cells

Article
Galectin-3 Is Associated with Cardiac Fibrosis and an Increased
Risk of Sudden Death
Mingma D. Sherpa 1,† , Swati D. Sonkawade 1,† , Vinesh Jonnala 1 , Saraswati Pokharel 2 , Mahyar Khazaeli 3 ,
Yan Yatsynovich 1 , Mohamad A. Kalot 1 , Brian R. Weil 1 , John M. Canty, Jr. 1 and Umesh C. Sharma 1, *

1 Division of Cardiology, Department of Medicine, Jacob’s School of Medicine and Biomedical Sciences,
University at Buffalo, 875 Ellicott Street, Buffalo, NY 14203, USA; mdsherpa@buffalo.edu (M.D.S.);
canty@buffalo.edu (J.M.C.J.)
2 Division of Thoracic Pathology and Oncology, Department of Pathology, Roswell Park Comprehensive
Cancer Center, Buffalo, NY 14203, USA
3 Department of Pathology, Jacob’s School of Medicine and Biomedical Sciences, Buffalo, NY 14203, USA
* Correspondence: sharmau@buffalo.edu; Tel.: +1-716-829-2663; Fax: +1-716-854-1840
† These authors contributed equally to this work.

Abstract: Background: Myocardial fibrosis is a common postmortem finding among individuals

with Sudden Cardiac Death (SCD). Numerous in vivo and in vitro studies have shown that increased
galectin-3 (gal3) expression into the myocardium is associated with higher incidence of fibrosis.
Although elevated gal3 expression is linked with myocardial fibrosis, its role in predicting the risk
of SCD is unknown. Methods: We reviewed the clinical datasets and post-mortem examination of
221 subjects who had died suddenly. We examined myocardial pathology including the extent of
cardiac hypertrophy, fibrosis, and the degree of coronary atherosclerosis in these subjects. In a select
group of SCD subjects, we studied myocardial gal3 and periostin expression using immunohisto-
chemistry. To further examine if a higher level of circulating gal3 can be detected preceding sudden
death, we measured serum gal3 in a porcine model of subtotal coronary artery ligation which shows
Citation: Sherpa, M.D.; Sonkawade, an increased tendency to develop lethal cardiac arrhythmias, including ventricular tachycardia or
S.D.; Jonnala, V.; Pokharel, S.; fibrillation. Results: Of the total 1314 human subjects screened, 12.7% had SCD. Comparison of
Khazaeli, M.; Yatsynovich, Y.; Kalot, age-matched SCD with non-SCD subjects showed that SCD groups had excessive myocardial fibrosis
M.A.; Weil, B.R.; Canty, J.M., Jr.;
involving both the left ventricular free wall and interventricular septum. In pigs with subtotal coro-
Sharma, U.C. Galectin-3 Is
nary artery ligation and SCD, we detected significantly elevated circulating gal3 levels approximately
Associated with Cardiac Fibrosis and
10 days preceding the SCD event. Immunohistochemistry showed increased myocardial gal3 and
an Increased Risk of Sudden Death.
Cells 2023, 12, 1218. https://
periostin expression in pigs that died suddenly, compared to the controls. Conclusion: Our study
doi.org/10.3390/cells12091218 shows that increased gal3 is associated with a higher risk of myocardial fibrosis and the risk of SCD.
This supports the importance of larger translational studies to target gal3 to prevent cardiac fibrosis
Academic Editors: José
and attenuate the risk of SCD.
Abad-Rodríguez and Alonso-Miguel
Higuero
Keywords: biomarkers; cardiac fibrosis; galectin-3; inflammation; mortality; risk stratification;
Received: 20 March 2023 sudden cardiac death
Revised: 15 April 2023
Accepted: 21 April 2023
Published: 23 April 2023
1. Introduction
Sudden cardiac death (SCD) from ventricular arrhythmia is a leading cause of mortality
worldwide [1–3]. There are more than 356,000 out-of-hospital SCDs annually in the United
Copyright: © 2023 by the authors.
States with nearly 90% being fatal [4]. Currently, there is a paucity of literature on the
Licensee MDPI, Basel, Switzerland.
clinico-pathological determinants of SCD. Consequently, we lack definite SCD surveillance
This article is an open access article
distributed under the terms and
guidelines to monitor those at risk. Reduced left ventricular ejection fraction (LVEF) is
conditions of the Creative Commons
currently the primary prognostic factor for predicting SCD, which may not be a sufficient
Attribution (CC BY) license (https://
approach since less than 20% of SCD is seen in those with severely reduced LV function
creativecommons.org/licenses/by/ (LVEF < 35%) [5]. Therefore, there is a strong need for developing more effective approaches
4.0/). to identifying risk-prone subjects. Prior studies demonstrated that the majority of SCD

Cells 2023, 12, 1218. https://doi.org/10.3390/cells12091218 https://www.mdpi.com/journal/cells

Cells 2023, 12, 1218 2 of 14

victims had abundant myocardial fibrosis in post-mortem analysis [6]. In fact, myocardial
fibrosis is now considered as the most common finding associated with SCD in absence of
other contributory causes [7,8]. We previously published the first data on the function of
macrophage-expressed galectin-3 (gal3) in predicting advanced cardiac fibrosis [9].
Galectins are a family of beta-galactoside-binding proteins that share a consensus
sequence in the carbohydrate recognition domain [10]. Among 17 galectin families that
have been identified so far, gal3 is the most widely studied family member and can be
found intracellular in the cytoplasm and nucleus, as well as extracellularly in various
tissues [11–13]. Gal3 is secreted by macrophages, mast cells, other inflammatory cells, and
stimulates cells to release various growth factors, as well as pro-inflammatory cytokines [14].
Gal3 also localizes into the cardiomyocytes immediately after the induction of myocardial
ischemia [15]. We and several other groups previously documented a robust link between
gal3 and cardiac fibrosis, which led to the approval for gal3 testing by enzyme-linked
immunosorbent assays (ELISA) by the US Food and Drug Administration (FDA) [16–18].
In 2019, we reported data showing that circulating gal3 is associated with poor survival
in resuscitated patients after out-of-hospital cardiac arrest. Gal3 enhanced the predictive
utility when combined with other clinical covariates [19]. Studies have also examined
numerous other factors including LV end-diastolic volume index, creatinine, arterial pH,
serum lactate, length of resuscitation, type of rhythm at the time of arrest, and patient
location at the time of events as the predictors of poor outcome [20–22]. However, there
are limited data examining the predictive utility of both circulating and myocardial gal3
expression in myocardial fibrosis and SCD. In this study, we tested the hypothesis that
increased circulating gal3 levels could precede sudden cardiac death. In subjects who died
suddenly, we also examined the degree of cardiac fibrosis and myocardial gal3 expression,
with a hypothesis that increased gal3 is associated with the risk of sudden death. Accurate
risk stratification of SCD will facilitate earlier intervention and potentially improve post-
cardiac arrest outcomes.

