A Small Endogeneous Peptide Mitigates Myocardial Remodeling in a Mouse Model of Cardioselective Galectin-3 Overexpression

Abstract

Background:

Myocardial galectin-3 expression is associated with cardiac inflammation and fibrosis. Increased galectin-3 portends susceptibility to heart failure (HF) and death. There are no data reporting the causative role of galectin-3 to mediate cardiac fibro-inflammatory response and HF.

Methods:

We developed a cardioselective galectin-3 gain-of-function mouse (Gal3+/+) using α-MHC-promotor. We confirmed galectin-3-transgene expression with RT-PCR, and quantified cardiac/circulating galectin-3 with Western blot and immunoassays. We used Echocardiogram and cardiac MRI to measure cardiac volumes, function and myocardial velocities. Ex-vivo, we studied myocardial inflammation/fibrosis and downstream TGFβ1-mRNA expression. We examined the effects of acute myocardial ischemia in presence of excess galectin-3 by inducing acute myocardial infarction (AMI) in mice. Two subsets of mice including mice treated with Ac-SDKP (a galectin-3-inhibitor) and mice with genetic galectin-3 loss-of-function (Gal3−/−) were studied for comparative analysis of galectin-3 function.

Results:

Gal3+/+ mice had increased cardiac/circulating galectin-3. Gal3+/+ mice showed excess pericardial fat-pad, dilated ventricles and cardiac dysfunction, which was partly normalized by Ac-SDKP. Cardiac-MRI showed reduced myocardial contractile velocities in Gal3+/+. The majority of Gal3+/+ mice did not survive AMI, and the survivors had profound cardiac dysfunction. Myocardial histology of Gal3+/+ mice showed macrophage/mast-cell infiltration, fibrosis and higher TGFβ1-mRNA expression, which were mitigated by both galectin-3 gene deletion and Ac-SDKP administration.

Conclusion:

Our study shows that cardioselective galectin-3 overexpression leads to multiple cardiac phenotypic defects including ventricular dilation and cardiac dysfunction. Pharmacological galectin-3 inhibition conferred protective effects with reduction of inflammation and fibrosis. Our study highlights the importance of translational studies to counteract galectin-3 function and prevent cardiac dysfunction.

Introduction

Over the last decade, studies have shown an emerging role of galectin-3 in the pathophysiology of heart failure (HF). The first preclinical data on the myocardial galectin-3 expression in a model of HF was published by our group in 2004¹. In 2006, we showed that serum galectin-3 levels are elevated in patients with acute HF, and are prognostic of adverse outcomes over a 60-day period after presentation². This emerging usefulness of galectin-3 in predicting major cardiac outcomes led to its approval by the Food and Drug Administration (FDA) as a new biomarker of these outcomes³. Although galectin-3 is recognized as a pro-fibrotic marker, studies to-date have not been able to conclusively examine galectin-3-dependent myocardial fibrosis and HF, since the majority of these studies used cell-culture or recombinant galectin-3. Therefore, the question remains as to whether in vivo overexpression of galectin-3 can initiate cardiac inflammation, fibrosis, and HF progression.

Galectin-3 is a member of the β-galactoside-binding family^{4, 5}. Galectin-3 is secreted by macrophages, mast cells, and other inflammatory cells, and stimulates cells to release various growth factors, as well as pro-inflammatory cytokines⁶. Galectin-3 also localizes into the cardiomyocytes immediately after the induction of myocardial ischemia⁷. Recently, Frunza and associates reported that cardiomyocyte- but not macrophage-specific galectin-3 localization was associated with adverse remodeling and dysfunction⁸. They also reported an attenuation of the cardiac dilatation response in female galectin-3 knockout mice after aortic constriction⁸. Once released into the circulation, galectin-3 can also function as an extracellular cytokine to activate other cells by binding to their receptors, including to the high-affinity IgE-binding receptor (FcƐRI) on the mast cell surface⁵.

The intracellular galectin-3 has been shown to play critical roles in biological responses through its intracellular receptors^{8, 9}. Galectin-3 also induces cardiac fibroblast proliferation, potentially via increased expression of a pro-growth molecule, cyclin D1, and the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1)^{1, 9–11}. Over 17 members of the galectin family with significant structural homologies are known^{5, 12}. Functional redundancies between the members of galectin family make it challenging to study the exact role of galectin-3 in a genetic loss-of-function model. Therefore, we have generated a cardioselective galectin-3 gain-of-function mouse model to study the potential causative and mechanistic effects of galectin-3 in cardiac remodeling.

Previous studies examined N-acetyl-lactosamine, which blocks the carbohydrate binding of galectin-3, in rodent models of pressure overload^{13, 14}. While the effects were viewed as beneficial, no conclusive data were provided to support an improvement of LV ejection fraction, which is the primary end-point of our study. Searching for novel agents to counteract galectin-3 activity and prevent cardiac remodeling, we previously reported N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) as an endogenous inhibitor of cardiac fibrosis¹¹. The current study utilizes Ac-SDKP, which is an endogenous peptide that we believe has more robust inhibition of fibrosis and inflammation, and promises therapeutically significant effects.

