Very Low-Density Lipoproteins of Metabolic Syndrome Modulates STIM1, Suppresses Store-Operated Calcium Entry, and Deranges Myofilament Proteins in Atrial Myocytes

Figure 1. Effects of metabolic syndrome (MetS)-very low-density lipoproteins (VLDLs) on the expression of stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1) and the O-GlcNAcylation of STIM1. (a) Quantitative RT-PCR of STIM1 (n = 4 for each group). Reduced STIM1 mRNA in the MetS-VLDL-treated group (MetS) ($ P = 0.012, # P = 0.005). (b) Representative bands of western blots for STIM1 and Orai1 channel proteins. (c) Reduced STIM1 membrane protein expression in the MetS-VLDL group (n = 4 for each group; $ P = 0.025, # P = 0.021). (d) Orai1 channel membrane protein expression among groups (n = 4 for each group; P = 0.5223). (e,f) Representative immunoblots and densitometry analysis (n = 4 for each group). Although STIM1 protein expression was reduced, the O-GlcNAcylation (85 kDa, indicated by the arrow) was larger in the MetS group. $ P = 0.038 for the MetS group versus the control. All of the changes in the MetS group were absent in the normal-VLDL group (Normal). (g) Whole-cell O-GlcNAcylation immunoblotting showed enhanced O-GlcNAcylation of the nuclear, cytosol, and membranous protein fractions in the MetS groups. (g,h) The inhibition…

Figure 2. Suppressed store-operated Ca2+ entry (SOCE) following sarcoplasmic reticulum (SR) Ca2+ depletion in MetS-VLDL-treated HL-1 cardiomyocytes. (a–c) Representative ratiometric tracings in fluorescence measurements in control, normal-VLDL, and MetS-VLDL-treated HL-1 cells during SOCE testing. (d–e) Representative ratiometric tracings from STIM-1 inhibited HL-1 cells. SKF 96365, a STIM1 inhibitor. (f) Analysis of data from the ratiometric fluorescence 340:380 ratio for the peak response to thapsigargin/caffeine (TG/Caff) (n = 5 experiments; $ P = 0.024, # P < 0.001, * P < 0.01, all vs. control) and the peak of SOCE (n = 5 experiments; all # P < 0.001 vs. control; all * P < 0.001 vs. normal-VLDLs).

Figure 3. MetS-VLDLs suppressed calcineurin–nuclear factor of activated T-cells (NFAT) signaling pathways. (a) Representative bands of western blots (n = 4 for each group) for nuclear and cytosolic fractions of calcineurin, NFAT, and phosphorylated NFAT in proteins. (b) Reduced nuclear calcineurin in the MetS-VLDL group (MetS) (n = 4; $ P = 0.037 vs. control, # P = 0.04 vs. normal-VLDL group (Normal)). (c) Unchanged cytosolic expression of calcineurin protein (n = 4, P = 0.9377). (d) Reduced nuclear NFAT in the MetS group (n = 4; $ P = 0.007, # P = 0.009). (e) Increased phosphorylated NFAT in the cytoplasm of the MetS group (n = 4, $ P = 0.04). (f) Reduced calcineurin activity in the MetS group (n = 6; $ P = 0.0001, # P = 0.0001).

Figure 4. MetS-VLDLs altered myofilament protein expression and induced sarcomere derangement. (a) Phosphorylated cardiac myosin-binding protein C (cMyBPC), cardiac troponin I (TnI) and T (TnT), and myosin light chain 2 (MLC2) on 1D-gradient gels stained with ProQ Diamond reagent (left) and total protein expression shown on the gel subsequently stained with SYPRO Ruby (right). SKF 96365, a STIM1-inhibtor; FK-506, a calcineurin-inhibitor; n = 4 for each group. (b). Densitometry analyses from gels with ProQ and SYPRO staining. (c) ProQ Diamond staining gels of mouse atrial proteins (n = 3 for each group: Control; Normal-VLDL-injected mice, nVLDL; and MetS-VLDL-injected mice, msVLDL). (d) Densitometry analyses for phosphorylated cMyBPC, desmin, TnT, TnI, and MLC2. $ P < 0.05 msVLDL versus control; # P < 0.05 msVLDL versus nVLDL. (e,f) Western blot and densitometry analysis for desmin in atrial tissues. (g) Representative transmission electron microscopy (TEM) pictures (at 5000× magnification) showing disorganized Z lines (Z, highlighted with dashed lines) in the atrial tissue of mice receiving a MetS-VLDL injection (msVLDL) compared to normal Z lines in the controls and in mice receiving…

Figure 5. Lipotoxicity of VLDLs on mediating maladaptation of calcium regulation to derangement of sarcomere proteins in atrial myopathy. In metabolic syndrome (MetS), VLDLs undergo biochemical property changes and become different from VLDLs of normal conditions [23]. MetS-VLDLs reduce STIM1 expression and enhance O-GlcNAcylation on STIM1 protein. These changes in concert suppress SOCE and the downstream calcineurin–NFAT pathway, resulting in alteration of myofilament protein expression, disruption of sarcomere organization, and atrial myopathy [35]. The progression of atrial myopathy ultimately leads to atrial fibrillation.