2. Methods
2.1. Human Post-Mortem Studies
2.1.1. Study Design and Data Abstraction
The autopsy program at Kaleida Health, the local tertiary care hospital for the Buf-
falo Niagara Community, has an existing inventory of 1600 post-mortem specimens. We
screened 1314 subjects that underwent comprehensive post-mortem analysis and identified
221 subjects that died suddenly. Of the 221 subjects, 167 deaths were reported as SCD.
SCD was defined as non-traumatic, unexpected circulatory arrest occurring within 1 h
of the onset of symptoms in an apparently asymptomatic subject. The controls included
54 subjects who died from causes other than SCD. We abstracted the clinical, laboratory,
and imaging data from the hospital records of all subjects who met the inclusion criteria.
The demographics included age, race, gender, body mass index (BMI), and family history
of SCD. Clinical comorbidities included diabetes mellitus, hypertension, heart failure,
coronary artery disease, obesity, and prior history of revascularization. Location of arrest
(home/public/hospital), having an implantable cardioverter-defibrillator (ICD), length of
cardiopulmonary resuscitation, and time to the return of spontaneous circulation (ROSC)
were also included.

2.1.2. Postmortem Procedures

Minimum standards required for assessment of sudden death in the general popula-
tion were previously published [23]. All postmortem studies were structured examinations
according to previously published guidelines [23]. After getting the relevant clinical infor-
mation for the autopsy, an external examination of the body is done. Once a full autopsy is
performed to exclude common and uncommon extra-cardiac causes of SD, the standard
gross examination of the heart is conducted.
Cells 2023, 12, 1218 3 of 14

2.1.3. Tissue Processing and Storage

A complete transverse (short-axis) transection of the heart at the papillary muscle
insertion (mid-ventricular) level was made. Additionally, short-axis parallel slices of
ventricles at 1 cm interval from the apex to the bases including subendocardial, mid-
myocardial, and subepicardial myocardium were made. In cases of sudden death, a
clinical autopsy was done to determine whether the death was due to cardiac disease
or one of the many non-cardiac causes along with a clinical presentation on admission.
Triphenyltetrazolium (TTC) staining is used in the autopsy room for preliminary diagnosis
of acute myocardial infarction [24]. The heart is sliced transversally at a thickness of 1 mm
and immersed in neutral TTC solution for 15 min to 20 min at 37 ◦ C [25]. Once emptied
of blood, total heart weight and wall thickness measurements were obtained, and various
myocardial sections were stained with H & E [23]. Tissues were harvested, formalin-fixed,
and paraffin-embedded. The histopathology slides are retained for at least 10 years from the
time of tissue retrieval, following the guidelines set by the College of American Pathologists
(CAP) [26]. Representative sections of the right ventricle, left ventricle, and septum were
submitted for histological evaluation.

2.1.4. Myocardial Tissue Morphometry and Immunohistochemistry

The extent of myocardial fibrosis was visualized by Masson’s trichrome staining
(Thermo Scientific, Masson trichrome kit #22110648). Whole section images were obtained
from Leica Aperio VERSA whole slide imaging System at 63× magnifications (Multispec-
tral Imaging Suite, University at Buffalo). The total myocardial area and the area of positive
staining for fibrosis were quantified using a color deconvolution algorithm that calculates
the total stained area (percentage of weak, medium, and strong positive and negative stain-
ing) corresponding to the red and blue colors of the trichrome stain. The fibrosis percentage
is assigned a numerical output using a validated algorithm reported previously [27,28].
The system was carefully programmed to evaluate fibrosis, highlighted in bright blue
with Mallory trichrome staining in contrast to deep red-stained myocardium [28]. Visual
and morphometric histological analyses at basal, midventricular, and apical levels were
performed. Commonly, a score of 0 is given when fibrosis is absent. For any presence of
fibrosis, score + is equal to ≤30% of the examined myocardium, score ++ when present
in >30% to <60% of the examined myocardium, and score +++ when present in ≥60% of the
examined myocardium [28]. In our study, the presence of any fibrosis was considered sig-
nificant. The tissue was incubated with Anti-Galectin-3 (Abcam, Cambridge, MA; ab76245)
and Anti-Periostin (Abcam, Cambridge, MA; ab215199) at a working dilution of 1 in 100,
followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) secondary antibody at 1 in
250 dilutions. Nuclear DNA was labeled in blue with DAPI (Invitrogen, P36931). Images were
taken with an epifluorescence microscope (Leica DMi8 inverted) at 100× magnifications.

2.2. Porcine Model

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
University at Buffalo at New York.

2.2.1. Experimental Porcine Model of SCD

The initial surgical preparation has been previously described [24,25] but modified
in that proximal stenosis was placed on two of the three coronary arteries [23]. Briefly,
juvenile pigs were premedicated with a Telazol (tiletamine 50 mg/mL and zolazepam
50 mg/mL)/ketamine (100 mg/mL) mixture (0.037 mL/kg im) and given prophylactic
antibiotics (cephalothin 50 mg/kg iv and gentamicin 5 mg/kg im) [23]. A thoracotomy
was performed under isoflurane anesthesia (1–3%). The proximal LAD and left circumflex
coronary (LC) arteries were dissected free and instrumented with 1.5-mm (ID) Delra
constrictors [25]. The right coronary artery (RCA) that supplies the posterior descending
Cells 2023, 12, 1218 4 of 14

artery was not manipulated [23]. Pain was controlled with an analgesic (butorphanol
0.025 mg/kg im) and an intercostal nerve block (2% Marcaine) [23].

