Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis

Abstract

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 VO(2). 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-3beta [GSK3beta], a 20-fold up-regulation of beta myosin heavy chain RNA and elevated G(s)alpha/G(i)alpha 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 GSK3beta, 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.

Fig.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 Methods. DDC fed mice demonstrated early fatigue (A), lower VO₂ (B) and high RER (D) when compared to 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 (□) and DDC (■)

Fig.2. DDC-fed mouse hearts have altered electrocardiographic, echocardiographic and hemodynamic parameters

(A) denotes representative M-mode cardiac 2DEcho 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 (QTc) as analyzed by rhythm strips, systolic (SBP) and mean (MBP) blood pressures as calculated in unsedated mice using tail-cuff, shortening fractions (%FS), ejection fractions (%EF) and calculated end diastolic volume (LVEDV) of the left ventricle by 2DEcho. (* p<0.05; n=6). Note: Chow (□) and DDC (■)

Fig.3. Cardiac hypertrophy and hypertrophic signaling in the DDC-fed heart

(A) Representative pictures of cardiac MR short axis mid-ventricular 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 the body weight (see Table.1). (B) shows qRTPCR analysis of key genes involved in cardiac hypertrophy. Note upregulation of β-MyHC, BNP and eEF2. (C) shows representative immunoblots for AKT, Ser473-phospho-AKT, GSK3β and Ser9-phospho-GSK3β with α-tubulin (loading control). (D) shows densitometric analysis of the respective bands normalized to α-tubulin and analyses of the degree of phosphorylation (pAKT/AKT and pGSK3b/GSK3b). (*p<0.05; n=5-7) Chow (□) and DDC (■)

Fig.4. Elevated G_sα/ G_iα ratios in the hearts of DDC-fed mice

(A) shows QPCR results of βAR-1 and βAR-2 RNA standardized to GAPDH and (B) depicts densitometric analysis of βAR-1and βAR-2 protein expression normalized to α-tubulin (C) Immunoblot pictures of whole heart incubated with antibodies for G_sα, G_iα and α-tubulin. (D) shows densitometric analysis of the respective bands normalized to α-tubulin. (*p<0.05; n=5-7) Note: Chow (□) and DDC (■)

Fig.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) denotes representative immunoblot results of equally loaded isolated membrane protein samples probed with antibodies for GLUT-1 and GLUT-4 proteins. (E) shows 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-ATPase membrane protein and Ponceau staining. (*p<0.05; n=5; and # p<0.05; n=3, Students t test.) Note: Chow (□) and DDC (■)

Fig. 6. Reduced fatty acid oxidation in hearts of DDC-fed mice

(A) shows decreased levels of serum NEFA levels and leptin levels in DDC fed mice. (B) shows QPCR results of key genes regulating fatty acid oxidation. (C) shows immunoblots of membranes incubated with antibodies for AMPKα and Thr172-phospho-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) shows membranes incubated with antibodies for ACC, Ser79-phospho-ACC and UCP-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 (□) and DDC (■). (E) shows representative pictures of Oil-Red-O stained frozen sections of the hearts showing no evidence of lipid accumulation in the DDC fed cardiac muscle.

Fig. 7. TGR5 and role for bile acids

(A) shows immunoblot of 4 whole heart tissues [H] showing a band detected at 37kDa using TGR5 antibody with spleen (S) as a positive control. (B) shows PCR 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) shows presence of TGR5 (red staining) in the OCT 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) shows bar graph comparing QPCR results of TGR5 RNA (standardized to GAPDH) and TGR5 protein expression (normalized to α-tubulin) between chow (□) and DDC fed (■) groups and expressed as fold change compared to chow group. (E) depicts circulating serum bile acids in DDC fed mice. (*p<0.05; Results: Mean±SD; (n=5) Note: Chow (□) and DDC (■)) (F) shows representative immunoblot results from n=3 experiments of equally loaded protein samples from isolated neonatal cardiomyocytes (0.5×10⁶ cells/well) incubated with DMSO as vehicle (Con), TCDCA (100μM) and LCA (10μM) for 4 hours. Membranes were incubated with antibodies for AKT, Ser473-phospho-AKT, GSK3β and Ser9-phospho-GSK3β with α-tubulin as the loading control. (G) depicts 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 DMSO control; n=3).