Association of Lipid Composition and Unsaturated Fatty Acids of VLDL with Atrial Remodeling in Metabolic Syndrome

doi:10.21203/rs.3.rs-1718690/v1

Background: Subjects with metabolic syndrome (MetS) commonly have atrial remodeling, which indicates a risk for atrial fibrillation.

Objective: This study determined MetS-related changes in lipid components in very-low-density lipoprotein (VLDL), which has been shown causing atrial remodeling, and the effect of statins on these changes, and the correlation between atrial remodeling and VLDL lipid compositions.

Methods: Blood samples were collected from 12 non-MetS and 27 sex- and age-matched MetS subjects. Fourteen patients with MetS (MetS–off statin) discontinued statin therapy 14 days before the study, while the remaining 13 remained on it (MetS–on statin). The VLDLs were isolated and processed for mass-based lipid profiling. Lipidomic analyses were performed and associated with atrial remodeling markers measured using standard echocardiography and electrocardiography.

Results: Compared with the VLDL components of the non-MetS group, glucosyl/galactosyl ceramide, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, and triglycerides were enriched in the MetS–off statin group. The statin therapy attenuated all abnormally abundant lipid classes in MetS, except for triglycerides. In addition, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, and triglycerides were significantly correlated with atrial dilatation, and the latter two were also correlated with the PR interval. Enrichment of double bonds, which indicates unsaturated fatty acids, was also significantly correlated with atrial remodeling, and P-wave duration.

Conclusion: This study suggests that the pathological lipid payload of MetS-VLDL may contribute to atrial remodeling in patients.

metabolic syndrome (MetS)

Very low-density lipoprotein (VLDL)

lipidomics

atrial remodeling

atrial cardiomyopathy

triglycerides

Metabolic syndrome (MetS) is a cluster of disorders that is strongly linked to increased risks and adverse outcomes of cardiovascular diseases, including atrial fibrillation (AF), the most prevalent arrhythmia [1] [2–5]. The pathogenic mechanisms by which MetS leads to atrial remodeling and AF development have also been clearly identified [4–8]. Central obesity, an important feature of MetS, is strongly correlated with the incidence of AF and is associated with a 50% increase in AF risk [9]. Nevertheless, the contribution of dyslipidemia to AF has been contradictory in clinical observational studies. Possible explanations include different study designs, populations, age ranges, and sex differences in lipid metabolism. A recent meta-analysis indicated that total cholesterol and low-density lipoprotein (LDL) cholesterol are inversely correlated with incident AF [10]. Nevertheless, the pleiotropic effects of statins, the most commonly used lipid-lowering drugs for preventing and treating AF have been consistently reported [11, 12].

Emerging evidence shows the pathogenic roles of very low-density lipoproteins (VLDL) in the pathogenesis of atrial remodeling, progression and thromboembolic complications in AF [13–15]. In our previous studies, the in vivo effects of VLDL extracted from patients with MetS were examined in mice and showed excess lipid accumulation and apoptosis in the atria with greater left atrial size and vulnerability to AF [16]. Mechanistic findings include delayed intra-cardiac conduction velocities, modulated gap junctions [17], disrupted calcium regulation, and derangements in sarcomere proteins [18]. The aforementioned findings were not observed with VLDL isolated from healthy volunteers without MetS, suggesting that the alteration of lipid components in the VLDL of MetS may be responsible for its ability to promote lipotoxicity in the atrium.

Most AF in MetS develops insidiously, while structural and electrical changes within the atria are building and progressive over a relatively long period of time (mostly years to a couple of decades) [19–21]. Structural remodeling of the atrium is demonstrated by chamber dilatation, myocardial inflammation, apoptosis, and increased tissue fibrosis [22]. Left atrial (LA) size is an important marker of AF and cardiovascular outcomes [23]. Structural and functional changes in the LA can be quantified using echocardiography. Electrical remodeling includes expressional and functional changes in ionic channels, delayed conduction velocity, and abnormally triggered electrical activity [24]. The P-wave duration and PR interval of electrocardiography are parameters reflecting atrial electricity and conduction, and are correlated with the development of AF [25, 26]. Echocardiography and electrocardiography were used to evaluate atrial structural and electrical remodeling in this study.

