. RD, a regular diet; HFD, and a high-fat diet. * p < 0.05, two-tailed t-test.

Figure 4

Effects of diet-induced obesity on the action potential characteristics of atrial myocytes. (A) Representative basal action potentials from left and right atrial myocytes isolated from mice fed regular and high-fat diets at 1-Hz stimulation. (B) Amplitude, overshoot, resting membrane potential (RMP), and maximum upstroke velocity (dV/dt_max) of myocytes from mice fed regular (black) and high-fat (red) diets in the presence and absence of isoproterenol (ISO) treatment. (C) Action potential duration (APD) to 25, 50, and 90% of repolarization. For these experiments, we used the left atrium of 6 animals, and the right atrium of 5 animals fed a regular diet; we used the left and right atria of 5 mice fed a high-fat diet. RD, regular diet; HFD, high-fat diet; LA, left atrium; RA, right atrium. For mice fed a regular diet, we used N = 6 mice, n = 12 cells from the left atrium, and N = 5 animals, n = 12 cells from the right atrium. For mice fed a high-fat diet, we used N = 5 animals, n = 12 cells from the left atrium, and N = 5 mice, n = 25 cells from the right atrium. * p < 0.05, two-tailed t-test.

Figure 5

Effects of diet-induced obesity on the action potential characteristics of left atrial myocytes upon isoproterenol treatment. (A) Representative basal action potentials from left atrial myocytes isolated from mice fed regular and high-fat diets at 1-Hz stimulation; measurements are shown at basal and isoproterenol (ISO) treatment conditions. (B) Amplitude, overshoot, resting membrane potential (RMP), and maximum upstroke velocity (dV/dt_max) of myocytes isolated from left atria. (C) Changes in APD₂₅ and APD₅₀ in response to isoproterenol (ISO) in mice fed regular and high-fat diets. RD, regular diet; HFD, high-fat diet. N = 6 mice, n = 12 cells; * p < 0.05, ** p < 0.01, two-tailed t-test.

Figure 6

Diet-induced obesity does not induce changes in inward Ca²⁺ and outward K⁺ currents in atrial myocytes. (A) Representative I_Ca traces from left atrial myocytes from mice fed regular and high-fat diets at baseline (basal) and upon isoproterenol treatment. (B) I_Ca current-voltage (I/V) relationship at baseline (basal) and under adrenergic stimulation; we used N = 3 mice, n = 6 cells from mice fed a regular diet, N = 3 mice, n = 10 cells from obese mice. (C) Representative I_K traces from left atrial myocytes from mice fed regular and high-fat diets at baseline (basal) and after isoproterenol treatment. (D) I_K current-voltage (I/V) relationship before and after isoproterenol treatment; we used N = 3 mice, n = 8 cells from mice fed a regular diet, N = 3 mice, n = 10 cells from obese mice. The inserts in panels B and D show the voltage/pulse protocols that were applied to record the total Ca²⁺ and K⁺ currents, respectively. RD, regular diet; HFD, high-fat diet.

Figure 7

DAD incidence is enhanced in atrial myocytes isolated from obese mice. (A) Representative recordings of atrial myocytes isolated from mice fed (A) a regular diet and (B) a high-fat diet. Recordings are shown in the absence and presence of isoproterenol. (C) Quantification of the DAD formation events. Under current clamp conditions, cells were paced at a frequency of 2 Hz. The incidence of DADs was analyzed over a 2 min recording period and compared across the experimental groups. RD, regular diet; HFD, high-fat diet. RD, regular diet; HFD, high-fat diet. For each group, we recorded DADs from N = 5 mice and n = 8 cells. Analysis was performed using ANOVA with Tukey’s post hoc test; **** p < 0.0001; ns, not significant.

Figure 8

Expression of Ca²⁺-handling proteins in atrial tissue induced by diet-induced obesity. (A) Western blot analysis of proteins involved in intracellular Ca²⁺ cycling in atria, including the ryanodine receptor (RyR), the L-type Ca²⁺ channel (Ca_v1.2), SERCA2a, the Na⁺/Ca²⁺ exchanger (NCX1) and PLN. (B) Quantification of protein expression by Western blot analysis of the Ca²⁺-handling proteins shown in panel A. Protein expression was normalized against GAPDH in all atrial tissue samples; N = 8 mice. (C) Western blotting of PLN phosphorylation in atrial tissue of mice fed regular and high-fat diets, with and without isoproterenol (ISO) treatment. (D) Protein quantification of phosphorylated PLN in atrial lysates; N = 4 mice. RD, regular diet; HFD, high-fat diet. Statistical differences were tested using ANOVA with Tukey post hoc test * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 9

Effects of a high-fat diet on the intracellular Ca²⁺ transient in atrial myocytes. (A) Ca²⁺ transient amplitude and (B) Ca²⁺ transient decay (τ) measured in atrial myocytes isolated from mice fed regular and high-fat diets at 1- and 2-Hz stimulation. Changes in (C) Ca²⁺ transient amplitude and (D) Ca²⁺ transient decay (τ) in response to isoproterenol for each group (regular and high-fat diets) at 1- and 2-Hz stimulation. Changes in these parameters are relative to the basal signal, i.e., before isoproterenol treatment. Data are presented as a violin plot, where dashed lines represent quartiles, full lines represent the median, and widths represent the number of individuals with the same value of the measured parameter. RD, regular diet; HFD, high-fat diet. N = 9 mice fed a regular diet, N = 8 mice fed a high-fat diet. Statistical differences were tested using the Mann–Whitney U-test; * p < 0.05, ** p < 0.01, **** p < 0.0001; ns, not significant.

Figure 10

Effects of a high-fat diet and small-molecule allosteric SERCA2a regulators on the Ca²⁺ transient decay in myocytes. Ca²⁺ transient decay (τ) measured at (A) 1 Hz and (B) 2 Hz field stimulation in myocytes isolated from mice fed a regular and high-fat diet. We compared the changes in τ with those induced by three potent SERCA2a activators (Yakuchinone A, 6-paradol, and Alpinoid D) on myocytes isolated from non-obese mice. Data are presented as a violin plot, where dashed lines represent quartiles, full lines represent the median, and widths represent the number of individuals with the same value of the measured parameter. RD, regular diet; HFD, high-fat diet. N = 9 mice fed a regular diet, N = 8 mice fed a high-fat diet, and N = 8 mice for the small-molecule treatments. Statistical differences were tested using ANOVA followed by a Tukey post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Figure 11

Transcriptomic analysis of atria from mice fed regular and high-fat diets. (A) Volcano plot of log₂ (fold change) on the x-axis plotted against -log₁₀ adjusted p-value on the y-axis showing upregulated (red) and downregulated (blue) genes induced by diet-induced obesity compared to a regular diet. (B) Pathway analysis showing the gene ontology biological process that is affected by a high-fat diet within the atria and sorted by the adjusted p-value. The color of the bar shows fold change, and the circle size indicates the number of differentially expressed genes in each biological process. (C) Heat map of the 59 downregulated and 24 upregulated genes within the atria of mice fed a high-fat diet; N = 5 mice per group.