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. 2021 Nov 26:12:771940.
doi: 10.3389/fphar.2021.771940. eCollection 2021.

Enhancing Fatty Acids Oxidation via L-Carnitine Attenuates Obesity-Related Atrial Fibrillation and Structural Remodeling by Activating AMPK Signaling and Alleviating Cardiac Lipotoxicity

Yudi Zhang  1 , Yuping Fu  1 , Tiannan Jiang  2 , Binghua Liu  3 , Hongke Sun  1 , Ying Zhang  1 , Boyuan Fan  1 , Xiaoli Li  1 , Xinghua Qin  3 , Qiangsun Zheng  1
Affiliations

Enhancing Fatty Acids Oxidation via L-Carnitine Attenuates Obesity-Related Atrial Fibrillation and Structural Remodeling by Activating AMPK Signaling and Alleviating Cardiac Lipotoxicity

Yudi Zhang et al. Front Pharmacol. .

Abstract

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical setting. Its pathogenesis was associated with metabolic disorder, especially defective fatty acids oxidation (FAO). However, whether promoting FAO could prevent AF occurrence and development remains elusive. In this study, we established a mouse model of obesity-related AF through high-fat diet (HFD) feeding, and used l-carnitine (LCA, 150 mg/kg⋅BW/d), an endogenous cofactor of carnitine palmitoyl-transferase-1B (CPT1B; the rate-limiting enzyme of FAO) to investigate whether FAO promotion can attenuate the AF susceptibility in obesity. All mice underwent electrophysiological assessment for atrial vulnerability, and echocardiography, histology and molecular evaluation for AF substrates and underlying mechanisms, which were further validated by pharmacological experiments in vitro. HFD-induced obese mice increased AF vulnerability and exhibited apparent atrial structural remodeling, including left atrial dilation, cardiomyocyte hypertrophy, connexin-43 remodeling and fibrosis. Pathologically, HFD apparently leads to defective cardiac FAO and subsequent lipotoxicity, thereby evoking a set of pathological reactions including oxidative stress, DNA damage, inflammation, and insulin resistance. Enhancing FAO via LCA attenuated lipotoxicity and lipotoxicity-induced pathological changes in the atria of obese mice, resulting in restored structural remodeling and ameliorated AF susceptibility. Mechanistically, LCA activated AMPK/PGC1α signaling both in vivo and in vitro, and pharmacological inhibition of AMPK via Compound C attenuated LCA-induced cardio-protection in palmitate-treated primary atrial cardiomyocytes. Taken together, our results demonstrated that FAO promotion via LCA attenuated obesity-mediated AF and structural remodeling by activating AMPK signaling and alleviating atrial lipotoxicity. Thus, enhancing FAO may be a potential therapeutic target for AF.