2.2.2. Use of Porcine Model for Gradual Coronary Occlusion

The pig heart is most closely analogous to the human heart in terms of size, anatomy,
and physiology [26,27]. The myocardium of a pig’s heart does not possess extensive col-
lateral circulation similar to the human heart [28]. Because of this, the pig heart does not
tolerate acute coronary occlusion well, but it can tolerate a gradual coronary occlusion.
If a coronary artery is slowly occluded, it can be used as a model for chronic myocardial
ischemia, chronic myocardial infarction, and heart failure [29]. Left ventricular systolic
function was quantified using M mode fractional shortening (<25% was considered im-
paired) and ejection fraction was measured using Simpson’s biplane method, as described
previously [30]. The porcine model developed a mildly reduced ejection fraction and
had SCD within 1–4 months after coronary instrumentation. This clinical translation was
important since the majority of SCA presents with severe, yet asymptomatic coronary
artery disease (CAD) with minimal or no reduction of cardiac function.

2.2.3. Myocardial Periostin Staining

Flash frozen 10µ thick pig heart LAD sections were fixed with 4% paraformaldehyde,
followed by 0.1% Triton X-100 permeabilization and 1% BSA/10% normal goat serum
tissue blocking. The tissue was incubated with Anti-Periostin (Abcam, Cambridge, MA;
ab215199) at a working dilution of 1 in 100, followed by Goat Anti-Rabbit IgG H&L (Alexa
Fluor® 594) secondary antibody at 1 in 250 dilutions. Nuclear DNA was labelled in blue
with DAPI (Invitrogen, P36931). Images were taken with an epifluorescence microscope
(Leica DMi8 inverted) at 100× magnifications. Periostin is often expressed by cardiac
myofibroblast and was used as a marker of a dynamic scar.

2.2.4. Myocardial Collagen Content

The extent of total myocardial fibrosis was visualized by trichrome staining (Thermo
Scientific™ Richard-Allan Scientific™ Masson Trichrome Kit #22110648). Whole sec-
tion images were obtained from Leica Aperio VERSA whole slide imaging System at
63× magnifications (Multispectral Imaging Suite, University at Buffalo). The total myocar-
dial area and the area of positive staining for fibrosis were quantified using color deconvolu-
tion algorithms. Briefly, whole section images were opened in Aperio ImageScope analysis
software [v12.4.0.5043]. From the toolbar, the rectangular box was drawn around the tissue
to define the area of interest, and a negative pen tool was used to remove peripheral fibrosis
and unwanted tissue section. The pre-existing positive pixel count v9 algorithm was used
for a color deconvolution to separate stains. Other parameters were set with manual scoring
on a number of high-power fields and intensity thresholds. The algorithm calculates the
total stained area (percentage of weak, medium, and strong positive and negative staining)
corresponding to the red and blue colors of the trichrome stain. The fibrosis percentage
was assigned a numerical output using a validated algorithm reported previously [31].

2.2.5. Circulating and Myocardial Galectin-3 Measurements

For circulating gal3 measurement, we used a pre-coated gal3 (LSBio, Seattle, WA
kit #LSF32161) specific 96 well strip microplates ELISA kit. In our quantitative enzymes-
linked assays, Gal3 ELISA was performed in triplicate well using pig serum samples
collected on baseline and post-instrumentation. This colorimetric quantitative sandwich
ELISA measures the optical density, which was normalized with pre-determined standard
gal3 concentration according to the manufacturer’s recommendation [32]. The porcine
myocardial tissue was incubated with Anti-Galectin-3 (Abcam, Cambridge, MA; ab76245)
at a working dilution of 1 in 100, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor®
594) secondary antibody at 1 in 250 dilutions. Nuclear DNA was labeled in blue with DAPI
Cells 2023, 12, 1218 5 of 14

(Invitrogen, P36931). Images were taken with an epifluorescence microscope (Leica DMi8
inverted) at 100× magnifications.

2.2.6. Statistical Analyses

We analyzed the data using standard descriptive statistics along with mean and
standard deviation for continuous variables and frequencies for categorical variables. We
tested the association between the categorical variables and hard outcomes using Chi-
square or Fisher exact test as appropriate. Data are expressed as mean ± standard deviation
and percentage (%) for continuous and categorical variables, respectively. The odds ratio
(OR) and 95% confidence interval (CI) were calculated for each independent variable with
a significant p-value. Differences were considered to be significant when p < 0.05. Statistical
data analysis was performed using SPSS version 23.0 (IBM Corp, Armonk, NY, USA, 2015).

3. Results
3.1. Clinical Characteristics
The mean age of the 221 subjects at the time of death was 62.4 ± 15.6 years. There
were 59.7% whites, 33.9% blacks, 3.6% Hispanics, and 1.4% other races. Of 139 men, 81%
of them had cardiac causes of death, whereas 19% had non-cardiac causes of death. The
socio-demographic variables are shown in Table 1. In 82 females, 67% had cardiac causes of
death, whereas 33% had non-cardiac causes of death. The mean body weight in our subjects
was 90.6 ± 29.6 kg. The mean heart weight for an average 87-kg man ranges from 277 to
481 g [33]. In our study, subjects with sudden cardiac death had the mean heart weight
of 549.2 ± 150.3 g while those with death from non-cardiac causes had a heart weight of
493.2 ± 103.8 g (p = 0.013). The mean LV thickness of the men who died from cardiac causes
was 1.8 ± 0.4 cm, as compared with 1.6 ± 0.4 cm for those who died from non-cardiac
causes (p = 0.005). Those who died from cardiac causes had higher heart weight, lower EF,
increased LV thickness, and higher BMI as shown in Table 2. The place of arrest (hospital
vs. home vs. public) did not show any significant differences. We have summarized the
demographic, clinical characteristics, and circumstances surrounding SCD in Table 1.

Table 1. Socio-demographic and baseline characteristics of the study subjects who died suddenly
from cardiac or non-cardiac causes along with concomitant clinical variables and cardiac tissue
morphometry.

Categorical Variables Non-Cardiac Cause of Cardiac Cause of Total

p-Value
Death (n = 54) Death (n = 167) (n = 221)
Female 27 (33%) 55 (67%) 82
Sex 0.024 *
Male 27 (19%) 112 (81%) 139
Family History of SCA 1 (5%) 21 (95%) 22 0.068
Chest pain before Arrest 6 (12%) 44 (88%) 50 0.001 *
0 45 (28%) 113 (72%) 158
1 2 (14%) 12 (86%) 14
NYHA Class 2 1 (11%) 8 (89%) 9 0.169
3 1 (10%) 9 (90%) 10
4 0 (0%) 11 (100%) 11
Revascularization 5 (10%) 43 (90%) 48 0.025 *
LBBB 1 (20%) 4 (80%) 5 0.480
RBBB 7 (37%) 12 (63%) 19 0.268
Cells 2023, 12, 1218 6 of 14

Table 1. Cont.

