Visceral Adipose Tissue Promotes Pressure-Induced Heart Failure Associated with Circulating Fatty Acids

Visceral adipose tissue (VAT) is the main source of circulating fatty acids (FAs) that provides the energy substrate for the heart. Till now, studies have not shown a clear association between individual circulating FAs and heart failure (HF). In this study, we aimed to investigate the changes in circulating FAs in HF mice and their association with VAT by removing epididymal white adipose tissue (eWAT). Here, we found that the serum levels of four fatty acids, namely arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid, were signicantly decreased in pressure-induced HF mice via transverse aortic constriction (TAC) surgery accompanied with cardiac enlargement and brosis. Most importantly, removal of eWAT in mice led to a signicant decrease in the levels of the above-mentioned fatty acids without any signicant difference between the HF and sham groups. Accordingly, cardiac enlargement and brosis were signicantly alleviated. We concluded that VAT excision alleviated TAC-induced cardiac failure by decreasing serum arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels.


Introduction
Heart failure (HF) is a major public health issue prevalent in approximately 2% of the population in Western countries and is associated with high morbidity and mortality rates despite pharmacological and non-pharmacological treatment [1]. Evidence suggests that development of HF is associated with abnormalities in cardiac energy metabolism [2]. The failing heart shows an energy de cit with lower levels of adenosine triphosphate (ATP) than does a normal healthy heart, probably because of altered energy substrate availability and impaired mitochondrial oxidative capacity [3]. In the healthy adult heart, the majority of ATP is produced by mitochondrial oxidation of fatty acids. Long-chain fatty acids are crucial oxidizable metabolic substrates for the heart tissue. After esteri cation into phospholipids, longchain fatty acids work as building blocks of cellular membranes and participate in associated signal pathways [4]. However, circulating long-chain FAs are heterogeneous, and the speci c FAs mainly utilized in cardiomyocyte metabolism during HF development remains unknown.
Changes in the cardiac environment cause cardiac cells to release regulatory peptides which have pathophysiological roles in organs distal to the heart, such as adipose tissue, muscle, and liver, where cell death, growth, brosis, and remodeling are regulated [5,6]. Natriuretic peptides are regulatory peptides released from the heart. In addition to mediating natriuresis, diuresis, and vasodilation, natriuretic peptides enhance lipolysis and lipid mobilization [7,8]. Moreover, it has also been reported that these natriuretic peptides induce changes in white adipose tissue (WAT) depots promoting the development of brown adipose tissue (BAT) characteristics (such as increased thermogenesis and energy expenditure), a phenomenon termed browning or beiging [9]. These ndings provide further evidence of crosstalk between the heart and the peripheral adipose tissues. VAT is not only responsible for lipid storage by lipogenesis, but also for breakdown of fatty acids (FAs) by lipolysis, consequently generating substrates for energy metabolism via β-oxidation [10]. FAs derived from VAT via lipolysis mainly serve as oxidizable substrates for the heart, and may be responsible for HF. We hypothesized that surgically removing VAT may decrease some kinds of circulating FAs levels, thus preventing HF.
In this study, we aimed to investigate the circulating long-chain fatty acids levels in pressure-induced cardiac failure models to elucidate the potential FAs mainly utilized in cardiomyocyte metabolism. In addition, we examined whether VAT excision could alleviate pressure-induced HF through changes in the levels of some FAs. This study will provide a new idea for the prevention and treatment of heart failure in the future.

Animals
Male C57BL/6J wild type mice were purchased from Slac Laboratory Animals (Shanghai, China), and were studied at 10 weeks of age. Animals were housed at 22°C with a 12-h light/12-h dark cycle with free access to water and standard chow. The study conforms to the relevant guidelines and regulations of the Bio-X Institutes of Shanghai Jiao Tong University (Shanghai, China) and was performed after securing approval from the Animal Care and Use Committee of Shanghai Xinhua Hospital a liated to Shanghai Jiao Tong University School of Medicine (Shanghai, China).