This study performed lipidomic analysis of VLDLs isolated from 12 non-MetS and 27 sex/age-matched MetS subjects. About half of the participants with MetS discontinued statin therapy. The objectives of this study were as follows: firstly, to determine the changes in lipid species in the VLDL of MetS; second, to determine if statins can improve the lipid component changes in VLDL of MetS; and finally, to determine if there is any correlation between the lipid component of VLDL and atrial remodeling in MetS.

Study subjects

To determine the differences in lipid species of VLDL between MetS and non-MetS subjects, and to determine the lipid-lowering drug, that is, statin on lipid species of VLDL, this study enrolled participants at a single medical center. Those with any of the followings were excluded: significant coronary heart disease, myocardial infarction, congenital heart diseases, heart failure, significant heart valve diseases, cerebrovascular diseases, cancers, insulin therapy, and pregnant or breastfeeding women. Those who met any 3 of the following 5 components were defined as having MetS: (1) central obesity (waist ≥ 80 cm for women and ≥ 90 cm for men); (2) raised blood pressure (BP) (systolic BP ≥130mmHg or diastolic BP ≥85 mmHg or treatment of previously diagnosed HTN); (2) raised fasting glucose (≥100 mg/dL or diagnosed type 2 DM); (3) elevated triglycerides (≥150mg/dL or on triglyceride-lowering treatment); and (4) reduced HDL-C (< 50 mg/dL for women and < 40 mg/dL for men). Among the participants, 12 non-MetS and 27 MetS subjects were selected based on age and sex-matched. Fourteen patients with MetS (the MetS–off statin group) were requested to discontinue all lipid lowering drugs, that are statins, 14 days prior to the study visit for sample and data collection, while the remaining 13 patients with MetS were informed to continue ordinary medicine including statins (the MetS–on statin group). The study protocol was approved by the Kaohsiung Medical University Hospital Institutional Review Board (IRB) (KMUHIRB-E(I)-20170256) and registered with trial registration number ISRCTN 69295295 (retrospectively registered on June 9, 2020). All subjects signed an informed consent form before participating in the study. The study adhered to the principles of the Declaration of Helsinki. Each participant also underwent measurements of height, body weight, abdominal and hip circumferences, blood pressure, and heart rate at the study visit. Medical records, if available, were reviewed, and medication use was recorded.

VLDL isolation and lipid profiling for lipidome analysis

All study subjects were instructed to fast before last midnight until 20 mL venous blood draws were completed and collected in BD VACUETTE® EDTA tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for subsequent VLDL isolation as described previously [14]. Briefly, the blood samples were centrifuged, separated from blood cells, and plasma was maintained for high-speed centrifugation at 10,000 rpm for 1 h to remove the upper chylomicrons. The ultracentrifuge at 40,000 rpm at 4°C for 24 h resulted in separated VLDL (density range of 1.006–1.063 g/mL) on the top. The VLDL samples were then subjected to lipid profiling using Lipotype GmbH (Dresden, Germany) [27].

Nomenclature

Lipid names and abbreviations were used: Cer, ceramide; Chol, cholesterol; DAG, diacylglycerol; HexCer, glucosyl/galactosyl ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; and their respective lysospecies lyso-PA (LPA), lyso-PC (LPC), lyso-PE (LPE), lyso-PI (LPI), and lyso-PS (LPS); and their ether derivatives PC O-, PE O-, LPC O-, and LPE O-; SE, sterol ester; SM, sphingomyelin; SLs, sphingolipids; and TAG, triacylglycerols (also named triglycerides). Lipid species were annotated according to their molecular composition as follows: (lipid class)-(sum of carbon atoms in fatty acids):(sum of double bonds in fatty acids);(sum of hydroxyl groups in the long-chain base and the fatty acid moiety) (for example, SM-32:2;1). Where available, individual fatty acid compositions following the same rules are given in brackets (for example, 18:1;0-24:2;0).