Keywords: AMPK (5′-AMP activated kinase); atrial fibrillation; fatty acids oxidation; l-carnitine; lipotoxicity; obesity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
LCA inhibits obesity–induced AF. (A) Experimental protocols of this study. (B) Representative AF induction by trans-esophageal burst pacing. (C,D) Analysis of AF frequency and duration. (E) Three-limb-lead electrocardiogram at baseline. (F,G) Analysis of P-wave. (H–L) Analysis of SNRTmax, cSNRTmax and ERP detected by programmed cardiac stimulation. n = 10 per group. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; FAO, fatty acids oxidation; Parea, P-wave area; P max, P-wave duration; AF, atrial fibrillation; ERP, effective refractory period; SNRT, sinus node recovery time; cSNRT, corrected sinus node recovery time.
FIGURE 2
FIGURE 2
LCA suppresses atrial remodeling in obese mice. (A) Representative echocardiographic images of LA among the groups. (B) Measurements of LA diameter and (C) LA filling volume detected by 2D-guided M-mode imaging. The SI and AP were obtained in a long-axis view, and ML was assessed in parasternal short-axis view. LA filling volume was calculated using the formula: LA filling volume = (4π×SI×AP×ML)/(3 × 2× 2 × 2). (D) Representative sections of WGA staining. (E) Representative sections of H&E staining. (F) Quantitative analysis of cellular morphology by ImageJ. (G) Relative mRNA levels of β-MHC using RT-qPCR. (H,I) Subcellular localization of Cx43 and immunohistochemistry. Triangles indicate lateralized Cx43. (J) Protein expression of Cx43 using Western blot. (K,L) Interstitial fibrosis (Arrow) using Masson’s trichrome staining. (M,N) Representative images and analysis of TGF-β signaling-associated proteins (TGF-β, α-SMA, collagen Ⅰ and collagen Ⅲ) using Western blot. (O) Relative mRNA expression levels of the fibrosis-related genes (TGF-β, α-SMA, Smad3, Smad7, collagen Ⅰ and collagen Ⅲ) using RT-qPCR. Scale bar: 50 μm n = 10 (A–C) or 4 (D–O) per group. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; FAO, fatty acids oxidation; LA, left atrium; SI, superoinferior dimension; AP, anteroposterior dimension; MI, mediolateral dimension; WGA, wheat Germ Agglutinin; H&E, hematoxylin-eosin; β-MHC, β–cardiac myosin heavy chain; Cx43, connexin-43; TGF-β, transform growth factor-β; α-SMA, α-smooth muscle actin; Smad, drosophila mothers against decapentaplegic protein.
FIGURE 3
FIGURE 3
LCA enhances FAO, thereby inhibits cardiac lipotoxicity by alleviating atrial steatosis in obese mice. (A) Representative sections of lipid accumulation (Arrow) using oil red O staining. (B) Quantitative analysis of lipid accumulation by ImageJ. (C) Schematic diagram illustrating cardiac lipid metabolism. (D) Quantitative analysis of the transcription of lipid uptake and transportation-related genes (CD36, FABP-pm, FABP3) by RT-PCR. (E) Representative image and quantitative analysis of CD36 by Western blot. (F) Representative images of membrane translocation of CD36 using immunohistochemistry. (G) Quantitative analysis of sarcolemma CD36 contents by ImageJ. (H) Quantitative analysis of the transcription of CPT1B using RT-qPCR. (I) Representative Image and quantitative analysis of CPT1B by Western blot. (J) Quantitative analysis of the transcription of PGC1α using RT-qPCR. (K,L) Representative images and quantitative analysis of FAO-related regulators (AMPK and PGC1α) by Western blot. Scale bar: 50 μm n = 4 per group. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; FAs, fatty acids; FAO, fatty acids oxidation; AMPK, AMP-activated protein kinase; CD36, FAT; CPT1B, carnitine palmitoyltransferase-1B; PGC1α, peroxisome proliferator-activated receptor γ coactivator1α; FABP-pm, plasma membrane fatty acid-binding protein; FABP3, fatty acid binding protein 3; p-, phoso-.
FIGURE 4
FIGURE 4
LCA inhibits cardiac oxidative stress and mitigates inflammation. Representative images and analysis of the subcellular localization of the oxidation products of (A,B) MitoSOX and (C,D) DHE. Red: oxidation products-staining. Scar: 10 μm n = 4. (E,F) Comparisons of serum MDA and SOD using commercially available kits among the groups. (G,H) Levels of atrial MDA and SOD normalized to tissue protein concentration. (I) Schematic diagram illustrating NRF2-related signals. (J,K) Localization of NRF2 in atria by confocal immune-cyto-chemical analysis: Blue: nucleus (DAPI); Red: NRF2-staining; Pink: merge of blue and red indicated nuclear localization of NRF2 (Arrow). Scar: 30 μm n = 4. (L,M) Representative images and quantitative analysis of anti-oxidative system involved protein expressions (NRF2 and SOD2) using Western blot. (N,O) Representative images and analysis of oxidative DNA damage by 8-OH-dG staining. Green: 8-OH-dG -staining; Blue: DAPI. Scar: 50 μm n = 4. (P,Q) Representative images and analysis of DNA damage by TUNEL staining. Red: TUNEL-staining; Blue: DAPI; Pink: merge. Scar: 50 μm n = 4. Comparisons of (R) serum LDH and serum (S) CK-MB using commercially available kits. n = 8. (T) Quantitative analysis of the expression of inflammation-related genes in the atria using RT-qPCR. (U) Representative images and quantitative analysis of expression and phosphorylation of NFκB using Western blot. