Sirtuin signalling governs calcium homeostasis and mitochondrial function in metabolic syndrome cardiomyocytes

Figures

• 

  FIGURE 1. Metabolic characterization of experimental animals. (a) Terminal body weight. (b) Terminal fasting blood glucose level. (c) IPGTT. (d) Serum triglyceride level. Data are presented as the mean ± SD. Individual data points represent individual animals. Sample sizes were as follows: Con (n = 8) and MetS (n = 7). *P < 0.05 vs. Con. Abbreviations: Con, control; IPGTT, intraperitoneal glucose tolerance test; MetS, metabolic syndrome.

• 

  FIGURE 2. MMP and ROS production in experimental groups. (a) MMP. (b) ROS levels. Data are presented as the mean ± SD. Individual data points represent individual cells obtained from separate cell isolations. Sample sizes were as follows: MMP, Con (n = 11 cells), EX527 (n = 7 cells), MetS (n = 15 cells) and SRT1720 (n = 13 cells); ROS, Con (n = 17 cells), EX527 (n = 12 cells), MetS (n = 18 cells) and SRT1720 (n = 12 cells). *P < 0.05 vs. Con; #P < 0.05 vs. MetS. Abbreviations: Con, control; MetS, metabolic syndrome; MMP, mitochondrial membrane potential; ROS, reactive oxygen species.

• 

  FIGURE 3. Intracellular Ca2+ homeostasis in isolated cardiomyocytes from experimental groups. (a) Representative [Ca2+]i recording from cardiomyocytes. (b) Baseline [Ca2+]i. (c) Electrically evoked Ca2+ transients during cardiomyocyte contraction. (d) Peak [Ca2+]i response following caffeine application, reflecting total sarcoplasmic reticulum Ca2+ content. Data are presented as the mean ± SD. Individual data points represent individual cells obtained from independent cell preparations. Sample sizes were as follows: baseline [Ca2+]i and electrically evoked Ca2+ transients, Con (n = 12 cells), EX527 (n = 14 cells), MetS (n = 35 cells) and SRT1720 (n = 20 cells); caffeine response experiments, Con (n = 6 cells), EX527 (n = 7 cells), MetS (n = 10 cells) and SRT1720 (n = 9 cells). *P < 0.05 vs. Con; #P < 0.05 vs. MetS. Abbreviations: Con, control; MetS, metabolic syndrome.

• 

  FIGURE 4. Alterations in Ca2+‐handling proteins during metabolic stress. Representative western blot images and quantitative analyses of PLB phosphorylation and expression, SERCA2, RyR phosphorylation and expression, and CaMKII phosphorylation and expression in isolated cardiomyocytes from experimental groups. (a) Phosphorylated phospholamban (p‐PLB; Ser16). (b) Total phospholamban (PLB). (c) p‐PLB/PLB ratio. (d) Phosphorylated ryanodine receptor (p‐RyR; Ser2808). (e) Total ryanodine receptor (RyR). (f) p‐RyR/RyR ratio. (g) Phosphorylated CaMKII (p‐CaMKII; Thr286). (h) Total CaMKII. (i) p‐CaMKII/CaMKII ratio. Data are presented as the mean ± SD. Individual data points represent independent biological samples. Sample sizes were as follows: p‐PLB, Con (n = 4), EX527 (n = 3), MetS (n = 4) and SRT1720 (n = 4); PLB, Con (n = 8), EX527 (n = 6), MetS (n = 4) and SRT1720 (n = 7); p‐RyR, Con (n = 6), EX527 (n = 6), MetS (n = 4) and SRT1720 (n = 4); RyR, Con (n = 4), EX527 (n = 4), MetS (n = 4) and SRT1720 (n = 3); p‐CaMKII, Con (n = 7), EX527 (n = 5), MetS (n = 5) and SRT1720 (n = 5); CaMKII, Con (n = 7), EX527 (n = 6), MetS (n = 6) and SRT1720 (n = 6). *P < 0.05 vs. Con; #P < 0.05 vs. MetS.…

• 

  FIGURE 5. Activation of apoptotic signalling pathways in response to metabolic stress. Representative western blot images and quantitative analyses of anti‐apoptotic Bcl‐2, pro‐apoptotic Bax, and the apoptotic index expressed as the Bcl‐2/Bax ratio in isolated cardiomyocytes from experimental groups. (a) Bcl‐2. (b) Bax. (c) Bcl‐2/Bax ratio. (d) Beclin‐1 protein expression as an indicator of autophagy‐related signalling. Data are presented as the mean ± SD. Individual data points represent independent biological samples. Sample sizes were as follows: Bcl‐2, Con (n = 4), EX527 (n = 3), MetS (n = 3) and SRT1720 (n = 4); Bax, Con (n = 7), EX527 (n = 6), MetS (n = 6) and SRT1720 (n = 6); Beclin‐1, Con (n = 4), EX527 (n = 4), MetS (n = 4) and SRT1720 (n = 3). *P < 0.05 vs. Con; #P < 0.05 vs. MetS. Abbreviations: Con, control; MetS, metabolic syndrome.

• 

  FIGURE 6. Schematic summary of the proposed mechanisms linking metabolic stress, sirtuin signalling and cardiomyocyte injury. The transition from a healthy cardiomyocyte to an apoptotic state is illustrated. Both metabolic stress and pharmacological inhibition of SIRT1 by EX527 suppress sirtuin activity, whereas SRT1720 activates this pathway. Reduced SIRT1 activity promotes CaMKII phosphorylation, a key regulator of intracellular Ca2+ homeostasis. Activated CaMKII enhances RyR2 phosphorylation, increasing SR Ca2+ leak and elevating cytosolic Ca2+ levels. Although CaMKII activation would normally increase PLB phosphorylation, PLB phosphorylation was markedly reduced in MetS and EX527‐treated cardiomyocytes, impairing SERCA‐mediated Ca2+ reuptake into the SR. This imbalance reduces SR Ca2+ content while increasing cytosolic Ca2+ concentration. The dissociation between CaMKII activation and PLB hypophosphorylation might reflect altered phosphatase activity during metabolic stress. In parallel, reduced SIRT1 activity impairs mitochondrial homeostasis, promoting mitochondrial dysfunction and increased ROS production, which further exacerbates Ca2+ dysregulation and apoptotic signalling.…