Stearoyl-CoA Desaturase-1 (SCD1) Augments Saturated Fatty Acid-Induced Lipid Accumulation and Inhibits Apoptosis in Cardiac Myocytes

Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS) generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis

FA and glucose oxidations in SCD1-overexpressed cardiac myocytes.

<p>(A): FA oxidation was significantly increased in rat neonatal cardiac myocytes treated with 100 µM palmitic acid (PA). (B): On the other hand, glucose oxidation was significantly reduced in cardiac myocytes treated with PA (100 µM). (C): PA (50 to 100 µM) induced excessive FA oxidation was attenuated in cardiac myocytes transduced with a MOI of 20 of Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). (D): PA (50 to 100 µM) -induced suppression of glucose oxidation recovered in cardiac myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). BSA was used as a vehicle. Each sample was counted in a scintillation counter (cpm) and data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA concentration. (E); Phosphorylation levels of AMPK and ACC decreased in cardiac myocytes transduced with an MOI of 20 of Ad-SCD1 in comparison with control cells transduced with Ad-LacZ. Oligomycin (1 µM) was used as an AMPK and ACC activator.</p

A proposed model for the protective role of SCD1 against FA-induced cardiac lipotoxicity.

<p><i>Adaptive stage</i>: SFAs uptake increases in the myocardium in obesity and diabetes, and SCD1 expression is induced to convert SFAs to MUFAs. Increased SCD1 expression in turn inhibits FA oxidation and apoptosis through an inhibition of AMPK/ACCβ activity and DAG/ceramide synthesis, which protect the heart from lipotoxic cardiomyopathy. <i>Maladaptive stage</i>: excessive humoral and nutrient stimuli increase oxidative stress which reduces SCD1 expression. As a consequence, pathways leading to FA oxidation and apoptosis are facilitated and lipotoxic cardiomyopathy occurs.</p

Biometric and echocardiographic parameters of rats fed the control or HS diet.

<p>Data are the means ± SD.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p><p>SBP, systolic blood pressure; DBP, diastolic blood pressure; IVSd, Interventricular septal end-diastolic dimension; LVEdD or LVEsD, left ventricular end-diastolic or systolic diameter; LVPWd, left ventricular posterior wall thickness in diastole; EF, ejection fraction; FS, fractional shortening; E/A, transmitral flow ratio.</p

Plasma fatty acid composition of rats fed the control or HS diet for 3 months.

<p>Data are the means ±SD, and are presented as percent of total fatty acids.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p

Saturated FA-induced apoptosis, DAG and ceramide level in SCD1-overexpressed cardiac myocytes.

<p>Cardiac myocytes were infected with Ad-LacZ as a control or Ad-SCD1 at an MOI of 20, and simultaneously treated with palmitic acid (PA) or stearic acid (SA). (A): Cleaved-caspase 3 and caspase 3 protein levels were analyzed by Western blotting. 250 µM of PA or SA induced cleaved-caspase 3 levels, and these inductions were attenuated in myocytes transduced with Ad-SCD1. (B): Caspase 3 and 7 activities were increased with PA or SA (100 to 250 µM) in cardiac myocytes, and inhibited in cells transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ(□). Values are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA or SA concentration. (C): To detect cell death via DNA fragmentation, left panels illustrate TUNEL fluorescent staining (green) and right panel illustrates nuclei (DAPI: blue). Scale bar = 200 µm. 250 µM of PA or SA induced TUNEL-positive cells in neonatal rat cardiac myocytes transduced with Ad-LacZ, and these TUNEL-positive cells were reduced by Ad-SCD1 transduction. (D) and (E): Both (D) DAG and (E) ceramide levels in lipid extracts were significantly increased by the addition of PA or SA (250 µM), and these increases of DAG and ceramide were attenuated in myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). Each sample was counted in a scintillation counter (cpm). An arbitrary value of 1.0 was assigned to cells transduced with Ad-LacZ by the addition of BSA. Data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA or SA concentration.</p

Expression of SCD1 in HS diet rat hearts and in obese and diabetes patients' hearts.

<p>(A) and (B): Semi-quantitative RT-PCR (A) and real-time RT-PCR (B) showed the increased expression of SCD1 after 3 months in HS diet rat hearts in comparison with normal fed rat hearts. GAPDH was measured as an internal control. mRNA of the normal fed group was normalized to a value of 1, and the mRNA level in the HS-fed group is shown relative to the control level. Values are reported as the means ± SD. N = 6–8, **p<0.01 vs. control. (C): Semi-quantitative RT-PCR of SCD1, lipoprotein lipase (LPL), fatty acid synthase (FAS), acyl-CoA synthase (ACS), elongation of long chain fatty acid member 6 (Elovl6), peroxisome proliferator coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor α (PPARα), pyruvate dehydrogenase kinase isozyme 1 and 4 (PDK1, PDK4) in HS- or normal fed rat hearts after 3 months. Expression levels of other lipid biosynthesis genes or FA oxidative genes were similar between two groups at the same time point. (D) to (G): Pathology of SCD1 in HS- or normal fed rat heart. HE-stained sections (D) and (E) and Masson's trichrome staining (F) and (G) of rat myocardium after 3 months of feeding. (D), (F): Normal fed; (E), (G): High-sucrose fed. (H) and (I): Immunohistochemical staining showed increased expression of SCD1 after 3 months in HS-fed rat hearts (I), while minimal SCD1 staining was observed in control hearts (H). (J) and (K): Although SCD1 was not detectable in cardiac myocytes of normal healthy subject (J) using immunohistochemical staining, SCD1 expression was increased in obese and diabetes patients' hearts (K). Counterstaining was performed with 2% methyl green. Scale bar = 200 µm.</p

Heart tissue fatty acid composition of rats fed the control or HS diet for 3 months.

<p>Data are the means ±SD, and are presented as percent of total fatty acids.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p

Effect of several humoral factors on SCD1 mRNA expression in neonatal rat cardiac myocytes.

<p>Real-time RT-PCR of SCD1 expressions in neonatal rat cardiac myocytes stimulated with (A) angiotensin II (0.1 to 1 µM), endothelin-1 (0.01 to 0.1 µM), norepinephrine (1 to 10 µM), lipopolysaccharide (10 to 50 ng/ml) for 24 h, (B) hypoxia (3 to 9 h) and (C) Hydrogen peroxide (H₂O₂: 0.1 to 50 µM) for 24 h. High dose (10–50 µM) of hydrogen peroxide significantly inhibited SCD1 mRNA expression in neonatal rat cardiac myocytes. Each SCD1 expression is presented relative to the gene expression of GAPDH and control is normalized to a value of 1. Values are reported as the means ± SD. **p<0.01 vs. control.</p

Involvement of SCD1 and lipid accumulation in cardiac myocyte.

<p>(A) to (F): Neonatal rat cardiac myocytes were infected with Ad-LacZ or Ad-SCD1 at a multiplicity of infection of 20. Oil red O staining showed that TG accumulation was markedly increased in myocytes transduced with Ad-SCD1 (D to F) in comparison with cells transduced with Ad-LacZ (A to C). 100 µM (B and E) or 250 µM (C and F) addition of palmitic acid (PA) was used as a substrate of SCD1, and BSA was used as a vehicle. Scale bar = 100 µm.</p