Categorical Variables Non-Cardiac Cause of Cardiac Cause of Total

p-Value
Death (n = 54) Death (n = 167) (n = 221)
LV Fibrosis 20 (16%) 108 (84%) 128 0.001 *
RV Fibrosis 3 (19%) 13 (81%) 16 0.583
Septal Fibrosis 3 (5%) 55 (95%) 58 0.000 *
Coronary Atherosclerosis 37 (20%) 150 (80%) 187 0.000 *
Minimal/Mild CAD 15 (45%) 18 (55%) 33 0.000 *
Acute MI 15 (14%) 93 (86%) 108 0.000 *
Results in Table 1 are expressed as absolute numbers (percentage). *, p < 0.05. CAD, coronary atherosclerotic
disease; CHF, congestive heart failure; LBBB, left bundle branch block; LV, left ventricle; MI, myocardial infarc-
tion; NYHA, New York Heart Association; RBBB, right bundle branch block; RV, right ventricle; SCA, sudden
cardiac arrest.

Table 2. Risk factors, clinical data, and significant laboratory values of the study subjects who died
suddenly from cardiac or non-cardiac causes.

Non-Cardiac Cause of Death Cardiac Cause of Total

Continuous Variables p-Value
(n = 54) Death (n = 167) (n = 221)
Age 62.4 ± 17.8 62.3 ± 14.9 62.4 ± 15.6 0.972
Weight 100.2 ± 41.5 87.5 ± 24 90.6 ± 29.6 0.006 *
Height 1.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 0.386
BMI 36.2 ± 15 31 ± 8 32.3 ± 10.4 0.001 *
BSA 2.1 ± 0.4 2 ± 0.3 2 ± 0.4 0.034
Ejection Fraction 57.3 ± 9.8 40.5 ± 18 42.9 ± 18.1 0.004 *
Systolic Blood Pressure 103.5 ± 40.3 95.5 ± 44.1 97.4 ± 43.2 0.392
Diastolic Blood
62.2 ± 33.6 55.3 ± 28.7 56.9 ± 29.9 0.292
Pressure
Heart Rate 101.2 ± 25.5 84.6 ± 31.2 88.4 ± 30.7 0.013 *
Creatinine 2.4 ± 2.1 2.4 ± 2.2 2.4 ± 2.2 0.982
Troponin 0.9 ± 2 34.6 ± 153.1 26.3 ± 133.6 0.224
Myoglobin 1242.4 ± 2527 1342.3 ± 4151.2 1327.8 ± 3942.7 0.942
CK-MB 283 ± 753.2 100.5 ± 188.3 135.7 ± 367.5 0.140
LV Thickness 1.6 ± 0.4 1.8 ± 0.4 1.7 ± 0.4 0.005 *
RV Thickness 0.6 ± 0.7 0.6 ± 0.3 0.6 ± 0.4 0.668
Septal Thickness 1.5 ± 0.4 1.7 ± 0.5 1.7 ± 0.4 0.291
Mitral Valve 10.2 ± 1.2 10.3 ± 1.4 10.2 ± 1.4 0.681
Aortic Valve 7.1 ± 1.2 7.5 ± 1 7.4 ± 1 0.060
Tricuspid Valve 12.1 ± 1.5 11.9 ± 2.2 12 ± 2.1 0.642
Pulmonic Valve 8.2 ± 1.1 8.1 ± 1.1 8.1 ± 1.1 0.869
Heart Weight 493.2 ± 103.8 549.2 ± 150.3 535.9 ± 142.4 0.013
Results in Table 2 are expressed as mean ± SD, with comparisons made by student T-tests or Chi-squared tests,
as appropriate. *, p < 0.05. BMI, body mass index; BNP, brain natriuretic peptide; BSA, body surface area;
CK-MB, creatinine kinase-myoglobin binding; HDL, high-density lipoproteins; HTN, hypertension; ICD, im-
plantable cardioverter defibrillator; LDL, low-density lipoprotein; LV, left ventricle, LVEF, left ventricular ejection
fraction; MI, myocardial infarction; NYHA, New York Heart Association; PEA, pulseless electrical activity; RV,
right ventricle.
Cells 2023, 12, 1218 7 of 14

Categorical variables in Table 3 show that left ventricular fibrosis (OR: 3.02, 95% CI:
1.62–5.61, p < 0.005), septal fibrosis (OR: 8.35, 95% CI: 2.71–26.37, p < 0.005), coronary
atherosclerosis (OR: 4.05, 95% CI: 1.87–8.82, p < 0.005), acute myocardial infarction (OR:
3.27, 95% CI: 1.71—6.24, p < 0.005), and chest pain (OR: 3.98, 95% CI: 1.57—9.74, p < 0.005)
were top five important factors in subjects who died from SCD. Patients with septal fibrosis
(OR: 8.35, 95% CI: 2.71–26.37, p < 0.005) were eight-fold more likely to die suddenly from
cardiac than non-cardiac causes. A total of 95% of subjects with septal fibrosis died from
SCD in comparison to 84% of subjects with left ventricular fibrosis. The data for spatial
fibrosis in relation to the region of the heart were derived from the autopsy report. The
presence of coronary atherosclerosis was seen in 80% of SCD. By multivariate analysis,
diabetes (OR: 2.59, 95% CI: 1.257–5.200, p = 0.028) and hypertension (OR: 2.69, 95% CI:
1.394–5.095, p = 0.013) were both independently associated with SCD.

Table 3. Clinical variables and cardiac tissue morphometry data of the study subjects who died
suddenly from cardiac or non-cardiac causes.

