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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cardiac dysfunction is a major cause of morbidity and mortality in patients with end-stage liver disease; yet the mechanisms remain largely unknown. We hypothesized that the complex interrelated impairments in cardiac structure and function secondary to progression of liver diseases involve alterations in signaling pathways engaged in cardiac energy metabolism and hypertrophy, augmented by direct effects of high circulating levels of bile acids. Biliary fibrosis was induced in male C57BL/6J mice by feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) supplemented diet. After 3 weeks, mice underwent live imaging (dual energy x-ray absorptiometry [DEXA] scanning, two-dimensional echocardiography [2DE], electrocardiography, cardiac magnetic resonance imaging), exercise treadmill testing, and histological and biochemical analyses of livers and hearts. Compared with chow-fed mice, DDC-fed mice fatigued earlier on the treadmill, with reduced VO2. Marked changes were identified electrophysiologically (bradycardia and prolonged QT interval) and functionally (hyperdynamic left ventricular [LV] contractility along with increased LV thickness). Hearts of DDC-fed mice showed hypertrophic signaling (activation of v-akt murine thymoma viral oncogene/protein kinase B [AKT], inhibition of glycogen synthase kinase-3β [GSK3β], a 20-fold up-regulation of β myosin heavy chain RNA and elevated Gsα/Giα ratio. Genes regulating cardiac fatty acid oxidation pathways were suppressed, along with a threefold increase in myocardial glycogen content. Treatment of mouse cardiomyocytes (which express the membrane bile acid receptor TGR5) with potent natural TGR5 agonists, taurochenodeoxycholic acid and lithocholic acid, activated AKT and inhibited GSK3β, similar to the changes seen in DDC-fed mouse hearts. This provides support for a novel mechanism whereby circulating natural bile acids can induce signaling pathways in heart associated with hypertrophy. Conclusion: Three weeks of DDC feeding-induced biliary fibrosis leads to multiple functional, metabolic, electrophysiological, and hypertrophic adaptations in the mouse heart, recapitulating some of the features of human cirrhotic cardiomyopathy. Hepatology 2010;51:2097–2107

Cardiac dysfunction in cirrhosis (cirrhotic cardiomyopathy) is characterized by multiple electrical, physiological, and structural responses in the heart, including left ventricular (LV) hypertrophy with impaired relaxation, baseline hyperdynamic contractility, conduction abnormalities, and an attenuated cardiovascular response to stress.1 The development of cirrhotic cardiomyopathy increases the risk of death in patients with cirrhosis and is associated with increased risks of hepatorenal syndrome, serious postoperative complications, and dysrhythmias.2, 3 In addition to increased mortality, cardiomyopathy is a co-morbid contributor to exercise fatigue in patients with cirrhosis.4, 5

Rodent models of liver injury (mainly common bile duct ligation [CBDL] and carbon tetrachloride treatment) have revealed critical insights into some of the molecular mechanisms underlying cirrhotic cardiomyopathy. Increased cholesterol/phospholipid ratio within the cardiomyocyte cell membrane, impaired β-adrenergic receptor (β-AR) function and abnormal gene expression of β-adrenergic signaling pathway members contributes to attenuated contractile response to inotropic stress in cirrhosis.6, 7 In addition, carbon monoxide, hemoxygenase, nitric oxide, endogenous cannabinoids, bile acids (BA), and endotoxins have been implicated in the pathophysiology of diminished cardiac function in cirrhotic rodents.8 Although these mediators help explain some of the attenuated cardiac contractility in cirrhosis, the causes of hyperdynamic contractility, hypertrophy, and rhythm disturbances, key features of baseline cardiac phenotype in cirrhosis in adults and children, remain essentially unknown.9, 10

Alteration in cardiac energy metabolism, especially substrate utilization, plays a major role in the physiology of cardiac function in health, disease, and hypertrophy. Often metabolic changes precede structural and functional changes in the heart.11, 12 Dysregulation in cardiac energetics has been proposed as one of the potential mechanisms underlying cirrhotic cardiomyopathy.13 Detailed investigations of whole body energetics, characterization of cardiac hypertrophy, and altered metabolism in human and animal models have been limited and could provide a critical insight into the pathophysiology of cirrhotic cardiomyopathy. In addition, it is likely that several pathways are engaged before the development of cirrhosis, although the timeline and mediators remain to be determined.