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; DHE, dihydroethidium; MDA, molondialdehyde; SOD, superoxide dismutase; NRF2, nuclear erythroid 2 p45-related factor 2; p-, phoso-; SOD2, manganese superoxide dismutase, superoxide dismutase 2; 8-OH-Dg, 8-hydroxydeoxyguanosine; IL, interleukin; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; NFκB, the nuclear factor kappa B; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB.
FIGURE 5
FIGURE 5
LCA restores glucose and insulin homeostasis. (A) Plasma glucose levels in the IPGTT. (B) Fast blood glucose levels among groups. (C) Analysis of AUCglucose during IPGTT. (D) Plasma glucose levels in the ITT. (E) Random blood glucose levels among groups. (F) Analysis of inverse AUCglucose during ITT. (G) Representative images and quantitative analysis of expression and phosphorylation of Akt by Western blot. (H,I) Representative images of membrane translocation of GLUT4 using immunohistochemistry and quantitative analyzed by ImageJ. (J) Representative images and quantitative analysis of the expression of GLUT4 by Western blot. (K) Quantitative analysis of the expression of glucose metabolism-related genes (GLUT1, GLUT4, HK2, PFKM, PKM2, and PDK4) using RT-qPCR. (L,M) Glycogen accumulation (Arrow) demonstrated by Periodic acid-Schiff staining. (N) Schematic diagram illustrating glucose metabolism. Scale bar: 50 μm n = 4 for in vivo experiments and 10 for in vitro experiments in each group. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, ¥ p < 0.05 HFD vs others, $ p < 0.05 HFD + LCA vs others, # p < 0.05 STD/STD + LCA vs HFD/HFD + LCA. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; AUC, area under the curve; Akt, protein kinase B; p-, phoso-; GLUT, glucose transporter; HK2, hexokinase2; PFKM, phosphofructokinase; PKM2, pyruvate kinase isozyme type M2; PDK4, pyruvate dehydrogenase kinase 4; p-, phoso-.
FIGURE 6
FIGURE 6
Pharmacological inhibition of AMPK via CC attenuated LCA-conferred beneficial effects in palmitate-treated primary atrial cardiomyocytes. (A) Schematic diagram illustrating the cell isolation, culture and treatment. (B) FAO rate in different treatment groups. The measure of substrate utilization after 18°C unsaturated fatty acid (Oleate, 100 μM) addition was normalized with maximal O2 consumption in Control cells. (C) Representative images and (D) quantitative analysis of the expression of FAO-related proteins using Western blot. (E) Cellular MDA concentrations among the groups. (F) Representative images and (G) quantitative analysis of the expression of oxidative stress-related proteins (NRFS and SOD2) and inflammation-related protein (NFκB) using Western blot. n = 3 or 4 each group. Two-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. CC, Compound C; LCA, l-carnitine; PA, palmitate; FAO, fatty acids oxidation; MDA, molondialdehyde; NRF2, nuclear erythroid 2 p45-related factor 2; AMPK, AMP-activated protein kinase; CD36, FAT; CPT1B, carnitine palmitoyltransferase-1B; p-, phoso-; PGC1α, peroxisome proliferator-activated receptor γ coactivator1α; NRF2, Nuclear factor erythroid 2-related factor 2; NFκB, the nuclear factor kappa B; SOD2, manganese superoxide dismutase, superoxide dismutase 2.
FIGURE 7
FIGURE 7
Hypothetical mechanisms of LCA-conferred cardio-protection against obesity-related AF. Possible mechanism proposed in this study: HFD-induced imbalance between FAs uptake and expenditure causes lipid accumulation and lipotoxicity, which triggers a set of chain reactions, including oxidative stress, DNA damage, inflammation and insulin resistance, and finally contributing to atrial remodeling and greater propensity for AF. Promotion of FAO via LCA combats obesity-induced AF by reducing myocardial lipotoxicity, alleviating atrial remodeling, including left atrial dilatation, cardiac hypertrophy, gap junction remodeling and interstitial fibrosis. Mechanistically, LCA supplement amended lipid metabolism through AMPK-dependent pathway. STD, standard diet; HFD, high-fat diet; LCA, l-carnitine; FAO, fatty acids oxidation; AF, atrial fibrillation; AMPK, AMP-activated protein kinase; CD36, FAT; CPT1B, carnitine palmitoyltransferase-1B; p-, phoso-; PGC1α, peroxisome proliferator-activated receptor γ coactivator1α.
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