Non-Cardiac
Cardiac Cause of Odds Ratio (Via
Categorical Variables Cause of Death Total (n = 221) p-Value 95% CI
Death (n = 167) Baptista—Pike)
(n = 54)
Septal Fibrosis 3 (5%) 55 (95%) 58 0.000 * 8.35 2.706, 26.370
Coronary
37 (20%) 150 (80%) 187 0.000 * 4.05 1.869, 8.815
Atherosclerosis
Minimal/Mild CAD 15 (45%) 18 (55%) 33 0.000 * 0.21 0.096, 0.470
Acute MI 15 (14%) 93 (86%) 108 0.000 * 3.27 1.707, 6.242
LV Fibrosis 20 (16%) 108 (84%) 128 0.001 * 3.02 1.626, 5.606
Aspirin 9 (11%) 75 (89%) 84 0.000 * 4.56 2.114, 10.200
Chest pain before
6 (12%) 44 (88%) 50 0.001 * 3.98 1.566, 9.743
Arrest
Statin 11 (13%) 74 (87%) 85 0.002 * 3.50 1.632, 7.551
HTN 29 (19%) 124 (81%) 153 0.013 * 2.69 1.394, 5.095
Revascularization 5 (10%) 43 (90%) 48 0.025 * 3.45 1.326, 8.439
DM 12 (14%) 71 (86%) 83 0.028 * 2.59 1.257, 5.200
Results in Table 3 are expressed as absolute numbers (percentage), with comparisons made by student T-tests or
Chi-squared tests, as appropriate. *, p < 0.05. CAD, coronary atherosclerotic disease; DM, diabetes mellitus; HDL,
high-density lipoproteins; HTN, hypertension; LV, left ventricle; MI, myocardial infarction.

3.2. Increased Myocardial Fibrosis and gal3 Expression in SCD Subjects

The results of Masson’s Trichrome-stained tissue sections (Figure 1A–C) demonstrated
total percentage area of fibrosis was significantly higher in the sudden cardiac death
subjects than in the non-cardiac sudden deaths (% Fibrosis: Non-SCD, 13.3% ± 3.4%; SCD,
19.3% ± 3.9%; p = 0.0383, n = 4–8). Within the same study group, we noted increased
gal3 expression [gal3 (CTF): Non-SCD, 12,260 ± 769.2; SCD, 22,806.6 ± 2448.2; p = 0.0247,
n = 4–6] (Figure 2B,D) and periostin expression [periostin (CTF): Non-SCD, 7706.2 ± 479.9;
SCD, 9769.6 ± 431.9; p = 0.0345, n = 4–6] in myocardial autopsy specimen obtained from
SCD subjects compared to controls (Non-SCD) group.
Cells 2023, 12, 1218
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Figure 1. Comparison of myocardial fibrosis in control (non-SCD) vs. sudden cardiac death (SCD).
Figure 1. Comparison of myocardial fibrosis in control (non-SCD) vs. sudden cardiac death (SCD).
Trichrome
Figure staining highlights
1. Comparison collagen
of myocardial content
fibrosis (blue staining)
in control in the
(non-SCD) vs. LV myocardium
sudden in SCD
cardiac death and
(SCD).
Trichrome staining highlights collagen content (blue staining) in the LV myocardium in SCD and
control subjects.
Trichrome (A,B)
staining Representative
highlights images of
collagen content the
(blueleft ventricular
staining) sections
in the LV from non-SCD
myocardium and SCD
in SCD and
control subjects.
subjects,subjects. (A,B)
respectively. Representative images of the left ventricular sections from non-SCD and
control (A,B) (C) Quantitativeimages
Representative analysis
of of
themyocardial collagen
left ventricular volume
sections fromfraction.
non-SCD N and
= 4 (non-
SCD
SCD subjects,
cardiac deaths) respectively. (C)
and 8 (sudden Quantitative analysis
cardiac death). of myocardial collagen volume fraction. N =4
subjects, respectively. (C) Quantitative analysisScale bar: 50 µm,collagen
of myocardial magnification
volume×400.
fraction. N = 4 (non-
(non-cardiac deaths) and 8 (sudden cardiac death). Scale bar: 50 µm, magnification ×400.
cardiac deaths) and 8 (sudden cardiac death). Scale bar: 50 µm, magnification ×400.

Figure 2. Comparison of myocardial periostin and galectin-3 in control (non-SCD) vs. sudden car-
diac
Figuredeath (SCD) groups.
2. Comparison Immunohistochemical
of myocardial periostin andanalysis of formaldehyde-fixed,
galectin-3 in control (non-SCD)paraffin-embed-
vs. sudden car-
Figure
ded 2. Comparison
post-mortem of myocardial
human heart periostin
sections with and galectin-3
periostin in control
(A,B,C) (non-SCD)(D,E,F)
and galectin-3 vs. sudden cardiac
in non-SCD
diac death (SCD) groups. Immunohistochemical analysis of formaldehyde-fixed, paraffin-embed-
death
vs. SCD.(SCD) groups.
Increasedhuman Immunohistochemical
periostin expression analysis
(p < 0.05) of formaldehyde-fixed,
in myocardial autopsy paraffin-embedded
specimen obtained from
ded post-mortem heart sections with periostin (A,B,C) and galectin-3 (D,E,F) in non-SCD
post-mortem
control human
(non-SCD) heart
groups sections
vs. SCD with periostin
subjects shown (A–C)
in the and
bar galectin-3
diagram in (D–F)
the in
right
vs. SCD. Increased periostin expression (p < 0.05) in myocardial autopsy specimen obtained from non-SCD
upper vs.
panel. SCD.
Sim-
ilarly, increased
Increased galectin-3
periostin expression
expression (p < (p <
0.05) 0.05)
in in myocardial
myocardial autopsy
autopsy specimen
specimen obtained
obtained
control (non-SCD) groups vs. SCD subjects shown in the bar diagram in the right upper panel. Sim- from
from con-
control
trol (non-SCD)
(non-SCD)
ilarly, increased groups
groups vs. and SCD subjects
SCDexpression
galectin-3 subjects (p <is0.05)
shown shown
in the in the
bar bar diagram
diagram
in myocardial in thein
autopsy the right
right upper
specimen lower
panel.
obtained panel. CTF,
Similarly,
from con-
corrected
increased
trol total
(non-SCD) fluorescent
galectin-3
groups andintensity.
expression (p <Scale
0.05)
SCD subjects isbar:
in 50 µm,
myocardial
shown magnification
in the autopsy
bar ×400.
specimen
diagram obtained
in the right lowerfrom control
panel. CTF,
corrected
(non-SCD)total fluorescent
groups and SCDintensity.
subjectsScale bar: 50inµm,
is shown themagnification
bar diagram in×400.
the right lower panel. CTF,
3.3. Increased Myocardial Fibrosis in a Porcine Model with SCD
corrected total fluorescent intensity. Scale bar: 50 µm, magnification ×400.
3.3. Increased Myocardial
These porcine Fibrosis
models in a Porcine
developed Model
mildly with SCD
reduced ejection fraction and had SCD
3.3. Increased
within Myocardial
1–3 months Fibrosis in a Porcine Model with SCD
These porcine after
modelscoronary instrumentation
developed with
mildly reduced sub-total occlusion
ejection fraction of LAD
and had SCDand
within These
1–3 porcine
left circumflex
months models
coronary developed
after vessels.
coronary mildly reducedwith
Toinstrumentation
study myocardial ejection fraction
fibrosis, and hadofSCD
we occlusion
sub-total performed a LAD within
quantita-
and
1–3 months
tive
left after
evaluation
circumflex ofcoronary instrumentation
cardiacvessels.
coronary collagen study with
Tocontent sub-total
in Masson’s
myocardial occlusion
we of LAD and
trichrome-stained
fibrosis, aleft
tissue
performed circum-
sections.
quantita-
tive evaluation of cardiac collagen content in Masson’s trichrome-stained tissue sections.
Cells 2023, 12, 1218 9 of 14