Here, we report that cardioselective galectin-3 expression leads to left ventricular dilation, pericardial fatty infiltration and progressive cardiac dysfunction. Mice with cardiac galectin-3 overexpression poorly tolerate myocardial ischemia. We also report that treatment with a small peptide, Ac-SDKP, limits the fibro-inflammatory cascade triggered by galectin-3. Our study highlights a need for translational studies to counteract galectin-3 function, and consequently prevent cardiac dysfunction.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request. The animal care and experimental protocols followed US National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committees of the Roswell Park Comprehensive Cancer Center (RPCCC) and University at Buffalo at New York.

Galectin-3 genetically engineered models:

We studied newly developed galectin-3 overexpression mice (Gal3+/+, 12–15 weeks old), with corresponding age- and strain-matched wild-type controls. Additional experimental groups included B6.Cg-Lgals3^tm1Poi/J galectin-3 knockout mice and appropriate controls purchased from Jackson laboratory.

Galectin-3 pharmacotherapy model:

To study the effects of pharmacological inhibition of galectin-3, a subgroup of Gal3+/+ mice underwent 12-week-long Ac-SDKP peptide therapy. Ac-SDKP was administered continuously at 3.2mg/kg/day doses for 12 weeks using subcutaneously inserted ALZET® Mini-Osmotic Pumps as described previously by our group¹⁵. After the completion of the treatment protocol, animals were sacrificed (with CO₂ overexposure) and organs were harvested for additional histopathological and molecular analysis.

Cardioselective galectin-3 overexpression using α-myosin heavy chain promotor:

Cardiomyocyte-specific galectin-3 overexpression (Gal3+/+) mice were developed on C57BL/6 strain using 9.052 bp linearized cDNA (pRP[Exp]-αMHC_long>mLgals3[BC145419]) construct downstream to α-myosin heavy chain promotor (Cyagen Biosciences Inc., and Roswell Park Comprehensive Cancer Center, Buffalo, NY) as shown in Figure 1, A.

Figure 1. Validation of the transgene expression in α-MHC promotor-restricted gal-3 expression mice.

Panel A is a vector Map for α-MHC-directed galectin-3 expression on One Shot® Stbl3™ Chemically Competent E. coli cloning host. Panel B is a representative image showing PCR products detecting the presence of transgene in Gal3+/+ mice. “Neg” and “Pos” denote lanes with negative and positive PCR products. Panel C shows representative results from the Western blot of protein isolated from cardiac tissue homogenate. Gal3+/+ mice (N=4) showed increased gal-3 protein expression (26 kDa), which was not seen in WT mice (N=4). Bands visualized at 124 kDa represents vinculin loading control. Panel D shows galectin-3 mRNA expression levels in Gal3+/+ (N=7) and WT (N=7) mice measured by real-time PCR (*, p=0.002). Panel E shows serum galectin-3 expression levels in WT (N=3) and Gal3+/+ (N=4) mice (*, p=0.04). “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific Gal3 overexpression transgenic mice. “N” denotes number of mice in each group.

Confirmation of myocardial galectin-3 gene and protein expression

Gene expression by real-time quantitative PCR:

Snap-frozen cardiac tissues (30 mg) were used for mRNA isolation using AllPrep DNA/RNA/Protein Mini Kit (Qiagen#80004). NanoDrop 2000 Spectrophotometer was used to determine protein and RNA concentrations. Real-time PCR was performed as described previously¹⁶. mRNA expression changes were measured in the mice heart tissue for galectin-3. The primer sequences used for real-time PCR are shown in Supplemental Table 1. Expression levels for galectin-3 gene relative to GAPDH were calculated using the threshold cycle difference (CT) method.

Circulating galectin-3 levels

Anti-galectin-3 enzyme-linked immune assays:

The serum galectin-3 levels were measured using mouse-specific Horseradish peroxidase-dependent galectin-3 ELISA kit (EMLGALS3, Thermo Fisher Scientific, Waltham, MA). Serum samples were diluted to 1:50 and sandwich ELISA was performed according to the manufacturer’s instructions.

Phenotyping of galectin-3 overexpression mice:

The phenotypic effects of cardioselective galectin-3 overexpression were monitored for up to 6 months with periodic monitoring of growth and activity of these mice. Transthoracic echocardiogram was used for the initial assessment of cardiac function. High-field cardiac MRI was used to further characterize myocardial tissue and measure cardiac function prior to euthanasia.

Transthoracic echocardiography:

Transthoracic echocardiogram was used to evaluate in vivo cardiac structure and function (Vivid E9 17-in2-dimensional BT12 mouse echo probe, model I13L, 5.8 to 14 MHz; General Electric Inc.) in lightly sedated mice. M-mode echocardiography was performed in the parasternal long-axis view for the measurement of LV dimensions, and then in the anterior short-axis view to evaluate LV ejection fraction. Two‐dimensional guided images of the LV were acquired in the long and short axes to measure LV cavity dimensions, anterior and posterior wall thicknesses, fractional shortening, and heart rate, as described previously by our group ¹⁷.

