Letrozole Accelerates Metabolic Remodeling through Activation of Glycolysis in Cardiomyocytes: A Role beyond Hormone Regulation
Estrogen receptor-positive (ER+) breast cancer is the most common molecular subtype—accounting for approximately 80% of all breast cancer cases—that predominantly affects premenopausal women . Anti-estrogen therapy is recommended as the primary adjuvant treatment for breast cancer patients after surgery . Aromatase inhibitors (AIs)—including anastrozole and letrozole—and selective estrogen receptor modulators (SERMs), such as tamoxifen, are the commonly used hormonal drugs . Currently, AI-based therapy is preferred over tamoxifen-based therapy as AIs are associated with a lower tumor recurrence rate than SERMs [ , , ]. It has been previously established that AIs have better therapeutic efficacy against ER+ breast cancer than SERMs. As a first line of breast cancer therapy, chemotherapy drugs are toxins and can induce cardiac toxicity . As a result of chemotherapy, heart function could reduce and prevent enough oxygen and nutrients from supplying the body . Along with chemotherapy drugs, anti-estrogenic therapy could be selected in ER+ breast cancer patients. Aromatase inhibitors are helpful for patients who have gone through menopause because they stop most estrogen production in the body, including in ovaries and fat . However, cohort studies have reported that patients taking AIs—rather than tamoxifen—are more predisposed to cardiovascular diseases, including myocardial infarction and heart failure [ , , ]. The underlying cause has been attributed to changes in lipid metabolism in the heart in response to AI-induced reduction in circulating estrogen levels . This perspective seems reasonable, as estrogens play a role in energy and metabolic homeostasis . Thus, the reduced circulating levels of estrogens can induce lipid metabolic changes . This altered lipid metabolism could adversely impact heart function, as the heart’s high energy demands are met through fatty acid (FA) oxidation, which accounts for 60–80% of ATP production . If the heart is not supplied with ATP from FA oxidation, glucose consumption increases (a lower ATP-yielding process in comparison) to meet energy needs [ , , ]. Subsequently, cardiac metabolism is remodeled to use glucose as the primary source of energy, thereby promoting functional heart failure—including compensatory hypertrophy—due to unmet energy needs [ , , , ]. In line with this background, our study showed hypertrophic changes in the left ventricle and metabolic energy changes associated with decreased FA oxidation and increased glycolysis in the heart of female rats exposed to the AI letrozole. Although both AIs and tamoxifen are involved in estrogen receptor signaling, a previous cohort study revealed that AIs seem to be associated with more aggressive adverse effects on the heart compared to tamoxifen. Eventually, we intend to reveal the mechanism by which letrozole adversely affects cardiac metabolism and whether this mechanism involves its function as a hormone regulator or as an independent molecule, and to elucidate the mechanism by which cardiac metabolic remodeling leads to cardiac structural remodeling. 