Cells 2023, 12, x FOR PEER REVIEW 9 of 14

flex coronary vessels. To study myocardial fibrosis, we performed a quantitative evaluation
of cardiac collagen content in Masson’s trichrome-stained tissue sections. Total percentage
area of
Total fibrosis was
percentage significantly
area higher
of fibrosis was in the porcine
significantly models
higher in theofporcine
coronary ligations
models that
of coro-
died suddenly (% Fibrosis: Non-SCD, 12.01% ± 1.8%; SCD, 25.0% ± 4.09%;
nary ligations that died suddenly (% Fibrosis: Non-SCD, 12.01% ± 1.8%; SCD, 25.0% ± p = 0.0361,
n = 5–6).
4.09%; p = The sudden
0.0361, death
n = 5–6). Thegroup
suddenalsodeath
showed increased
group expression
also showed of periostin
increased in the
expression of
myocardium of the animals that died suddenly compared to controls [Periostin
periostin in the myocardium of the animals that died suddenly compared to controls [Per- (CTF):
Non-SCD,
iostin (CTF):8736 ± 629.5;8736
Non-SCD, SCD,± 629.5; ± 888.2;
11,453SCD, p =±0.0463,
11,453 888.2; pn==0.0463,
5–6] (Figure 3B,D).
n = 5–6] (Figure 3B,D).

Figure 3. Comparison of myocardial fibrosis burden in porcine model of control (non-SCD) vs. sud-
Figure 3. Comparison of myocardial fibrosis burden in porcine model of control (non-SCD) vs.
den cardiac death (SCD). (A,B,C) Representative images of trichrome-stained left ventricular sec-
sudden cardiac death (SCD). (A–C) Representative images of trichrome-stained left ventricular
tions in pigs with non-SCD and SCD, respectively. Bar diagram in the right upper panel shows
sections in pigs
significantly with non-SCD
increased collagen and SCD,fraction
volume respectively. Bar
in these diagram
animals. in theRepresentative
(D,E,F) right upper panel shows
images of
significantly increased collagen volume fraction in these animals. (D–F) Representative
periostin immunohistochemical-stained left ventricular sections in pigs with non-SCD and SCD. Bar images of
periostininimmunohistochemical-stained
diagram left ventricular
the right lower panel shows significantly sections
increased in pigslevels
periostin with in
non-SCD and SCD.
these animals. N
=Bar
5–6diagram
per group. CTF,
in the corrected
right total fluorescent
lower panel intensity.increased
shows significantly Scale bar:periostin
200 µm, levels
magnification ×100.
in these animals.
N = 5–6 per group. CTF, corrected total fluorescent intensity. Scale bar: 200 µm, magnification ×100.
3.4. Mild Reduction in Cardiac Function in a Porcine Model with SCD
3.4. Mild Reduction in Cardiac Function in a Porcine Model with SCD
The clinical data were obtained during follow-up at 3 and 4 months post coronary
The clinicaltodata
instrumentation were
create obtained
a gradual during occlusion
coronary follow-upmodel
at 3 and 4 months
similar post coronary
to humans. The re-
instrumentation to create a gradual coronary occlusion model similar
sults in porcine models showed that LVEF and LVFS showed mean LVEF: 57.5 to humans. The
± 3.5% and
results in porcine models showed that LVEF and LVFS showed mean LVEF: 57.5
mean LVFS: 30.2 ± 3.5% at 3 months post-surgery vs. LVEF: 57.4 ± 9.0% and mean LVFS: ± 3.5%
and ±mean
31.5 4.7% LVFS: 30.2 ±post-surgery.
at 4 months 3.5% at 3 months post-surgery
The results vs. (without
in controls any±coronary
LVEF: 57.4 9.0% andinstru-
mean
LVFS: 31.5 ± 4.7% at 4 months post-surgery. The results in controls (without any coronary
mentation) showed a mean LVEF: 65.4 ± 4.3%. The results in controls (without any coro-
instrumentation) showed a mean LVEF: 65.4 ± 4.3%. The results in controls (without any
nary instrumentation) showed a mean LVEF: 65.4 ± 4.3%. (Supplemental data includes
coronary instrumentation) showed a mean LVEF: 65.4 ± 4.3%. (Supplemental data includes
two avi files showing the mild reduction in EF based on the induced chronic ischemia
two avi files showing the mild reduction in EF based on the induced chronic ischemia
created post-instrumentation.)
created post-instrumentation.)
3.5.
3.5. Increased
IncreasedCirculating
Circulating and
and Myocardial
Myocardial Galectin-3
Galectin-3 in
in Porcine
Porcine Model
Model with
with SCD
SCD
We
We found that higher gal3 levels were detected approximately10
found that higher gal3 levels were detected approximately 10days
daysprior
priorto tosub-
sub-
jects with VT/VF cardiac arrest compared to 30–40 days before sudden cardiac
jects with VT/VF cardiac arrest compared to 30–40 days before sudden cardiac death death [Ga-
lectin-3 (pg/mL):
[Galectin-3 survivors,
(pg/mL): 257.5 257.5
survivors, ± 18.83; SCD, 458.1
± 18.83; SCD,±458.1
22.8; ±
p <22.8;
0.0001,
p < n0.0001,
= 5–9]nas= seen
5–9] in
as
Figure 4A and B. Figure 4C,D are the representative immunofluorescence images of ga-
lectin-3-stained pig left ventricular sections with non-SCD and SCD. The bar diagram (Fig-
ure 4E) shows a significantly increased level of galectin-3 in these animals (p < 0.0197).
Cells 2023, 12, 1218 10 of 14

