SREBP1 is significantly up-regulated in diabetic myocardium and may be associated lipid accumulation.
We found that db/db mice began to develop significant hyperlipidemia in the early stage of diabetes (Fig. 1A). The damage of hyperlipidemia to organs is mainly reflected in the ectopic deposition of lipid in organs, resulting in lipotoxicity. Especially for diabetes, cardiac lipotoxicity is one of the important causes of cardiovascular events 12. Therefore, we used transmission electron microscopy (TEM) and oil red O staining to detect the lipid content of the heart tissue of the mice. As shown in Fig. 1B-C, the heart of db/db mice showed a large accumulation of lipid droplets. According to previous research reports, prevention of cardiac lipid accumulation by inhibiting cardiac SREBP activation may be an effective strategy to prevent cardiac lipotoxicity13. However, the role of SREBP1 in diabetic heart lipid accumulation is not clear. Next, we examined the expression of SREBP1 in the heart. We found that the expression of SREBP1 was significantly up-regulated in the heart of db/db mice (Fig. 1D-G). We used immunofluorescence to detect the expression of SREBP1 and lipid content (Nile red staining) in the heart. We found that lipid accumulation was accompanied by a significant activation of SREBP1 (Fig. 1H). Collectively, SREBP1 is significantly up-regulated in diabetic myocardium and may be associated lipid accumulation.
SREBP1 expression positively correlated with the cardiac dysfunction in human heart specimens
To further confirm that the same metabolic pathways as db/db mouse hearts also exist in the human heart, we then used human heart specimens to analyze the correlation between SREBP1 and cardiac dysfunction. Heart specimens from two categories of decedents (normal and T2DM patients) who were age and sex matched were then collected (Supplementary Table 1). T2DM patients presented the most severe glycogen deposition and cardiac fibrosis, as well as higher expression of both SREBP1 and 4-HNE compared with those of normal groups (Fig. 2A-H). Linear regression analysis of the 22 heart specimens revealed that the glycogen content significantly correlated with SREBP1 expression (Fig. 2I). Additionally, SREBP1 expression was significantly correlated with the extent of cardiac fibrosis (Fig. 2J), 4-HNE expression (Fig. 2K). Taken together, these data provided profound evidence that, under the pathological conditions of T2DM, aberrant expression of SREBP1 correlated with glycogen deposition and fibrosis, lipid peroxidation in human hearts.
Long-term statins administration worsens cardiac dysfunction and pathological remodeling in type 2 diabetic mice
In order to treat hyperlipidemia and lipotoxicity of target organs, the current clinical basic lipid-lowering drugs are statins. However, the effect of long-term administration of statins on cardiac lipids and cardiac function in diabetes is not yet clear. In a 40 weeks treatment period, we selected atorvastatin and rosuvastatin, the first-line clinically lipid-lowering drugs, as a representative drug to treat three classic diabetic mice on a daily basis (Fig. 3A). After 40 weeks of statins administration, we evaluated cardiac function in each group of db/db mice by echocardiography. In the statins-treated group, both the ejection fraction and fractional shortening were significantly decreased compared with those in the Db group (Fig. 3B to D, Supplementary Table 1). We observed that serum BNP level was also significantly increased after statins treatment (Fig. 3E), which is an important cardiac test in diagnosing heart failure14.
To further examine the functional status of cardiomyocytes in db/db mice and statins-treated db/db mice, we subjected freshly isolated primary cardiomyocytes from db/m, db/db and Db + ATO group mice to a single-cell contraction assay using an electrical stimulator (Fig. 3F-I to VI). We found that the sarcomere shortening trace and sarcomere peak shortening of isolated cardiomyocytes had a similar trend to the ejection fraction in vivo, suggesting that the contraction of isolated cardiomyocytes measured ex vivo responded to the ejection fraction in vivo (Fig. 3F-II). Notably, although there was no significant difference in resting sarcomere length between the three groups of mouse cardiomyocytes, Db + ATO group cardiomyocytes exhibited lower levels of sarcomere shortening (Fig. 3F-II), together with a lower velocity during both the contraction (decreased by 15.8%) and relaxation (decreased by 17.6%) phases (Fig. 3F- IIl to IV) compared with those of db/db mouse cardiomyocytes. Figure 3F-V to Vl also reflect further diminished contractility in cardiomyocytes in the Db + ATO group.