The goal of this study was to characterize the live mouse cardiac phenotype and explore the extrahepatic molecular consequences in the heart of a recently described mouse model of biliary fibrosis.14 We found multiple impairments in cardiac function and structure in mice with biliary fibrosis, along with distinct changes in cell signaling pathways leading to hypertrophy and altered metabolism. Finally, evidence is provided that supports a contributing role for elevated circulating levels of bile acids to altered cardiac signaling pathways in biliary fibrosis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals and Diet.

Biliary fibrosis was induced by feeding 6-week-old to 8-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) with 0.1% 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) (Sigma-Aldrich, St. Louis, MO) supplemented chow (Harlan-Teklad Inc., Madison, WI) for 3 weeks. Age-matched male C57BL/6J mice fed isocaloric chow were used as controls for all experiments. Mice were weighed twice per week. Feeds and bedding were weighed and changed twice per week to provide an estimate of food intake. Mice were fed ad libitum and had free access to water. Food was withdrawn 4 hours before experiments, and all experiments were done in accordance with Institutional Animal Care and Use Committee–approved protocols at Baylor College of Medicine (BCM).

Anthropometric Measurements.

After 3 weeks, DDC and control mice underwent dual energy x-ray absorptiometry scanning (GE-Lunar PIXImus, Madison, WI) to evaluate lean mass, fat mass, bone mineral density, bone mineral content, and percent fat content (Mouse Phenotype Core [MPC] facility, BCM, Houston, TX).

Exercise Tolerance and Energy Expenditure.

After 3 weeks, mice were exposed to a standard exercise protocol on a treadmill integrated with metabolic chamber (Oxymax Deluxe VO2/VCO2 System Columbus Instruments, Columbus, OH) (See Supporting Information). Indirect calorimetry was performed at baseline, during exercise, and for 5 minutes after treadmill was stopped. Oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were analyzed from the gas sampled (MPC).

Cardiac Parameters.

Continuous electrocardiograms (EC Genie, Mouse Systems Inc., Quincy, MA) and tail-cuff blood pressures (six-channel NIBP system with BPMONWIN software [IITC Life Science Inc., Woodland Hills, CA]) were recorded noninvasively in unsedated mice. Two-dimensional echocardiography (2DE) was performed in the MPC on sedated mice (Vevo 770 Digital RF, VisualSonic Inc. Toronto, CN). Cardiac magnetic resonance imaging (Bruker Biospin, Billerica, MA) was performed in sedated mice and analyzed by a blinded investigator as described previously15 (See Supporting Information).

Serum Analyses.

Sera and anti-coagulated blood were collected from the inferior vena cava and analyzed for alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, total and conjugated bilirubin levels, and complete blood count, respectively (Cobas Integra 400+; Roche) at the Center of Comparative Medicine (BCM). Serum bile acid levels and nonesterified fatty acid levels were evaluated by colorimetric methods (BioQuant Inc, San Diego, CA. & Roche). Serum leptin and tumor necrosis factor alpha levels were measured using commercial enzyme-linked immunosorbent assay kits (RayBiotech Inc., Norcross, GA).

Cardiac and Liver Histology.

Hearts and liver were rapidly isolated and flash frozen in liquid nitrogen, or used for subsequent histological analyses—routine hematoxylin-eosin and Masson-Trichrome (for collagen). Optimal cutting temperature compound–embedded frozen sections were stained with Oil Red-O (for lipids) and periodic acid-Schiff (PAS) for glycogen. Immunohistochemistry for TGR5 was performed on frozen hearts using rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO.) at a 1:200 dilution. Liver sections were stained with routine hematoxylin-eosin. All histological studies were performed by the Texas Gulf Coast Digestive Disease Center. Myocardial glycogen content was compared enzymatically as described previously16 and histologically using image analysis (see Supporting Information).

Quantitative Real Time Polymerase Chain Reaction.

RNA levels were analyzed by quantitative real-time polymerase chain reaction (primer sequences in Supporting Information Table S1), as described previously,17 using SYBR Green (Applied Biosystems, Foster City, CA.). Relative RNA expression was calculated by delta Cycle threshold (Ct) method. Expression of target genes were normalized to internal standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and reported as fold change compared with chow.

