Proteome Alterations in Cardiac Fibroblasts: Insights from Experimental Myocardial Infarction and Clinical Ischaemic Cardiomyopathy

Abstract

Ischaemic heart disease (IHD) is a chronic condition that can cause pathological cardiac remodelling and heart failure (HF). In this study, we sought to determine how cardiac fibroblasts were altered post-experimental myocardial infarction (MI). Female C57BL6 mice underwent experimental MI by permanent left coronary artery ligation. Cardiac fibroblasts were isolated from extracted heart tissue of experimental MI mice and subsequently treated with the pro-fibrotic cytokine, TGF-β, for 24 h and analysed using high throughput LC-MS/MS analysis. Findings were validated using mass spectrometry data generated from human left ventricular tissue analysis, which were collected from patients with ischaemic cardiomyopathy (ISCM) and age/sex-matched patients without clinical HF (NF). Proteomic analysis revealed significant protein expression changes in mouse cardiac fibroblasts after MI. These changes were most pronounced at 1 month post-MI, compared to earlier time points (3 days and 1 week). TGF-β treatment profoundly affected fibroblast cells extracted from MI mice, indicating a heightened sensitivity to pro-fibrotic factors after myocardial injury. Extracellular matrix (ECM) proteins significantly altered in MI fibroblasts following TGF-β treatment were significantly associated with cardiac remodelling. Notably, Lox was significantly changed in both isolated fibroblasts treated with TGF-β from experiment MI mice and human ISCM. Isolated cardiac fibroblasts from MI mice are more susceptible to developing pathogenic traits following TGF-β treatment than isolated fibroblasts from normal heart tissue. ECM proteins associated with these enhanced fibroblast activities and functions are evident. These altered proteins may play a functional role in MI-associated cardiac dysfunction.

Full text

Academic Editor: Andrea Sonaglioni
Received: 4 March 2025
Revised: 16 April 2025
Accepted: 17 April 2025
Published: 18 April 2025
Citation: Russell-Hallinan, A.; Tonry,
C.; Kerrigan, L.; Edgar, K.; Collier, P.;
McDonald, K.; Ledwidge, M.; Grieve,
D.; Karuna, N.; Watson,C. Proteome
Alterations in Cardiac Fibroblasts:
Insights from Experimental
Myocardial Infarction and Clinical
Ischaemic Cardiomyopathy. Int. J.
Mol. Sci. 2025,26, 3846. https://
doi.org/10.3390/ijms26083846
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Article
Proteome Alterations in Cardiac Fibroblasts: Insights
from Experimental Myocardial Infarction and Clinical
Ischaemic Cardiomyopathy
Adam Russell-Hallinan 1, † , Claire Tonry 1,†, Lauren Kerrigan 1, Kevin Edgar 1, Patrick Collier 2,
Ken McDonald 3,4, Mark Ledwidge 3,4, David Grieve 1, Narainrit Karuna 1, 5,* and Chris Watson 1,4,*
1Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast,
Belfast BT9 7BL, Northern Ireland, UK; adamrh.ucd@gmail.com (A.R.-H.); claire.tonry@qub.ac.uk (C.T.);
l.kerrigan@qub.ac.uk (L.K.); kevin.edgar@qub.ac.uk (K.E.); david.grieve@qub.ac.uk (D.G.)
2Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA
3STOP-HF Unit, St Vincent’s University Hospital Healthcare Group, D04 T6F4 Dublin, Ireland;
kenneth.mcdonald@ucd.ie (K.M.); mark.ledwidge@ucd.ie (M.L.)
4UCD Conway Institute, School of Medicine, University College Dublin, D04 V1W8 Dublin, Ireland
5Department of Pharmaceutical Care, Faculty of Pharmacy, Chiang Mai University,
Chiang Mai 50200, Thailand
*Correspondence: nkaruna01@qub.ac.uk (N.K.); chris.watson@qub.ac.uk (C.W.)
†These authors contributed equally to this work.
Abstract: Ischaemic heart disease (IHD) is a chronic condition that can cause pathological
cardiac remodelling and heart failure (HF). In this study, we sought to determine how
cardiac fibroblasts were altered post-experimental myocardial infarction (MI). Female
C57BL6 mice underwent experimental MI by permanent left coronary artery ligation.
Cardiac fibroblasts were isolated from extracted heart tissue of experimental MI mice and
subsequently treated with the pro-fibrotic cytokine, TGF-
β
, for 24 h and analysed using high
throughput LC-MS/MS analysis. Findings were validated using mass spectrometry data
generated from human left ventricular tissue analysis, which were collected from patients
with ischaemic cardiomyopathy (ISCM) and age/sex-matched patients without clinical
HF (NF). Proteomic analysis revealed significant protein expression changes in mouse
cardiac fibroblasts after MI. These changes were most pronounced at 1 month post-MI,
compared to earlier time points (3 days and 1 week). TGF-
β
treatment profoundly affected
fibroblast cells extracted from MI mice, indicating a heightened sensitivity to pro-fibrotic
factors after myocardial injury. Extracellular matrix (ECM) proteins significantly altered
in MI fibroblasts following TGF-
β
treatment were significantly associated with cardiac
remodelling. Notably, Lox was significantly changed in both isolated fibroblasts treated
with TGF-
β
from experiment MI mice and human ISCM. Isolated cardiac fibroblasts from
MI mice are more susceptible to developing pathogenic traits following TGF-
β
treatment
than isolated fibroblasts from normal heart tissue. ECM proteins associated with these
enhanced fibroblast activities and functions are evident. These altered proteins may play a
functional role in MI-associated cardiac dysfunction.
Keywords: myocardial infarction; ischaemic heart disease; proteomics; TGF-
β
; cardiac
remodelling; extracellular matrix
1. Introduction
Myocardial infarction (MI) typically results from acute plaque rupture within the
coronary arteries that supply oxygenated blood to the cardiac tissue and is a major cause of
Int. J. Mol. Sci. 2025,26, 3846 https://doi.org/10.3390/ijms26083846

Int. J. Mol. Sci. 2025,26, 3846 2 of 16
morbidity and mortality worldwide [
1
]. Acute MI has a high mortality rate and is difficult
to diagnose and treat due to variable symptoms and sudden and unpredictable onset [
2
].
Although cardiac troponin is considered a useful clinical marker for myocardial injury,
it takes time for cardiac troponin to be released into the bloodstream, which can lead to
delayed diagnosis. Moreover, cardiac troponin can be elevated due to complications other
than MI [
2
,
3
]. There is a need for additional biomarkers to help us better understand
MI and the subsequent healing response, as well as a need for more effective therapeutic
options to reverse the damage caused by MI. The majority of FDA-approved drugs for
various diseases, including cardiovascular diseases, usually target human proteins [
4
].
Therefore, characterising pathophysiological changes to the cardiac proteome could reveal
novel protein targets for therapeutic intervention.
The loss of perfusion to the affected area of cardiac tissue during MI causes cardiomy-
ocyte necrosis, which cannot be compensated for due to the poor regenerative capacity of
cardiomyocytes. Instead, tissue repair requires mobilisation and activation of fibroblasts
to the injured site, which leads to deposition of fibrotic tissue to help maintain structural
integrity; however, overproduction can result in cardiac dysfunction and heart failure
(HF) [
1
,
5
–
7
]. Hence, cardiac fibroblasts are seen to play a prominent role in the evolution of
ischaemic heart disease (IHD) to more advanced stages of HF [
8
,
9
]. Clinically, it would be
beneficial to identify patients with perturbations in cardiac fibroblast activity and progres-
sive fibrotic remodelling, as these patients could benefit from an intervention that targets
fibroblast cells [1,10].
This study aimed to investigate the effect of MI on cardiac fibroblasts by comprehen-
sively characterising resulting changes in the proteome of these cells from the different
time points post-infarct induction (inflammatory phase, proliferative phase, and remod-
elling/maturation phase) along with examining the changes in response to pro-fibrotic
activation by TGF-
β
. It is anticipated that these data could highlight potential therapeu-
tic targets for better management of MI-induced complications and also reveal protein
biomarkers of HF risk post-MI.
2. Results
2.1. Proteomic Characterisation of Myocardial Infarction Reveals Unique Proteomic Profile of
Chronic Injury
Female C57BL6 mice underwent experimental MI by permanent left coronary artery
ligation, which resulted in a reduction in left ventricular ejection fraction (Figure 1A).
Mice were sacrificed after 3 days, 1 week and 1 month post-surgery (Figure 1A) to re-
flect the phases of cardiac wound healing post-infarct, including the inflammatory phase,
proliferative phase and the remodelling/maturation phase [
6
]. Heart tissue from MI and
sham experimental mice was used to generate primary fibroblasts. Protein was subse-
quently extracted from primary fibroblast cells and processed for high throughput liquid
chromatography–mass spectrometry, resulting in over 5000 proteins identified across all
samples. Principal component analysis revealed some overlap in MI cardiac fibroblast
proteome profiles at ‘Day 3’ and ‘Week 1’ post-MI, suggesting an ‘acute’ MI phenotype.
Notably, a distinct proteome profile was observed in MI cardiac fibroblasts at 1 month
post-MI (Figure 1B). The later time point was considered to be reflective of a more ‘chronic’
MI phenotype. Proteins that are significantly differentially expressed in response to experi-
mental MI cardiac fibroblasts were identified at 3 days (423 proteins), 1 week (239 proteins),
and 1 month (125 proteins), compared to sham cardiac fibroblasts. Among them, two
differentially expressed proteins were identified between MI and sham cardiac fibroblasts
at all three time points (Figure 1C). Both proteins—Neuroplastin and Trans-acting tran-
scription factor 1—were significantly elevated in MI cardiac fibroblasts at all time points