Lipid extraction for MS lipidomics

Lipids were extracted using a two-step chloroform–methanol procedure. Samples were spiked with an internal lipid standard mixture containing cardiolipin (CL), 16:1/15:0/15:0/15:0; Cer, 18:1;2/17:0; DAG, 17:0/17:0; HexCer, 18:1;2/12:0; LPA, 17:0; LPC, 12:0; LPE, 17:1; LPG, 17:1; LPI, 17:1; LPS, 17:1; PA, 17:0/17:0; PC, 17:0/17:0; PE, 17:0/17:0; PG, 17:0/17:0; PI, 16:0/16:0; PS, 17:0/17:0; cholesterol ester (CE), 20:0; SM, 18:1;2/12:0;0; TAG, 17:0/17:0/17:0; and Chol. After the extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Each first-step dry extract was resuspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, v/v/v), and each second-step dry extract was resuspended in 33% ethanol solution of methylamine/chloroform/methanol (0.003:5:1, v/v/v). All liquid handling steps were performed using the Hamilton Robotics STARlet robotic platform with an anti-droplet control feature for organic solvent pipetting.

MS data acquisition

The samples were analyzed by direct infusion on a Q-Exactive mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a TriVersa NanoMate ion source (Advion BioSciences, Inc. Ithaca, NY, USA). Samples were analyzed in both positive and negative ion modes with a resolution of 280,000 at m/z = 200 for MS and 17,500 for MS/MS experiments in a single acquisition. MS/MS was triggered by an inclusion list encompassing the corresponding MS mass ranges scanned in 1-Da increments. Both MS and MS/MS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC and PC O- as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. Only MS was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts; and Chol as an ammonium adduct of an acetylated derivative [28].

Data analysis and post-processing

Lipid identification using LipotypeXplorer (2) was performed on the unprocessed mass spectra. For the MS-only mode, lipid identification was based on the molecular masses of intact molecules. MS/MS mode included the collision-induced fragmentation of lipid molecules, and lipid identification was based on both the intact masses and the masses of the fragments. Prior to normalization and further statistical analysis, lipid identification was filtered according to mass accuracy, occupation threshold, noise, and background [29]. The List of identified lipids and their intensities were stored in a database optimized for the particular structure inherent to lipidomic datasets. The intensity of the lipid class-specific internal standards was used for lipid quantification [30]. The identified lipid molecules were quantified by normalization to a lipid class-specific internal standard. The amounts of individual lipid molecules (species or subspecies, in p-moles) of a given lipid class were summed to yield the total amount of the lipid class. The amounts of the lipid classes were normalized to the total lipid amount, yielding mol. % per total lipids.

Lipidomic data processing

The processed lipidomic data were analyzed using LipidSig [31]. In brief, the lipid profiling data of each sample were scale-normalized to the total amount of lipids. Lipids classes with at least a two-fold change between the groups were identified as significantly changed in MetS or by the use of lipid-lowering drugs. Fisher’s exact test was then conducted to test the enrichment of the significantly changed lipids in each lipid class (such as PC, PE, and LPC). In addition, Spearmen’s correlation was used to examine the correlations between the lipid amount and clinical factors.

Laboratory testing for biochemical data

Biochemical data were obtained from the Department of Laboratory Medicine at Kaohsiung Medical University Hospital according to standard laboratory procedures. Technicians who performed the tests were blinded to the participants’ identity and data, including glucose, hemoglobin A1c (HbA1c), total cholesterol, triglycerides, VLDL, LDL-cholesterol, high-density lipoprotein (HDL)-cholesterol, alanine aminotransferase (ALT), creatinine, and uric acid.

Echocardiographic assessment

Echocardiography was performed for the measurement of left atrium (LA) diameter, maximum volume, and minimum volume by an experienced cardiologist using a transthoracic cardiac probe (Vivid 9E; General Electric Medical Systems, Horten, Norway), according to the standards of the American Society of Echocardiography [32]. LA volumes and total emptying fraction (EF) of the LA were derived using the modified Simpson’s method. Raw data were assessed while the researchers were blinded to the other data.

Measurement of electrocardiographic (ECG) parameters

Twelve-lead ECG was performed by medical technicians during the study visit. One experienced technician who was blinded to clinical information and data measured all subjects’ ECG parameters including P wave durations, PR intervals, QRS width, QTc intervals in lead II, and the duration and terminal force of P waves in lead V1 [33]. Only regular sinus rhythm was included for measurement and any arrhythmias, and bundle branch block, obvious ST-T abnormalities, and atrioventricular blocks were discarded.