2.1. Letrozole Promotes Hypertrophic Cardiomyopathy of the Left Ventricle (LV) in Letrozole-Treated Rats (LTZ-Rats) To investigate whether letrozole acts as a causative factor of cardiovascular diseases—as is statistically indicated—slow-release letrozole pellets were inserted subcutaneously in female rats ( A). After exposing the rats to 0.1 mg/kg/day of letrozole for 4 weeks, the principal female reproductive organs, i.e., the ovaries and the uterus, were assessed to check whether the circulating estrogen levels were reduced. The organs were expected to show features of polycystic ovarian syndrome in response to reduced estrogen levels [ , , ]. As expected, the ovaries became more swollen and reddish, and the diameter of the uterus became shrunken, in LTZ-rats compared to those in vehicle-treated rats (Veh-rats) ( B). Microscopic observation showed that the swollen ovaries bore multiple cysts and the endometrial thickness was reduced in the shrunken uterus of LTZ-rats compared to Veh-rats ( B). A quantitative analysis indicated that the ovary weight to body weight ratio was increased in LTZ-rats compared to that in Veh-rats ( C). In contrast, uterus weight to body weight ratio decreased in LTZ-rats compared to that in Veh-rats (42%, p < 0.05), along with a significant decrease in endometrial thickness in LTZ-rats compared to Veh-rats (49%, p < 0.05) ( D,E). Subsequently, quantitative RT-PCR analysis was performed. In accordance with the role of letrozole as an AI, mRNA expression levels of estrogen-responsive genes—including prostaglandin D2 synthase ( Ptgds ), small heterodimer partner ( Shp ) and signal transducer and activator of transcription 3 ( Stat3 )—within the uterine tissues remarkably decreased in LTZ-rats compared to those in Veh-rats ( Ptgds , 4%, p < 0.05) ( Shp , 20%, p < 0.05) ( Stat3 , 61%, p < 0.05) ( F). The reduced levels of circulating estrogens, as indicated by the above mentioned factors, resulted in an increase in the body weight of LTZ-rats compared to that of Veh-rats (1.30-fold, p < 0.05) ( G). However, despite the increased body weight, no hematological differences—blood glucose, triglyceride (TG), and total cholesterol (TCHO) levels—were observed between Veh-rats and LTZ-rats ( H). Following validation of the LTZ-challenged animal model, we examined the pathophysiological condition of the heart. The nuclei of LV cardiomyocytes were flatter in LTZ-rats than in Veh-rats, and the cytoplasm-to-nucleus ratio occupied the same area in LTZ-rats, compared to Veh-rats (86%, p < 0.05) ( A). As the above histological characteristics pertain to hypertrophic cardiomyopathy , we examined the expression levels of heart failure markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Interestingly, the expression levels of both ANP and BNP were significantly higher in LTZ-rats than in Veh-rats (ANP, 4.92-fold, p < 0.05) (BNP, 2.26-fold, p < 0.05) ( B). Subsequently, we performed quantitative RT-PCR to analyze the mRNA expression levels of cardiac physio-functional genes, including ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 ( Atp2a2 ), myosin heavy chain, α isoform ( Myh6 ), and myosin heavy chain, β isoform ( Myh7 ). The mRNA expression level of Atp2a2 , an indicator of cardiac contractility, increased in LTZ-rats compared to that in Veh-rats (1.88-fold, p < 0.05) ( C). Additionally, Myh6 and Myh7 expression, reflecting increased cardiomyocyte size and LV wall thickening, respectively, increased in LTZ-rats compared to Veh-rats ( Myh6 , 1.19-fold, p < 0.05) ( Myh7 , 1.28-fold, p < 0.05) ( C). We also analyzed the mRNA expression of these three genes in vitro using the H9c2 cell line and observed a similar increase in expression in letrozole-treated cells (LTZ-cells) compared to that in vehicle-treated cells (Veh-cells) ( Atp2a2 , 1.20-fold, p < 0.05) ( Myh6 , 1.19-fold, p < 0.05) ( Myh7 , 1.28-fold, p < 0.05) ( D). However, no significant change in mRNA expression levels was observed in cells treated with ethynylestradiol (E2-cells) and testosterone (T-cells) ( D). These histological and molecular alterations in cardiomyocytes indicate that letrozole may induce hypertrophic cardiomyopathy of the LV. 2.2. Letrozole Decreases FA Consumption in Cardiomyocytes While Regulating Estrogen Levels Next, we looked at the reasons underlying the hypertrophic changes in letrozole-exposed hearts, considering that cardiac structural remodeling, including cardiac hypertrophy, might develop in response to altered energy metabolism . We first looked for traces of altered lipid metabolism related to TG accumulation and FA uptake and oxidation, as FA serves as the primary energy source for the heart . TG levels in the hearts were higher in LTZ-rats than in Veh-rats (2.4-fold, p < 0.05) ( A). Conversely, Western blotting revealed that the expression of cluster of differentiation 36 (CD36), a plasma membrane protein facilitating FA uptake in cells, significantly decreased in LTZ-rats compared to that in Veh-rats (41%, p < 0.05) ( B). Additionally, quantitative RT-PCR indicated that the expression of adipose triglyceride lipase ( A tgl ), an enzyme that hydrolyzes TGs to diacylglycerols and serves as the rate-limiting step of lipolysis, decreased in LTZ-rats compared to that in Veh-rats (59%, p < 0.05) ( C). In line with the expression of Atgl , the expression of genes associated with β-oxidation, including carnitine palmitoyl transferase I ( Cpt1 ), very long-chain acyl-CoA dehydrogenase ( Acadvl ) and medium-chain acyl-CoA dehydrogenase ( Acadm ) also decreased in LTZ-rats compared to that in Veh-rats ( Cpt1 , 75%, p < 0.05) ( Acadvl , 81%, p < 0.05) ( Acadm , 73%, p < 0.05) ( C). To determine whether these changes in lipid metabolism were attributed to reduced estrogen levels or to letrozole, an in vitro assay—using LTZ-, E2-, and T-treated H9c2 cells—was performed. CD36 was expressed at higher levels in E2-cells than in Veh-cells (1.23-fold, p < 0.05) ( D). The transcript-level expression of A tgl and β-oxidation genes, including Cpt1 , Acadvl and Acadm, was also higher in E2-cells than in Veh-cells ( Cpt1 , 2.16-fold, p < 0.05) ( Acadvl , 1.36-fold, p < 0.05) ( Acadm , 1.58-fold, p < 0.05) ( E). However, no significant changes in protein and mRNA expression were observed in LTZ-cells compared to Veh-cells, even though Cpt1 level increased in T-cells compared to Veh-cells (1.53-fold, p < 0.05) ( E). These findings indicate increased TG accumulation and decreased TG degradation and FA oxidation, thereby suggesting that FA consumption by cardiomyocytes is lower in the presence of letrozole-induced reduced estrogen levels. 2.3. Letrozole Induces Enhanced Glucose Consumption in Cardiomyocytes Independent of Its Role in Modulating Estrogen Levels After looking at the changes in lipid metabolism, we examined glucose metabolism to determine whether glucose consumption increased to compensate for the decrease in FA consumption. The expression levels of hexokinase 2 (HK2)—which catalyzes the first step of glycolysis —and pyruvate kinase muscle form 2 (PKM2)—which catalyzes the last step of glycolysis —increased in LTZ-rats compared to those in Veh-rats (HK2, 1.82-fold, p < 0.05) (PKM2, 1.42-fold, p < 0.05) ( A). Additionally, the expression of pyruvate dehydrogenase (PDH)—the enzyme that converts pyruvate to acetyl-CoA —also increased in LTZ-rats compared to Veh-rats (2.03-fold, p < 0.05) ( A). From an in vitro assay using the H9c2 cell line, the expression levels of HK2, PKM2, and PDH were found to be higher in LTZ-cells compared to those in Veh-cells (HK2, 2.00-fold, p < 0.05) (PKM2, 1.24-fold, p < 0.05) (PDH, 1.86-fold, p < 0.05) ( B). Although the expression of PKM2 and/or PDH also increased in E2-cells and T-cells compared to that in Veh-cells (E2-cells, PKM2, 1.16-fold, p < 0.05) (E2-cells, PDH, 1.35-fold, p < 0.05) (T-cells, PDH, 1.55-fold, p < 0.05), the extent of the increase was more notable in LTZ-cells than in E2-cells and T-cells ( B). To determine whether letrozole induces increased glucose consumption in cardiomyocytes independent of its role in reducing estrogen levels, we performed an in vitro assay using H9c2 cells, wherein the cells were exposed to high and low concentrations of glucose. In the presence of high glucose concentrations (HG), the expression levels of HK2, PKM2, and PDH increased in letrozole-treated cells (HG-LTZ-cells) compared to Veh-cells (HG-Veh-cells) (HK2, 1.73-fold, p < 0.05) (PKM2, 1.30-fold, p < 0.05) (PDH, 1.61-fold, p < 0.05) ( A). When exposed to low glucose concentrations (LG), the expression of HK2 alone increased in LTZ-cells (LG-LTZ-cells) compared to that in Veh-cells (LG-Veh-cells) (2.19-fold p < 0.05) ( A). Subsequently, we performed glycolysis stress tests for quantitatively monitoring the glycolysis rate as an indicator of glucose consumption. In LTZ-cells, the extracellular acidification rate (ECAR) substantially increased compared to that in Veh-cells (2.17-fold p < 0.05) ( B). These findings indicate that letrozole enhances glycolysis in cardiomyocytes, independent of its role in modulating estrogen levels, to compensate for decreased FA oxidation. 

