Editor: Salvatore Piano, University of Padova Faculty of Medicine and Surgery, Italy
Equal contributors to this study.
Galectin-3 plays a key pathogenic role in cardiac hypertrophy and heart failure. The present study aimed to investigate the effects of galectin-3 on cardiomyopathy – related factors and cardiac contractility in a rat model of cirrhotic cardiomyopathy.
Rats were divided into two sets, one for a functional study, the other for cardiac contractile-related protein evaluation. There were four groups in each set: sham operated and sham plus
Galectin-3 and collagen I/III ratio were significantly increased in cirrhotic hearts. TNFα and BNP were significantly increased in BDL serum and heart compared with sham controls. Galectin-3 inhibitor significantly decreased galectin-3, TNFα, and BNP in cirrhotic hearts but not in sham controls. N-Lac also significantly improved the blood pressure, and systolic and diastolic cardiomyocyte contractility in cirrhotic rats but had no effect on sham controls.
Increased galectin-3 in the cirrhotic heart significantly inhibited contractility via TNFα. Inhibition of galectin-3 decreased the cardiac content of TNFα and BNP and reversed the decreased blood pressure and depressed contractility in the cirrhotic heart. Galectin-3 appears to play a pathogenic role in cirrhotic cardiomyopathy.
• Galectin-3 inhibits cardiac contractility.
• Galectin-3 is increased in patients and rat models with cirrhosis.
• Galectin-3 inhibition improved cardiac contractility in cirrhotic hearts.
• Galectin-3 is a potential therapeutic target in cirrhotic cardiomyopathy.
Cirrhosis is associated with several cardiovascular disturbances. These disturbances include cardiac contractile dysfunction when challenged, and decreased peripheral vascular resistance and arterial pressure. Despite the baseline increase in cardiac output, cardiac function in patients with cirrhosis is abnormal in several respects. Patients show attenuated systolic and diastolic contractile responses to stress stimuli, electrophysiological repolarization changes including prolonged QT interval, and enlargement or hypertrophy of cardiac chambers. This constellation of cardiac abnormalities is termed cirrhotic cardiomyopathy [
The mechanisms underlying impaired cardiac contractile responsiveness to stressful stimuli in cirrhotic cardiomyopathy remain incompletely clarified. Galectin‐3, a beta‐galactoside‐binding lectin, is predominantly expressed by activated macrophages. We previously showed that macrophages/monocytes are increased in the murine cirrhotic heart [
Rabbit polyclonal anti-galectin-3 antibody, goat polyclonal anti-COL I antibody, rabbit polyclonal anti-COL III antibody and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Donkey anti-goat immunoglobulin (horseradish peroxidase conjugated, HRP) and goat anti-rabbit immunoglobulin (HRP) were from Santa Cruz and were used as a secondary antibody. Isoproterenol was purchased from Sigma (St. Louis, MO, USA). Other reagents were purchased from Sigma, Bio-Rad (Hercules, CA, USA), or Fisher Scientific (Waltham, MA, USA) and were the highest available grade.
Male Sprague-Dawley rats between 200 and 250 g, bred by Charles River (St Laurent, Canada), were used in the present study. The protocol was approved by the Faculty of Medicine Animal Experimentation Committee in the University of Calgary Cumming School of Medicine in accordance with guidelines established by the Canadian Council on Animal Care. Cirrhosis was induced according to previously described methods [
Two sets of rats were used in this study, one to study cirrhotic cardiomyopathy – related protein changes and another for a functional study. There were four groups (n=6 in each group) in each set: sham operation and sham plus galectin-3 inhibitor (
Western blots were performed as previously described [
Hearts embedded in paraffin were cut to 7 µm sections which were de-paraffined by xylene and graded concentrations of alcohol. Antigens were retrieved by citrate buffer. H2O2 (3%) was used to eliminate the endogenous peroxidase activity. After washing, the sections were blocked with 5–10% normal goat serum. Subsequently, the slices were incubated with rabbit anti-rat antibody at 4°C overnight. Following the wash in phosphate buffered saline, slices were incubated with goat anti-rabbit, biotin-labelled secondary antibody for one hour at room temperature. After an additional wash with PBS, slices were incubated with Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA). The color was developed with DAB reagent (Vector Laboratories), hematoxylin was used for counterstain. Spleen was used as a positive control.