Statistical analysis

All continuous variables (mean ± standard deviation) were compared among the three groups: non-MetS, MetS–off statin, and MetS–on statin by ordinary one-way ANOVA test followed by multiple comparisons using Tukey’s test. The results were considered statistically significant based on a P-value of < 0.05. Statistical analyses were performed using the statistical package in GraphPad Prism (version 9; GraphPad Software, Inc., San Diego, CA, USA) software system and SPSS statistical software (version 22; IBM Corp., Armonk, NY, USA) and SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Spearmen’s correlation was performed to determine the correlation between specific lipids and each parameter of cardiac remodelling (for the atrium and the ventricle) and the MetS.

Characteristics of study subjects

Among the 12 non-MetS subjects, 14 MetS–off statin subjects, and 13 MetS–on statin subjects, sex, and age were matched (Table 1). Compared with the non-MetS group, the two MetS groups had greater obesity parameters, including BMI, waist circumference, and hip circumference. Similarly, systolic and diastolic blood pressures were higher in the MetS groups. There were no significant differences in markers for renal function creatinine or liver function ALT levels. In common lipid profiles, while cholesterol levels were similar among groups, the MetS–off statin group had significantly lower HDL-C (37.0 ± 7.9 mg/dL vs. non-MetS 63.8 ± 15.8 mg/dL, p < 0.0001), higher triglycerides (235.1 ± 177.0 mg/dL vs. non-MetS 88.7 ± 21.1 mg/dL, p = 0.006) values and higher uric acid (6.4 ± 1.4 mg/dL vs. non-MetS 5.1 ± 0.9 mg/dL, p = 0.0234).

Table 1

Characteristics of study subjects
	Non-MetS (n = 12)	MetS–off statin (n = 14)	MetS–on statin (n = 13)	P value
Age (years)	48.4 ± 7.8	54.0 ± 8.8	54.0 ± 7.8	0.1528
Men: women (n:n)	7:5	7:7	8:5
BMI (kg/m2)	21.5 ± 2.4	30.1 ± 4.5*	30.2 ± 2.1^¶	< 0.0001
Waist circumference (cm)	78.9 ± 8.0	99.5 ± 9.4*	103.8 ± 9.2^¶	< 0.0001
Hip circumference (cm)	95.1 ± 4.0	103.9 ± 9.0*	105.5 ± 6.9^¶	0.0015
Systolic BP (mmHg)	113.3 ± 13.8	141.1 ± 15.6*	150.1 ± 19.0^¶	< 0.0001
Diastolic BP (mmHg)	69.0 ± 8.5	86.5 ± 10.2*	93.8 ± 11.3^¶	< 0.0001
Fasting glucose (mg/dL)	87.0 ± 7.1	119.7 ± 26.9*	110.3 ± 25.9^¶	0.0020
HbA1c (%)	5.5 ± 0.3	6.7 ± 1.5	6.3 ± 0.7	0.0712
Total cholesterol (mg/dL)	169.2 ± 25.2	183.6 ± 34.6	168.6 ± 25.4	0.3291
Triglyceride (mg/dL)	88.7 ± 21.1	235.1 ± 177.0*	173.3 ± 60.5	0.0084
VLDL (mg/dL)	2.7 ± 3.1	27.1 ± 25.1*	18.7 ± 9.3^¶	0.0018
LDL-Cholesterol (mg/dL)	105.4 ± 25.4	119.5 ± 28.6	105.3 ± 23.1	0.2761
HDL-Cholesterol (mg/dL)	63.8 ± 15.8	37.0 ± 7.9*	44.6 ± 9.4^¶	< 0.0001
ALT (IU/L)	22.6 ± 10.6	30.8 ± 14.6	24.9 ± 9.8	0.1727
Creatinine (µIU/mL)	0.78 ± 0.11	0.78 ± 0.16	0.76 ± 0.17	0.9644
Uric acid (mg/dL)	5.1 ± 0.9	6.4 ± 1.4*	5.6 ± 1.2	0.0292
Abbreviations:
MetS, metabolic syndrome; DC, discontinued; BMI, body mass index; BP, blood pressure. ALT, alanine aminotransferase; VLDL, very-low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Structural remodeling of LA demonstrated by echocardiography