To investigate whether letrozole acts as a causative factor of cardiovascular diseases—as is statistically indicated—slow-release letrozole pellets were inserted subcutaneously in female rats ( A). After exposing the rats to 0.1 mg/kg/day of letrozole for 4 weeks, the principal female reproductive organs, i.e., the ovaries and the uterus, were assessed to check whether the circulating estrogen levels were reduced. The organs were expected to show features of polycystic ovarian syndrome in response to reduced estrogen levels [ , , ]. As expected, the ovaries became more swollen and reddish, and the diameter of the uterus became shrunken, in LTZ-rats compared to those in vehicle-treated rats (Veh-rats) ( B). Microscopic observation showed that the swollen ovaries bore multiple cysts and the endometrial thickness was reduced in the shrunken uterus of LTZ-rats compared to Veh-rats ( B). A quantitative analysis indicated that the ovary weight to body weight ratio was increased in LTZ-rats compared to that in Veh-rats ( C). In contrast, uterus weight to body weight ratio decreased in LTZ-rats compared to that in Veh-rats (42%, p < 0.05), along with a significant decrease in endometrial thickness in LTZ-rats compared to Veh-rats (49%, p < 0.05) ( D,E). Subsequently, quantitative RT-PCR analysis was performed. In accordance with the role of letrozole as an AI, mRNA expression levels of estrogen-responsive genes—including prostaglandin D2 synthase ( Ptgds ), small heterodimer partner ( Shp ) and signal transducer and activator of transcription 3 ( Stat3 )—within the uterine tissues remarkably decreased in LTZ-rats compared to those in Veh-rats ( Ptgds , 4%, p < 0.05) ( Shp , 20%, p < 0.05) ( Stat3 , 61%, p < 0.05) ( F). The reduced levels of circulating estrogens, as indicated by the above mentioned factors, resulted in an increase in the body weight of LTZ-rats compared to that of Veh-rats (1.30-fold, p < 0.05) ( G). However, despite the increased body weight, no hematological differences—blood glucose, triglyceride (TG), and total cholesterol (TCHO) levels—were observed between Veh-rats and LTZ-rats ( H). Following validation of the LTZ-challenged animal model, we examined the pathophysiological condition of the heart. The nuclei of LV cardiomyocytes were flatter in LTZ-rats than in Veh-rats, and the cytoplasm-to-nucleus ratio occupied the same area in LTZ-rats, compared to Veh-rats (86%, p < 0.05) ( A). As the above histological characteristics pertain to hypertrophic cardiomyopathy , we examined the expression levels of heart failure markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Interestingly, the expression levels of both ANP and BNP were significantly higher in LTZ-rats than in Veh-rats (ANP, 4.92-fold, p < 0.05) (BNP, 2.26-fold, p < 0.05) ( B). Subsequently, we performed quantitative RT-PCR to analyze the mRNA expression levels of cardiac physio-functional genes, including ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 ( Atp2a2 ), myosin heavy chain, α isoform ( Myh6 ), and myosin heavy chain, β isoform ( Myh7 ). The mRNA expression level of Atp2a2 , an indicator of cardiac contractility, increased in LTZ-rats compared to that in Veh-rats (1.88-fold, p < 0.05) ( C). Additionally, Myh6 and Myh7 expression, reflecting increased cardiomyocyte size and LV wall thickening, respectively, increased in LTZ-rats compared to Veh-rats ( Myh6 , 1.19-fold, p < 0.05) ( Myh7 , 1.28-fold, p < 0.05) ( C). We also analyzed the mRNA expression of these three genes in vitro using the H9c2 cell line and observed a similar increase in expression in letrozole-treated cells (LTZ-cells) compared to that in vehicle-treated cells (Veh-cells) ( Atp2a2 , 1.20-fold, p < 0.05) ( Myh6 , 1.19-fold, p < 0.05) ( Myh7 , 1.28-fold, p < 0.05) ( D). However, no significant change in mRNA expression levels was observed in cells treated with ethynylestradiol (E2-cells) and testosterone (T-cells) ( D). These histological and molecular alterations in cardiomyocytes indicate that letrozole may induce hypertrophic cardiomyopathy of the LV. 