Int. J. Mol. Sci. 2025,26, 3846 3 of 16
(Figure 1D,E). Fifty-nine proteins related to the extracellular matrix (ECM) were measured
in the mass spectrometry dataset. Maladaptive ECM proteins were most evident at 1 month
post-MI (Figure 1F,G), which indicates that proteome changes at 1 month are related to the
established pathological cardiac remodelling that occurs post-MI.
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 3 of 16
differentially expressed proteins were identified between MI and sham cardiac fibroblasts
at all three time points (Figure 1C). Both proteins—Neuroplastin and Trans-acting tran-
scription factor 1—were significantly elevated in MI cardiac fibroblasts at all time points
(Figure 1D,E). Fifty-nine proteins related to the extracellular matrix (ECM) were measured
in the mass spectrometry dataset. Maladaptive ECM proteins were most evident at 1
month post-MI (Figure 1F,G), which indicates that proteome changes at 1 month are re-
lated to the established pathological cardiac remodelling that occurs post-MI.
Figure 1. Proteomic characterisation of myocardial infarction experimental mice. Female C57BL6
mice underwent experimental myocardial infarction (MI) by permanent left coronary artery liga-
tion. Mice were sacrificed on day 3, 1 week and 1 month post-surgery. (A) Heart tissue from n = 3
(day 3), n = 6 (week 1 and 1 month) mice were used to generate primary fibroblasts, which were
treated with the pro-fibrotic cytokine TGF-b or DMSO (control) for 24 h before mass spectrometry
analysis using dia-PASEF acquisition. (B) Principal component analysis of resulting mass spectrom-
etry data reveals a unique proteomic profile in untreated (control) fibroblasts from MI mice at 1
month. (C) Welch’s t-test is applied to identify significant protein expression changes between MI
and sham cardiac fibroblast cells on day 3, 1 week, and 1 month. (D,E) Two proteins (Neuroplastin
and Trans-transcription factor1) are significantly elevated in MI mice at all time points. (F,G) In
week 1 and 1 month, the average fold changes of extracellular matrix proteins (n = 59) are trending
higher in MI cardiac fibroblasts. Bar charts show the mean ± standard error of the mean (SEM).
Coloured dot plots (F) represent individual samples, and black dots (G) represent individual extra-
cellular matrix (ECM) proteins. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, non-significant.
Figure 1. Proteomic characterisation of myocardial infarction experimental mice. Female C57BL6
mice underwent experimental myocardial infarction (MI) by permanent left coronary artery ligation.
Mice were sacrificed on day 3, 1 week and 1 month post-surgery. (A) Heart tissue from n = 3 (day 3),
n=6
(week 1 and 1 month) mice were used to generate primary fibroblasts, which were treated with
the pro-fibrotic cytokine TGF-b or DMSO (control) for 24 h before mass spectrometry analysis using
dia-PASEF acquisition. (B) Principal component analysis of resulting mass spectrometry data reveals a
unique proteomic profile in untreated (control) fibroblasts from MI mice at 1 month. (C) Welch’s t-test
is applied to identify significant protein expression changes between MI and sham cardiac fibroblast
cells on day 3, 1 week, and 1 month. (D,E) Two proteins (Neuroplastin and Trans-transcription factor1)
are significantly elevated in MI mice at all time points. (F,G) In week 1 and 1 month, the average
fold changes of extracellular matrix proteins (n = 59) are trending higher in MI cardiac fibroblasts.
Bar charts show the mean
±
standard error of the mean (SEM). Coloured dot plots (F) represent
individual samples, and black dots (G) represent individual extracellular matrix (ECM) proteins.
*p< 0.05; ** p≤0.01; *** p≤0.001; ns, non-significant.
2.2. Pathway Dysregulation in Chronic MI Model
To understand the signature proteome of chronic MI phenotype, we revealed proteins
that were significantly differentially expressed between MI and sham cardiac fibroblasts at
1 month post-MI but not at day 3 and week 1 post-MI (Figure 2A,B). These significantly
expressed proteins at 1 month post-MI vs. 1 month sham cardiac fibroblasts were signif-
icantly associated with a biological process related to cell motility and migration, RNA

Int. J. Mol. Sci. 2025,26, 3846 4 of 16
metabolism and processing (Figure 2A,B). Interestingly, while the top ten enriched KEGG
pathways were most significantly associated with 1 month post-MI marked by fold enrich-
ment, the same pattern of fold enrichment was observed for all 10 pathways at day 3 and
week 1 time points. The majority of the top ten enriched KEGG pathways associated with
proteins de-regulated at 1 month post-MI are also enriched by proteins that are significantly
differentially expressed at Day 3. These pathways and associated proteins may, therefore,
have a fundamental role in the onset and sustained pathology of MI-associated cardiac
dysfunction (Figure 2C).
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 4 of 16
2.2. Pathway Dysregulation in Chronic MI Model
To understand the signature proteome of chronic MI phenotype, we revealed pro-
teins that were significantly differentially expressed between MI and sham cardiac fibro-
blasts at 1 month post-MI but not at day 3 and week 1 post-MI (Figure 2A,B). These sig-
nificantly expressed proteins at 1 month post-MI vs. 1 month sham cardiac fibroblasts
were significantly associated with a biological process related to cell motility and migra-
tion, RNA metabolism and processing (Figure 2A,B). Interestingly, while the top ten en-
riched KEGG pathways were most significantly associated with 1 month post-MI marked
by fold enrichment, the same paern of fold enrichment was observed for all 10 pathways
at day 3 and week 1 time points. The majority of the top ten enriched KEGG pathways
associated with proteins de-regulated at 1 month post-MI are also enriched by proteins
that are significantly differentially expressed at Day 3. These pathways and associated
proteins may, therefore, have a fundamental role in the onset and sustained pathology of
MI-associated cardiac dysfunction (Figure 2C).
Figure 2. Pathway dysregulation in chronic myocardial infarction experimental model. (A,B) Gene
Ontology analysis reveals biological processes (BP), cellular compartments (CC) and molecular
functions (MF) that are more de-regulated by significantly down and upregulated genes 1 month
post-myocardial infarction. (C) The top most enriched Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways (mouse) associated with protein changes at 1 month post-MI are also de-regu-
lated at day 3 post-MI. ‡ = common up and downregulated genes in D3 and 1m datasets; ¥ = common
up and downregulated genes at all time points.
Figure 2. Pathway dysregulation in chronic myocardial infarction experimental model. (A,B) Gene
Ontology analysis reveals biological processes (BP), cellular compartments (CC) and molecular
functions (MF) that are more de-regulated by significantly down and upregulated genes 1 month
post-myocardial infarction. (C) The top most enriched Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways (mouse) associated with protein changes at 1 month post-MI are also de-regulated
at day 3 post-MI.
‡
= common up and downregulated genes in D3 and 1m datasets; ¥ = common up
and downregulated genes at all time points.
2.3. Effect of TGF-βTreatment on MI Cardiac Fibroblasts
Comparison of protein expression changes in MI cardiac fibroblasts with and without
TGF-βtreatment, a pro-fibrotic cytokine at day 3, week 1 and month 1 post-MI revealed a
large number of differentially expressed proteins at each time point (Figure 3A). The protein
expression of serine/threonine protein kinase, doublecortin-like kinase 1 (Dclk1) and
anthrax toxin receptor 1 (Antxr1) significantly decreased in response to TGF-
β
treatment of
MI cardiac fibroblasts at all time points (Figure 3B,C). Furthermore, prolyl endopeptidase-
like protein (Prepl) protein expression increased in MI cardiac fibroblasts, compared with
sham cardiac fibroblasts in response to TGF-
β
treatment at week 1 and month 1, and this
negatively correlated with left ventricular mass (Figure 3D). Altogether, this underlines