Echocardiography was performed for each study subjects to measure LA size in diameter, volumes, and function in the EF which indicates atrial contractility (Fig. 1A–F). Compared with the non-MetS group, the MetS groups, no matter off or on statin had significant larger LA chamber size with greater diameters (LA diameter, non-MetS 3.2 ± 0.3 cm vs. MetS-off statin 4.4 ± 0.4 cm vs. MetS-on statin 4.3 ± 0.3 cm, P < 0.0001) and greater volumes (LA maximum volume, non-MetS 45.2 ± 9.5 mL vs. MetS-off statin 81.9 ± 13.9 mL vs. MetS-on statin 77.5 ± 18.9 mL, P < 0.0001; LA minimum volume, non-MetS 28.1 ± 7.5 mL vs. MetS-off statin 39.3 ± 10.8 mL vs. MetS-on statin 42.0 ± 11.3 mL, P = 0.0065). Interestingly, the EF of LA was significantly greater in the MetS-off statin group than in the non-MetS group (LAEF, non-MetS 38.1 ± 9.5% vs. MetS-off statin 53.3 ± 9.7% vs. MetS-on statin 40.9 ± 15.7%, P = 0.0041).

Atrial electrical remodeling revealed by electrocardiography

Electrocardiography was performed on each study subjects to measure various P-wave parameters (Fig. 1). The PR interval was significantly wider in the MetS-off statin group than in the non-MetS group (176.1 ± 19.0 msec vs. 156.2 ± 15.4 msec, P = 0.00138), whereas the P wave duration (in leads II and V1), terminal force of P waves, QRS width and QTc interval were not significantly different among groups (Fig. 1G–L).

Lipid components of VLDL are different between non-MetS and MetS groups and the changes are blunted by statin treatment

The lipid components of VLDL were determined using mass spectrometry. In the comparison analysis, the most different lipid species were found between non-MetS and MetS–off statin: DAG 16:0, LPC, LPE, PI, Cer, and TAG were significantly higher in the MetS–off statin group than in the non-MetS group, whereas CE, PC, PC-O, PE-O, and DAG 18:0 were significantly lower in the MetS–off statin group than in the non-MetS group (Fig. 2A). Compared to non-MetS, the MetS–on statin had lipid species in VLDL higher with LPE, PC-O, DAG 16:0, Cer, PE, and TAG but lower with PC, PC-O, PE-O, DAG 18:0, and PI (Fig. 2B). The comparison between the two MetS, the –off and –on statin groups, indicated the effect of statins on lipid species of VLDL that PC-O, PC, PE-O, PI, long chain TAG (46:0, 46:1, 48:0, 48:1, and 50:1), and CE were significantly reduced but Cer, DAG 18:0–2, PE, Chol, and TAG 54:4, 54:5, and 55:4 were significantly increased (Fig. 2C). PCA demonstrated the patterns of lipid species in VLDL among the three groups (Fig. 2D).

Changes of lipid structural and functional categories in VLDL of MetS

The relative abundance of lipid components was analyzed and compared between groups with lipid classes (Fig. 3A–C), structural categories (Fig. 3D–F), and functional categories (Fig. 3G–I). The comparison between MetS–off statin and non-MetS revealed changes in MetS without the effects of statins. Sixteen lipid classes were identified, and cardiolipin was not detectable in the VLDLs of the study participants. Comparing to the non-MetS group, the MetS–off statin group had a significantly greater abundance of HexCer, LPC, LPE, and TAG, but less Chol and DAG in VLDL. Comparing the five lipid structural categories to non-MetS, the MetS–off statin group was more abundant with glycerolipids and less abundant with sterols (Fig. 3D). Comparing the three functional categories to non-MetS, the MetS–off statin group had more lysolipids and storage lipids and fewer membrane lipids (Fig. 3G). The comparison between the MetS–on statin and the MetS–off statin groups revealed the effects of statins (Figure C, F, I). Comparing to the MetS–off statin group, the MetS–on statin group had increased Chol, DAG, and reduced HexCer in lipid classes, increased sterols, increased membrane lipids, and decreased storage lipids. The comparison between the MetS–on statin and the non-MetS groups revealed residual differences in the lipid components of VLDL upon statin treatment (Fig. 3B, E, H). Compared to non-MetS, MetS–on statin still had more TAG but less PC-O and less PE-O in lipid classes, more glycerolipids in structure categories, more sterols and less membrane lipids in functional categories.