Next, we looked at the reasons underlying the hypertrophic changes in letrozole-exposed hearts, considering that cardiac structural remodeling, including cardiac hypertrophy, might develop in response to altered energy metabolism . We first looked for traces of altered lipid metabolism related to TG accumulation and FA uptake and oxidation, as FA serves as the primary energy source for the heart . TG levels in the hearts were higher in LTZ-rats than in Veh-rats (2.4-fold, p < 0.05) ( A). Conversely, Western blotting revealed that the expression of cluster of differentiation 36 (CD36), a plasma membrane protein facilitating FA uptake in cells, significantly decreased in LTZ-rats compared to that in Veh-rats (41%, p < 0.05) ( B). Additionally, quantitative RT-PCR indicated that the expression of adipose triglyceride lipase ( A tgl ), an enzyme that hydrolyzes TGs to diacylglycerols and serves as the rate-limiting step of lipolysis, decreased in LTZ-rats compared to that in Veh-rats (59%, p < 0.05) ( C). In line with the expression of Atgl , the expression of genes associated with β-oxidation, including carnitine palmitoyl transferase I ( Cpt1 ), very long-chain acyl-CoA dehydrogenase ( Acadvl ) and medium-chain acyl-CoA dehydrogenase ( Acadm ) also decreased in LTZ-rats compared to that in Veh-rats ( Cpt1 , 75%, p < 0.05) ( Acadvl , 81%, p < 0.05) ( Acadm , 73%, p < 0.05) ( C). To determine whether these changes in lipid metabolism were attributed to reduced estrogen levels or to letrozole, an in vitro assay—using LTZ-, E2-, and T-treated H9c2 cells—was performed. CD36 was expressed at higher levels in E2-cells than in Veh-cells (1.23-fold, p < 0.05) ( D). The transcript-level expression of A tgl and β-oxidation genes, including Cpt1 , Acadvl and Acadm, was also higher in E2-cells than in Veh-cells ( Cpt1 , 2.16-fold, p < 0.05) ( Acadvl , 1.36-fold, p < 0.05) ( Acadm , 1.58-fold, p < 0.05) ( E). However, no significant changes in protein and mRNA expression were observed in LTZ-cells compared to Veh-cells, even though Cpt1 level increased in T-cells compared to Veh-cells (1.53-fold, p < 0.05) ( E). These findings indicate increased TG accumulation and decreased TG degradation and FA oxidation, thereby suggesting that FA consumption by cardiomyocytes is lower in the presence of letrozole-induced reduced estrogen levels. 

After looking at the changes in lipid metabolism, we examined glucose metabolism to determine whether glucose consumption increased to compensate for the decrease in FA consumption. The expression levels of hexokinase 2 (HK2)—which catalyzes the first step of glycolysis —and pyruvate kinase muscle form 2 (PKM2)—which catalyzes the last step of glycolysis —increased in LTZ-rats compared to those in Veh-rats (HK2, 1.82-fold, p < 0.05) (PKM2, 1.42-fold, p < 0.05) ( A). Additionally, the expression of pyruvate dehydrogenase (PDH)—the enzyme that converts pyruvate to acetyl-CoA —also increased in LTZ-rats compared to Veh-rats (2.03-fold, p < 0.05) ( A). From an in vitro assay using the H9c2 cell line, the expression levels of HK2, PKM2, and PDH were found to be higher in LTZ-cells compared to those in Veh-cells (HK2, 2.00-fold, p < 0.05) (PKM2, 1.24-fold, p < 0.05) (PDH, 1.86-fold, p < 0.05) ( B). Although the expression of PKM2 and/or PDH also increased in E2-cells and T-cells compared to that in Veh-cells (E2-cells, PKM2, 1.16-fold, p < 0.05) (E2-cells, PDH, 1.35-fold, p < 0.05) (T-cells, PDH, 1.55-fold, p < 0.05), the extent of the increase was more notable in LTZ-cells than in E2-cells and T-cells ( B). To determine whether letrozole induces increased glucose consumption in cardiomyocytes independent of its role in reducing estrogen levels, we performed an in vitro assay using H9c2 cells, wherein the cells were exposed to high and low concentrations of glucose. In the presence of high glucose concentrations (HG), the expression levels of HK2, PKM2, and PDH increased in letrozole-treated cells (HG-LTZ-cells) compared to Veh-cells (HG-Veh-cells) (HK2, 1.73-fold, p < 0.05) (PKM2, 1.30-fold, p < 0.05) (PDH, 1.61-fold, p < 0.05) ( A). When exposed to low glucose concentrations (LG), the expression of HK2 alone increased in LTZ-cells (LG-LTZ-cells) compared to that in Veh-cells (LG-Veh-cells) (2.19-fold p < 0.05) ( A). Subsequently, we performed glycolysis stress tests for quantitatively monitoring the glycolysis rate as an indicator of glucose consumption. In LTZ-cells, the extracellular acidification rate (ECAR) substantially increased compared to that in Veh-cells (2.17-fold p < 0.05) ( B). These findings indicate that letrozole enhances glycolysis in cardiomyocytes, independent of its role in modulating estrogen levels, to compensate for decreased FA oxidation. 