Int. J. Mol. Sci. 2025,26, 3846 5 of 16
an increased sensitivity to pro-fibrotic stimulation by TGF-
β
in cardiac fibroblasts isolated
from MI experimental hearts. Then, we specifically studied proteins related to crosslinked
collagen fibres, namely protein-lysine 6-oxidase (Lox) and Lysyl oxidase homolog 3 (Loxl3).
Both Lox and Loxl3 were significantly upregulated in MI cardiac fibroblasts in response to
TGF-
β
treatment (Figure 4A,B). These proteins are significantly associated with a number of
other collagen proteins, indicating their potential role in the pathogenesis of cardiac fibrosis
(Figure 4C). To confirm the pathogenic association with cardiac fibrosis, we found that
expression of Lox was significantly elevated in patients with ISCM compared to patients
without clinical HF (Figure 4D,E). Expression of Loxl3 was elevated in ISCM cardiac tissue;
however, this increase was not determined as statistically significant.
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 16
2.3. Effect of TGF-β Treatment on MI Cardiac Fibroblasts
Comparison of protein expression changes in MI cardiac fibroblasts with and without
TGF-β treatment, a pro-fibrotic cytokine at day 3, week 1 and month 1 post-MI revealed a
large number of differentially expressed proteins at each time point (Figure 3A). The pro-
tein expression of serine/threonine protein kinase, doublecortin-like kinase 1 (Dclk1) and
anthrax toxin receptor 1 (Antxr1) significantly decreased in response to TGF-β treatment
of MI cardiac fibroblasts at all time points (Figure 3B,C). Furthermore, prolyl endopepti-
dase-like protein (Prepl) protein expression increased in MI cardiac fibroblasts, compared
with sham cardiac fibroblasts in response to TGF-β treatment at week 1 and month 1, and
this negatively correlated with left ventricular mass (Figure 3D). Altogether, this under-
lines an increased sensitivity to pro-fibrotic stimulation by TGF-β in cardiac fibroblasts
isolated from MI experimental hearts. Then, we specifically studied proteins related to
crosslinked collagen fibres, namely protein-lysine 6-oxidase (Lox) and Lysyl oxidase hom-
olog 3 (Loxl3). Both Lox and Loxl3 were significantly upregulated in MI cardiac fibroblasts
in response to TGF-β treatment (Figure 4A,B). These proteins are significantly associated
with a number of other collagen proteins, indicating their potential role in the pathogen-
esis of cardiac fibrosis (Figure 4C). To confirm the pathogenic association with cardiac
fibrosis, we found that expression of Lox was significantly elevated in patients with ISCM
compared to patients without clinical HF (Figure 4D,E). Expression of Loxl3 was elevated
in ISCM cardiac tissue; however, this increase was not determined as statistically signifi-
cant.
Figure 3. Effect of TGF-β treatment on MI cardiac fibroblasts. (A) Welch’s t-test is applied to identify
significant protein changes as a result of TGF-β treatment of MI cardiac fibroblasts at day 3, 1 week
Figure 3. Effect of TGF-
β
treatment on MI cardiac fibroblasts. (A) Welch’s t-test is applied to identify
significant protein changes as a result of TGF-
β
treatment of MI cardiac fibroblasts at day 3, 1 week
and 1 month time points. (B–D) Three of the commonly up- and downregulated proteins (Dclk1,
Antrx1 and Prepl) at all time points in response to MI. Bar charts show the mean
±
standard error of
the mean (SEM). * p< 0.05; ns, non-significant.

Int. J. Mol. Sci. 2025,26, 3846 6 of 16
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 16
and 1 month time points. (B–D) Three of the commonly up- and downregulated proteins (Dclk1,
Antrx1 and Prepl) at all time points in response to MI. Bar charts show the mean ± standard error of
the mean (SEM). * p < 0.05; ns, non-significant.
Figure 4. MI sensitises cardiac fibroblasts to pro-fibrotic stimulation by TGF-β. (A,B) The extracel-
lular matrix proteins Lox and Loxl3 are significantly elevated in MI cardiac fibroblasts after TGF-β
treatment. (C) Lox and Loxl3 are significantly associated with a number of collagen proteins (red =
negative correlation; green = positive correlation). (D) Lox and (E) Loxl3 are also elevated in human
ISCM, compared to patients without clinical heart failure. Bar plots represent the mean ± standard
error of the mean (SEM). * p < 0.05; ** p ≤ 0.01; ns, non-significant.
2.4. In Silico Validation of Proteomic Changes in Cardiac Fibroblasts
Mass spectrometry data generated previously by Shah et al. [9] was used to validate
signature proteomic changes observed in this study. With a similar approach to assessing
cardiac fibroblasts isolated from MI heart mice, Shah et al. [9] performed mass spectrom-
etry analysis of cardiac fibroblasts generated from the hearts of mice subject to experi-
mental MI and sacrificed after 7 days. Thirty-eight percent of proteins identified in our
dataset were also identified in Shah et al.’s dataset [9] (Figure 5A). The protein Neuroplas-
tin, which was significantly elevated in MI cardiac fibroblasts at all time points in our
study, was also significantly associated with MI in the Shah et al.’s dataset [9] (Figure
5B,C). Furthermore, Lox was found to be substantially increased in cardiac fibroblasts
generated from the site of MI infarct, compared to remote regions (Figure 5D,E). This un-
derlines further evidence of a potential fundamental role of Lox in driving the pro-fibrotic
response of MI-injured cardiac fibroblasts. Protein collagen, type VI, alpha 1 (Col6a1) was
significantly downregulated in MI cardiac fibroblasts in response to TGF-β treatment; this
is also significantly downregulated in response to MI in the Shah et al.’s dataset [9] (Figure
5F,G).
Figure 4. MI sensitises cardiac fibroblasts to pro-fibrotic stimulation by TGF-
β
. (A,B) The extra-
cellular matrix proteins Lox and Loxl3 are significantly elevated in MI cardiac fibroblasts after
TGF-
β
treatment. (C) Lox and Loxl3 are significantly associated with a number of collagen pro-
teins (
red = negative
correlation; green = positive correlation). (D) Lox and (E) Loxl3 are also ele-
vated in human ISCM, compared to patients without clinical heart failure. Bar plots represent the
mean ±standard error of the mean (SEM). * p< 0.05; ** p≤0.01; ns, non-significant.
2.4. In Silico Validation of Proteomic Changes in Cardiac Fibroblasts
Mass spectrometry data generated previously by Shah et al. [
9
] was used to validate
signature proteomic changes observed in this study. With a similar approach to assessing
cardiac fibroblasts isolated from MI heart mice, Shah et al. [
9
] performed mass spectrometry
analysis of cardiac fibroblasts generated from the hearts of mice subject to experimental
MI and sacrificed after 7 days. Thirty-eight percent of proteins identified in our dataset
were also identified in Shah et al.’s dataset [
9
] (Figure 5A). The protein Neuroplastin, which
was significantly elevated in MI cardiac fibroblasts at all time points in our study, was also
significantly associated with MI in the Shah et al.’s dataset [
9
] (Figure 5B,C). Furthermore,
Lox was found to be substantially increased in cardiac fibroblasts generated from the
site of MI infarct, compared to remote regions (Figure 5D,E). This underlines further
evidence of a potential fundamental role of Lox in driving the pro-fibrotic response of
MI-injured cardiac fibroblasts. Protein collagen, type VI, alpha 1 (Col6a1) was significantly
downregulated in MI cardiac fibroblasts in response to TGF-
β
treatment; this is also
significantly downregulated in response to MI in the Shah et al.’s dataset [
9
] (Figure 5F,G).