Cardiac magnetic resonance imaging:

We used 4.7 Tesla preclinical magnetic resonance imaging (MRI) scanner using a 35 mm internal diameter, quadrature transceiver coil, and the ParaVision 3.0.2 acquisition platform (Bruker Biospin, Billerica, MA), as described previously by our group¹⁵. The detailed description of data acquisition and post-processing is provided as supplemental material.

Cardiac morphometry and myocyte nuclear density:

Hematoxylin and eosin (H&E) stained myocardial tissue sections were used to quantify myocyte diameter by counting at least 150–200 cells from the whole heart tissue, covering both ventricles and interventricular septum. Whole heart images were obtained from Leica Aperio VERSA whole slide imaging System at 63x magnifications (Multispectral Imaging suite, University at Buffalo). The detailed description was previously reported by Weil et al ¹⁸, and is also provided as supplemental material.

Myocardial macrophage infiltration:

Formalin-fixed 4-μm-thick paraffin-embedded sections were placed on charged slides, and dried at 60°C for one hour. Slides were cooled to room-temperature and added to the Dako Omnis autostainer, where they were deparaffinized and rehydrated. Sections were incubated for 30 minutes in Flex TRS high pH (Dako; catalog GV80411–2) for target retrieval. Slides were then incubated with anti-CD163 antibody (Abcam, ab182422, 1:300 dilution) for 30 minutes. Rabbit Envision (Agilent K4003, 30 minutes) followed by a Dab chromogen (5 minutes) were used for the visualization of macrophages.

Myocardial mast cell infiltration:

Toluidine blue staining was done to quantify mast cells using BioGenex Toluidine blue stain kit (#SS057). Whole heart images were obtained from the Aperio ScanScope CS System and the number of mast cells (purple cytoplasmic stained cells) were counted in the myocardium using a counter pen tool in Aperio ImageScope software by two blinded observers.

Coronary vascular and peri-vascular collagen content:

The extent of total myocardial fibrosis was visualized by trichrome staining (Thermo Scientific™ Richard-Allan Scientific™ Masson Trichrome Kit #22110648). The total myocardial area and the area of positive staining for fibrosis were quantified using color deconvolution algorithms. The detailed description is provided as supplemental material.

Ischemic stress with acute myocardial infarction:

To study the effects of ischemic stress in galectin-3 overexpression mice compared with wildtype control, we induced acute myocardial infarction (MI) by permanent ligation of left anterior descending (LAD) coronary artery. Acute MI was induced in both wildtype (N=5) and Gal3+/+ (N=9) mice (age 14–15 weeks) by using our study protocol described previously ¹⁹. Briefly, mice were anesthetized with ketamine (1 mg/kg intramuscular) and xylazine (5 mg/kg subcutaneous) and were intubated to undergo a ligation procedure (6–0 prolene) of the LAD. The chest wall was closed with 5–0 silk sutures, and the mice were left to recover at 30°C. Sham surgeries were executed in the same manner, excluding coronary artery ligation.

Statistical Analyses:

The sample size calculations were based on assessment of changes in imaging (15% change in EF measured by Echocardiogram or cardiac MRI, with a CV of 10%) and fibrosis (10% change in interstitial collagen content assessed with trichrome staining, with CV of 30%) as the primary endpoints. A sample size of 6–10 experiments achieves 65–89% power (at a two-sided significance level of 0.05) to detect such effects. The secondary endpoint was a 10% change in LV peak systolic velocities measured by cardiac MRI, which was also appropriately powered with this sample size. Analysis plan: Quantitative endpoints were summarized by group using the mean and standard deviation (SD) with 95% confidence intervals. When appropriate, the endpoints were modeled as a function of treatment group (wild-type control, Gal3−/−, Gal3+/+ and Pep-Gal3+/+) using 1-way ANOVA models; with between-group comparisons made using Tukey’s multiple comparison tests. All model/test assumptions were verified graphically using quantile-quantile and residual plots, with transformations applied as appropriate. All tests were two-sided at a nominal significance level of 0.05.

Results

Increased cardiac and circulating galectin-3 levels in α-MHC-directed galectin-3 gain-of-function model:

First, α-MHC-directed galectin-3 expression in the diploid cloning host was confirmed with repeated sequencing of the transgene vector (Figure 1, A). Cardioselective galectin-3 overexpression founder mice (Gal3+/+) were validated by genomic DNA analysis as shown in the representative figure 1, B (N=7). Of the 80 pups screened, 8 were confirmed to be positive. The transgene positive mice were then cross-bred with strain-matched C57Bl6 mice. Gal3+/+ mice had 5 copies of transgene per diploid genome. Western blot showed abundant myocardial galectin-3 expression in the Gal3+/+ transgenic mice compared to wild-type (WT) controls (Figure 1, C, N=4). Additional analysis using real-time PCR showed a significant increase of galectin-3 mRNA in Gal3+/+ (N=7) compared to WT (N=7) mice hearts (p=0.002, N=7) (Figure 1, D). Importantly, enzyme-linked immunosorbent assay also showed increased circulating galectin-3 levels in Gal3+/+ mice sera [WT (N=3), 19.23±3.5; Gal3+/+ (N=4), 47.83±17.7; p=0.04] (Figure 1, E).