Previous cohort studies on breast cancer indicate that AIs increase the risk of heart failure by up to 86% and cardiovascular mortality up to 50%, compared with SERMs such as tamoxifen [ , , ]. The underlying cause has been linked to lipid metabolism changes in the heart, induced by decreasing levels of circulating estrogens . However, not many research groups have focused on the direct influence of letrozole in cardiac metabolism. After exposure to letrozole for four weeks, the rats’ hearts showed metabolic energy changes involving decreased FA oxidation and increased glycolysis. Taking into account the fact that AIs induce more adverse hypertrophic changes in the heart compared to tamoxifen, letrozole may function to mediate such changes, along with modulating the estrogen levels. To confirm the reduced estrogen levels in the animal model post letrozole exposure, we assessed the female reproductive organs, i.e., the ovaries and the uterus. When the subcutaneously administered pellets of letrozole were released into the body, the reduced levels of circulating estrogens induced in turn were reflected in the form of atrophy of the uterus, as letrozole exposure represents hypoestrogenism and hyperandrogenism in females . LTZ-rats showed multi-ovarian cysts and a light uterus with a thinning endometrium, similar to the findings in our previous studies [ , , ]. Furthermore, we also observed decreased expression of estrogen-responsive genes in the uterus, along with increased body weight. Although no significant changes in hematological profiles, including blood glucose, TG and TCHO, were observed in LTZ-rats, this might be considered to be due to a shortened letrozole exposure duration, unlike our previous studies. Nevertheless, these findings indicate that estrogen levels were sufficiently reduced in the animal model. Next, we examined whether letrozole induced cardiac structural remodeling. It is interesting to note that cardiomyocytes within the LV had a higher cytoplasm-to-nucleus ratio and flatter nuclei in LTZ-rats compared to those in the Veh-rats, an observation that indicates structural remodeling . Moreover, this histological observation was supported by increased expression levels of heart failure markers , such as ANP and BNP, and physio-functional markers, such as Atp2a2 , Myh6 , and Myh7 , which indicate contractility , cardiomyocyte size  and LV wall thickness , respectively. In parallel with the animal study, H9c2 cardiomyocytes treated with letrozole also showed increased expression of the above three physiological markers. However, the two sex steroid hormones, ethynylestradiol (E2) and testosterone (T), failed to change these physiological markers. These results suggest that letrozole could directly make the heart vulnerable to cardiac hypertrophy. It has been reported that cardiac structural remodeling is mediated by a shift in cardiac energy metabolism, where glucose is utilized as the primary energy source instead of FAs [ , , , ]. LTZ-rats showed cardiac hypertrophy and TG accumulation, and decreased β-oxidation, TG degradation, and intracellular uptake of FA. As the estrogen receptor acts as a transcription factor for the peroxisome proliferator-activated receptor ( PPAR ) α promoter , the expression of PPARα-regulated β-oxidative transcripts including Cpt1 , Acadvl and Acadm decreased in the hearts of LTZ-rats where the circulating estrogen levels were low . The expression of A tgl —associated with TG degradation—was also decreased due to reduced estrogen receptor signaling , and reduced A tgl led to excess TG accumulation in the heart . Furthermore, TG accumulation prevents the intracellular uptake of FA by CD36 in cardiomyocytes . These series changes in lipid metabolism were also observed in E2-treated H9c2 cells, but not in LTZ-treated cells. These findings suggest that estrogens can modulate lipid metabolism by regulating estrogen receptor signaling and PPARα signaling, and thereby, might induce cardiac structural remodeling via altered lipid metabolism. Therefore, it also might be intimated that PPARα agonists, including fenofibrate as an activator of fatty acid oxidation, might have an effect as a target drug for ER+ breast cancer, although this needs to be justified through further study . In the presence of decreased FA oxidation, the heart increases glucose consumption to meet the energy demands during pathological conditions . LTZ-rats showing decreased FA oxidation displayed increased glycolysis. As a causative factor of this increase, estrogens themselves could be excluded because estrogens are known to stimulate glycolysis . It is expected for the cause to be a compensatory mechanism from estrogen-induced decreases in FA oxidation, and/or to be independent of its role in modulating estrogen levels. Interestingly, this induced glucose consumption was more notable in H9c2 cells treated with letrozole than in E2-treated cells. This suggests that letrozole increased glycolysis, independent of its role in modulating estrogen levels. To determine whether letrozole enhances glucose consumption in cardiomyocytes, we performed a glucose addition assay and glycolysis stress test using H9c2 cells. The LTZ-cells showed increased glycolysis in the presence of high glucose concentration and increased ECAR, which can directly evaluate the amount of glucose consumed by the cells quantitatively . This demonstrates that letrozole facilitates glucose consumption in cardiomyocytes independently in response to an estrogens-induced decrease in FA oxidation. This finding indicates that letrozole increases glycolysis to compensate for decreased FA oxidation and independently accelerates glucose consumption. Our study demonstrated that letrozole, an AI, modulates cardiac metabolic remodeling in two ways. One is via estrogen-mediated lipid metabolism, and the other is via estrogen-independent glycolysis. Reduced estrogen levels induced by letrozole suppress fatty acid consumption, and the insufficient energy is compensated for through enhanced glucose consumption in cardiomyocytes. Additionally, letrozole directly induces glucose consumption, while estrogens itself do not mediate it. In such cardiac metabolic remodeling conditions, the heart is unable to meet its high energy demands, and therefore, is predisposed to cardiac structural remodeling, including concentric hypertrophy. To prevent cardiac remodeling, we suggest that ER+ breast cancer patients receiving letrozole should abstain from a carbohydrate-rich diet and medicate with an activator of fatty acid oxidation for facilitating fatty acid oxidation, at least during the letrozole treatment. 