Int. J. Mol. Sci. 2025,26, 3846 7 of 16
Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 16
Figure 5. In silico validation of proteomic changes in cardiac fibroblasts. (A) Publicly available mass
spectrometry data were accessed through ProteomeXchange for validation of observed protein expres-
sion changes (Shah et al. [
9
]). (B–G) Neuroplastin (Nptn), Lox and collagen type Vi alpha 1 (Col6A1)
were verified as being significantly associated with MI. Bar plots represent the
mean ±standard
error
of the mean (SEM). * p< 0.05; ** p≤0.01; **** p≤0.0001; ns, non-significant.

Int. J. Mol. Sci. 2025,26, 3846 8 of 16
3. Discussion
Over the last decade, HF prevalence has significantly increased. Although prompt
urgent revascularisation and effective treatment strategies have greatly reduced acute MI
mortality, IHD continues to be a leading cause of HF [
11
,
12
]. After a pathogenic myocardial
injury, the heart undergoes a complex process of structural and functional remodelling
through several processes, including inflammatory and fibrotic responses [
13
]. While
cardiac troponin is the most commonly used cardiac enzyme for MI diagnosis, it does
not peak until 18 to 24 h after symptom onset and can be detected in the blood for up
to 14 days [
14
]. However, detecting biomarkers related to myocardial damage at a more
extended time point might be challenging. Therefore, this is an opportunity to provide
additional markers that have been sought in order to provide better risk stratification in
both acute and chronic phases.
A previous study showed increasing numbers of differentially expressed proteins
responding to experimental MI at sequential time points (10 min, 1 h, 6 h, 24 h, and 72 h) [
2
].
Therefore, we provide important data on pathogenic remodelling within cardiac fibroblasts
beyond the early inflammatory and proliferative phases until the remodelling/maturation
phase. In our study, a distinct proteome signature between the ‘acute’ MI phase (3 days and
1 week) and the ‘chronic’ MI phase (1 month) is evident. Moreover, the current study reveals
that maladaptive ECM protein expression was most pronounced 1 month after the MI event.
These results support the concept of ‘transition from infarction to remodelling’ [
13
,
15
]. After
acute MI, pressure and volume overload increase wall stress, impairing left ventricular
function, and immune cell infiltration triggers scar formation and cardiomyocyte loss. In
the chronic phase, persistent inflammation and other factors drive extracellular matrix
expansion and ongoing remodelling [
13
,
16
]. Interestingly, we found neuroplastin and
trans-acting transcription factor 1 were increased in MI cardiac fibroblast at all timeframes.
Neuroplastin is upregulated under ER stress and can induce inflammation via NF-kB
activation [
17
], while trans-acting transcription factor 1 (also known as Sp1) is associated
with MI and cardiac hypertrophy [18,19].
It is becoming increasingly understood that activation of fibroblasts in the context
of cardiac injury leads to the establishment of a persistent pro-fibrotic cellular phenotype
enhancing cardiac fibrosis [
20
,
21
]. To understand this further, we studied isolated cardiac
fibroblasts from both MI and sham environments and their response to pro-fibrotic cy-
tokine, TGF-
β
. The serine/threonine protein kinases are linked to multiple cardiovascular
diseases such as ischemia-reperfusion injury, HF, and MI [
22
]. However, the specific role
of doublecortin-like kinase 1 (Dclk1) in cardiovascular diseases is limited. It has been
reported that specific deletion of Dclk1in macrophages has demonstrated a reduction in
cardiac hypertrophy, myocardial fibrosis and atherosclerotic plaques [
23
,
24
]. An additional
study has provided evidence that reducing Dclk1 in the context of diabetic cardiomyopathy
through genetic knockout or inhibitors can reduce cardiac fibrosis [
25
]. Translating these
studies to our findings would suggest that decreased Dclk1 in response to TGF-
β
treatment
in isolated MI fibroblasts could be a mechanism to balance and modulate a pro-fibrotic
response. Further functional studies of Dclk1 in MI are required to elucidate its true role in
this context.
Anthrax toxin receptor 1 deficiency promotes fibroblast senescence and links to the
ECM and cell-matrix adhesion process [
26
,
27
]. Moreover, prolyl endopeptidase-like protein
was increased in our isolated fibroblasts treated with TGF-
β
from MI mice, and this protein
primarily involves mitochondrial function, and deficiency in prolyl endopeptidase-like
protein can lead to mitochondrial dysfunction, which may impact cardiac health and com-
plications [
28
,
29
]. However, our isolated cardiac fibroblast treated with TGF-
β
increased
in prolyl endopeptidase-like protein, suggesting multifunctional roles in pathogenic MI.

Int. J. Mol. Sci. 2025,26, 3846 9 of 16
We notably found that the protein levels of Lox and Loxl3 were elevated at week 1 and
1 month in isolated cardiac fibroblasts treated with TGF-
β
from MI mice, and these pro-
teins centrally involve crosslinking and stabilising collagen and elastin fibres, including
cardiac remodelling [
30
,
31
]. Increased expression of Lox was subsequently validated in
both clinical samples from patients with ISCM and in cardiac fibroblasts from the site of
MI infarct [
9
]. Changes in Lox and Loxl3 are associated with different collagen protein
expressions. Therefore, these highlight the important roles of Lox and Loxl3 in pathogenic
cardiac remodelling. Collagen cross-linking occurs through two distinct pathways: an
enzymatic process facilitated by enzymes from the transglutaminase or LOX families and a
nonenzymatic (promoted by advanced glycation end-products; AGEs) process [
32
,
33
]. Evi-
dence from human and animal studies suggests that dysregulated LOX/LOXL isoenzyme
function or expression has been linked to cardiovascular diseases [
32
,
34
]. In a prospective
study, elevated circulating soluble LOX-1 is associated with the risk for first-time MI [
35
].
Furthermore, the upregulation of LOX isoforms (LOX and LOXL1–4), accompanied by a
significant accumulation of mature collagen fibres within the infarcted region, was con-
firmed in experimental MI mice [
36
]. Taken together, these results, along with our findings,
highlight potential roles for LOX and LOXL modulation as biomarkers related to cardiac
remodelling following MI and as potential therapeutic targets for post-MI recovery. Ad-
ditionally, we found that Col6a1 was significantly decreased in our dataset (isolated MI
cardiac fibroblasts stimulated by TGF-
β
vs. isolated sham cardiac fibroblasts stimulated
by TGF-
β
) and in the external dataset from Shah et al. [
9
]. Col6 is a nonfibrillar collagen
highly expressed in developing and adult hearts [37]. Previous research has reported that
following infarction, Col6 was elevated
in vivo
and induced myofibroblast differentia-
tion [
38
,
39
]. Col6a deficiency can be protective by limiting the extent of fibrosis and scar
formation, potentially improving cardiac function after the injury [
40
]. Therefore, further
experimental studies are necessary to elucidate the specific roles of Col6a1 and to provide
mechanistic insights that distinguish Col6a1 from other collagen subtypes. From a clinical
perspective, noninvasive techniques such as speckle tracking echocardiography (STE) may
help to identify myocardial areas with reduced deformation due to myocardial fibrosis.
Lower myocardial deformation in cardiac chambers is associated with a greater extent of
myocardial fibrosis within the heart walls. STE analysis offers clinicians a noninvasive
method to detect areas of myocardial fibrosis in both coronary artery disease (CAD) [
41
]
and non-CAD [
42
] contexts. The inclusion of STE analysis in translational studies would
enhance the phenotyping of cardiac remodelling.
Cardiac fibroblasts undergo temporal phenotypic changes throughout the wound
healing response, including post-MI. Cardiac fibroblast activation and how they respond to
external signals may vary in the context of MI, as indicated by the differential response of
MI-derived fibroblasts to TGF
β
in our study. Cardiac fibroblasts exhibit different cellular
phenotypes and physiological roles at varying times post-MI [
43
–
45
]. These changes
include the inflammatory phase with activation of various cytokines (e.g., IL-1
β
, IL6)
and chemokines (e.g., CXCL-8, CCL-2) [
46
–
48
], the proliferative phase and maturation
phase, which are important processes to induce transdifferentiation of fibroblasts into
myofibroblasts and generation of ECM proteins through anti-inflammatory cytokines (e.g.,
IL-10) and pro-fibrotic factors (e.g., TGF-
β
1) [
49
–
51
]. Ultimately, our findings support the
understanding of the post-MI fibroblast response and may reveal insights into cardiac
remodelling and identify novel targets to improve treatments.
Altogether, our study with cross-sectional follow-up following an MI event provides
protein signatures related to myocardium damage and underlines the unique response of
cardiac fibroblasts to pro-fibrotic cytokines. The differential proteomic response to pro-
fibrotic stimuli in those cells previously exposed to a post-MI microenvironment poses