seen in Figure 4A,B. Figure 4C,D are the representative immunofluorescence images of galec-
Cells 2023, 12, x FOR PEER REVIEW tin-3-stained pig left ventricular sections with non-SCD and SCD. The bar diagram 10 of 14
(Figure 4E) shows a significantly increased level of galectin-3 in these animals (p < 0.0197).

Figure 4. Comparison of circulating gal3 levels in porcine model (SCD vs. survivors). Panel (A)
Figure 4. Comparison of circulating gal3 levels in porcine model (SCD vs. survivors). Panel (A) shows
shows comparison of circulating galectin-3 levels in survivors (N = 9) vs. SCD group (N = 5) approx-
comparison
imately of circulating
3 months after thegalectin-3
coronarylevels in survivors
intervention. (N (B)
Panel = 9)shows
vs. SCD thegroup (N = 5) approximately
time-course of circulating
3 months after
galectin-3 the coronary
increase with higher intervention. Panel~10
levels detected (B)days
shows the time-course
before ventricular of circulating galectin-3
tachycardia/ventricular
increase with
fibrillation highercardiac
(VT/VF) levels detected ~10 daystobefore
arrest, compared ventricular
the mean tachycardia/ventricular
galectin-3 levels measured in thefibrillation
pigs that
had no cardiac
(VT/VF) arrest.
cardiac N =compared
arrest, 3–11, *, p to
= 0.0036 10 days
the mean before levels
galectin-3 cardiacmeasured
arrest vs.in20the
days before
pigs that cardiac
had no
arrest
cardiac inarrest.
SCD pigs,
N = †, p = 0.0002
3–11, 10 days10before
*, p = 0.0036 cardiaccardiac
days before arrest in SCDvs.
arrest vs.20survivors pigs.cardiac
days before (C,D) shows
arrest
representative immunofluorescence images of galectin-3-stained pig left ventricular
in SCD pigs, †, p = 0.0002 10 days before cardiac arrest in SCD vs. survivors pigs. (C,D) shows sections with
non-SCD and SCD. The bar diagram (E) in the lower panel shows a significantly increased
representative immunofluorescence images of galectin-3-stained pig left ventricular sections with level of
galectin-3 in these animals. N = 5 per group. CTF, corrected total fluorescent intensity. Scale bar: 50
non-SCD and SCD. The bar diagram (E) in the lower panel shows a significantly increased level of
µm, magnification ×400.
galectin-3 in these animals. N = 5 per group. CTF, corrected total fluorescent intensity. Scale bar:
50 µm, magnification ×400.
4. Discussion
4. Discussion
Our study reports several novel observations. Firstly, we found that circulating gal3
playsOura critical
studyrole in risk
reports stratifying
several novelSCD by showing
observations. increased
Firstly, circulating
we found gal3 levels
that circulating 10
gal3
days
playsprior to SCD
a critical role as compared
in risk to 30SCD
stratifying days.
by Similarly, immunohistochemical
showing increased circulating gal3 analysis
levels
showed
10 days increased
prior to SCD periostin and trichrome
as compared to 30 (markers of fibrosis)
days. Similarly, in those with SCD. Unlike
immunohistochemical analy-
other clinicalincreased
sis showed biomarkers, gal3 hasand
periostin pro-fibrotic
trichrome activity and of
(markers canfibrosis)
lead to loss of cardiac
in those with func-
SCD.
tion
Unlike[9]. other
We have previously
clinical showngal3
biomarkers, that has
in patients with acute
pro-fibrotic activitymyocardial
and can leadinfarction,
to losscir-
of
cardiac function
culating [9]. We haveispreviously
gal3 overexpression associated shown that in
with major patients
adverse with acute myocardial
cardiovascular outcomes
infarction,
[32]. circulating gal3
We characterized theoverexpression
role of gal3, both is associated with in
in vitro and major adverse
vivo, in ourcardiovascular
prior studies
outcomesIt[32].
[9,31,32]. is nowWe important
characterized the role of
to recognize gal3,
that both in increased
sustained vitro and in vivo,ofincirculating
levels our prior
studies
gal3 can[9,31,32]. It is nowmechanism
be an aberrant important to recognize
during that sustained
the transition fromincreased levelsContinued
SCA to SCD. of circulat-
ing gal3 can be
surveillance an aberrant
of elevated mechanism
gal3 levels canduring the transition
be a useful predictorfrom SCA to SCD.
in identifying theContinued
high-risk
population for SCD. Likewise, increased serum and myocardial gal3 proteinthe
surveillance of elevated gal3 levels can be a useful predictor in identifying high-risk
levels were
population
noted for SCD. Likewise,
in a translational increased
preclinical model serum and myocardial
of coronary gal3 protein levels
microembolism-induced acutewere
my-
noted in injury
ocardial a translational preclinical
[32]. In addition to model of coronary
the prognostic rolemicroembolism-induced
of gal3 demonstrated inacute my-
patients
with ACS and coronary microembolism-induced acute MI model [32], our study adds to
the field by demonstrating a new association between circulating gal3 levels and SCD. We
have also shown that myocardial gal3 expression closely follows the expression of other
molecules representing myocardial fibrosis, such as periostin.
Cells 2023, 12, 1218 11 of 14