Immunoblotting.

Proteins from homogenized whole hearts and membranes were extracted using standard procedures.17 After gel electrophoresis and immunoblotting, the gels were analyzed for expression of various proteins with specific antibodies (see Supporting Information, Table S2). Equal protein loading was confirmed by α-tubulin (Sigma-Aldrich, St. Louis, MO) for whole hearts or sodium-potassium adenosine triphosphatase plasma membrane antibody (Abcam, Cambridge, MA) and Ponceau staining for membrane extracts. Results were analyzed by densitometry (Kodak software, Scientific Imaging Systems; New Haven, CT) and reported as fold change compared with chow.

Isolation of Neonatal Cardiomyocytes and Bile Acid Treatments.

Cardiomyocytes were isolated from 2-day-old to 3-day-old neonatal C57BL/6J mouse pups (Cellutron Life Sciences, Baltimore, MD) as described previously18 (Supporting Information). Isolated cardiomyocytes (0.5 × 106cells/well) were incubated with 100 μM sodium taurochenodeoxycholic acid (TCDCA), 10 μM sodium lithocholic acid (LCA) (Sigma-Aldrich, St. Louis, MO), or vehicle (dimethylsulfoxide [DMSO]). After 4 hours, experiments were terminated with ice-cold phosphate-buffered saline, cells were lysed, and proteins were extracted and analyzed by immunoblotting. Each experiment was performed in duplicate and repeated three times (n = 3). For each set of experiment, cardiomyocytes were isolated and pooled from 15 to 20 neonatal hearts.

Statistical Analysis.

All data are presented as means ± SD (unless specified). Data were analyzed using nonparametric Mann-Whitney test (unless specified). All statistical calculations were done using the PRISM 5.0 software program (Graph-Pad Prism, San Diego, CA). P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

DDC-Fed Mice Exhibit Early Fatigue, Decreased Whole Body Oxygen Consumption, and Increased RER.

Three weeks of DDC-feeding led to biliary fibrosis and changes in serum indices of liver disease consistent with those noted by other investigators14 (Supporting Information Fig. S2). DDC-fed C57BL/6J mice lost weight and demonstrated changes in body composition, mainly an approximately 30% decrease in lean and fat mass, without a change in percent fat or bone mineral content (Supporting Information Fig. S1). On the metabolic treadmill, DDC-fed mice were exhausted earlier than their chow-fed counterparts (Fig. 1A). In addition, indirect calorimetry performed continuously during exercise showed that DDC-fed mice have decreased VO2, decreased peak VO2, and an increased RER (RER = VCO2/VO2) (Fig.1B, D). Thus, for whole body composition and exercise parameters, DDC-fed mice have several key similarities to those seen in patients with biliary cirrhosis.4, 5

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Figure 1. DDC-fed mice have limited tolerance for exercise and altered oxygen utilization: Mice were challenged with acute maximal exercise on a treadmill. Time to exhaustion, VO2, VCO2, and RER were measured by indirect calorimetry as reported in Materials and Methods. DDC fed mice demonstrated early fatigue (A), lower VO2 (B) and high RER (D) when compared with chow-fed mice at each time point. VCO2 did not did not differ between the groups. (*P < 0.05; Results: Mean ± SEM; n = 6; Mann-Whitney for each time point). Note: Chow (white) and DDC (black).

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The Hearts of DDC-Fed Mice Are Hypertrophic and Exhibit Altered Electrical and Functional Parameters.

Unsedated DDC-fed mice exhibited bradycardia, prolonged QT intervals, and higher systolic blood pressure (Fig. 2B). 2DE revealed hyperdynamic contractility evidenced by an approximately 25% increase in both ejection fractions and shortening fractions (Fig. 2A, B), similar to those seen in patients with cirrhosis or in infants with biliary atresia and portal hypertension.5, 9, 10 Cardiac magnetic resonance imaging of DDC-fed mice showed an approximately 25% increase in both LV and right ventricular ejection fractions compared with chow-fed hearts (Table 1). Cardiac mass index, LV free wall, and interventricular septal thickness were significantly increased in DDC-fed mouse hearts (Fig. 3A and Table 1). LV stroke volume index tended to be higher in the DDC-fed mice (Table.1).