Int. J. Mol. Sci. 2025,26, 3846 10 of 16
potential therapeutic targets to alleviate aberrant cardiac remodelling and HF development.
Our study contains limitations. The murine model of MI was conducted using female mice,
and the human dataset within our research uses only males. It is important to note that
sex differences, which are well known in the context of myocardial remodelling, such as
hormone regulation and immune/inflammatory regulation [
52
], could potentially influence
the translation of research findings into clinical studies. An example of this has been
demonstrated where estrogen supplementation in non-castrated male mice led to improved
cardiac function and attenuated cardiac remodelling after MI [
53
]. To overcome this hurdle,
we validated our results using the dataset from Shah et al. [
9
], which was generated using
both male and female mice. This suggests that changes in the candidate proteins identified
in this study, which are associated with myocardial injury, occur independently of sex.
While our study highlights the potential role of Lox and Loxl3 in cardiac fibroblasts and
cardiac remodelling post-MI, we did not investigate the experimental impact of these
candidate proteins in this study. Modulation of these extracellular proteins using genetic or
pharmacological approaches, specifically in fibroblasts, may help elucidate their precise
roles and impact in ischaemic cardiomyopathy.
4. Materials and Methods
4.1. Human Data and Analysis
Left ventricular (LV) tissue samples were collected from male patients who underwent
orthotropic cardiac transplantation for ischaemic cardiomyopathy (ISCM) (n = 9). Matched
control patients were non-failing hearts (n = 9) who died of noncardiac causes. The
Institutional Review Board at the Cleveland Clinic provided ethical permission for the use
of tissue and data collecting. Descriptions of patient demographics and clinical features
were previously reported [
54
]. Proteomic sample preparation and analysis of LV tissue
samples were previously described [
55
]. Briefly, using a timsTOF Pro (Bruker, Billerica, MA,
USA) quadrupole time-of-flight mass spectrometer, which integrates trapped ion mobility
separations coupled online via a Captivespray electrospray source (Bruker) to a nanoElute
(Bruker) nanoflow liquid chromatography system, mass spectrometry (MS) proteomics
data were obtained. Then, Spectronaut software version 18 was utilised to generate the
spectral library, and an independent Student’s t-test was applied to obtain differences
between groups with FDR correction. All pvalues < 0.05 were significant changes [55].
4.2. In Vivo Model of Experimental MI
Female C57Bl/6 mice (8–10 week old) were obtained from Charles River UK (Harlow,
UK) and underwent experimental MI by permanent left coronary artery ligation, as de-
scribed previously [
56
]. Briefly, a 7-0 suture was passed under the left anterior descending
(LAD) coronary artery and permanently tied using a single interrupted suture. Sham
mice underwent the full surgical procedure, except the 7-0 suture was not tied off after
passing under the LAD. Mice were randomly selected to undergo sham or MI surgery. After
successful surgical induction of MI, mice were allocated to their designated time points
and sacrificed at 3 days, 1 week and 1 month post-surgery. Surgical-matched sham mice
from the same day were used as the control groups at each time point. This experimental
MI model was previously well-established for MI characteristics in a previous study [
57
].
All animal work was conducted under guidelines established by Directive 2010/63/EU
of the European Parliament on the protection of animals used for scientific purposes and
UK Home Office regulations. All experimental protocols were approved by the Biological
Services Unit at Queen’s University Belfast.

Int. J. Mol. Sci. 2025,26, 3846 11 of 16
4.3. Echocardiography Data Acquisition and Data Analysis
After 3 days, 7 days and 28 days post-op, mice were anaesthetised with 1.5% isoflu-
rane in oxygen and imaged in the supine position using a Vevo 770 ultrasound system
with high-frequency 45 MHz RMV707B scanhead (VisualSonics, Toronto, ON, Canada).
Core temperature was maintained at 37
◦
C, and heart rates were kept consistent between
experimental groups (400–500 BPM). Electrocardiogram (ECG) was derived using Limb
electrodes. Standard 2D echocardiographic images were obtained from the parasternal
long-axis view for assessment of left ventricular dimensions and function.
4.4. Cardiac Fibroblast Isolation and Culture
After cardiac imaging, animals were sacrificed, and cardiac fibroblasts were isolated
from the entire left ventricle as previously described [
58
]. Briefly, excised cardiac tissue
was rinsed, minced and then digested by collagenase II (600 U/mL, 17101015; Gibco™,
Waltham, MA, USA) and DNase I solution (60 U/mL, 18047019; Invitrogen™, Waltham,
MA, USA) in Hanks-buffered saline solution (14025092; Gibco™, Waltham, MA, USA).
After 1 h incubation at 37
◦
C, with mechanical dissociation applied every 15 min, the
entire cell suspension was filtered through a 30
µ
m cell strainer, centrifuged, and resus-
pended in Dulbecco’s Modified Eagle Medium (DMEM; 11965092; Gibco™, Waltham, MA,
USA) supplemented with 10% fetal bovine serum (FBS; Gibco™, Waltham, MA, USA)
and 1
×
antibiotic–antimycotic solution (15240-062; Gibco™, Waltham, MA, USA). Cells
were subsequently transferred to T25 flasks and incubated under standard cell culture
conditions (37
◦
C; 5% CO
2
). The cells from P1 were used for the experiments. To assess
pathogenic response within isolated cardiac fibroblasts from MI and sham mice, isolated
cardiac fibroblasts in serum-free media were treated with TGF-
β
(5
µ
g/mL (R&D Systems,
Minneapolis, MN, USA, 7754-BH-005/C)) for 24 h. Cell pellets were collected and stored at
80 ◦C until further analysis.
4.5. Sample Preparation for Mass Spectrometry Analysis
Primary cardiac fibroblast cells were lysed in Urea lysis buffer (8M Urea, 0.1M Tris-Cl
pH 8.0). Protein samples were quantified using the bicinchoninic acid (BCA) assay (23227;
Thermo Scientific™, Waltham, MA, USA). Twenty-five
µ
g of protein was denatured in a
final concentration of 10 mM 1,4-dithiothreitol (DTT) (Roche, Mannheim, Germany) and
alkylated with 14 mM iodoacetamide (IAA) (I1149, Sigma-Aldrich, Gillingham, UK). Protein
was digested with a 1:50 protein–enzyme ratio of Trypsin (V5111; Promega, Madison, WI,
USA). Peptides were dried down under a vacuum, resuspended in 1% trifluoroacetic acid
(TFA) (302031, Sigma-Aldrich, Gillingham, UK) and then de-salted on C18-packed stage-tip
columns. Briefly, C18 stage-tips were activated with 50
µ
L of 50% acetonitrile (AcN; 34851;
Sigma-Aldrich, Gillingham, UK)/0.1% TFA. The stage-tips were washed with 1% TFA
before adding ~8
µ
g of peptide in 1% TFA. After a further two wash steps with 1% TFA,
the peptide was eluted from the stage-tip in 25
µ
L of 50% AcN/0.1% TFA. All digested
samples were pooled based on experimental groups and fractionated using the Pierce™
High-pH Reversed-Phase Peptide Fractionation Kit (84868; Thermo Scientific™, Waltham,
MA, USA), as per the manufacturer’s instructions. Prior to mass spectrometry analysis,
peptide samples were dried down under a vacuum, re-constituted in 0.1% formic acid
(F0507; Sigma-Aldrich, Gillingham, UK) and loaded onto EvoTips™ (Odense, Denmark),
as per manufacturer’s instructions.
4.6. Mass Spectrometry Analysis
All samples were analysed as part of one experimental run on a timsTof Pro mass
spectrometer (Bruker Daltonics, Billerica, MA, USA) connected to an Evosep One liquid