Significant correlation of VLDL lipid components with LA size and function

To test our hypothesis that VLDL lipid components can affect LA remodeling, Spearman’s rank-order correlation was used to examine the correlation of each lipid class with cardiac remodeling markers, including LA diameter, maximum volume, empty fraction of LA, LV end diastole volume, and LV mass index, and parameters of electrical remodeling, such as P wave duration, PR interval, P wave terminal force, QRS width and QTc interval (Fig. 4A). Lipid classes were also analyzed for MetS scores, BMI, and HR (Fig. 4A–B). The MetS score was positively correlated with LPC, LPE, and TAG and inversely correlated with Chol (Fig. 4B). The increased diameter and maximum volume indicate LA enlargement, which is a hallmark of structural remodeling [34]. The results showed that LPC, LPE, and TAG were significantly associated with the LA maximum volume (Fig. 4A). LPE and TAG were also significantly correlated with the PR interval, indicating intra-atrial conduction velocity (Fig. 4A). The PR interval was inversely correlated with Chol, HexCer and LPC O- (Fig. 4C). LPE and LPC were also correlated with the LV mass index, which indicates ventricular hypertrophy. Consistently, MetS score, LA maximum volume, and LV mass were positively correlated with LPC and LPE. TAG alone was positively correlated with the MetS score, BMI, LA diameter, maximum volume, and PR interval. These findings together suggest that the lipid components of VLDL can affect cardiac remodeling, particularly the structural and electrical remodeling of LA.

Alteration of TAG (triglyceride) total length and association of unsaturated fatty acids with cardiac remodeling

To elucidate whether any TAG changes in VLDL were correlated with MetS score and cardiac remodeling, the total length of TAG fatty acids was compared between the non-MetS and the MetS–off statin groups (Fig. 5A–B). Spearman’s correlation was used to test the correlation of the number of double bonds in TAG with the MetS score and cardiac remodeling markers (Fig. 5C). With three fatty acids, mostly in the form of TAG, the abundance of total length with 48, 50, 52, 55, 56, and 60 carbons was greater in the MetS–off statin group than in the non-MetS group (Fig. 5A). The presence of three double bonds, which indicates that the unsaturated fatty acids of TAG were positively correlated with the MetS score, BMI, and LA remodeling markers, including LA diameter, maximum volume, emptying volume, and EF, that is, LA contractility. However, the presence of only a single double bond, which indicates a lower degree of unsaturated fatty acids in TAG, was negatively correlated with MetS scores and markers for obesity and cardiac remodeling. Interestingly, the extreme richness of double bonds is positively correlated with P-wave duration, which indicates the required time for completing all myocardial action potentials in the entire and bilateral atria. This finding suggests that richness of unsaturated fatty acids affect atrial remodeling with respect to chamber dilatation, the emptying function, and regulation of the action potentials.

The main findings from this study are as follows: (1) Compared with the VLDL of the non-MetS group, the MetS–off statin group had significantly greater lipid content with HexCer, LPC, LPC-O, LPE, and TAG, and less Chol and DAG. (2) Except for TAG, increased lipid classes in the VLDL of MetS subjects were significantly attenuated by statin therapy. (3) The abundance of the lipid classes LPC, LPE, and TAG in VLDL was positively correlated with atrial dilatation, which was rather inversely correlated with the richness of Chol in VLDL. (4) The abundances of LPE and TAG in VLDL were significantly correlated with the PR interval. (5) Double bonds of unsaturated fatty acids were significantly correlated with atrial remodeling markers, including diameter, volume, emptying function, and P-wave duration. Together, these findings together suggest that modification of lipid components in VLDL affects atrial remodeling and may be responsible for increased AF risks in MetS.

Increased lipid components with LPC, LPE, and HexCer in VLDL and atrial remodeling

LPC is derived from the cleavage of phosphatidylcholine (PC) by phospholipase A2 [35]. LPC can induce proinflammatory cytokines, oxidative stress, and apoptosis in endothelial cells and vascular smooth muscle and can also affect intracellular Ca²⁺ balance. LPC has been reported to play a role in atherosclerosis, diabetes, myocardial infarction, and Alzheimer’s disease [35, 36]. The increased LPC content in the VLDL of MetS can possibly affect atrial Ca²⁺ regulation and induce oxidative stress in atrial myocytes. To our knowledge, this is the first study to report a correlation between LPC and atrial remodeling in MetS.