Next, we assessed myocardial pathological remodeling, which are often associated with diastolic and systolic dysfunction. Histopathologic analysis of the myocardial tissue revealed cardiomyocyte cell degeneration and inflammatory infiltrates (Fig. 3G). Employing TEM, hearts from control animals showed a typical myofibrillar arrangement, while cardiomyocyte degeneration was observed in db/db mice, especially in the statins-treated db/db mice (Fig. 3H). Almost all myofibril constituents were affected, with an evident lack of myofibril integrity disarray and even more serious myofibril breakage, lysis and necrosis. Additionally, mitochondria were swollen and underwent cristolysis, that is, the lysis and breakage of mitochondrial cristae. Mitochondrial disposition was also altered, while db/m control mice showed an organized arrangement and mitochondria were clearly visible, with a normal round shape, but db/db mice administered statins displayed large accumulations of mitochondria throughout the whole cell, and several were degenerated15. Taken together, these findings suggest that statins administration significantly worsens both the contractile and relaxation functions of cardiomyocytes in statins-treated db/db mice.
Simultaneously, HE, PAS and Masson staining showed no significant pathological changes in the hearts of db/m mice after long-term statins administration (Supplementary Fig. 1A - c). Moreover, we observed the same heart pathological changes in KK-ay diabetic mice and STZ-induced diabetic mice (Supplementary Fig. 2A-D). These results suggested that long-term statins administration worsens cardiac dysfunction and pathological remodeling in type 2 diabetic mice, but not in db/m control mice.
Interestingly, from the above results, we observed that the statins-treated db/db mice exhibited hallmarks of myocardial functional damage, including progressive reductions in relaxation and contractility recorded by echocardiography, and single-cell contraction assays. Next, we explored the cardiac structural changes in db/db mice. As shown in Supplementary Fig. 3A to B and 3 F, we quantified CD68-positive macrophage and interleukin (IL)-1 beta expression in the myocardium. CD68-positive macrophage invasion and IL-1 beta are inflammatory inducers in cardiac disease. We found that CD68-positive macrophages invasion and IL-1 beta expression significantly increased in the statins-treated db/db mice. In addition to inflammation, cardiac fibrotic remodeling is also relevant cardiac structural change. We found that statins treatment in db/db mice significantly promoted cardiac interstitial fibrosis (Supplementary Fig. 3C to E, G to I). Collectively, long-term statins treatment induces cardiac structural changes in db/db mice.
Statins decreased serum cholesterol and LDL-C but were associated with diabetes progression in T2DM
In order to further explore the effect of statins on diabetes, we conducted a small-scale retrospective clinical study. The lipid profile and other clinical characteristics of outcomes of T2DM (n = 291) and T2DM treated with statins (n = 108) are shown in Supplementary Table 3. Statins users had filled prescriptions for statins for a mean duration of 4.34 years. Statins users showed significantly lower serum levels of total cholesterol and LDL-C than those in the control group (Supplementary Table 3). At the same time, we observed a significant decrease in AST in statins users, but did not observe an appreciable change in appreciably for renal function and echocardiography. Interestingly, we found that statins users had significantly increased diabetes indicators such as OGTT-2 h, HbA1c, and fasting C-peptide levels, which means that statins use increases IR in type 2 diabetes. Our results are also consistent with several RCTs5,16, observational studies17, and animal studies18. IR has been shown to increase the risk of diabetic complications, and long-term statins administration has been shown to accelerate the progression of diabetic nephropathy in diabetic mice by increasing IR in our previous study11. With the advancement of diabetes treatment technology, previously hidden cardiovascular complications have gradually become the main cause of death in diabetes. Therefore, more attention needs to be paid to the effects of long-term statins therapy on diabetes-related cardiac insufficiency. Although this retrospective study found that statins therapy appeared to have no significant effect on echocardiography (P > 0.05), but we observed a significant increase in creatine kinase (CK) and high-sensitivity cardiac troponin (hs-cTnT) in statins users (Supplementary Table 3). Elevated hs-TnT and CK is strongly associated with progressive myocardial damage19,20, which is more sensitive than echocardiography in detecting myocardial injury. These findings suggest that statins use was associated with an increased risk of IR and a higher risk of diabetes progression, especially myocardial injury.