TNFα and BNP were measured by ELISA using a commercially available kit (Biosource, Camarillo, CA, USA for TNFα and Novus Biologicals, Littleton, CO, USA for BNP). The serum was used directly; left ventricles were homogenized in ice-cold phosphate-buffered saline and centrifuged at 2,000 g, 4°C for 10 minutes. The supernatants were placed on ice. Protein concentration was determined with Bio-Rad protein assay using bovine serum albumin as standard. The assay of TNFα and BNP for serum and supernatants from left ventricles was performed as follows. Samples and standard (50 μL) were pipetted into wells precoated with rat TNFα-specific antibody. Biotinylated anti-TNFα or anti-BNP solution (50 μL) was added to each well, plate was covered and incubated at room temperature for 1.5 hours (BNP, 2 hours). The liquid was then discarded, and the wells were washed with washing buffer for four times. Streptavidin-peroxidase (100 μL) was added to each well and incubated at room temperature for 45 minutes (for BNP, SP conjugate, 50 μL), and the washing process was repeated four times. Subsequently, 100 μL stabilized chromogen was added to each well in a dark room and incubated for 20 minutes (50 μL for BNP, incubation time was 25 minutes). Finally, 100 μL stop solution (50 μL for BNP) was added and the wells were read at 450 nm with a spectrometer (Molecular Devices, Menlo Park, CA, USA).
Under isoflurane anesthesia, a polygraph recorder (Grass Instruments, West Warwick, RI, USA) was used to measure blood pressure via a femoral artery cannula. The blood pressure was measured twice, 3 weeks and 4 weeks after surgery. We adjusted the isoflurane concentration to record the highest mean blood pressure.
Ventricular myocytes were isolated using methods that have been described previously [
The protocol of measuring systolic and diastolic contractility of isolated cardiomyocytes was performed as previosly described [
Results are expressed as means±standard deviation. For analysis of two independent groups, a Student’s t-test was used. For analysis of three or more independent groups, one-way ANOVA with
Ventricular galectin-3 protein expression in each group was confirmed by immunohistochemistry (
Western blot analysis also showed that the expression of galectin-3 in left ventricular tissue was significantly increased in BDL rats compared with sham rats (galectin-3/GAPDH, 0.2±0.05 vs. 0.82±0.18,
Collagen I was significantly increased, and collagen III significantly decreased in cirrhotic left ventricles; the ratio of collagen I/III was significantly increased from 0.44±0.04 in sham operated rats to 1.83±0.28 (
TNFα levels in cardiac homogenates showed significant increases in the BDL cirrhotic rats compared with those of sham-operated controls (310.4±33.5 vs. 189.9±20.9 pg/mg,
BNP levels in cardiac homogenates showed significant increases in the BDL cirrhotic rats compared with those of sham-operated controls (18.6±3.8 ng/g vs. 10.6±5.0 ng/g,
The isolated cardiomyocyte contractility studies confirmed attenuated systolic and diastolic contractility of cardiomyocytes from cirrhotic rats. The maximum velocity of systolic contraction was significantly decreased in cirrhotic cardiomyocytes compared with controls (5.76±1.43 vs. 7.76±1.90 µm/s,
There were no significant changes of blood pressure in sham, and BDL groups measured twice, at 3 weeks after surgery, and 4 weeks after surgery. N-Lac significantly reversed the decreased blood pressure in BDL rats, not in sham controls (
To our knowledge, the current study is the first to examine a possible regulatory role of galectin-3 in cirrhotic cardiomyopathy. Galectin-3 is a protein encoded by the
Several studies have clarified a key role of galectin-3 in physiological and pathophysiological processes of the heart. van Kimmenade and colleagues [
Cardiac dysfunction is latent in cirrhotic patients. However, when challenged, the cardiac dysfunction manifests, leading to increased morbidity and mortality in cirrhotic patients who undergo procedures or surgeries such as transjugular intrahepatic portosystemic shunt and liver transplantation [
The evidences of involvement of galectin-3 in cirrhotic cardiomyopathy are as follows: 1) galectin is produced by monocytes/macrophages, and we previously showed that monocytes/macrophages are increased in cirrhotic hearts [
Effective treatments that improve cardiac function in patients with cirrhosis are not yet available. Conventional remedies for other heart diseases, such as vasodilators, are not applicable because of the significant baseline vasodilatation in cirrhotic patients. Similarly, angiotensin-converting enzyme inhibitors are also not usable due to the marked vasodilating effect which may precipitate profound hypotension. Direct inotropic agents such as cardiac glycosides were proven ineffective in improving any contractile parameters [
The present study demonstrated that galectin-3 indeed was increased in the cirrhotic heart; this upregulation paralleled the cardiac content of TNFα. Although the inhibition of galectin-3 did not significantly change the ratio of cardiac collagen I/III, or liver fibrosis, this may have been due to the short time frame of galectin-3 application (only three doses). However, N-Lac, a galectin-3 inhibitor, not only downregulated galectin-3 but also decreased the cardiac level of TNFα. Another biomarker related to cardiac function, BNP, was also increased in both heart and serum in BDL rats, and N-Lac significantly decreased BNP levels in both heart and serum from cirrhotic animals but not sham controls. These BNP results along with the improvement of blood pressure in cirrhotic rats all support the notion that the N-lac improves cardiac function in cirrhotic cardiomyopathy. Therefore our results imply that galectin-3 inhibition may be a useful therapeutic target for cirrhotic cardiomyopathy.