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Figure 2. DDC-fed mouse hearts have altered electrocardiographic, echocardiographic, and hemodynamic parameters: (A) Representative M-mode cardiac 2DE pictures of chow-fed and DDC-fed mice showing hyperdynamic LV in DDC-fed mice. Bar graph in (B) denotes heart rate (HR), QT interval, corrected QT interval as analyzed by rhythm strips, systolic (SBP), and mean (MBP) blood pressures as calculated in unsedated mice using tail-cuff, shortening fractions, ejection fractions, and calculated end diastolic volume (LVEDV) of the left ventricle by 2DE. (*P < 0.05; n = 6). Note: Chow (white) and DDC (black).

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Table 1. Cardiac Parameters from MRI
Cardiac ParametersCHOW (n = 5) (Results ± SD)DDC (n = 5) (Results ± SD)P Value
  1. Values obtained for the respective cardiac parameters after quantitative analysis of cardiac MRI. (Statistics: Student t test).

LV ejection fraction (%)48.03 ± 4.766.9 ± 3.70.0002
RV ejection fraction (%)49.7 ± 1.3562.12 ± 4.50.007
Posterior wall thickness (mm)0.71 ± 0.040.88 ± 0.0350.0001
Septal thickness (mm)0.92 ± 0.061.168 ± 0.0360.0001
Cardiac mass (Normalized to body weight)0.0021 ± 0.00030.0037 ± 0.00070.008
LVEDV (mL)0.054 ± 0.0040.043 ± 0.0090.0547
LVESV (mL)0.025 ± 0.0060.014 ± 0.0040.009
LV stroke volume index (mL/g)1.44 ± 0.11.75 ± 0.30.07
RV stroke volume index (mL/g)1.10 ± 0.141.4 ± 0.340.13
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Figure 3. Cardiac hypertrophy and hypertrophic signaling in the DDC-fed heart: (A) Representative pictures of cardiac MRI short axis midventricular slices in diastole shows chow-fed hearts (CH) on the left and DDC-fed hearts on the right. Concentric LV hypertrophy in DDC hearts is evident on these images, showing increased LV posterior wall and septal thickness. This was verified by quantitative analysis of the posterior wall, septal thickness, and LV mass normalized to body weight (Table 1). (B) Quantitative real-time polymerase chain reaction analysis of key genes involved in cardiac hypertrophy. Note up-regulation of β-MyHC, brain natriuretic peptide and eukaryotic elongation factor-2. (C) Representative immunoblots for AKT, phospho-Ser473-AKT, GSK3β and phospho-Ser9-GSK3β with α-tubulin (loading control). (D) Densitometric analysis of the respective bands normalized to α-tubulin and analyses of the degree of phosphorylation (pAKT/AKT and pGSK3β/GSK3β). (*P < 0.05; n = 5-7). Chow (white) and DDC (black).

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DDC-Induced Biliary Cirrhosis Leads to Multiple Alterations in Cardiac Hypertrophy Signaling Pathways and Expression of Relevant Target Genes.

Central among the many changes associated with cardiac hypertrophy in a variety of mouse models are activation of v-akt murine thymoma viral oncogene/protein kinase B (AKT) and inhibition of glycogen synthase kinase-3β (GSK3β).19 DDC feeding resulted in a 1.5-fold increase in cardiac levels of phospho-Ser473-AKT levels (activation) and a 1.75-fold increase in phospho-Ser9-GSK3β (inhibition) (Fig. 3C, D), both associated with increased expression of genes responsible for cardiac hypertrophy.20 Expression of several, but not all, key genes involved in cardiac hypertrophy were up-regulated: notably, β myosin heavy chain (βMyHC; 20-fold), brain natriuretic peptide (2-fold), and eukaryotic elongation factor-2 (1.8-fold) (Fig. 3B and Supporting Information Fig. S3A).

β-Adrenergic Receptor Signaling.

Several researchers have identified altered β-adrenergic signaling in cirrhosis, linked to reductions in either β-adrenergic receptor (βAR) expression7 or impairments in postreceptor signaling pathways.6 Myocardial contractility is governed in large part by signaling through βAR-1 and βAR-2 and their associated co-regulatory Gsα and Giα subunits. DDC-feeding did not change RNA or protein expression of either β-ARs (Fig. 4). However, a significant suppression of Giα, with a modest activation of Gsα, led to a twofold increase in the Gsα/Giα ratio.