Enlarged LV chamber and pericardial fatty changes in galectin-3 overexpression mice:

Compared to wild-type controls, mice with cardioselective galectin-3 overexpression had grossly enlarged hearts (Figure 2, A–B). Longitudinal microscopic sections of hematoxylin and eosin (H&E) stained heart sections of Gal3+/+ mice showed dilated LV chambers compared to WT controls (Figure 2, C–D). Unexpectedly, Gal3+/+ mice had yellowish-discoloration of thoracic wall and pericardial lining, which was not seen in WT mice (Figure 2, E–F). There were significant changes in systolic (SBP) blood pressures in Gal3+/+ (N=8) compared to wild-type mice (N=8), p=0.04. We did not see any changes in diastolic blood pressures (DBP) (Figure 2, G). Additionally, galectin-3 transgenic mice (N=8) showed a tendency to have a higher heart-to-body-weight ratio compared to wildtype controls (N=7), although the difference was not statistically significant (Figure 2, H).

Figure 2: Gross examination findings in cardiac-specific galectin-3 overexpressing transgenic mice hearts.

Panel A and B show representative gross examination image of WT and Gal3+/+ mice, respectively. Panel C and D are representative images of hematoxylin and eosin- stained heart tissue sections from WT and Gal3+/+ mice, respectively. Gal3+/+ mice displayed dilation of LV chambers compared to WT controls. Panel E and F are representative images showing thoracic wall, epicardial and pericardial surfaces of WT and Gal3+/+ mice, respectively. A distinct yellowish-discoloration was noticed in Gal3+/+ mice, which was not seen in WT controls. Panel G shows the average systolic (*, p=0.04, N=8) and diastolic (p=0.3, N=8) blood pressure measured in these mice. Panel H shows heart to body weight ratios in WT (N=7) and Gal3+/+ (N=8) mice. “SBP” denotes systolic blood pressure, “DBP” denotes diastolic blood pressure, “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice and “N” denotes number of mice in each group. Scale bar represents 2 mm.

Echocardiographic evidence of LV dilatation and dysfunction in galectin-3 overexpression mice, and its prevention by chronic subcutaneous Ac-SDKP infusion:

We examined the changes in the cardiac dimensions and volumes by transthoracic echocardiogram as described previously²⁰. Echocardiogram of Gal3+/+ mice showed enlarged LV chamber volume with reduced LV ejection fraction [(EDV: WT (N=8), 0.094±0.032; Gal3+/+(N=8), 0.126±0.034, p=0.23and EF %: WT (N=8), 71.73±8.7; Gal3+/+(N=8), 57.09±3.8, p=0.009)]. Chronic subcutaneous infusion of Ac-SDKP, a galectin-3 inhibitor peptide in Gal3+/+ mice (N=7), reversed galectin-3 overexpression associated phenotypes (EDV%, p=0.04 and EF%, p=0.0005) compared to untreated Gal3+/+ mice as shown in Figure 3, A and B.

Figure 3: Comparison of cardiac function using echocardiogram and cardiac MRI:

Panel A and B shows ejection fraction (EF %) and End diastolic volume (EDV) calculated in WT (N=8), Gal3+/+ (N=8) and Pep-Gal3+/+ (N=7) mice using echocardiogram. *, p < 0.05 WT vs. Gal3+/+ mice and #, p < 0.05 Pep-Gal3+/+ vs. Gal3+/+ mice. Panel C-H show cardiac MRI data and representative images. Panel C demonstrates myocardial velocity curves illustrating changes in the initial generation of contractile forces over time. D, bar graph showing peak systolic velocity changes in WT (N=5), Gal3+/+ (N=5) and Gal3−/− (N=5) mice. *, p =0.006 WT vs. Gal3+/+ mice and †, p=0.003 Gal3−/− vs. Gal3+/+ mice. Panel E-F, representative short-axis cine images of WT mice. E, End systole, F, End diastole. G-H, Representative short-axis cine images of galectin-3 overexpressing transgenic mice. G, End systole, H, End diastole. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice, “Gal3−/−” denotes galectin-3 knock out mice and “Pep-Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice treated with Ac-SDKP peptide. “N” denotes number of mice in each group.

Cardiac MRI showing abnormal cardiac function with reduced myocardial peak-systolic velocities in galectin-3 overexpression mice, with normal functional indices in wild-type controls and galectin-3 knockouts:

The 2D echocardiographic data was further validated by high-field cardiac MRI in a subset of experimental mice. Gal3+/+ mice showed reduced LV systolic function compared to WT controls [(EF%: Gal3+/+ (N=5), 47.46±9.5; WT (N=5), 63.49±4.0 and p=0.007)] (Table 1). Importantly, galectin-3 knockout mice had normal ejection fraction [(EF%: Gal3−/− (N=5), 64.26±5.5, p=0.005, Gal3−/− vs. Gal3+/+,)], The velocity mapping algorithm of ECG-gated MRI images demonstrated reduced peak systolic contractile velocities in galectin-3 overexpression mice compared to wild-type controls and galectin-3 knockout mice [v_S (cm/s): WT (N=5), −1.299±0.1; Gal3+/+ (N=5), −0.925±0.12, p=0.006 vs. Gal3+/+ and Gal3−/− (N=5), −1.339±0.22, p=0.003 vs. Gal3+/+] (Figure 3, C–D). Representative cardiac MRI images are shown in Figure 3, E–H.