LPE originates from the exogenous lysophospholipid metabolic pathway. After uptake, lyso-PE is a major source of PE through acylation by an acyl-CoA-dependent acyltransferase Ale 1p [37]. The A1e 1P activity is enriched in the mitochondrial-associated membranes. The trafficking pathway for LPE to reach mitochondria remains unknown [38]. PE metabolism has been associated with Alzheimer's disease, Parkinson's disease, and nonalcoholic liver disease [35]. We suggest that the increased LPE component in VLDL may also affect the mitochondrial function in atrial cardiomyocytes.

Another significantly increased lipid class in VLDL is HexCer, in which ceramide undergoes a monohexosyl substitution by specific ceramide synthase/synthases [39]. Monohexosylcermide (HexCer) is essential for cellular physiological functions such as myelinogenesis in the nervous system, epidermal permeability, cell proliferation and apoptosis [39]. Ceramide is shown associated with primary cardiomyopathy and secondary cardiometabolic disorders, such as diabetic cardiomyopathy [40–42].

While the Lands cycle has been described by William E.M. Lands [43] as a conversion process between lysophospholipids and phospholipids, it is recognized as an important mechanism for neutral lipid consumption in cells. Lipid droplets (LD) are the playground of the Lands cycle [44, 45] and are colocalized in the endoplasmic reticulum (ER) where VLDL package takes place [46]. LD served to the ER with LPC/LPE during the VLDL packing. The LPC/LPE enrichment in the MetS-VLDL particle suggests a trajectory of pathological hepatocytes producing VLDL, that has been supported by other studies. In a study by Rong et al., genetic defects in lpcat3 (LD enzyme) in mice resulted in arachidonic acid, LPC, and TAG reduction in VLDL particles [47]. Hepatic lpcat3 function might be through microsomal TAG transfer during VLDL production [48]. On the other hand, in the intestinal lumen, enhanced postprandial lysophospholipids absorption can suppress hepatic fatty acid oxidation, lead to increased VLDL synthesis and tissue lipid deposition, and improve diet-induced hyperlipidemia [49].

The negative correlation of cholesterol with atrial dilatation and PR interval

This study found negative correlations between cholesterol level with atrial dilatation and the PR interval. The abundance of cholesterol in VLDL was also negatively correlated with the MetS score, BMI, LA emptying volume, and LV volume. This study finding may partially explain why cholesterol level has a paradoxical association with incident AF in clinical observational studies [50]. Unlike the major effects of statins on the reduction of LDL-cholesterol, statins increase the relative abundance of cholesterol in the VLDL. Further studies are required to elucidate the underlying mechanisms.

Statins partially normalize lipid component changes in VLDL of MetS

In our study, statins reduced the abnormally abundant LPC, LPC O-, LPE, and HexCer in the VLDL of MetS. Another study also showed that statins normalized the plasma lipidome of 12 MetS males in comparison with 12 healthy controls [51]. Consistently, statins can shift the lipid components of MetS toward being more similar to non-MetS. The reduction in LPC, which can induce inflammation and oxidative stress may explain the mechanism underlying the anti-inflammation effects of statins [52]. This may also partially explain the benefit of statin therapy in reducing cardiovascular events [53]. Although statins can restore the aforementioned lipid classes in MetS, TAG remains abnormally abundant. VLDL is a major apoB-containing lipoproteins. Our findings regarding the effects of statins on the VLDL lipidome are consistent with the previous recognition of apoB and TAG as residual cardiovascular risk factors [54, 55]. The effects of statins on restoring VLDL lipid component changes in MetS may explain the long-term benefits of statin in reducing the risk of AF [11, 56].

Changes of TAG in VLDL

Clinical observational studies have also reported elevated TAG levels in AF patients [57, 58]. This study is the first to disclose increased long-chain TAG and the strong correlation of three double bonds of unsaturated fatty acids with atrial remodeling. This finding partially explains the increased risk of AF in patients with MetS and elevated TAG levels. The liver is the major organ that produces and secrets VLDL. We suggest that the alteration of TAG in VLDL is derived from fatty liver that commonly coexists in subjects with MetS. Non-alcoholic human fatty liver has been found to increase the activity of hepatic stearol-CoA desaturase (SCD)-1, which converts saturated fatty acids to monosaturated fatty acids as a major substrate for the synthesis of TAG and other lipids [59]. In contrast, the inverse correlation of single double bonds of fatty acids with MetS score, BMI, and almost all atrial and ventriclular dilatation markers may indicate a lower degree of SCD-1 activity.