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Figure 4. Elevated Gsα Giα ratios in the hearts of DDC-fed mice: (A) shows quantitative polymerase chain reaction results of βAR-1 and βAR-2 RNA standardized to GAPDH. (B) Densitometric analysis of βAR-1and βAR-2 protein expression normalized to α-tubulin. (C) Immunoblot pictures of whole heart incubated with antibodies for Gsα, Giα, and α-tubulin. (D) Densitometric analysis of the respective bands normalized to α-tubulin. (*P < 0.05; n = 5-7) Note: Chow (white) and DDC (black).

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Glycogen Accumulation and Altered Fatty Acid Metabolism in the Hearts of DDC-Fed Mice.

Cardiac histology showed increased staining with PAS (Fig. 5A). Quantitative image analysis and biochemical analysis of cardiac tissue showed a threefold increase in myocardial glycogen content (Fig. 5B, C) after DDC feeding. Both glucose transporter protein (GLUT)-1 and GLUT-4 membrane protein levels, which are responsible for most cellular glucose uptake, were increased in DDC-fed mouse hearts. RNA levels of GLUT-1, but not GLUT-4, were elevated (Fig. 5D, E). Serum free fatty acids (a major energy substrate for a healthy resting heart), and leptin levels (a key regulator of cardiac fatty acid oxidation [FAO]),21 were, respectively, 60% and 80% reduced in DDC-fed mice (Fig. 6A). Expression of heart-type fatty acid binding protein, mitochondrial-carnitine palmitoyl transferase-2, short chain acyl-coA-dehydrogenase, and uncoupling protein-3—key genes responsible for cardiac FAO—were suppressed in DDC-fed mice (Fig. 6B). CD36, mitochondrial-carnitine palmitoyl transferase-1, medium acyl-coA-dehydrogenase, long acyl-coA-dehydrogenase, and very long acyl-coA-dehydrogenase RNA levels did not significantly change with DDC feeding (data not shown). DDC feeding induces activation of cardiac acetyl-coenzyme A carboxylase (ACC, via an approximately 50% decrease in phospho-ACC, Fig. 6D) which has been shown to contribute to suppression of FAO.22 AMPKα protein levels were unchanged by DDC feeding (Fig. 6C). Finally, uncoupling protein-3, which is involved in cardiac FAO23 and remodeling,24 was approximately 50% suppressed after DDC-feeding at both RNA and protein levels (Fig. 6B, D). This reduction in FAO was not linked to changes in myocardial lipid accumulation (Fig. 6E).

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Figure 5. Increased glucose transporter expression and glycogen deposition in hearts of DDC-fed mice: Representative frozen sections (A) of heart (n = 5/group) after PAS staining shows increased glycogen deposition in DDC hearts compared with chow-fed hearts. Myocardial glycogen content was quantified enzymatically (B) and by blinded colorimetric image analysis to quantify percent staining (C) by PAS. (D) Representative immunoblot results of equally loaded isolated membrane protein samples probed with antibodies for GLUT-1 and GLUT-4 proteins. (E) Gene expressions of GLUT-1 and GLUT-4 standardized to GAPDH and (lower row) densitometric analysis of GLUT-1 and GLUT-4 bands. Equal loading confirmed by sodium-potassium-adenosine triphosphatase membrane protein and Ponceau staining. (*P < 0.05; n = 5; and #P < 0.05; n = 3, Student t test.) Note: Chow (white) and DDC (black).

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Figure 6. Reduced fatty acid oxidation in hearts of DDC-fed mice. (A) Decreased levels of serum nonesterified fatty acid levels levels and leptin levels in DDC fed mice. (B) Quantitative polymerase chain reaction results of key genes regulating fatty acid oxidation. (C) Immunoblots of membranes incubated with antibodies for AMPKα and phospho-Thr172-AMPKα, with bar graph below showing densitometric analysis of the respective bands standardized to α-tubulin and analysis of the degree of phosphorylation of AMPKα. (D) Membranes incubated with antibodies for ACC, phospho-Ser79-ACC, and uncoupling protein-3 and α-tubulin as loading control, with bar graph showing densitometric analysis of the respective bands standardized to α-tubulin and analysis of the degree of phosphorylation of ACC. (*P < 0.01; n = 5) Note: Chow (white) and DDC (black). (E) Representative pictures of Oil-Red-O–stained frozen sections of the hearts showing no evidence of lipid accumulation in the DDC-fed cardiac muscle.

