Liver X Receptor Agonist AZ876 Induces Beneficial Endogenous Cardiac Lipid Reprogramming and Protects Against Isoproterenol‐Induced Cardiac Damage

Background: It is known that dietary intake of polyunsaturated fatty acids may improve cardiac function. However, relatively high daily doses are required to achieve sufficient cardiac concentrations of beneficial omega-3 fatty acids. The liver X receptor (LXR) is a nuclear hormone receptor and a crucial regulator of lipid homeostasis in mammals. LXR activation has been shown to endogenously reprogram cellular lipid profiles toward increased polyunsaturated fatty acids levels. Here we studied whether LXR lipid reprogramming occurs in cardiac tissue and exerts cardioprotective actions. Methods and Results: Male 129SV mice were treated with the LXR agonist AZ876 (20 mu mol/kg per day) for 11 days. From day 6, the mice were injected with the nonselective beta-agonist isoproterenol for 4 consecutive days to induce diastolic dysfunction and subendocardial fibrosis while maintaining systolic function. Treatment with isoproterenol led to a marked impairment of global longitudinal strain and the E/e' ratio of transmitral flow to mitral annular velocity, which were both significantly improved by the LXR agonist. Histological examination showed a significant reduction in isoproterenol-induced subendocardial fibrosis by AZ876. Analysis of the cardiac lipid composition by liquid chromatography-high resolution mass spectrometry revealed a significant increase in cardiac polyunsaturated fatty acids levels and a significant reduction in saturated fatty acids by AZ876. Conclusions: The present study provides evidence that the LXR agonist AZ876 prevents subendocardial damage, improves global longitudinal strain and E/e' in a mouse model of isoproterenol-induced cardiac damage, accompanied by an upregulation of cardiac polyunsaturated fatty acids levels. Cardiac LXR activation and beneficial endogenous cardiac lipid reprogramming may provide a new therapeutic strategy in cardiac disease with diastolic dysfunction

Adipose tissue ATGL modifies the cardiac lipidome in pressure-overload-induced left ventricular failure

Adipose tissue lipolysis occurs during the development of heart failure as a consequence of chronic adrenergic stimulation. However, the impact of enhanced adipose triacylglycerol hydrolysis mediated by adipose triglyceride lipase (ATGL) on cardiac function is unclear. To investigate the role of adipose tissue lipolysis during heart failure, we generated mice with tissue-specific deletion of ATGL (atATGL-KO). atATGL-KO mice were subjected to transverse aortic constriction (TAC) to induce pressure-mediated cardiac failure. The cardiac mouse lipidome and the human plasma lipidome from healthy controls (n = 10) and patients with systolic heart failure (HFrEF, n = 13) were analyzed by MS-based shotgun lipidomics. TAC-induced increases in left ventricular mass (LVM) and diastolic LV inner diameter were significantly attenuated in atATGL-KO mice compared to wild type (wt) -mice. More importantly, atATGL-KO mice were protected against TAC-induced systolic LV failure. Perturbation of lipolysis in the adipose tissue of atATGL-KO mice resulted in the prevention of the major cardiac lipidome changes observed after TAC in wt-mice. Profound changes occurred in the lipid class of phosphatidylethanolamines (PE) in which multiple PE-species were markedly induced in failing wt-hearts, which was attenuated in atATGL-KO hearts. Moreover, selected heart failure-induced PE species in mouse hearts were also induced in plasma samples from patients with chronic heart failure. TAC-induced cardiac PE induction resulted in decreased PC/PE-species ratios associated with increased apoptotic marker expression in failing wt-hearts, a process absent in atATGL-KO hearts. Perturbation of adipose tissue lipolysis by ATGL-deficiency ameliorated pressure-induced heart failure and the potentially deleterious cardiac lipidome changes that accompany this pathological process, namely the induction of specific PE species. Non-cardiac ATGL-mediated modulation of the cardiac lipidome may play an important role in the pathogenesis of chronic heart failure

Assessment of Myocardial Microstructure in a Murine Model of Obesity-Related Cardiac Dysfunction by Diffusion Tensor Magnetic Resonance Imaging at 7T