Table 1.

Cardiac MRI-based comparison of left ventricular (LV) dimensions, volume and function in galectin-3 gain and loss function mice

Parameters	WT (N=5)	Gal3−/− (N=5)	Gal3+/+ (N=5)
EDD (mm)	4.38 ± 0.34	4.44 ± 0.48 (p=1.0 vs. WT)	4.63 ± 0.42 (p=0.6 vs. WT; p=0.7 vs. Gal3−/−)
ESD (mm)	2.65 ± 0.3	2.64 ± 0.33 (p=1.0 vs. WT)	3.37 ± 0.59 (p=0.05 vs. WT; p=0.05 vs. Gal3−/−)
FS (%)	39.65 ± 3.3	40.37 ± 4.7 (p=1.0 vs. WT)	27.76 ± 6.7 (p=0.008 vs. WT; p=0.005 vs. Gal3−/−)
Mid-ventricular EDV (mm3)	15.14 ± 2.4	15.61 ± 3.2 (p=1.0 vs. WT)	16.96 ± 3.1 (p=0.6 vs. WT; p=0.7 vs. Gal3−/−)
Mid-ventricular ESV (mm3)	5.57 ± 1.3	5.55 ± 1.4 (p=1.0 vs. WT)	9.12 ± 3.1 (p=0.05 vs. WT; p=0.05 vs. Gal3−/−)
Mid-ventricular SV (mm3)	9.57 ± 1.3	10.06 ± 2.2 (p=0.9 vs. WT)	7.84 ± 0.6 (p=0.2 vs. WT; p=0.10 vs. Gal3−/−)
Mid-ventricular EF (%)	63.49 ± 4	64.26 ± 5.5 (p=1 vs. WT)	47.46 ± 9.5 (p=0.007 vs. WT; p=0.005 vs. Gal3−/−)

EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening; EDV, end-diastolic volume; ESV, end-systolic volume; SV, stroke volume; EF, ejection fraction. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice, “Gal3−/−” denotes galectin-3 knock out mice. “N” denotes number of mice in each group.

Cardiomyocyte hypertrophy with reduced nuclear density in galectin-3 overexpression mice:

To examine the effects of excess galectin-3 on cardiomyocytes, we counted nuclear density and measured myocyte diameter in H&E stained myocardial sections. We found a significantly reduced myocyte nuclear density in Gal3+/+ mice compared to controls [(myocyte nuclei/mm²: WT (N=6), 1649±373.3; Gal3+/+ (N=7), 1161±381.1, p=0.04 (Figure 4, A–C)]. On the contrary, the cardiomyocyte size was higher in Gal3+/+ mice (p=0.02 WT vs. Gal3+/+ mice, N=8) (Figure 4, D).

Figure 4: Effects of Galectin 3 over-expression in cardiomyocyte size and nuclear density:

Panel A and B demonstrate representative images of H&E-stained myocardial tissue sections from WT and Gal3+/+ mice respectively. Panel C shows quantification of nuclear density in WT (N=6) and Gal3+/+ (N=7) mice, *, p =0.04 WT vs. Gal3+/+ mice and Panel D shows cardiomyocyte diameter in WT (N=8) and Gal3+/+ (N=8) mice. *, p =0.02 WT vs. Gal3+/+ mice. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice. “N” denotes number of mice in each group. Scale bar: 40µm, magnification x 630.

Attenuation of myocardial macrophage and mast cell infiltration in galectin-3 knockout and Ac-SDKP infusion models:

We evaluated myocardial macrophage infiltration by immunohistochemical staining with CD163 antibody, which labels monocyte and mature macrophages (Figure 5, A–E). CD163 staining revealed cardiac macrophage accumulation in Gal3+/+ mice compared to WT controls [(macrophages/cm³: WT (N=8), 88±26.6; Gal3+/+ (N=7), 171±47.3; p=0.0005)]. Modulation of galectin-3 by genetic loss-of-function (Gal3−/−) or pharmacologic modulation with Ac-SDKP therapy significantly reduced CD163 positive cell infiltration (macrophages/cm³: Gal3−/− (N=7), 74±35.6, p=0.0001 vs. Gal3+/+ and Pep-Gal3+/+ (N=6), 118±19.8, p=0.05 vs. Gal3+/+)]. These cells were distributed singly or in small cluster throughout the myocardium.