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TGR5 and Bile Acid Signaling in Mouse Heart.

As a first step to identify the molecular mediators of altered cardiac myocyte function in biliary fibrosis, we focused on exploring roles for bile acids (BA) and the recently discovered G protein coupled receptor for BAs, TGR5.25 DDC-fed mice have approximately 50-fold elevated circulating levels of serum bile acids compared with chow-fed mice (Fig. 7E), at concentrations capable of activating TGR5.26 When activated by extracellular BAs, including taurine-conjugated primary BAs, this G protein coupled receptor initiates G protein containing signaling pathways in several metabolically active tissues.27 However, little is known about a role for cardiac TGR5. Immunoblot and quantitative real-time polymerase chain reaction analysis of RNA indicates that TGR5 is present in mouse heart and isolated cardiomyocytes (Fig. 7A, B). Immunohistochemistry indicates its presence in both endothelium and cardiomyocytes (Fig. 7C). Cardiac TGR5 RNA and protein expression did not change in DDC-fed mouse hearts (Fig. 7D). To study whether there are direct effects of BAs (natural TGR5 agonists) on cardiomyocytes, we exposed neonatal mouse cardiomyocyte cultures to TCDCA (EC50 for TGR5 of 1.9 μM) and LCA (EC50 for TGR5 of 0.58 μM).26 After incubation for 4 hours, TCDCA (100 μM) as well as LCA (10 μM) activated AKT and inhibited GSK3β, similar to the changes in vivo after DDC feeding (compare Fig. 7G to Fig. 3).

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Figure 7. TGR5 and role for bile acids: (A) Immunoblot of four whole heart tissues [H] showing a band detected at 37 kDa using TGR5 antibody with spleen (S) as a positive control. (B) Polymerase chain reaction analysis of flash frozen whole heart (H), isolated neonatal cardiomyocytes (CM), spleen [S] as positive control, and water [W] used as a negative control. Note TGR5 band at 126 base pair besides molecular weight marker (M). (C) Presence of TGR5 (red staining) in the optimal cutting temperature compound fixed flash-frozen hearts by immunohistochemistry using rabbit polyclonal antibody for TGR5 (Bar = 20 μm thick). No staining seen in the isotype control–stained heart. (D) Bar graph comparing quantitative polymerase chain reaction results of TGR5 RNA (standardized to GAPDH) and TGR5 protein expression (normalized to α-tubulin) between chow-fed (white) and DDC-fed (black) groups and expressed as fold change compared with the chow-fed group. (E) Circulating serum bile acids in DDC-fed mice. (*P < 0.05; Results: Mean ± SD; (n = 5). Note: Chow (white) and DDC (black). (F) Representative immunoblot results from n = 3 experiments of equally loaded protein samples from isolated neonatal cardiomyocytes (0.5 × 106 cells/well) incubated with dimethylsulfoxide as vehicle (Con), TCDCA (100 μM), and LCA (10 μM) for 4 hours. Membranes were incubated with antibodies for AKT, phospho-Ser473-AKT, GSK3β, and phospho-Ser9-GSK3β with α-tubulin as the loading control. (G) Bar graphs showing densitometric analysis of the degree of activation (phosphorylation) of AKT (p-AKT/AKT) and inhibition (phosphorylation) of GSK3β (p-GSK3β/GSK3β). (*P < 0.05; Statistics: Mann-Whitney as compared with dimethylsulfoxide control; n = 3).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Extrahepatic consequences of cirrhosis such as cirrhotic cardiomyopathy strongly impact morbidity and mortality.8 In this study, we report that the DDC-fed model of biliary fibrosis, cholestasis, and liver injury recapitulates many of the cardiac abnormalities seen in human cirrhotic cardiomyopathy. In addition, changes were identified in multiple signaling pathways that could potentially provide insight into the development of cirrhotic cardiomyopathy. DDC-fed mice have multiple alterations in exercise physiology (early fatigability, decreased VO2, and increased RER), cardiac rhythm (bradycardia and prolonged QT interval), cardiac morphology (LV and septal hypertrophy), and cardiac function (increased LV ejection and shortening fractions) that model most of the essential elements of overt cardiac dysfunction in patients with cirrhosis.5, 8, 9, 28, 29 At the molecular level, these hearts exhibit activation of AKT and inhibition of GSK3β pathways, both of which contribute to hypertrophic and hyperdynamic changes in the heart.30-35 Although cardiac expression of β-ARs did not change, Giα was significantly reduced, with a twofold increase in Gsα/Giα ratio, altering βAR signal transduction.36