Experimental models
Ten weeks old C57BL/6J male mice (20-25g) were divided into two groups. In one group, mice were anesthetized with 80-100 mg/kg ketamine and 5-10 mg/kg xylazine intraperitoneally, and then the epididymal white adipose tissue (eWAT) and the main visceral adipose tissue (VAT) were removed. The other group served as normal controls with eWAT reserved. Both groups were then randomly divided into sham and heart failure (HF) groups. The HF mice were established by transverse aortic constriction (TAC). Brie y, mice were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally), and after opening the chest wall, the thoracic aorta was identi ed after blunt dissection through the intercostal muscles. Silk suture (7-0) was placed around the transverse aorta and tied around a 26-gauge blunt needle, which was subsequently removed. Sham-operated mice underwent a similar surgical procedure without aortic constriction. After 11 weeks, cardiac structure and function were determined by echocardiography. Mice were then euthanized under iso urane anesthesia by cervical dislocation, and the hearts were weighed and collected for further analysis.

Echocardiography
Echocardiography was performed 11 weeks after TAC under the guidance of M-shaped curves of twodimensional ultrasonic echocardiogram. Every 3 continuous means of the cardiac cycle were selected as the test value. Some cardiac diameters such as fractional shortening (FS), diastolic left ventricular posterior wall (LVPWd), and diastolic left ventricular internal diameter (LVIDd) were quanti ed as previously described [11] 2.4 Processing of specimens All mice were weighed before opening the chest wall. The heart was weighed after being taken out, leaving the left ventricle intact (including the inter-ventricular septum) for further weighing. Then, the weight index of the left ventricle (weight of the left ventricle/the body weight) was calculated. Mouse serum was isolated from whole blood, and blood samples were incubated for 60 min at room temperature to induce coagulation. Afterwards, samples were centrifuged at 5000 rpm for 5 min at 4˚C and the supernatant (serum) was collected and stored at -80℃ until further analysis.

Hematoxylin and eosin/Masson's staining
Heart tissue samples were formalin-xed, para n-embedded and stained with Hematoxylin/Eosin (HE) and Masson. Cardiac myocyte cross sectional area was assessed using HE-stained and Masson-stained sections of myocardium. After staining, the myocytes were cut into transverse sections and the perimeter of the cell borders of 50 myocytes were measured using CellSens (Olympus). Myocytes were selected in up to ten 400x microscopic elds each separated into 20 equally sized squares. Only one transversally myocyte per square was measured to increase representativeness.

Quantitative real time PCR (qRT-PCR)
Total RNA was isolated from heart samples with TRIzol reagent (Invitrogen; Thermo Fisher Scienti c, Inc.), puri ed with the RNEasy Kit (Qiagen, Hilden, Germany), and reverse transcribed using the oligo (dT) primers with the Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche diagnostics, Shanghai, china), as previously described [12]. Samples were analyzed for mRNA levels using SYBRgreen Master Mix (cat. no. 04887352001; Roche diagnostics), and ampli cation was monitored using the CF X96 Real-Time PCR system (Bio-Rad, Munich, Germany) with the following thermocycling conditions: 5 s at 95˚C followed by 30 s at 60˚C for 42 cycles. The reaction mixture was composed of 10 µl SYBR premix Ex Taq II, 0.2 µl primers, 8.8 µl H 2 O, and 1 µl DNA. Data were normalized to the expression levels of GAPDH (internal control) and were presented as arbitrary units normalized to wild type expression levels. Primer sequences in the present study are presented in Table 1.

Analysis of fatty acids
Mouse serum was isolated from whole blood. For FAs serum pro ling, 100 μl of murine serum was hydrolyzed under alkaline-methanolic conditions, neutralized and diluted in methanol (1:10) containing internal FA standards. Analysis of fatty acids was performed using HPLC (Agilent 1200 HPLC system), coupled with an Agilent 6460 triple quad mass was performed, as previously described [13]. Serum concentration of FFA was measured using a HR-NEFA kit (WAKO), as previously described [13].
Triglycerides (TG) in serum were determined using a triglyceride assay kit (DiaSys GmbH), according to the manufacturer's instructions.

Statistical analysis
Quantitative data were expressed as mean ± standard error of the mean (SEM). Comparison of mean values between groups was evaluated by the following tests: two-way ANOVA followed by post Bonferroni tests , two-way repeated measures ANOVA followed by post Bonferroni tests, one-way ANOVA followed by Tukey's or Bonferroni multiple comparison test, or unpaired t tests, as appropriate, and data were analyzed with GraphPad Prism software. Exact value of number is provided for each type of experiments. Statistical signi cance was assumed at P< 0.05. Vertical lines in the histograms indicate standard error of the mean (SEM).