Figure 5: Assessment of myocardial macrophage and mast cell infiltration:

Panel A-D demonstrate representative images of CD163 stained myocardial macrophages from WT (N=8), Gal3−/− (N=7), Gal3+/+ (N=7) and Pep-Gal3+/+ (N=6) mice, respectively. Brown staining represents CD163 positive macrophages. Panel E shows quantification of myocardial macrophages. *, p=0.0005 WT vs. Gal3+/+; †, p=0.0001 Gal3−/− vs. Gal3+/+ and #, p=0.05 Pep-Gal-3+/+ vs. Gal3+/+. Panel F-I demonstrate representative images of toluidine blue-stained myocardial tissue sections from WT (N=8), Gal3−/− (N=5), Gal3+/+ (N=7) and Pep-Gal3+/+ (N=6) mice, respectively. Mast cells are highlighted by purple staining of cytoplasmic granules. Panel J shows quantification of myocardial mast cells. *,p=0.0006 WT vs. Gal3+/+ and † represents p=0.005 Gal3−/− vs. Gal3+/+ mice, and #, p=0.001 Pep-Gal3+/+ vs. Gal-3+/+. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice, “Gal3−/−” denotes galectin-3 knock out mice and “Pep-Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice treated with Ac-SDKP. “N” denotes number of mice in each group. Scale bar: 20µm, magnification x 400.

We also examined the degree of mast cell infiltration in the whole myocardium (short-axis sections) with a toluidine blue stain (Figure 5, F–J). Compared to wild-type controls, a 2-fold increase in the number of infiltrating mast cells was noted in Gal3+/+ mice. The mast cell infiltration was significantly attenuated in galectin-3 knockout and Ac-SDKP treated mice [(mast cells/cm³: WT (N=8), 10±2.1; Gal3−/− (N=5), 11±3.3; Gal3+/+ (N=7), 21±5.7 and Pep-Gal3+/+ (N=6), 10±6.1, p=0.0006, WT vs. Gal3+/+; p=0.005, Gal3−/− vs. Gal3+/+ and p=0.001, Gal3+/+ vs. Pep-Gal3+/+)].

Increased myocardial collagen content in galectin-3 overexpression mice and its prevention by Ac-SDKP infusion:

To study myocardial fibrosis, we performed quantitative evaluation of cardiac collagen content in Masson’s Trichrome stained tissue sections. Total percentage area of fibrosis was significantly higher in the Gal3+/+ mice than in the WT control and Gal3−/− groups [(% collagen volume fraction: WT (N=7), 2.1±0.6; Gal3−/− (N=6), 2.7±1.1; Gal3+/+ (N=8), 4.4±1.1, p=0.002, WT vs. Gal3+/+ and p=0.03, Gal3−/− vs. Gal3+/+)]. Patchy distribution of interstitial collagen was noted in both anterior and posterior myocardial segments in Gal3+/+ mice. Administration of Ac-SDKP showed a tendency to lower the interstitial collagen content [(% collagen volume fraction: Pep-Gal3+/+ (N=6), 3.0±1.3, p=0.08 vs. Gal3+/+)] (Figure 6, A–E).

Figure 6: Comparison of interstitial collagen deposition in the myocardium: Panel.

A-D show representative images of Masson’s trichrome-stained WT (N=7), Gal3−/− (N=6), Gal3+/+ (N=8) and Pep-Gal3+/+ (N=6) mice tissue, respectively. Blue staining represents interstitial collagen content. Panel E shows quantification of collagen volume fraction. *, p=0.002 WT vs. Gal3+/+, †, p=0.03 Gal3−/− vs. Gal3+/+ mice. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice, “Gal3−/−” denotes galectin-3 knock out mice and “Pep-Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice treated with Ac-SDKP peptide. “N” denotes number of mice in each group. Scale bar: 20µm, magnification x 400.

Increased TGFβ1 expression by galectin-3 overexpressing mice, with trends towards its prevention by Ac-SDKP infusion:

To further investigate the profibrotic effects of galectin-3 overexpression in mouse heart tissues, we compared the mRNA expression of TGFβ1 in cardiac tissue homogenate from Gal3−/− (N=7), Gal3+/+ (N=8), Pep-Gal3+/+ (N=7) and WT (N=7) controls. Cardiac homogenate from Gal3+/+ mice showed a robust increase of TGFβ1expression (7.8±4.7-fold changes, p=0.0006, N=7–8) compared to WT controls. Also, reduced TGFβ1expression were noted in Gal3−/− mice compared to Gal3+/+ (p=0.003). There were no significant changes in the expression of cardiac TGFβ1 mRNA in Gal3−/− vs. wild-type controls. Additionally, Ac-SDKP treatment showed a tendency to reduce TGFβ1 expression in Gal3+/+ mice compared to untreated Gal3+/+ mice, although the difference was not statistically significant (Figure 7).

Figure 7: Effects of galectin-3 overexpression on TGFβ1 expression in mouse myocardial tissue: TGFβ1 mRNA expression was analyzed by qPCR in mouse heart tissue.