Int. J. Mol. Sci. 2025,26, 3846 12 of 16
chromatography system (EvoSep BioSystems, Odense, Denmark). A reversed-phase C18
Endurance column using the 30 SPD method was used for peptide separation. Data-
dependent acquisition mode (DDA) was used for the analysis of high pH-reversed phase
fractionated sample pools to generate data for the spectral library. Data independent
acquisition mode (DIA) was used to analyse individual samples. Trapped ion mobility
spectrometry (TIMS) mode was used for data acquisition. Parallel accumulation serial
fragmentation (PASEF) was used to select trapped ions for ms/ms. Regular intervals
throughout the pooled sample digests were analysed as quality control.
4.7. Data Analysis
Fragpipe (version 22.0) software (https://github.com/Nesvilab/FragPipe accessed
on 11 August 2024) was used to generate spectral library files from the acquired DDA data.
DIA-NN (version 1.9.2) software (https://github.com/vdemichev/DiaNN accessed on 14
August 2024) was used to process raw (.d) diaPASEF data files for a spectral library building,
protein identification and quantification. Data processing was performed using R (version
4.4.1) software. Raw data were log-transformed, and proteins with >70% missing values
across all samples were removed. Missing data for the remaining protein were imputed
by sampling values from a normal distribution. To compare means between two groups,
Welch’s t-test was employed. All tests were two-tailed with a predefined significance level
of p< 0.05. Pathway analysis was carried out using clusterProfiler version 4.12.6 [
59
] based
on ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. Statistical
analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA,
USA).
5. Conclusions
These current findings open avenues for drug repurposing and the design of novel
therapeutic strategies aimed at modulating key proteins involved in fibrosis progression
after myocardial injury. By identifying specific proteomic alterations associated with
maladaptive remodelling, our study also paves the way for the development of biomarker-
driven precision medicine approaches. These biomarkers may serve as indicators of post-MI
progression, predictors of therapeutic response, or even surrogate endpoints in clinical
studies.
Author Contributions: Conceptualisation, A.R.-H., C.W. and D.G.; methodology, A.R.-H., L.K. and
K.E.; validation, C.T. and N.K.; formal analysis, C.T. and N.K.; resources, P.C., K.M., M.L., D.G. and
C.W.; data curation, C.T.; writing—original draft preparation, C.T., N.K. and A.R.-H.; writing—review
and editing, C.T., N.K., A.R.-H. and C.W.; supervision, C.W.; project administration, C.W.; funding
acquisition, C.W. All authors have read and agreed to the published version of the manuscript.
Funding: Funding support was provided by the British Heart Foundation (PG/18/21/33599), Heart
Research UK (RG2662/17/19), and the European Society of Cardiology (ESC) First Contact Initiative
Grant (2019 award—generously hosted by the lab of Dr. Borja Ibanez at the Centro Nacional de
Investigaciones Cardiovasculares, Madrid).
Institutional Review Board Statement: The ethics committee at Queen’s University Belfast (QUB
AWERB) provided approval for the animal studies described here (Study code—ARH2021.06.05.21,
Approval Date—7 May 2021). All work was conducted under DoH licence PPL2821 and in accordance
with ASAP 1986 regulations. The study conformed to the principles outlined in the Declaration of
Helsinki, and patients gave informed consent. Ethical approval for human data collection and the
use of human tissue was obtained from the Cleveland Clinic Institutional Review Board (IRB2378; 1
February 2017).

Int. J. Mol. Sci. 2025,26, 3846 13 of 16
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript:
IHD ischaemic heart disease
HF heart failure
TGF-βTranscription Growth Factor Beta
LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry
ISCM ischaemic cardiomyopathy
NF Non-failure
MI myocardial infarction
ECM extracellular matrix
FDA Food and Drug Administration
LV left ventricular
MS mass spectrometry
LAD left anterior descending
UK United Kingdom
BPM Beats per minute
ECG Electrocardiogram
DMEM Dulbecco’s Modified Eagle Medium
FBS fetal bovine serum
BCA Bicinchoninic acid assay
DTT 1.4-dithiothreitol
IAA iodoacetamide
TFA trifluoroacetic acid
AcN acetonitrile
DDA Data dependent acquisition
DIA Data independent acquisition
TIMS trapped ion mobility spectrometry
PASEF Parallel accumulation serial fragmentation
KEGG Kyoto Encyclopedia of Genes and Genomes
RNA Ribonucleic acid
Dclk1 doublecortin-like kinase 1
Antxr1 anthrax toxin receptor 1
Prepl prolyl endopeptidase-like protein
Lox protein-lysine 6-oxidase
Loxl3 Lysyl oxidase homolog 3
Col6a1 Protein collagen, type VI, alpha 1
ER Endoplasmic reticulum
NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells
References
1.
Venugopal, H.; Hanna, A.; Humeres, C.; Frangogiannis, N.G. Properties and Functions of Fibroblasts and Myofibroblasts in
Myocardial Infarction. Cells 2022,11, 1386. [CrossRef] [PubMed]
2.
Wu, J.; Yan, J.; Hua, Z.; Jia, J.; Zhou, Z.; Zhang, J.; Li, J.; Zhang, J. Identification of molecular signatures in acute myocardial
infarction based on integrative analysis of proteomics and transcriptomics. Genomics 2023,115, 110701. [CrossRef] [PubMed]
3. Kanderian, A.S.; Francis, G.S. Cardiac troponins and chronic kidney disease. Kidney Int. 2006,69, 1112–1114. [CrossRef]