Statins-induced cardiac lipid metabolism disorder contributes to cardiac dysfunction, in which the SREBP lipogenesis pathway plays an important role
Of particular interest for our focus is what the factors are that cause cardiac dysfunction. We therefore hypothesized that statins altered cardiac metabolic effects in db/db mice. To address this, indirect calorimetry was performed after db/db mice had been on statins treatment for 40 weeks. There was a marked decrease in the respiratory exchange ratio (RER) in statins-treated db/db mice, calculated by the ratio of VO2/VCO2, compared with db/db and db/m control mice during the light or the dark cycle (Fig. 4A), indicating a change in the energy source from carbohydrates to proteins and lipids in the mice. In addition to the RER, the energy expenditure, energy balance, food consumed, and water consumed all showed obvious differences (Supplementary Fig. 4A to G). It suggests that statins may mainly alter cardiac lipid metabolism in db/db mice. Next, we evaluated the expression of fatty acid metabolism-related genes, including fatty acid uptake, fatty acid oxidation (FAO), and synthesis. Interestingly, we observed that the genes involved in lipolysis, fatty acid uptake and FAO were not significantly changed, while the SREBP1 lipogenesis pathway was significantly upregulated (Fig. 4B-C), highlighting that statins-induced cardiac dysfunction may be associated with the SREBP1 lipogenesis pathway.
Next, we investigated SREBP1 expression and localization in the heart. Consistent with the immunoblot results, we found that SREBP1-N was significantly upregulated and translocated to the nucleus in the myocardium of statins-treated db/db mice (Fig. 4D). SREBP1 and its downstream targeting enzymes such as acetyl coenzyme A carboxylase 1(ACC1), were also significantly upregulated in the myocardium of statins-treated db/db mice (Fig. 4E to H), which is consistent with western blotting results. Although SREBP1 as a principal transcription factor that regulate the expression of genes involved in FA synthesis21, but the role of endogenously synthesized FA in cardiomyocyte is unknown, one of the most important questions is whether cardiomyocyte can synthesize FA. Therefore, we focus on whether Atorvastatin can increase the synthesis of fatty acids in cardiomyocytes under the high-glucose condition.
Because it is difficult to verify lipid synthesis in cardiomyocytes in vivo, we used neonatal mouse primary cardiomyocytes (NMPCs) to verify in vitro whether statins treatment promotes increased lipids synthesis in cardiomyocytes under high glucose conditions. We next traced the metabolic fluxes in statins induced NMPCs and observed generally increased intermediates in fatty acid de novo synthesis pathways (Fig. 5A), further confirming that statins treatment promotes increased the de novo synthesis of FA in cardiomyocytes under high-glucose conditions. Taken together, these results indicate that statins caused cardiac dysfunction through endogenously synthesized FA, in which the SREBP lipogenesis pathway plays an important role.
Genetic knockdown of SREBP1 alleviated statins-induced cardiac dysfunction in diabetic mice
Endogenous FA Synthesis may drive cardiac lipid accumulation. Oil red O staining and electron microscopy analysis also confirmed that statins caused cardiac lipid accumulation in db/db mice (Fig. 5B - C). We further examined the lipid levels in the cardiac tissue of the mice in each group, and found that statins treatment mainly caused a significant increase in cardiac FFA and TG levels, while TCHO and LDL-C had no significant difference (Fig. 5D). Most importantly, we found that lipid accumulation was accompanied by aberrant high expression of SREBP1 through Nile red staining and immunofluorescence SREBP1 staining (Supplementary Fig. 5A). On the basis of the aberrantly high expression of SREBP1 in cardiomyocytes of statins-treated db/db mouse hearts, we next examined cardiac histology and cardiac systolic function in diabetic srebp1-deficient mice and their WT littermates in response to statins treatment. We first tested the genotype of srebp1-deficient mice and the expression of srebp1 in the hearts of diabetic mice to determine that srebp1 is reduced (Supplementary Fig. 6A - C). Indeed, we found that statins therapy had no influence on the ejection fraction or fractional shortening of diabetic srebp1-deficient mice compared to the STZ and STZ + ATO10 groups (Fig. 5E, H, I). Consistent with the echocardiographic results, statins therapy had no influence in cardiac fibrosis of diabetic srebp1-deficient mice compared to the STZ and STZ + ATO10 groups (Fig. 5F, J). Additionally, we detected the expression of the lipid peroxidation -associated phenotype markers 4-HNE, which confirmed that statins therapy had no influence on cardiac lipid peroxidation in diabetic srebp1-deficient mice (Fig. 5G, K). To date, we have shown that cardiac lipid accumulation is associated with aberrant high expression of SREBP1, and genetic knock down of SREBP1 alleviated statins-induced cardiac fibrosis and dysfunction in STZ-induced diabetic mice.