Removal of eWAT attenuates TAC-induced cardiac enlargement
To further evaluate the cardiac structure changes, mice were subjected to 11 weeks of pressure overload by transverse aortic constriction (TAC), or sham operations (Sham). The heart weights and cardiac characteristics are presented in Fig 1. Heart weight-to-body weight ratios (HW/BW) and weight index of the left ventricle (LW/BW) were signi cantly increased in TAC mice compared to sham mice (P<0.01), however, the ratios were signi cantly decreased in TAC mice with eWAT excision compared to TAC mice with eWAT reserved (P<0.05). Echocardiographic measurements revealed that LVPWd and LVIDd were signi cantly increased (P<0.05) while FS% was notably decreased (P<0.01) in TAC mice compared to sham mice. However, LVPWd showed a more pronounced decrease in eWAT excised mice compared to eWAT reserved mice following TAC (P<0.05). LVIDd diameters were not signi cantly altered between eWAT excision and eWAT reserved mice undergoing TAC. In addition, FS% was signi cantly increased in TAC mice with eWAT reserved compared to eWAT excised (P<0.05). These data showed that there was certain improvement in myocardial hypertrophy and cardiac e ciency after the intervention.
3.2 Removal of eWAT changes cardiac morphology in TAC-induced HF mice The cardiac morphological changes are presented in Fig 2. The size of the hearts increased markedly in size and weight compared to sham group at 11 weeks after TAC. TAC-induced cardiac enlargement was attenuated after eWAT excision. Accordingly, the myocardial area in cardiac cross section increased signi cantly in TAC-induced mice whereas this effect was diminished after eWAT excision. Cardiac brosis was signi cantly induced during pressure overload in HF mice as shown by Masson staining. In the TAC group, myocyte hypertrophy and ber derangement were found with a signi cant amount of collagen piling in the intercellular space. The above changes were effectively alleviated after eWAT removal.

Removal of eWAT decreases mRNA expression of ANP, COL1, and COL3A1 in pressure-induced HF mice
In keeping with the morphological changes described above, marker genes for cardiac failure such as the ANP, BNP, MYH7, CTGF, COL1, and COL3A1were markedly induced by TAC in WT-hearts (Fig. 3). To recognize potential FAs involved in the regulation of cardiac function in pressure-induced HF mice, we examined 24 kinds of FAs, triglycerides (TGs), and free circulating FAs (FFA) in serum. As depicted in TAC and sham groups presented in Fig. 4, in eWAT reserve mice, pressure overload resulted in a signi cant reduction of selected circulating FAs including arachidic acid (P<0.001), behenic acid (P<0.001), lignoceric acid (P<0.01), and docosapentaenoic acid (P<0.05) after TAC (Fig. 4K, R, V, U). The serum TG level was signi cantly increased (P<0.05). In addition, the serum FFA levels were not different between the two groups. Next, we measured whether the lack of eWAT regulated the release of these preferential FAs in a selective manner. We found that in sham and TAC groups, eWAT excision signi cantly attenuated the circulating arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels compared to eWAT reserve groups (P<0.05). But the serum TG and FFA were not different between the two groups. Most importantly, after excision of eWAT, the circulating arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels were not signi cantly different between the sham and TAC groups. Taken together, pressure-induced cardiac damage was accompanied by changes in circulating FAs among which arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid displayed the most apparent variation, and these changes can be reversed by deletion of eWAT.