DDC-feeding leads to suppression of key regulators of cardiac FAO, enhanced expression of membrane glucose transporters, and a threefold increase in glycogen content, suggesting a novel dysregulation in cardiac energetics and metabolism. Finally, a direct activation of AKT and inhibition of GSK3β is seen in isolated cardiomyocytes cultures by TCDCA and LCA (natural TGR5 ligands), providing support for a potential direct role for high levels of circulating BAs in altering cardiac signaling.

2DE and cardiac magnetic resonance imaging of DDC-fed mice show increased thickness of the LV free wall and interventricular septum, consistent with findings in hearts of adults37 as well as infants9, 10 with biliary cirrhosis. Several animal models of liver injury (carbon tetrachloride,38 CBDL,39 and now DDC feeding) lead to LV hypertrophy, which, in turn, can lead to diastolic dysfunction and the inability to compensate for increased demands (for example, systolic dysfunction when stressed). From a cell signaling perspective, several critical mediators have emerged as central to cardiac hypertrophy—AKT activation and GSK3β inhibition.40 Chronic activation of AKT leads to myocardial hypertrophy in mice via mechanisms including either inhibition of GSK3β19, 20 or activation of other signaling pathways.35 Transgenic mice overexpressing cardiac AKT demonstrate hypertrophy, hypercontractility, and an attenuated contractile response to dobutamine30 that mirror the cardiac impairments seen in patients with cirrhosis. In the DDC-fed mouse model, the heart displays several phenotypical and molecular features of physiological hypertrophy, such as hypercontractile LV, AKT activation, and up-regulation of eukaryotic elongation factor-241 as well as characteristics of pathologic hypertrophy such as “re-expression” of fetal growth genes β-MyHC and brain natriuretic peptide.42 Whether these changes reflect adaptive or maladaptive responses to liver injury/biliary fibrosis or response to vascular changes remains to be seen. Importantly, whether the remodeling is reversible in the DDC model as seen in patients with cirrhotic cardiomyopathy after liver transplantation remains to be determined.

Cardiopulmonary exercise testing of patients with cirrhosis revealed early fatigue, lower VO2, and lower maximal VO2.4, 5 DDC-fed mice showed similar physiological changes to humans throughout the course of exercise treadmill testing. Although not statistically significant, these mice demonstrated higher VCO2, which also contributed to the higher RER. High RER is often seen when carbohydrates are oxidized preferentially over lipids,43 suggesting that substrate utilization by metabolically active tissues, including the heart, is impaired in these mice.

A resting heart uses circulating fatty acids for 60% to 70% of its energy needs whereas glucose and lactate supply the remainder. The heart switches its substrate preference from fatty acids to glucose as a compensatory mechanism under stressors such as congestive heart failure, pathological hypertrophy, and cardiomyopathy.11, 12 Histological and biochemical evidence suggest a switch in cardiac substrate utilization favoring glycogen accumulation and reduced fatty acid oxidation in DDC-fed mice (Figs. 5 and 6). The presence of glycogen in the hearts of this model is surprising and intriguing and may be a contributor to structural and functional alterations rather than an innocent bystander. It is possible that in the DDC model, cardiomyocytes store glycogen as endogenous fuel in the face of shortage of exogenous fuel (fatty acids) to maintain vital functions as a survival mechanism. Energy efficiency (adenosine triphosphate/mole O2) improves when heart “switches” from fats to glycogen as preferred fuel for respiration.44 This could explain the hypercontractility in an “unstressed state.” During catecholamine stress, as in exercise, this endogenous glycogen is rapidly depleted, leading to decompensation—a hallmark of cirrhotic cardiomyopathy. Accumulation of minimal amounts of glycogen as seen in mice overexpressing 5′-AMP-activated protein kinase subunit gamma 2 leads to hypertrophy and increased contractility, whereas excessive glycogen deposition leads to severe cardiac dysfunction.45-47 At a molecular level, accumulation of glycogen can be explained by activation of AKT with resultant inactivation of GSK3β.44 At a physiological level, myocardial glycogen content is increased in fasting or as an adaptive response to stress “hibernation.”44 Overall, detailed mechanisms and consequences underlying the marked alterations in cardiac metabolism, along with changes in cell signaling pathways and their physiological relevance in the development of cardiomyopathy in this liver injury model, await further study.