4.1. Antibody Antibodies used in the present study are shown in below. 4.2. Animals Norway female rats were accommodated for one week and tested in the pathogen-free facility at Chungnam National University, in line with the Chungnam National University Animal Care Committee (202006A-CNU-104). We provided them with standard chow diet (LabDiet ® Rodent Diet 5001 contains as a percentage of total calories: 28% protein, 60% carbohydrate and 12% fat) and water ad libitum up to the age of eight weeks. At the age of four weeks, letrozole slow releasing pellets (0.1 mg/kg/day) were inserted subcutaneously in the nape. The standard chow diet was provided consistently for four weeks and the rats were then sacrificed. The number of rats used for the experiment was three and three for each group, designated Vehicle-rats (Veh-rats) and Letrozole-rats (LTZ-rats). 4.3. Hematological Measure Assay Blood glucose was measured using Accu-Chek Active [Model GB] (CR2032, Roche Diabetes Care GmbH, Mannheim, Germany). Triglyceride (TG) was evaluated with non-diluted serum. Serum TG level was measured with FUJI DRI-CHEM SLIDE (TG-P III) by DRI-CHEM4000 (Fuji Film, Tokyo, Japan). Identically, total cholesterol (TCHO) level was measured FUJI DRI-CHEM SLIDE (TCHO-P III), after serum was diluted by 1/5 fold with sterilized phosphate-buffered saline (PBS). 4.4. Hematoxylin & Eosin Staining Rats’ hearts were arranged in 10% formaldehyde solution and embedded in paraffin. The paraffinized samples were sectioned to 4 μm and stuck on slide glass. The samples were stained with haematoxylin and eosin (H&E) after being de-waxed and rehydrated. The stained samples were examined using a VM600 Digital Slide Scanning System (Motic, San Francisco, CA, USA). 4.5. Tissue Triglyceride Measure Assay LV tissues of the hearts were homogenized with PBS and centrifuged at 13,000 rpm for 10 min. Supernatants were discarded and pellets were dried. After drying, the pellets were re-suspended in 200 μL PBS. An amount of 50 μL of resuspension samples was dispensed in a 96 well plate and 150 μL of enzyme reagent (AM157S, ASAN PHARM. CO., LTD., Seoul, Korea) was added. The plate was incubated at 37 °C for 1 h. After 1 h, relative absorbance value of the incubated samples was measured at 550 nm using a microplate reader (Infinite 200 PRO, Tecan Life Sciences, Männedorf, Switzerland). 4.6. Cell Culture To validate in vivo experimental data, we performed in vitro assays using the H9c2 cardiomyocytes line. The cells were grown at 37 °C in a 5% CO 2 atmosphere and were cultured with Dulbecco’s modified Eagle’s medium (DMEM)-High glucose (LM 001-05, Welgene, Gyeongsan, Gyeongbuk, Korea) added to 5% fatal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Additionally, to remove endogenous steroid hormones, a starvation medium was used, composed of DMEM/F-12 (LM 002-05, Welgene) added to 2% charcoal dextran fetal bovine serum (CD-FBS) and 1% P/S. To assess fluctuations in lipid metabolism, we supplied fatty acids to cells. Fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) dissolved in absolute ethanol (K46253383 506, EMD Millipore Corp.) were treated and incubated for 48 h. Letrozole, ethynylestradiol and testosterone (Letrozole, L0248, Tokyo chemical industry CO. LTD., Tokyo, Japan) (Ethynylestradiol, E0037, Tokyo chemical industry CO. LTD) (Testosterone, T0027, Tokyo chemical industry CO. LTD) were dissolved in dimethyl sulfoxide (DMSO) (#MKCH9998, Sigma-Aldrich, St. Louis, MO, USA). The three reagents were added to 100 nM/L respectively and incubated for 24 h. 4.7. Glucose Addition Assay After H9c2 cells were cultured with DMEM-High glucose (LM 001-05, Welgene) added to 5% FBS and 1% P/S, the medium was changed to a low glucose medium (LM 001-17, Welgene), which contained glucose 1000 mg/L, and was added to 2% CD-FBS and 1% P/S, similar to the above starvation medium. Following additional supplementation with fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM), the cells were incubated for 48 h. At 24 h of incubation with the low glucose medium, extra glucose was supplemented to high glucose group cells using 45% D-glucose (LS 001-02, Welgene) to reach glucose concentrations of 4500 mg/L, and incubation was maintained for the remaining 24 h. 4.8. Glycolysis Stress Test After preparing H9c2 cells incubated in a starvation medium to which fatty acids were added for 48 h and treated with letrozole for an additional 24 h, the cells were further incubated with a low glucose medium (LM 001-17, Welgene) free from FBS and P/S for 1 h. The medium was changed to XFp running buffer (1035757-100, Agilent Technologies, Santa Clara, CA, USA), which contains the identical amounts of D-glucose, sodium pyruvate and L-glutamine as a low glucose medium, to decarboxylate the cells for 1 h. Glucose (25 mM) was added and extracellular acidification rate (ECAR) was measured using a Seahorse XFp glycolytic rate assay kit (103022-100, Agilent Technologies) and Seahorse XFp analyzer (S7802A, Agilent Technologies). 4.9. Total RNA Extraction and Real-Time Quantitative PCR Total RNA samples, including the LV tissues of the hearts and H9c2 cells, were extracted using TRIzol reagent (15596-026, Life Technologies, Carlsbad, CA, USA). cDNA was synthesized with 1.5 µg of total RNA samples and reverse transcriptase kit (SG-cDNAS100, Smartgene, Lausanne, Switzerland). Quantitative PCR (real-time PCR) was performed using Excel Taq Q-PCR Master Mix (SG-SYBR-500, Smartgene) and Stratagene Mx3000P (Agilent Technologies). All primers used for real-time PCR were produced by Bionics Inc. (Seoul, Korea) and are listed in . mRPLP0 was used as control for in vivo and in vitro samples. All experiments were run more than three times, and mRNA values were calculated based on the cycle threshold and monitored for an amplification curve. 4.10. Western Blotting Protein samples, including LV tissues of the hearts and H9c2 cells, were extracted using T-PER reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA), which is a protein lysis buffer. Through a Bradford assay, the sample concentration was quantified using PRO-Measure solution (#21011, Intron, Seoul, Korea). SDS-PAGE electrophoresis was performed on 10% polyacrylamide gels and the gels were transferred to a PVDF membrane. Following the transfer, the membranes were blocked using 3% BSA (9048-46-8, LPS Solution, Chisinau, Moldova) with diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies ( ) dissolved with 3% BSA were operated overnight in 4 °C. The membranes were washed three times with TBS-T and secondary antibodies ( ) were operated in identical fashion to primary antibodies. The blotting results were detected with ECL solution (XLS025-0000, Cyanagen, Bologna, Italy) and Chemi Doc (Fusion Solo, Vilber Lourmat, Marne-la-Vallée, France). 4.11. Statistical Analysis Data are reported as the mean ± standard deviation (SD). Using Graph Pad Software (Graph Pad Inc., San Diego, CA, USA), Student’s t -test and one-way ANOVA were performed to recognize differences between means. 