Background: Obesity exerts multiple deleterious effects on the heart that may ultimately lead to cardiac failure. This study sought to characterize myocardial microstructure and function in an experimental model of obesity-related cardiac dysfunction. Methods: Male C57BL/6N mice were fed either a high-fat diet (HFD; 60 kcal% fat, n = 12) or standard control diet (9 kcal% fat, n = 10) for 15 weeks. At the end of the study period, cardiac function was assessed by ultra-high frequency echocardiography, and hearts were processed for further analyses. The three-dimensional myocardial microstructure was examined ex vivo at a spatial resolution of 100 × 100 × 100 μm3 by diffusion tensor magnetic resonance imaging (DT-MRI) at 7T. Myocardial deformation, diffusion metrics and fiber tract geometry were analyzed with respect to the different myocardial layers (subendocardium/subepicardium) and segments (base/mid-cavity/apex). Results were correlated with blood sample analyses, histopathology, and gene expression data. Results: HFD feeding induced significantly increased body weight combined with a pronounced accumulation of visceral fat (body weight 42.3 ± 5.7 vs. 31.5 ± 2.2 g, body weight change 73.7 ± 14.8 vs. 31.1 ± 6.6%, both P < 0.001). Obese mice showed signs of diastolic dysfunction, whereas left-ventricular ejection fraction and fractional shortening remained unchanged (E/e’ 41.6 ± 16.6 vs. 24.8 ± 6.0, P < 0.01; isovolumic relaxation time 19 ± 4 vs. 14 ± 4 ms, P < 0.05). Additionally, global longitudinal strain was reduced in the HFD group (−15.1 ± 3.0 vs. −20.0 ± 4.6%, P = 0.01), which was mainly driven by an impairment in basal segments. However, histopathology and gene expression analyses revealed no myocardial fibrosis or differences in cardiomyocyte morphology. Mean diffusivity and eigenvalues of the diffusion tensor were lower in the basal subepicardium of obese mice as assessed by DT-MRI (P < 0.05). The three-dimensional fiber tract arrangement of the left ventricle (LV) remained preserved. Conclusion: Fifteen weeks of high-fat diet induced alterations in myocardial diffusion properties in mice, whereas no remodeling of the three-dimensional myofiber arrangement of the LV was observed. Obese mice showed reduced longitudinal strain and lower mean diffusivity predominantly in the left-ventricular base, and further investigation into the significance of this regional pattern is required

Deletion of ATGL in adipose tissue attenuates pressure overload-induced LV failure.

<p><b>A:</b> Representative images of the hearts. <b>B</b>: Heart weight (HW)/ body weight (BW) ratio (mean and SEM, n = 5–6). <b>C</b>: Representative microscopic cross-sections of the hearts stained with hematoxylin/ eosin (H/E). <b>D</b>: Myocardial area, calculated based on microscopic sections of heart tissue, stained with H/E, analogue to the images presented in C (mean and SEM, n = 5–6). <b>E</b>: Representative M-Mode images of the echocardiographic analysis. <b>F-J</b>: cardiac echocardiographic analysis of mice (mean and SEM, n = 7): <b>F</b>: Left-ventricular mass (LVM). <b>G</b>: LVM relative to tibia length (LVM/TL). <b>H</b>: Left-ventricular internal diameter in diastole (LVID-d). <b>I</b>: Ejection fraction [%] (EF). <b>J</b>: Fractional shortening [%] (FS). <b>K</b>: Analysis of mRNA expression of beta-cardiac myosin heavy chain isogene (βMHCH), qRT-PCR studies were carried out using total RNA isolated from LV tissue. Data are presented as x-fold over wt-sham mice (mean and SEM, n = 5–6). <b>L</b>: Representative microscopic cross-sections of the hearts stained with picrosirius red. <b>M</b>: Representative high magnification images from picrosirius red-stained sections. <b>N:</b> Cardiac fibrosis calculated based on microscopic sections of the heart tissue, stained with picrosirius red, analogue to the images presented in L: 0 = no fibrosis, 1 = mild fibrosis, 2 = moderate fibrosis, 3 = severe fibrosis (mean and SEM, n = 5–6). <b>O and P:</b> Analysis of mRNA expression of collagen (Col) 1a1 (O) and Col3 (P), qRT-PCR studies were carried out using total RNA isolated from LV tissue. Data are presented as x-fold over wt-sham mice (mean and SEM, n = 5–6).*p<0.05 vs. wt sham, **p<0.01 vs. wt sham, ***p<0.001 vs. wt sham, ****p<0.0001 vs. wt sham, $p<0.05 vs. wt TAC,$ $p<0.01 vs. wt TAC,$ $$ p<0.001 vs. wt TAC, $$ $$ p<0.0001 vs. wt TAC, ##p<0.01 vs. atATGL-KO sham; 2-way ANOVA (Bonferroni post-test).</p

Selected lipid species are altered in plasma samples from patients with HFrEF MS-based shotgun lipidomics analysis of human plasma samples from HFrEF-patients (n = 13) and non-HFrEF controls (n = 10).