Overexpression of galectin-3 significantly increased gene expression of TGFβ1 in Gal3+/+ (N=8) mice compared to WT (N=7) controls and Gal3−/− (N=7) mice; *, p=0.0006 WT vs. Gal3+/+; and †, p=0.003 Gal3−/− vs. Gal3+/+. Treatment with Ac-SDKP peptide in Gal3+/+ (N=7) mice showed tendency to reduce expression of TGFβ1 mRNA. “WT” denotes wild type control, “Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice, “Gal3−/−” denotes galectin-3 knock out mice and “Pep-Gal3+/+” denotes cardiomyocyte-specific gal3 overexpression transgenic mice treated with Ac-SDKP peptide. “N” denotes number of mice in each group.

Reduced tolerance to acute myocardial infarction by galectin-3 overexpression mice:

Mice with cardioselective galectin-3 expression poorly tolerated acute MI, with more than 80% mortality within a week after acute MI. Cardiac MRI in a survivor mouse showed severe asymmetric dilatation of the left ventricle, left ventricular wall thinning, swirling of the blood volume in the ventricular cavity suggesting severe low flow state, and a global ejection fraction of less than 10% (Supplemental figure 1).

Discussion

Although prior studies have shown a strong association between elevated circulating galectin-3 and adverse cardiac events, it is still unclear whether galectin-3 is causatively involved in mediating cardiac remodeling and HF. We report that mice with cardioselective galectin-3 overexpression demonstrate: 1) visible changes in cardiac morphology including dilated ventricles and excess pericardial fat pad, 2) reduced cardiac function with reduction in myocardial systolic contractile velocities, 3) myocardial fibro-inflammatory changes with cardiomyocyte nuclear loss, and 4) poor tolerance to myocardial ischemia. Chronic subcutaneous Ac-SDKP infusion results in robust inhibition of galectin-3-dependent myocardial remodeling. This is the first galectin-3 gain-of-function study that confirms the detrimental galectin-3 effects with the prevention of these effects through chronic Ac-SDKP infusion. This study emphasizes the need for additional preclinical studies to counteract galectin-3 in subjects susceptible to develop HF.

Cardiomyocyte-specific galectin-3 overexpression model allows for a better understanding of galectin-3-mediated myocardial pathology. A recently published study by Nguyen et al. reported galectin-3 upregulation in cardiomyocytes in a mouse model of dilated cardiomyopathy²¹. Although inflammatory cells (macrophages and mast cells) express galectin-3, there is accumulating evidence that cardiomyocytes also express galectin-3 in the fibrotic myocardium⁸. Although galectin-3 has developed from the bench to bedside as a biomarker, it is important to recognize that it has a ubiquitous expression source, and galectin-3 levels can alter under stress or immunomodulation²². For instance, a study by Grupper et al. showed no association between the circulating galectin-3 levels and post-cardiac transplant outcomes in 62 immunosuppressed patients²³. Importantly, the cardiac pathology prior to transplantation varied widely, and included both dilated and ischemic cardiomyopathies. On the contrary, Suárez-Fuentetaja et al. studied 122 cardiac transplant patients and reported that circulating gelectin-3 levels steadily decreased during the first year after transplant. Nevertheless, 1-year posttransplant galectin-3 serum levels that remained elevated were associated with increased long-term risk of death and graft failure²⁴.

After our initial publication on the risk-predictive role of galectin-3 in HF, the majority of studies focused on its biological effects in myocardial fibrotic remodeling^{1, 25}. In 2004, using a novel endomyocardial biopsy approach in rats, we demonstrated an early myocardial expression of galectin-3 mRNA in Ren-2 rats that later developed myocardial fibroinflammatory changes followed by decompensated HF¹. In 2019, we reported the clinical data showing the risk-predictive value of circulating galectin-3 measured at an early stage of their hospital stay in subjects resuscitated from out-of-hospital cardiac arrest²⁶. Although the initial FDA approval for galectin-3 was to aid prognosis in patients with HF, our current research showing abundant myocardial fibrosis, macrophage and mast cell infiltration, and TGF-β expression supports the concept that galectin-3 is possibly involved in the initial stages of the pathophysiology of myocardial remodeling, thus underscoring the importance of galectin-3 inhibition to prevent HF.

Calvier et al. further expanded this field of research to include vascular smooth cells, and examined the role of galectin-3 in aldosterone-induced vascular remodeling. Their data indicated that galectin-3 was necessary for the vascular fibro-inflammatory response²⁷. A second study by the same group used a model of experimental hyperaldosteronism, in which increased galectin-3 expression was strongly associated with cardio-renal fibrosis²⁸. These data are complementary to our current galectin-3 pharmacomodulation study, since there is evidence that Ac-SDKP prevents increased collagen deposition and cell proliferation in the heart and kidney in aldosterone-salt hypertensive models²⁹.