Antibodies used in the present study are shown in below. 

Norway female rats were accommodated for one week and tested in the pathogen-free facility at Chungnam National University, in line with the Chungnam National University Animal Care Committee (202006A-CNU-104). We provided them with standard chow diet (LabDiet ® Rodent Diet 5001 contains as a percentage of total calories: 28% protein, 60% carbohydrate and 12% fat) and water ad libitum up to the age of eight weeks. At the age of four weeks, letrozole slow releasing pellets (0.1 mg/kg/day) were inserted subcutaneously in the nape. The standard chow diet was provided consistently for four weeks and the rats were then sacrificed. The number of rats used for the experiment was three and three for each group, designated Vehicle-rats (Veh-rats) and Letrozole-rats (LTZ-rats). 

Blood glucose was measured using Accu-Chek Active [Model GB] (CR2032, Roche Diabetes Care GmbH, Mannheim, Germany). Triglyceride (TG) was evaluated with non-diluted serum. Serum TG level was measured with FUJI DRI-CHEM SLIDE (TG-P III) by DRI-CHEM4000 (Fuji Film, Tokyo, Japan). Identically, total cholesterol (TCHO) level was measured FUJI DRI-CHEM SLIDE (TCHO-P III), after serum was diluted by 1/5 fold with sterilized phosphate-buffered saline (PBS). 

Rats’ hearts were arranged in 10% formaldehyde solution and embedded in paraffin. The paraffinized samples were sectioned to 4 μm and stuck on slide glass. The samples were stained with haematoxylin and eosin (H&E) after being de-waxed and rehydrated. The stained samples were examined using a VM600 Digital Slide Scanning System (Motic, San Francisco, CA, USA). 

LV tissues of the hearts were homogenized with PBS and centrifuged at 13,000 rpm for 10 min. Supernatants were discarded and pellets were dried. After drying, the pellets were re-suspended in 200 μL PBS. An amount of 50 μL of resuspension samples was dispensed in a 96 well plate and 150 μL of enzyme reagent (AM157S, ASAN PHARM. CO., LTD., Seoul, Korea) was added. The plate was incubated at 37 °C for 1 h. After 1 h, relative absorbance value of the incubated samples was measured at 550 nm using a microplate reader (Infinite 200 PRO, Tecan Life Sciences, Männedorf, Switzerland). 