<p>A: Box plots show distribution of total mole percent values for each lipid class corrected for age and BMI (see supplementary methods). To test for differential changes a Mann-Whitney U test between HFrEF-patients and controls was performed. Adjusted p-values are indicated: *p<0.05, **p<0.01, **p<0.001. B: Estimated mean log2 fold change (HFrEF vs. control) vs. estimated mean mole percent of lipid species in the control group (see supplementary methods). Triangles represent significantly changed lipid species (FDR adjusted p-value < 0.1 and absolute value of log2-fold change ≥ 0.5), bubbles show those which are not significantly changed, size indicates log-transformed adjusted p-values. C: Bar graph shows the estimated log2-fold change (HFrEF vs. control) ± regression standard error of differentially changed lipid species (see B.). Colors represent log10 adjusted p-values as indicated. Lipid classes: Cer: ceramide, DAG: diacylglycerol, LPC: lyso-phosphatidylcholine, LPE: lyso-phosphatidylethanolamine, PC: phosphatidylcholine, PC O-: phosphatidylcholine-ether, PE: phosphatidylethanolamine, PE O-: phosphatidylethanolamine-ether, PI: phosphatidylinositol, SE: sterol ester, SM: sphingomyelin, ST: sterols, TAG: triacylglycerol.</p

Induction of pressure-mediated cardiac PE species is attenuated in atATGL-KO mice.

<p>MS-based shotgun lipidomics analysis of heart tissue samples (LV) isolated 11 weeks after intervention (sham or TAC) from wild-type (wt) or atATGL-KO mice. Lipid class denotations see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007171#pgen.1007171.g003" target="_blank">Fig 3</a>. <b>A-F:</b> Mean log2-fold change (TAC vs. sham) vs. mean mole percent of lipid species. Triangles represent significantly changed lipid species (FDR adjusted p-value < 0.1 and absolute value of log2-fold change ≥ 0.5); bubbles represent those which are not significantly changed; size indicates log-transformed adjusted p-values. <b>A, C, E:</b> wt-mice. <b>B, D, F:</b> atATGL-KO-mice. <b>G+H:</b> Significantly changed PC-PE ratios of matched FAs in wt-mice (<b>G</b>) or atATGL-KO mice (<b>H</b>). The mean ratio ± SEM is shown on a logarithmic scale, Mann-Whitney U test for TAC vs. sham: *p<0.05, **p<0.01 (adjusted) in wt-mice, no significant changes were found in at ATGL-KO mice. <b>I:</b> upper panels: WB analysis of heart lysates using antibodies against cleaved caspase 3 and Bcl-associated X protein (Bax); lower panel: WB analysis of HL-1 cardiomyocytes lysates from cells stimulated with vehicle (Veh) or fatty acid (FA) mix (C16:0, C18:1,C18:2 in different concentrations) using antibodies against cleaved caspase 3; loading control: β–actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).</p

Metabolic phenotype and analysis of blood FA-profile in wt- and atATGL-KO mice.

<p><b>A:</b> Intraperitoneal Glucose Tolerance Test (ipGTT), (n = 4–6, 2-way ANOVA (Bonferroni post-test) from AUC). <b>B</b>: Area under the curve (AUC) of ipGTT, (mean and SEM, n = 4–6, 2-way ANOVA (Bonferroni posttest)). <b>C</b>: Insulin Tolerance Test (ITT), (n = 7–8, 2-way ANOVA (Bonferroni posttest) from AUC). <b>D</b>: Area under the curve (AUC) of ITT, (mean and SEM, n = 7–8, 2-way ANOVA (Bonferroni posttest)). <b>E</b>: Profile of selected serum FAs in TAC-operated mice analyzed by rapid resolution HPLC/ Tandem MS. <b>F</b>: Serum level of non-esterified fatty acids (NEFAs) in wt-TAC and atATGL-KO-TAC mice. <b>G</b>: Serum level of triacylglyerols (TAGs) in wt-TAC and atATGL-KO-TAC mice. (mean and SEM, n = 5, or as indicated, unpaired t-test). ***p<0.001 vs. wt sham, $p<0.05 vs. wt TAC,$ $$ p<0.001 vs. wt TAC, $$ $$ p<0.001 vs. wt TAC.</p