Discussion
In this study, we aimed to investigate the serum FAs in pressure-induced HF mice with eWAT reserved and eWAT excised. Mice without aortic constriction that underwent a similar surgical procedure served as controls. We demonstrated for the rst time that from among multitudinous serum FAs, arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels were signi cantly decreased in pressureinduced cardiac failure model. In addition, removal of eWAT signi cantly alleviated cardiac structure and function in HF mice accompanied by a decrease in these four serum FAs levels, indicating non-cardiac tissues such as eWAT participate in the regulation of cardiac failure.
The role of WAT in HF development and cardiac metabolism has been previously studied, with most studies focusing mainly on cardiac lipase function [14,15]. In this study, we concentrated on VAT and the impact of adipose tissue lipolysis on the development of pressure-induced cardiac failure. This study revealed that the serum triacylglycerol (TG) level was signi cantly increased in pressure-induced HF mice compared to controls. Our ndings corroborated previous studies that reported a positive association between increased adipose tissue lipolysis and impairment of cardiac function in heart failure models [16,17]. Circulating FAs are liberated from WAT via hydrolysis of TG which is catalyzed by two major adipose tissue lipases, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), under hormonal control [18]. Deletion of ATGL in adipose tissue prevents pressure-induced LV failure by decreasing adipose tissue lipolysis [19]. Consistent with previous studies, our ndings showed that removal of eWAT signi cantly alleviated pressure-induced cardiac hypertrophy and brosis, as evidenced by decreased mRNA expression of cardiac hypertrophy and brosis genes ANP, COL1, and COL3A1.
Fatty acids are involved in post-translational modi cation of proteins through protein acylation and activation of protein kinases. Recent experimental ndings have indicated that several cardiac genes are under the control of fatty acids [20,21]. However, there are multiple FAs present in circulating bloodstream, and it is not clear which of them are mainly involved in cardiac energy metabolism. We analyzed serum FAs using HPLC, which was previously utilized in cardiovascular research for the identi cation of new molecular co-mediator of exercise-induced cardiac hypertrophy [13]. Our study found that serum arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels were signi cantly decreased in pressure-induced cardiac injury. Arachidic acid, behenic acid, and lignoceric acid are long-chain saturated FAs that are found in circulation. Circulating long-chain saturated FAs with 20 carbons or more are integrated biomarkers of diet and metabolism [22]. The long-chain saturated FAs are known components of ceramides that are involved in apoptosis and cardiac dysfunction [23,24]. The heart of genetically engineered mice with reduced ceramides containing long-chain saturated FAs showed increased brosis, endoplasmic reticulum stress, and apoptosis [25]. Lemaitre et al have identi ed an association of higher levels of plasma phospholipid very-long-chain saturated fatty acids with lower risk of incident HF in a prospective cohort of older American adults [22]. Similar results were obtained in our study, indicating that decreased arachidic acid, behenic acid, and lignoceric acid were probably involved in pressure-induced cardiac failure. In addition, we also found that the serum level of docosapentaenoic acid, a long-chain unsaturated fatty acid, was signi cantly decreased in pressure-induced HF mice. In addition, we found no increased levels of FAs in serum of pressure-induced heart failure mice. This eliminated the probability that these reduced fatty acids were converted to other fatty acids. Thus, we hypothesized that these four fatty acids are involved in cardiac function impairment during pressure overload. However, this needs to be veri ed by analyzing lipid class in cardiac tissue through further studies.
The current study found that mice after removal of eWAT showed a marked reduction of these four fatty acids in both normal and pressure-induced HF mice compared to mice with eWAT reserved, which indicated that these four fatty acids may be principally derived from eWAT. In addition, we observed a slightly, but not signi cantly, decreased TG and FFA levels. eWAT serves as a main VAT, which can more easily produce FFA than subcutaneous adipose tissue (SAT) because VAT has higher expression of adrenergic receptors 1, 2, and 3 on the cell membrane [26,27]. Surprisingly, no signi cant differences were found in circulating FFA and TG after eWAT removal in this study. One possible explanation for this might be that VST is mainly distributed around abdominal organs including epididymal adipose tissue, omental adipose tissue, mesenteric adipose tissue, retroperitoneal adipose tissue, perirenal adipose tissue, etc [28]. Although eWAT was removed in this study, other visceral adipose tissues may have also played a role in producing FFA. Another possible explanation might be because of the difference in the experimental period selected in this study. There are, however, other possible explanations that need to be further studied. The mechanisms underlying the differential impact of adipose tissue on cardiometabolic risk have started to be unraveled; the inability of the subcutaneous adipose tissue to expand in response to positive energy balance serves as an important mechanism that necessitates further studies on the subcutaneous adipose tissue.

Conclusion
Taken together, the results of this study demonstrate for the rst time that serum arachidic acid, behenic acid, lignoceric acid, and docosapentaenoic acid levels are signi cantly decreased in pressure-induced heart failure. Furthermore, we showed that the decreased levels of these four FAs can be signi cantly reversed by removing eWAT from mice before inducing HF. However, whether these four fatty acids accumulate in cardiomyocytes remains unknown. Further research should be carried out to explore the accumulation of these fatty acids in cardiomyocytes and the damage to the mitochondrial function of cardiomyocytes, so as to provide new therapeutic targets for clinical treatment of heart failure.

Declarations Data availability statement
The data used to support the ndings of this study are available with the corresponding author upon request.

Funding statement
This work was supported by the National Natural Science Foundation of China (grant number 81901990).

Declaration of Competing Interests
The authors declare that there are no con icts of interest.