Glucose-mediated glycosylation promotes SREBP-cleavage activating protein (SCAP) trafficking to the Golgi leading to SREBP activation in NMPCs
Up to this point, we next sought to investigate how statins treatment upregulates SREBP expression in cardiomyocytes and is translocated to the nucleus. Previous studies have shown that increasing intracellular glucose, promotes N-glycosylation of SCAP and consequent activation of SREBP1 in tumorigenesis22. To this end, we hypothesized that statins treatment increases the intracellular accumulation of glucose, and the consequent activation of SREBP1. To address this, we conducted PAS staining, and detection of the glucose content indicated massive glycogen deposition in cardiomyocytes of statins-treated db/db mice (Fig. 6A, C, E, F). We also observed that, in addition to the heart, glucose uptake and glycogen deposition were similarly increased in the kidneys and muscles of db/db mice (Supplementary Fig. 7A - B). We found that statins treatment significantly upregulated the expression of RAGE in cardiomyocytes (Fig. 6B to G, Supplementary Fig. 7C - D). Interestingly, 18F-FDG PET/CT in vivo demonstrated that statins treatment db/db mice exhibited no difference in cardiac glucose uptake compared with db/db control mice (Fig. 6D, H). Next, we used western blot to detect enzymes of glycolysis in the heart (Fig. 6I to J). We found that the key enzyme, the first step of glycolysis, hexokinase 2 (HK2) did not change significantly, but other downstream metabolic enzymes, such as phosphofructokinase (PFKM), pyruvate kinase M2 (PKM2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), significantly decreased in the statin treatment groups. At the same time, glycogen synthase 1 (GYS1) significantly increased, which suggested that statins might reduce glucose utilization, while an increase in the synthesis of glycogen in db/db mice (Fig. 6K). This effect was mediated, at least in part, through statins increases IR in db/db mice23. At the same time, we also detected the AKT-mTOR signaling pathway in the heart, which is related to IR, confirming that statins can activate the AKT-mTOR signaling pathway (Supplementary Fig. 8A - C). Collectively, under the pathological conditions of T2DM, cardiac glucose uptake remains unchanged, but statins treatment can increase cardiac IR, which impairs cardiac glucose utilization, leading to glycogen deposition in diabetic hearts.
To explore the mechanisms by which glucose enhances SCAP protein levels and activates SREBP, NMPCs were cultured in the absence or presence of glucose (25 mmol/L) and atorvastatin for 24 hours, and SREBP1 and SCAP processing was analyzed by immunoblot and immunofluorescence microscopy. Additionally, N-acetylglucosamine (GlcNAc; 20 mmol/l) facilitated N-glycosylation, and tunicamycin (Tuni; 1 mg/ml) was an effective inhibitor of N-glycosylation. The immunoblot data (Fig. 7A - B) showed that GlcNAc enhanced SCAP protein levels and promoted SREBP-1 cleavage; conversely, exposure of cells to Tuni reduced both SCAP protein levels and SREBP-1 cleavage. As expected, exposure of cells to glucose (25 mmol/L) with atorvastatin was as effective as treatment with GlcNAc at enhancing SCAP protein levels and promoting SREBP-1 cleavage, while adding Tuni at the same time inhibited these effects (Fig. 7A - B). In parallel, downstream targets for SREBP1, i.e., fatty acid synthase (FASN), ACC1, stearoyl-coenzyme A desaturase 1 (SCD1), were consistent with SREBP1 (Fig. 7A - B). Immunofluorescence imaging showed that under Tuni exposure conditions, even with supplementation with high glucose and atorvastatin, SREBP1 was still retained in the endoplasmic reticulum (ER) membrane as shown by co-staining with protein disulfide isomerase (PDI), an ER membrane protein. Converse results were observed with GlcNAc or high glucose with atorvastatin treatment (Fig. 7C). SREBP stability, transport to the Golgi and cleavage require the formation of a complex between SREBP and SCAP22,24. We observed that GlcNAc or glucose (25 mmol/L) with atorvastatin treatment had the same effect on SCAP and SREBP1 trafficking to the Golgi and subsequent SREBP1 activation, N-terminal fragment of epitope-tagged SREBP in the nuclear as shown by costaining with nuclear DAPI and Golgin, a Golgi protein marker (Fig. 7D - E). Opposite results were observed in the Tuni treatment.