Int. J. Mol. Sci. 2025,26, 3846 14 of 16
4.
Sun, Z.; Yun, Z.; Lin, J.; Sun, X.; Wang, Q.; Duan, J.; Li, C.; Zhang, X.; Xu, S.; Wang, Z.; et al. Comprehensive mendelian
randomization analysis of plasma proteomics to identify new therapeutic targets for the treatment of coronary heart disease and
myocardial infarction. J. Transl. Med. 2024,22, 404. [CrossRef]
5.
Masarie, F.E., Jr.; Miller, R.A. Medical Subject Headings and medical terminology: An analysis of terminology used in hospital
charts. Bull. Med. Libr. Assoc. 1987,75, 89–94.
6.
Prabhu, S.D.; Frangogiannis, N.G. The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to
Fibrosis. Circ. Res. 2016,119, 91–112. [CrossRef]
7.
Tani, H.; Sadahiro, T.; Yamada, Y.; Isomi, M.; Yamakawa, H.; Fujita, R.; Abe, Y.; Akiyama, T.; Nakano, K.; Kuze, Y.; et al. Direct
Reprogramming Improves Cardiac Function and Reverses Fibrosis in Chronic Myocardial Infarction. Circulation 2023,147,
223–238. [CrossRef]
8.
Ko, T.; Nomura, S.; Yamada, S.; Fujita, K.; Fujita, T.; Satoh, M.; Oka, C.; Katoh, M.; Ito, M.; Katagiri, M.; et al. Cardiac fibroblasts
regulate the development of heart failure via Htra3-TGF-beta-IGFBP7 axis. Nat. Commun. 2022,13, 3275. [CrossRef]
9.
Shah, H.; Hacker, A.; Langburt, D.; Dewar, M.; McFadden, M.J.; Zhang, H.; Kuzmanov, U.; Zhou, Y.Q.; Hussain, B.; Ehsan, F.; et al.
Myocardial Infarction Induces Cardiac Fibroblast Transformation within Injured and Noninjured Regions of the Mouse Heart. J.
Proteome Res. 2021,20, 2867–2881. [CrossRef]
10.
Humeres, C.; Frangogiannis, N.G. Fibroblasts in the Infarcted, Remodeling, and Failing Heart. JACC Basic Transl. Sci. 2019,4,
449–467. [CrossRef]
11.
Conrad, N.; Judge, A.; Tran, J.; Mohseni, H.; Hedgecott, D.; Crespillo, A.P.; Allison, M.; Hemingway, H.; Cleland, J.G.; McMurray,
J.J.V.; et al. Temporal trends and patterns in heart failure incidence: A population-based study of 4 million individuals. Lancet
2018,391, 572–580. [CrossRef] [PubMed]
12.
Martin, S.S.; Aday, A.W.; Almarzooq, Z.I.; Anderson, C.A.M.; Arora, P.; Avery, C.L.; Baker-Smith, C.M.; Barone Gibbs, B.; Beaton,
A.Z.; Boehme, A.K.; et al. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart
Association. Circulation 2024,149, e347–e913. [PubMed]
13.
Frantz, S.; Hundertmark, M.J.; Schulz-Menger, J.; Bengel, F.M.; Bauersachs, J. Left ventricular remodelling post-myocardial
infarction: Pathophysiology, imaging, and novel therapies. Eur. Heart J. 2022,43, 2549–2561. [CrossRef]
14.
Madsen, L.H.; Christensen, G.; Lund, T.; Serebruany, V.L.; Granger, C.B.; Hoen, I.; Grieg, Z.; Alexander, J.H.; Jaffe, A.S.; Van
Eyk, J.E.; et al. Time course of degradation of cardiac troponin I in patients with acute ST-elevation myocardial infarction: The
ASSENT-2 troponin substudy. Circ. Res. 2006,99, 1141–1147. [CrossRef]
15.
Zhang, Q.; Wang, L.; Wang, S.; Cheng, H.; Xu, L.; Pei, G.; Wang, Y.; Fu, C.; Jiang, Y.; He, C.; et al. Signaling pathways and targeted
therapy for myocardial infarction. Signal Transduct. Target. Ther. 2022,7, 78. [CrossRef]
16.
Sun, Y. Myocardial repair/remodelling following infarction: Roles of local factors. Cardiovasc. Res. 2009,81, 482–490. [CrossRef]
17.
Yagi, T.; Asada, R.; Kanekura, K.; Eesmaa, A.; Lindahl, M.; Saarma, M.; Urano, F. Neuroplastin Modulates Anti-inflammatory
Effects of MANF. iScience 2020,23, 101810. [CrossRef]
18.
Xie, R.; Zhang, Y.; Zhang, J.; Li, J.; Zhou, X. The Role of Circular RNAs in Immune-Related Diseases. Front. Immunol. 2020,11, 545.
[CrossRef]
19.
Yu, S.; Sun, Z.; Ju, T.; Liu, Y.; Mei, Z.; Wang, C.; Qu, Z.; Li, N.; Wu, F.; Liu, K.; et al. The m7G Methyltransferase Mettl1 Drives
Cardiac Hypertrophy by Regulating SRSF9-Mediated Splicing of NFATc4. Adv. Sci. 2024,11, e2308769. [CrossRef]
20.
Travers, J.G.; Kamal, F.A.; Robbins, J.; Yutzey, K.E.; Blaxall, B.C. Cardiac Fibrosis: The Fibroblast Awakens. Circ. Res. 2016,118,
1021–1040. [CrossRef]
21.
Daseke, M.J., 2nd; Tenkorang, M.A.A.; Chalise, U.; Konfrst, S.R.; Lindsey, M.L. Cardiac fibroblast activation during myocardial
infarction wound healing: Fibroblast polarization after MI. Matrix Biol. 2020,91–92, 109–116. [CrossRef] [PubMed]
22.
Wu, Y.; Zou, Y.; Song, C.; Cao, K.; Cai, K.; Chen, S.; Zhang, Z.; Geng, D.; Zhang, N.; Feng, H.; et al. The role of serine/threonine
protein kinases in cardiovascular disease and potential therapeutic methods. Biomed. Pharmacother. 2024,177, 117093. [CrossRef]
[PubMed]
23.
Yang, B.; Zhao, Y.; Luo, W.; Zhu, W.; Jin, L.; Wang, M.; Ye, L.; Wang, Y.; Liang, G. Macrophage DCLK1 promotes obesity-induced
cardiomyopathy via activating RIP2/TAK1 signaling pathway. Cell Death Dis. 2023,14, 419. [CrossRef]
24.
Huang, Z.; Shen, S.; Han, X.; Li, W.; Luo, W.; Lin, L.; Xu, M.; Wang, Y.; Huang, W.; Wu, G.; et al. Macrophage DCLK1 promotes
atherosclerosis via binding to IKKbeta and inducing inflammatory responses. EMBO Mol. Med. 2023,15, e17198. [CrossRef]
25.
Ji, L.; Yang, X.; Jin, Y.; Li, L.; Yang, B.; Zhu, W.; Xu, M.; Wang, Y.; Wu, G.; Luo, W.; et al. Blockage of DCLK1 in cardiomyocytes
suppresses myocardial inflammation and alleviates diabetic cardiomyopathy in streptozotocin-induced diabetic mice. Biochim.
Biophys. Acta Mol. Basis Dis. 2024,1870, 166900. [CrossRef]
26.
Besschetnova, T.Y.; Ichimura, T.; Katebi, N.; St Croix, B.; Bonventre, J.V.; Olsen, B.R. Regulatory mechanisms of anthrax toxin
receptor 1-dependent vascular and connective tissue homeostasis. Matrix Biol. 2015,42, 56–73. [CrossRef]