Ac-SDKP has been found to have macrophage-inhibitory and anti-fibrotic effects in some experimental models^{29, 30}. We have previously demonstrated that Ac-SDKP can exert anti-galectin-3 effects by inhibiting its pro-inflammatory effects at multiple stages of macrophage morphogenesis³⁰. In a rodent model, Liu et al. in 2008 co-infused galectin-3 along with a galectin-3 inhibitor (Ac-SDKP) into the pericardial sac. Their study demonstrated that exogenous galectin-3 infusion could induce myocardial fibrosis, whereas Ac-SDKP treatment attenuated these effects¹¹. In the current study, Ac-SDKP showed a tendency to lower myocardial collagen content in galectin-3 overexpressing mice although the effect was not robust enough to reach a statistical significance. This could be related to the fact that Ac-SDKP effectively prevents the development of fibrosis but it may not have significant effects in the reversal process. In 2013, a study by Yu and associates demonstrated reduced myocardial inflammation and fibrosis in galectin-3 null mice after pressure overload, but no major changes in the LV ejection fraction were noted¹³.

Although we expected galectin-3 expression to be restricted to the myocardial microenvironment, we found significantly increased levels of circulating galectin-3 in Gal3+/+ mice, indicating that galectin-3 is released from the cardiomyocytes under overexpressed conditions. Our findings support multiple published studies that have reported strong association between increased serum galectin-3 levels with reduced LV function and poor cardiovascular outcomes after acute MI and cardiac arrest^{26, 31, 32}.

We also observed that galectin-3 overexpression was associated with accentuated inflammatory changes in the myocardium. Specifically, there was a higher density of CD163-positive macrophages and mast cells in clusters distributed throughout the myocardium. Macrophages are typically recruited to sites where they can perform phagocytic as well as scavenger functions and generate pro-inflammatory, pro-fibrotic signals upon activation^33–35. Previous studies have reported galectin-3 as a marker of macrophage activation mainly because of its elevated surface expression in phagocytic macrophages^{36, 37}. Along these lines, Sano et al. showed that recombinant human galectin-3 induces monocyte migration in vitro, and it is chemotactic at high concentrations ³⁸.

In addition to macrophages, the role of mast cells has also been recognized in HF since 1960³⁹. Mast cells are activated by cross-linking of the IgE receptor⁴⁰. Mast cell degranulation products, such as chymase and tryptase, generate the active pro-fibrotic form of transforming growth factor-β1, which can mediate fibrosis³⁹. Similarly, an exogenous source of galectin-3 has been shown to activate mast cells⁴¹, ⁴², but the function of endogenous galectin-3 in cardiac fibrosis and HF had not been examined before. This connection is important, because evidence suggests that both macrophages and mast cells are activated in cardiac fibrosis and HF. We also noted mast cell infiltration in the cardioselective galectin-3 overexpressing transgenic mice myocardium compared to the wild-type controls.

Study Limitations:

Our study has a few limitations which can be overcome with future research. Our mouse model was generated using an α-MHC promotor, which is a constitutive promotor and allows for continual transcription of galectin-3. Although there are reports suggesting a susceptibility of HF in rats with higher basal galectin-3, an ideal approach would be an inducible promotor with strong and tightly controllable galectin-3 regulation. Additional challenges include a smaller size for validating circulating galectin-3 levels (N=3) and gated cardiac MRI (N=5) for the comparison of cardiac function in transgenic models. Although multiple complementary approaches were used for the genotypic confirmation and phenotypic characterization of galectin-3 overexpression, small sample size and a possibility of a Type I error remains to be one of the limitations of this study.

Conclusions and Future Implications.

Based on the above observations and published literature, we propose a possible explanation through which galectin-3 could exert the observed effects of myocardial fibro-inflammatory changes and contractile dysfunction. First, galectin-3 is known to activate cell cycle proteins involved in cardiac fibroblast growth and collagen production^{1, 10}. Second, the involvement of galectin-3 to induce macrophage-mast cell infiltration into the myocardium can lead to downstream activation of inflammatory pathways that can further release pro-inflammatory and pro-fibrotic mediators, which can be inhibited by a small therapeutic peptide, Ac-SDKP. Our results emphasize the importance of translational studies to rigorously assess the effects of galectin-3 for cardiac remodeling and test the potential benefits of our lead molecule Ac-SDKP so as to determine whether this merits progression to future translational studies.

WHAT IS NEW?

• We report the first preclinical study of galectin-3 gene modulation that examines fibro-inflammatory effects of cardioselective galectin-3 overexpression.

• Use of a small endogenous peptide, Ac-SDKP, reduces interstitial fibrosis, inhibits cardiac macrophage infiltration and preserves myocardial contractile function.

WHAT ARE THE CLINICAL IMPLICATIONS?

• Galectin-3 is the only molecule known to be upregulated in the myocardium before the development of cardiac dysfunction, suggesting its role at the early stage of the pathogenesis of cardiac inflammation and fibrosis.

• The galectin-3 inhibitory effects of Ac-SDKP can be extended to benefit larger groups, including subjects with cardiac fibrosis and heart failure, in which morbidity remains very high.

Non-standard Abbreviations and Acronyms

Ac-SDKP

N-acetyl-seryl-aspartyl-lysyl-proline

α-MHC

α-myosin heavy chain

FcƐRI

High-affinity IgE-binding receptor

Gal3

Galectin-3

Pep-Gal3+/+

Peptide-treated galectin-3 overexpressing mice

Vs

Myocardial peak-systolic velocity