High circulating levels of BAs, in the order of 50-fold to 100-fold higher than normal,48 are among the central components of cirrhosis, and biliary cirrhosis in particular. The primary BAs such as TCDCA and LCA, whose levels are elevated in cirrhosis, activate AKT and inhibit GSK3β in isolated cardiomyocyte cultures at concentrations seen in cirrhosis (Fig. 7), supporting a potential novel role in mediating hypertrophy, hypercontractility, and metabolic changes. BAs are multifunctional molecules that have recently been identified as true hormones,49 with intracellular (cell signaling cascades, nuclear receptors) and extracellular functions targeting cell surface receptors (TGR5).25 BA nuclear receptors and BA transporters are poorly expressed in the myocardium, but TGR5 is present (Fig. 7).50, 51 Thus, there is likely a pathway for circulating BAs to signal to cardiomyocytes extracellularly, through TGR5.52 These data along with others53, 54 are among the first to identify cardiomyocytes as a direct target for BA signaling, suggesting a strong role for BAs in the pathophysiology of cirrhotic cardiomyopathy. It is likely that the hypertrophy, hypercontractility, electrocardiographic disturbance, and altered cardiac metabolism are interlinked, and the “cross-talk” needs further analysis. It is also likely that BAs do not act alone; rather they act in concert with other circulating molecules (cytokines, endotoxin, chemokines, lipids, for example) to contribute to the ultimate changes seen in the cirrhotic heart.

Although head-to-head comparison between cardiac findings in the mouse DDC model and rat CBDL and rat carbon tetrachloride models are difficult secondary to distinct modes of injury, timelines, and species differences, there are some similarities of note. All three models demonstrate cardiac hypertrophy.38, 55 Attenuated contractility to inotropic stress in CBDL and carbon tetrachloride models has been shown to be secondary to downstream effects of decreased β-adrenergic signaling and up-regulated cannabinoid receptor signaling likely mediated by endotoxemia, high circulating tumor necrosis factor alpha, nuclear factor kappaB, high levels of hemoxygenase-1, and increased nitric oxide production. In contrast, cardiac physiology in the DDC mouse model is mostly hyperdynamic, with a robust up-regulation of cardiac hemoxygenase-1 RNA, similar to CBDL rat hearts56 (Supporting Information Fig. S4). There are other differences as well, and direct comparisons of the energetic, metabolic, and molecular phenotypes between these models in mice are needed to properly compare the consequences of these distinct models.

Among the more relevant issues is whether the mouse DDC model develops portal hypertension and systemic hemodynamic changes seen in some of the other models. There is some indirect evidence for this, via an increase in spleen weight index (Supporting Information Fig. S2); however, direct determination of portal pressures and hemodynamics will be needed to be certain. In fact, it may be even more significant that such changes in the hearts of these mice can develop with biliary fibrosis alone. Further studies are needed to properly define these important issues.

In conclusion, multiple interrelated impairments in cardiac cell biology and function are evident in the DDC-induced model of biliary fibrosis, which involve multiple metabolites and signaling pathways, including those engaged by high circulating levels of BAs. Investigating the mechanisms of the multiple metabolic, functional, and molecular signaling alterations induced by liver failure on cardiac function, along with analyzing in detail the contributions of systemic bile acids on cardiomyocyte biology, will help design rational targets to counteract the serious clinical cardiac consequences of cirrhosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank David Durgan, Ph.D., and Martin Young, Ph.D., for enzymatic glycogen analysis; Milton Finegold, M.D., and the Texas Gulf Coast Digestive Disease Center for histology; and Mary Michele Mariscalco, M.D., for critical review of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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