Previous studies have confirmed that SCAP contains a luminal region (a.a. 540–707) with two N-glycosylation sites that are protected from proteolysis when intact membranes are treated with trypsin25. This luminal fragment has a molecular weight of approximately 30 kDa and allows the resolution of individual glycosylation variants of SCAP by SDS-PAGE (Fig. 7F). Next, we wondered whether SCAP N-glycosylation is associated with atorvastatin treatment in human AC-16 cardiomyocytes. Within the physiological glucose concentration, N-glycans of SCAP showed that the apparent mass of the fewest trypsin protected fragments decreased (without glycans) after PNGase F digestion (Fig. 7F, Lane 1). Excess glucose and atorvastatin showed that the apparent mass of the most trypsin protected fragments decreased (without glycans) after PNGase F digestion (Fig. 7F, Lane 3). Tuni nearly abolished SCAP N- glycosylation, showing the two weaker bands of the most trypsin protected fragments, even in the presence of high glucose and atorvastatin stimulation. Taken together, these data demonstrate that under high glucose conditions, atorvastatin induced SCAP N-glycosylation and promoted SCAP trafficking to the Golgi, leading to SREBP activation.
Statins therapy combined with L-carnitine alleviates statins-induced cardiac dysfunction and pathological remodeling
We further explored how to alleviate cardiac dysfunction associated with statins therapy in db/db mice. Previous studies have shown that L-carnitine treatment enhances FAO and carnitine palmitoyltransferase I (CPTI) expression, furthermore L-carnitine supplementation might be an effective tool for improvement of glucose utilization in T2DM26, which is important in the pathogenesis of multiple cardiovascular disorders27. More importantly, L-carnitine significantly down-regulated the expressions of SREBP128. We found that the content of L-carnitine in the heart tissue of db/db mice was significantly lower than that of db/m control mice, while there was no significant difference after statins treatment compared with db/db control mice (Fig. 8A). We also supplemented db/db mice with L-carnitine (300 mg/kg) in the absence or presence of atorvastatin by oral gavage once daily. After supplementation with L-carnitine, we found that the content of L-carnitine in the heart tissue of db/db mice was significantly increased (Fig. 8A). At the same time, the ejection fraction and fractional shortening did not decrease significantly after db/db mice were supplemented with L-carnitine and atorvastatin (Fig. 8B to C). Histopathologic analysis of the myocardial tissue showed that supplementation with L-carnitine and atorvastatin effectively reduced myocardial injury, cardiac fibrosis and glycogen deposition (Fig. 8D – F, I). Then we detected the expression levels of SREBP1 and 4HNE in the heart. We confirmed that statins combined with L-carnitine could effectively reduce the expression of SREBP1 and alleviate cardiac lipotoxicity ((Fig. 8G – H, J - K). These results indicate that, under the pathological conditions of T2DM, statins therapy combined with L-carnitine may abolish statins-induced cardiac dysfunction and pathological remodeling.
Taken together, our results have shown that long-term statins administration accelerated cardiac endogenous fatty acid synthesis by enhancing the intracellular accumulation of glucose, promoting SCAP N-glycosylation and leading to SREBP-1 activation, which promotes cardiac lipotoxicity and cardiac dysfunction (Supplementary Fig. 9).