ocardial injury [32]. In addition to the prognostic role of gal3 demonstrated in patients
with ACS and coronary microembolism-induced acute MI model [32], our study adds to
the field by demonstrating a new association between circulating gal3 levels and SCD. We
have also shown that myocardial gal3 expression closely follows the expression of other
molecules representing myocardial fibrosis, such as periostin.
Secondly, increased left ventricular and septal fibrosis is associated with increased
mortality with SCD. There has been increasing evidence supporting the link between my-
ocardial fibrosis and ventricular arrhythmia [22,34]. Both human subjects and the porcine
model of SCD showed significant increase in percent fibrosis (Figures 1 and 3). Cardiac
fibroblasts have abundant intracytoplasmic and perinuclear gal3 receptors. Intrapericardial
infusion of gal3 leads to LV dysfunction and excess collagen I deposition [9]. Nevertheless,
we found RV fibrosis played a noncritical role in the pathogenesis of SCD. RV-free wall
fibrosis may worsen paradoxical sepal motion, causing RV-LV dyssynchrony, and quite
often may lead to supraventricular tachyarrhythmia, but malignant ventricular arrhythmias
are rare [26]. Most prior studies done on myocardial interstitium and fibrosis have focused
on LV. Therefore, the relevance of RV fibrosis to an arrhythmogenic substrate is limited
and needs to be further investigated. In our study, 50% of SCD subjects had recorded
arrhythmias (ventricular tachycardia, ventricular fibrillation, asystole, pulseless electrical
activity) prior to arrest. Increased interstitial deposits of collagen filaments act as insulating
barriers, supporting conduction slowing and conduction block [27]. Variations in cardiac
conduction and conduction block, resulting in re-entrant wave front of excitation, are the
classically recognized arrhythmic consequences of increased cardiac fibrosis [27].
Along with left ventricular fibrosis and septal fibrosis, other variables such as the
presence of coronary atherosclerosis, acute myocardial infarction, higher BMI, increased
heart weight, and LV thickness put subjects at higher risk for SCD. Gal3 was associated with
increased left ventricular mass [29]. Other groups have reported marked up-regulation of
gal3 in a pressure-overloaded heart, confirming hypertrophic cardiac remodeling [9,28].
Despite advances in the treatment and prevention of cardiovascular disease (CVD) and
reduction in total cardiovascular mortality, the incidence of SCD remains high. Emerging
evidence on gal3 may be able to help stratify patients who are at increased risk of SCD.
Medical optimization of other comorbidities such as hypertension and diabetes along with
regular follow-up will help lower the risk for SCD.

5. Study Limitations
This study has only a modest sample size as described above. Most of the patient
population was 59.7% Caucasian, despite attempts to include a diverse patient population
from multiple centers. Only 37% of subjects with SCD met the criteria. The staining was
only performed on limited samples depending on the time of autopsy and the quality of the
tissue specimen. The highest quality staining was obtained in these human subjects and the
porcine model. Furthermore, one might point to the other variables that can affect survival
in out-of-hospital cardiac arrest, including the medication provided during resuscitation
and initiation of hypothermia protocol. Nevertheless, even after the adjustment for the
majority of biomarkers and clinical variables, an elevated circulating gal3 level remains
as a significant predictor of poor cardiovascular outcome in these subjects with SCD. The
predictive role of circulating gal3 may be further enhanced when combined with other
clinical variables, which may help in further risk stratification and guide management in
these patients. Despite important advances over the last 10 years, ancillary examinations,
such as toxicology, microbiology, and sampling for genetics are, all too often, not adequately
performed to extensively study other factors involved in SCD [30].

6. Conclusions and Future Implication

Our results emphasize the importance of larger translational studies to assess the
effects of galectin-3 for cardiac remodeling, fibrosis, and SCD. Additionally, similar to gal3,
galectin-1 (gal1) and galectin-9 (gal9) are widely studied members of the galectin family.
Cells 2023, 12, 1218 12 of 14

Prior studies also have shown the involvement of gal1 and gal9 in sudden death and heart
failure. Studies have shown that gal1 promotes the development of atherosclerosis [35]
and gal9 participates in the regulation of atherosclerosis [36]. In our future studies, we aim
to continue our research with other galectin family members such as gal1 and gal9. Fur-
thermore, the heterogeneity of SCD pathophysiology, where acquired and genetic aspects
coexist in the development of fatal ventricular arrhythmias, can affect the strategies of risk
prevention [37]. Current guideline recommendations do not emphasize the importance
of advanced imaging techniques, including cardiac MRI, in the decision-making process
of ICD implantation [38–40]. This aspect represents the greatest potential field for future
research and clinical trials because of the high accuracy of MRI to identify endomyocardial
fibrosis, which acts as a substrate for tachyarrhythmias [37]. Emerging biomarkers such as
gal3 and advanced imaging-guided risk assessment may potentially change the landscape
and decision-making in the primary prevention of SCD.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/cells12091218/s1, Supplemental Table S1: Socio-Demographic and
baseline characteristics of the study subjects who died suddenly from cardiac or non-cardiac causes
along with concomitant clinical variables and cardiac tissue morphometry; Supplemental Table S2:
Risk factors, clinical data, and significant laboratory values of the study subjects who died suddenly
from cardiac or non-cardiac causes.
Author Contributions: M.D.S., data collection, analysis, and manuscript writing; V.J., clinical data
collection; S.D.S., laboratory testing and data analysis; S.P., data interpretation, conceptualization,
and funding; M.K., sample retrieval, data collection, and interpretation; Y.Y., data collection and
conceptualization; M.A.K., data analysis and interpretation; B.R.W., experimental design and data
analysis; J.M.C.J., conceptualization, feedback, and funding of the porcine studies; and U.C.S.,
conceptualization, manuscript editing, funding support, and overall supervision. All authors have
read and agreed to the published version of the manuscript.
Funding: UCS is supported by the Mentored Career Development Award K08HL131987 and
R01HL152090 award from the National Institutes of Health (NIH)/National Heart, Lung, and
Blood Institute (NHLBI). SP received support from NIH/NHLBI Research Grants (award num-
bers R21HL154028 and R01HL150266).
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the Institutional Review Board of the University at
Buffalo (protocol code 5438, date of approval 24 October 2021), and Institutional Animal Care and
Use Committee (protocol 35026N, date of approval 8 February 2022).
Informed Consent Statement: The study was conducted according to the guidelines of the Declara-
tion of Helsinki and approved by the Institutional Review Board of the University at Buffalo (protocol
code 5438, date of approval 24 October 2021), and Institutional Animal Care and Use Committee
(protocols 35026N and MED02011Y, date of approval 8 February 2022).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to the sensitivity of patient information.
Conflicts of Interest: The authors declare no conflict of interest.

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