To validate in vivo experimental data, we performed in vitro assays using the H9c2 cardiomyocytes line. The cells were grown at 37 °C in a 5% CO 2 atmosphere and were cultured with Dulbecco’s modified Eagle’s medium (DMEM)-High glucose (LM 001-05, Welgene, Gyeongsan, Gyeongbuk, Korea) added to 5% fatal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Additionally, to remove endogenous steroid hormones, a starvation medium was used, composed of DMEM/F-12 (LM 002-05, Welgene) added to 2% charcoal dextran fetal bovine serum (CD-FBS) and 1% P/S. To assess fluctuations in lipid metabolism, we supplied fatty acids to cells. Fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) dissolved in absolute ethanol (K46253383 506, EMD Millipore Corp.) were treated and incubated for 48 h. Letrozole, ethynylestradiol and testosterone (Letrozole, L0248, Tokyo chemical industry CO. LTD., Tokyo, Japan) (Ethynylestradiol, E0037, Tokyo chemical industry CO. LTD) (Testosterone, T0027, Tokyo chemical industry CO. LTD) were dissolved in dimethyl sulfoxide (DMSO) (#MKCH9998, Sigma-Aldrich, St. Louis, MO, USA). The three reagents were added to 100 nM/L respectively and incubated for 24 h. 

After H9c2 cells were cultured with DMEM-High glucose (LM 001-05, Welgene) added to 5% FBS and 1% P/S, the medium was changed to a low glucose medium (LM 001-17, Welgene), which contained glucose 1000 mg/L, and was added to 2% CD-FBS and 1% P/S, similar to the above starvation medium. Following additional supplementation with fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM), the cells were incubated for 48 h. At 24 h of incubation with the low glucose medium, extra glucose was supplemented to high glucose group cells using 45% D-glucose (LS 001-02, Welgene) to reach glucose concentrations of 4500 mg/L, and incubation was maintained for the remaining 24 h. 

After preparing H9c2 cells incubated in a starvation medium to which fatty acids were added for 48 h and treated with letrozole for an additional 24 h, the cells were further incubated with a low glucose medium (LM 001-17, Welgene) free from FBS and P/S for 1 h. The medium was changed to XFp running buffer (1035757-100, Agilent Technologies, Santa Clara, CA, USA), which contains the identical amounts of D-glucose, sodium pyruvate and L-glutamine as a low glucose medium, to decarboxylate the cells for 1 h. Glucose (25 mM) was added and extracellular acidification rate (ECAR) was measured using a Seahorse XFp glycolytic rate assay kit (103022-100, Agilent Technologies) and Seahorse XFp analyzer (S7802A, Agilent Technologies). 

Total RNA samples, including the LV tissues of the hearts and H9c2 cells, were extracted using TRIzol reagent (15596-026, Life Technologies, Carlsbad, CA, USA). cDNA was synthesized with 1.5 µg of total RNA samples and reverse transcriptase kit (SG-cDNAS100, Smartgene, Lausanne, Switzerland). Quantitative PCR (real-time PCR) was performed using Excel Taq Q-PCR Master Mix (SG-SYBR-500, Smartgene) and Stratagene Mx3000P (Agilent Technologies). All primers used for real-time PCR were produced by Bionics Inc. (Seoul, Korea) and are listed in . mRPLP0 was used as control for in vivo and in vitro samples. All experiments were run more than three times, and mRNA values were calculated based on the cycle threshold and monitored for an amplification curve. 

Protein samples, including LV tissues of the hearts and H9c2 cells, were extracted using T-PER reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA), which is a protein lysis buffer. Through a Bradford assay, the sample concentration was quantified using PRO-Measure solution (#21011, Intron, Seoul, Korea). SDS-PAGE electrophoresis was performed on 10% polyacrylamide gels and the gels were transferred to a PVDF membrane. Following the transfer, the membranes were blocked using 3% BSA (9048-46-8, LPS Solution, Chisinau, Moldova) with diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies ( ) dissolved with 3% BSA were operated overnight in 4 °C. The membranes were washed three times with TBS-T and secondary antibodies ( ) were operated in identical fashion to primary antibodies. The blotting results were detected with ECL solution (XLS025-0000, Cyanagen, Bologna, Italy) and Chemi Doc (Fusion Solo, Vilber Lourmat, Marne-la-Vallée, France). 

Data are reported as the mean ± standard deviation (SD). Using Graph Pad Software (Graph Pad Inc., San Diego, CA, USA), Student’s t -test and one-way ANOVA were performed to recognize differences between means. 

Our data indicate that letrozole is an independent molecule that can promote glucose oxidation as well as an estrogen blocker in cardiomyocytes. As the heart typically uses fatty acid as a primary energy source, increased glycolysis is a risk factor for heart health. Interestingly, letrozole increased glycolysis in cardiomyocytes, along with cardiac hypertrophy. We found out that the increase in glucose consumption resulted from two motions: one was a compensatory reaction involving estrogen-mediated reduced fatty oxidation and the other was hormone-independent increased glycolysis. This study proposes perspectives on why ER+ breast cancer patients receiving letrozole are vulnerable to cardiovascular disease and how to establish precautions against cardiac adverse effects from this drug. 