In conclusion, galectin-3 was significantly increased in the cirrhotic heart, and significantly inhibited contractility via TNFα. Inhibition of galectin-3 decreased the cardiac content of TNFα and reversed the depressed contractility in the cirrhotic heart. Galectin-3 appears to play a pathogenic role in cirrhotic cardiomyopathy and may be a potential new therapeutic strategy for cirrhotic cardiomyopathy.
Ki Tae Yoon, Hongqun Liu, Jing Zhang and Sojung Han are involved in model creation, data collection and analyses, manuscript drafting. Samuel S. Lee desigend this study. All authors approved the final version.
The authors have no conflicts to disclose.
We thank Dr. Yongxiang Chen for the help in immunohistochemistry.
Supplementary material is available at Clinical and Molecular Hepatology website (
Representative haematoxylin and eosin staining of livers (×200). There was significant structural damage in BDL liver. Short term treatment by N-Lac had no significant change on the liver pathology in BDL rats. (A) Sham. (B) Sham+N-Lac. (C) BDL. (D) BDL+N-Lac. BDL, bile duct ligation; Sham, sham operated; N-Lac,
epresentative Masson staining of livers (×100). There was significant fibrosis in BDL liver. Short term treatment by N-Lac had no significant attenuation of the fibrosis. (A) Sham. (B) Sham+N-Lac. (C) BDL. (D) BDL+N-Lac. BDL, bile duct ligation; Sham, sham operated; N-Lac,
bile duct ligation
brain natriuretic peptide
enzyme-linked immunosorbent assay
glyceraldehyde 3-phosphate dehydrogenase
haematoxylin and eosin
heart failure with preserved ejection fraction
horseradish peroxidase conjugated
horseradish peroxidase
Tris-buffered saline buffer
tumor necrosis factor alpha
Representative immunohistochemistry of galectin-3 protein expression in hearts (×200). (A) Sham. (B) BDL. (C) BDL+N-Lac. (D) Spleen was used as positive control. (A) Scant staining is visible in cardiomyocytes. (B) Significantly increased galectin-3 staining is predominantly in cardiomyocyte cytoplasm, although a few macrophages also stain. (C) N-Lac significantly decreased galectin-3 staining in BDL heart. N-Lac did not affect galectin-3 postive staining in heart from sham operated controls (data not shown). (D) Spleen shows expected heavy galectin-3 staining, as a positive control. Sham, sham operated; BDL, bile duct ligation; N-Lac,
(A) Representative Western blot analysis of galectin-3 protein expression in hearts. From left, 1st and 2nd lanes, sham-operated controls; 3rd and 4th lanes, sham+N-Lac, 5th and 6th lanes, BDL, 7th and 8th lanes, BDL+N-Lac. (B) Computerized optical densitometry showed that galectin-3 protein expression was significant increased in BDL heart, N-Lac significantly decreased galectin-3 protein expression in BDL, N-Lac did not affect galectin-3 protein expression in sham operated rats (n=6 in each group, *
(A) Representative Western blot analysis of collagen I/III protein expressions in hearts. From left, 1st and 2nd lanes, sham-operated controls; 3rd and 4th lanes, sham+N-Lac, 5th and 6th lanes, BDL, 7th and 8th lanes, BDL+N-Lac. (B) Computerized optical densitometry showed that the ratio of collagen I/III was shifted from collagen III dominant to
collagen I dominant in BDL heart, N-Lac had the tendency to decrease the ratio of collagen I/III in cirrhotic heart (n=6 in each group, *
The concentrations of TNFα in serum (A) and heart (B). From left, 1st and 2nd lanes, sham-operated controls; 3rd and 4th lanes, sham+N-Lac, 5th and 6th lanes, BDL, 7th and 8th lanes, BDL+N-Lac. The concentrations of TNFα in serum and heart were significantly increased in BDL, N-Lac significantly decreased TNFα concentrations in both serum and heart from cirrhotic rats (n=6 in each group, *
The concentrations of BNP in serum (A) and heart (B). The concentrations of BNP in serum and heart were significantly increased in BDL rats. N-Lac significantly decreased BNP concentrations in both serum and heart from cirrhotic rats (n=6 in each group, *
(A) Isoproterenol-stimulated maximal systolic velocity in isolated cardiomyocytes. Systolic velocity was significantly decreased in cirrhotic rats compared to the sham control group (
(A) Diatolic relaxation velocity in isolated cardiomyocytes under the environment of isoproterenol stimulation. Diatolic relaxation velocity was significantly decreased in cirrhotic rats compared to the sham control group (
Blood pressure changes. The blood pressure was significantly decreased in cirrhotic rats, N-Lac significantly increased blood pressure in BDL rats. N-Lac had no effect on blood pressure in sham rats. The two measurements had no significant difference in BDL rats (n=6 in each group, *