Int. J. Mol. Sci. 2025,26, 3846 15 of 16
27.
Przyklenk, M.; Karmacharya, S.; Bonasera, D.; Pasanen-Zentz, A.L.; Kmoch, S.; Paulsson, M.; Wagener, R.; Liccardi, G.; Schiavinato,
A. ANTXR1 deficiency promotes fibroblast senescence: Implications for GAPO syndrome as a progeroid disorder. Sci. Rep. 2024,
14, 9321. [CrossRef]
28.
Ramachandra, C.J.A.; Hernandez-Resendiz, S.; Crespo-Avilan, G.E.; Lin, Y.H.; Hausenloy, D.J. Mitochondria in acute myocardial
infarction and cardioprotection. EBioMedicine 2020,57, 102884. [CrossRef]
29.
Rosier, K.; McDevitt, M.T.; Smet, J.; Floyd, B.J.; Verschoore, M.; Marcaida, M.J.; Bingman, C.A.; Lemmens, I.; Dal Peraro, M.;
Tavernier, J.; et al. Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function. iScience 2021,
24, 103460. [CrossRef]
30. Kagan, H.M.; Trackman, P.C. Properties and function of lysyl oxidase. Am. J. Respir. Cell Mol. Biol. 1991,5, 206–210. [CrossRef]
31.
Rodriguez, C.; Martinez-Gonzalez, J. The Role of Lysyl Oxidase Enzymes in Cardiac Function and Remodeling. Cells 2019,8,
1483. [CrossRef] [PubMed]
32. Lopez, B.; Gonzalez, A.; Hermida, N.; Valencia, F.; de Teresa, E.; Diez, J. Role of lysyl oxidase in myocardial fibrosis: From basic
science to clinical aspects. Am. J. Physiol. Heart Circ. Physiol. 2010,299, H1–H9. [CrossRef] [PubMed]
33.
Heymans, S.; Gonzalez, A.; Pizard, A.; Papageorgiou, A.P.; Lopez-Andres, N.; Jaisser, F.; Thum, T.; Zannad, F.; Diez, J. Searching
for new mechanisms of myocardial fibrosis with diagnostic and/or therapeutic potential. Eur. J. Heart Fail. 2015,17, 764–771.
[CrossRef] [PubMed]
34.
Rodriguez, C.; Martinez-Gonzalez, J.; Raposo, B.; Alcudia, J.F.; Guadall, A.; Badimon, L. Regulation of lysyl oxidase in vascular
cells: Lysyl oxidase as a new player in cardiovascular diseases. Cardiovasc. Res. 2008,79, 7–13. [CrossRef]
35.
Schiopu, A.; Bjorkbacka, H.; Narasimhan, G.; Loong, B.J.; Engstrom, G.; Melander, O.; Orho-Melander, M.; Nilsson, J. Elevated
soluble LOX-1 predicts risk of first-time myocardial infarction. Ann. Med. 2023,55, 2296552. [CrossRef]
36.
Gonzalez-Santamaria, J.; Villalba, M.; Busnadiego, O.; Lopez-Olaneta, M.M.; Sandoval, P.; Snabel, J.; Lopez-Cabrera, M.; Erler, J.T.;
Hanemaaijer, R.; Lara-Pezzi, E.; et al. Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and
promote cardiac dysfunction. Cardiovasc. Res. 2016,109, 67–78. [CrossRef]
37.
Crossman, D.J.; Shen, X.; Jullig, M.; Munro, M.; Hou, Y.; Middleditch, M.; Shrestha, D.; Li, A.; Lal, S.; Dos Remedios, C.G.; et al.
Increased collagen within the transverse tubules in human heart failure. Cardiovasc. Res. 2017,113, 879–891. [CrossRef]
38.
Bryant, J.E.; Shamhart, P.E.; Luther, D.J.; Olson, E.R.; Koshy, J.C.; Costic, D.J.; Mohile, M.V.; Dockry, M.; Doane, K.J.; Meszaros, J.G.
Cardiac myofibroblast differentiation is attenuated by
α3
integrin blockade: Potential role in post-MI remodeling. J. Mol. Cell.
Cardiol. 2009,46, 186–192. [CrossRef]
39.
Naugle, J.E.; Olson, E.R.; Zhang, X.; Mase, S.E.; Pilati, C.F.; Maron, M.B.; Folkesson, H.G.; Horne, W.I.; Doane, K.J.; Meszaros, J.G.
Type VI collagen induces cardiac myofibroblast differentiation: Implications for postinfarction remodeling. Am. J. Physiol. Heart
Circ. Physiol. 2006,290, H323–H330. [CrossRef]
40.
Luther, D.J.; Thodeti, C.K.; Shamhart, P.E.; Adapala, R.K.; Hodnichak, C.; Weihrauch, D.; Bonaldo, P.; Chilian, W.M.; Meszaros,
J.G. Absence of type VI collagen paradoxically improves cardiac function, structure, and remodeling after myocardial infarction.
Circ. Res. 2012,110, 851–856. [CrossRef]
41.
Grenne, B.; Eek, C.; Sjoli, B.; Dahlslett, T.; Uchto, M.; Hol, P.K.; Skulstad, H.; Smiseth, O.A.; Edvardsen, T.; Brunvand, H. Acute
coronary occlusion in non-ST-elevation acute coronary syndrome: Outcome and early identification by strain echocardiography.
Heart 2010,96, 1550–1556. [CrossRef] [PubMed]
42.
Sonaglioni, A.; Albini, A.; Fossile, E.; Pessi, M.A.; Nicolosi, G.L.; Lombardo, M.; Anza, C.; Ambrosio, G. Speckle-Tracking
Echocardiography for Cardioncological Evaluation in Bevacizumab-Treated Colorectal Cancer Patients. Cardiovasc. Toxicol. 2020,
20, 581–592. [CrossRef] [PubMed]
43.
Turner, N.A. Inflammatory and fibrotic responses of cardiac fibroblasts to myocardial damage associated molecular patterns
(DAMPs). J. Mol. Cell. Cardiol. 2016,94, 189–200. [CrossRef]
44.
Shinde, A.V.; Frangogiannis, N.G. Fibroblasts in myocardial infarction: A role in inflammation and repair. J. Mol. Cell. Cardiol.
2014,70, 74–82. [CrossRef]
45.
van Nieuwenhoven, F.A.; Turner, N.A. The role of cardiac fibroblasts in the transition from inflammation to fibrosis following
myocardial infarction. Vasc. Pharmacol. 2013,58, 182–188. [CrossRef]
46.
Hwang, M.W.; Matsumori, A.; Furukawa, Y.; Ono, K.; Okada, M.; Iwasaki, A.; Hara, M.; Miyamoto, T.; Touma, M.; Sasayama,
S. Neutralization of interleukin-1beta in the acute phase of myocardial infarction promotes the progression of left ventricular
remodeling. J. Am. Coll. Cardiol. 2001,38, 1546–1553. [CrossRef]
47.
Turner, N.A.; Das, A.; Warburton, P.; O’Regan, D.J.; Ball, S.G.; Porter, K.E. Interleukin-1alpha stimulates proinflammatory cytokine
expression in human cardiac myofibroblasts. Am. J. Physiol. Heart Circ. Physiol. 2009,297, H1117–H1127. [CrossRef]
48. Chen, B.; Frangogiannis, N.G. Chemokines in Myocardial Infarction. J. Cardiovasc. Transl. Res. 2021,14, 35–52. [CrossRef]
49.
Chen, W.; Frangogiannis, N.G. Fibroblasts in post-infarction inflammation and cardiac repair. Biochim. Biophys. Acta 2013,1833,
945–953. [CrossRef]

Int. J. Mol. Sci. 2025,26, 3846 16 of 16
50.
Dobaczewski, M.; Bujak, M.; Li, N.; Gonzalez-Quesada, C.; Mendoza, L.H.; Wang, X.F.; Frangogiannis, N.G. Smad3 signaling
critically regulates fibroblast phenotype and function in healing myocardial infarction. Circ. Res. 2010,107, 418–428. [CrossRef]
51.
Dobaczewski, M.; de Haan, J.J.; Frangogiannis, N.G. The extracellular matrix modulates fibroblast phenotype and function in the
infarcted myocardium. J. Cardiovasc. Transl. Res. 2012,5, 837–847. [CrossRef] [PubMed]
52.
DeLeon-Pennell, K.Y.; Lindsey, M.L. Somewhere over the sex differences rainbow of myocardial infarction remodeling: Hormones,
chromosomes, inflammasome, oh my. Expert Rev. Proteom. 2019,16, 933–940. [CrossRef]
53.
Cavasin, M.A.; Sankey, S.S.; Yu, A.L.; Menon, S.; Yang, X.P. Estrogen and testosterone have opposing effects on chronic cardiac
remodeling and function in mice with myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 2003,284, H1560–H1569.
[CrossRef]
54.
Glezeva, N.; Moran, B.; Collier, P.; Moravec, C.S.; Phelan, D.; Donnellan, E.; Russell-Hallinan, A.; O’Connor, D.P.; Gallagher, W.M.;
Gallagher, J.; et al. Targeted DNA Methylation Profiling of Human Cardiac Tissue Reveals Novel Epigenetic Traits and Gene
Deregulation Across Different Heart Failure Patient Subtypes. Circ. Heart Fail. 2019,12, e005765. [CrossRef]
55.
Tonry, C.; Linden, K.; Collier, P.; Ledwidge, M.; McDonald, K.; Collins, B.C.; Watson, C.J. Proteomic Characterisation of Heart
Failure Reveals a Unique Molecular Phenotype for Hypertrophic Cardiomyopathy. Biomedicines 2024,12, 1712. [CrossRef]
56.
Zamilpa, R.; Zhang, J.; Chiao, Y.A.; de Castro Bras, L.E.; Halade, G.V.; Ma, Y.; Hacker, S.O.; Lindsey, M.L. Cardiac wound healing
post-myocardial infarction: A novel method to target extracellular matrix remodeling in the left ventricle. Methods Mol. Biol. 2013,
1037, 313–324. [PubMed]
57.
Robinson, E.; Tate, M.; Lockhart, S.; McPeake, C.; O’Neill, K.M.; Edgar, K.S.; Calderwood, D.; Green, B.D.; McDermott, B.J.; Grieve,
D.J. Metabolically-inactive glucagon-like peptide-1(9-36)amide confers selective protective actions against post-myocardial
infarction remodelling. Cardiovasc. Diabetol. 2016,15, 65. [CrossRef]
58.
Mouton, A.J.; Ma, Y.; Rivera Gonzalez, O.J.; Daseke, M.J., 2nd; Flynn, E.R.; Freeman, T.C.; Garrett, M.R.; DeLeon-Pennell,
K.Y.; Lindsey, M.L. Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to
angiogenesis. Basic Res. Cardiol. 2019,114, 6. [CrossRef]
59.
Wu, T.; Hu, E.; Xu, S.; Chen, M.; Guo, P.; Dai, Z.; Feng, T.; Zhou, L.; Tang, W.; Zhan, L.; et al. clusterProfiler 4.0: A universal
enrichment tool for interpreting omics data. Innovation 2021,2, 100141. [CrossRef]
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