Considering the complexity of lipid metabolism, further studies are needed to elucidate the pathological role of lipid classes that are found significant for atrial dilatation and conduction, including TAG. The effects of highly unsaturated fatty acids on altering the atrial action potentials will be another interesting topic for studying the mechanisms. [59]

Study Limitations

However, some limitations of this study cannot be avoided. One is the limited grant resources and small sample size, which might lead to a high risk of a type I error. Second, the blood samples were collected in the morning post an over 8 h fasting period, and the lipidome of VLDL shown in this study might not be unapplicable for postprandial VLDLs.

The findings of this study further delineate the pathogenic role of VLDL lipotoxicity, which is derived from altered lipid components with increased LPC, LPE, and unsaturated fatty acids in TAG. Our results call for mechanistic studies of lipotoxicity in atrial cardiomyopathy and cardiometabolic disorders.

AF: atrial fibrillation; Cer, ceramide; DAG, diacylglycerol; HexCer, glucosyl/galactosyl ceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lyso-PC;

LPE, lyso-PE; O-, ether derivatives; TAG, triacylglycerol; VLDL, very low-density lipoprotein; MetS, metabolic syndrome.

Ethics approval and consent to participate

Our study was approved by the Kaohsiung Medical University Hospital Institutional Review Board. This study was conducted with adherence to the principles of the Declaration of Helsinki. Signed informed consent was obtained from every subject prior to engaging in any research.

Consent for publication

Not applicable.

Availability of data and materials

All the re-identified data are available upon reasonable request ([email protected]).

Competing interests

"The authors declare no conflict of interest."

Funding

This study was supported by Kaohsiung Medical University Hospital (KMUH109-9R11 and KMUH110-0R11), Taiwan Ministry of Science and Technology Grants (MOST 109-2327-B-039-002 and MOST 109-2314-B-037-111-MY3), KMU Global Networking Talent Plan (110KMUOR01), National Health Research Institutes (NHRI-EX107-10724SC, NHRI-EX108-10724SC, NHRI-EX109-10724SC, and NHRI-EX110-10724SC), and China Medical University Hospital (DMR-110-231).

Author contributions

All authors have approved the submitted version and agree to be personally accountable for their own contributions. Author Contributions: Conceptualization, H.C. Lee; Data curation, H.C. Lee, W.C. Cheng and W.L. Ma; Formal analysis, W.C. Cheng; Funding acquisition, H.C. Lee and W.L. Ma; Investigation, H.C. Lee, W.C. Cheng, W.L. Ma, Y.H. Lin, S.J. Shin, and Y.H. Lin; Methodology, H.C. Lee, W.C. Cheng, and W.L. Ma; Project administration, H.C. Lee; Resources, H.C. Lee; Software, H.C. Lee and W.C. Cheng; Supervision, H.C. Lee; Validation, H.C. Lee, W.C. Cheng and W.L. Ma; Visualization, H.C. Lee, W.C. Cheng and W.L. Ma; Writing – original draft, H.C. Lee; and Writing – review & editing, H.C. Lee, W.C. Cheng, W.L. Ma, S.J. Shin, and Y.H. Lin.

Acknowledgments

The authors thank Miss Chung-Ya Wang for VLDL sample isolation and purification.

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No competing interests reported.

Association of Lipid Composition and Unsaturated Fatty Acids of VLDL with Atrial Remodeling in Metabolic Syndrome

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results

Characteristics of study subjects

Structural remodeling of LA demonstrated by echocardiography

Atrial electrical remodeling revealed by electrocardiography

Lipid components of VLDL are different between non-MetS and MetS groups and the changes are blunted by statin treatment

Changes of lipid structural and functional categories in VLDL of MetS

Significant correlation of VLDL lipid components with LA size and function

Alteration of TAG (triglyceride) total length and association of unsaturated fatty acids with cardiac remodeling

Discussions

Increased lipid components with LPC, LPE, and HexCer in VLDL and atrial remodeling

The negative correlation of cholesterol with atrial dilatation and PR interval

Statins partially normalize lipid component changes in VLDL of MetS

Study Limitations

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1