Pro‐inflammatory cytokines as emerging molecular determinants in cardiolaminopathies

Abstract

Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies strictly associated with significant deterioration of the electrical and contractile function of the heart. Despite the continuous flow of biomedical evidence, linking cardiac inflammation to heart remodelling in patients harbouring lmna mutations is puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belonging to four different families carrying pathogenic lmna mutations segregating with cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n = 11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony‐stimulating factor (G‐CSF) and interleukin 6 (IL‐6) were found in all affected patients’ sera. In addition, elevated levels of Interleukins (IL) IL‐1Ra, IL‐1β IL‐4, IL‐5 and IL‐8 and the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were measured in a large subset of patients associated with more aggressive clinical manifestations. Finally, the expression of the pro‐inflammatory 70 kDa heat shock protein (Hsp70) was significantly increased in serum exosomes of patients harbouring the lmna mutation associated with the more severe phenotype. Overall, the identification of patient subsets with overactive or dysregulated myocardial inflammatory responses could represent an innovative diagnostic, prognostic and therapeutic tool against Lamin A/C cardiomyopathies.

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J Cell Mol Med. 2021;25:10902–10915.wileyonlinelibrary.com/journal/jcmm
1 | INTRODUCTION
Mutations in lmna gene encoding intermediate filament proteins of
the inner n uclear membra ne Lamin A/C (LMNA ) cause tissue- specif ic
systemic diseases collectively known as laminopathies. Cardiac lami-
nopathies encompass a wide spectrum of clinical entities with high
penetrance and (usually) adult onset. Although dilated cardiomyopa-
thy with conduction defects (DCM- CD) is the most prevalent pheno-
type, different mutational sites might correlate with different clinical
manifestations spanning from conduction disorders, frequent atrial
fibrillation and life- threatening ventricular arrhythmias, with nor-
mal or altered ventricular systolic function.1,2 This surprising large
Received: 22 July 20 21
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Revised: 16 Septemb er 2021
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Accepted: 23 September 2021
DOI: 10.1111/jcmm.16975
ORIGINAL ARTICLE
Pro- inflammatory cytokines as emerging molecular
determinants in cardiolaminopathies
Andrea Gerbino1 | Cinzia Forleo2 | Serena Milano1 | Francesca Piccapane1 |
Giuseppe Procino1 | Martino Pepe2 | Mara Piccolo2 | Piero Guida3 | Nicoletta Resta4 |
Stefano Favale2 | Maria Svelto1 | Monica Carmosino5
This is an op en acces s article unde r the terms of the Creat ive Commo ns At tri bution License, which permits use, distr ibution and reproduc tion in any medium ,
provide d the original wor k is properly cited.
© 2021 The Authors. Journal of Cellular and Molecular Medicine publish ed by Foundation fo r Cellular and Molecular Medicine and Joh n Wiley & Sons Ltd.
Gerbi no and Forleo con tribut ed equally to thi s work as first au thors.
1Department of Biosciences,
Biotechnologies and Biopharmaceutics,
University of Ba ri, Bar i, Ital y
2Department of Emergency and Organ
Transplantation, Cardiology Unit,
University of Ba ri Aldo M oro, Bar i, Ital y
3Regional Gener al Hospital “F. Miulli”,
Acquaviva delle Fonti, Italy
4Division of Medical Genetics,
Depar tment of B iomedical Sciences and
Human On colog y, University of Bar i Aldo
Moro, Bari, Italy
5Depar tment of S cience s, Unive rsity of
Basilicata, Potenza, Italy
Correspondence
Monica Carmosino, Department of
Science s, Unive rsity of Basilicata, Potenza,
Italy.
Email: monica.carmosino@unibas.it
Funding information
This work w as supported by funding
from ‘Carmosino19LAMINOPATIE’ and
‘Carmosino20RIL’ to Monica Carmosino
and from t he CLUSTER TECNOLOG ICO
REGIONALE ‘DICLIMA X’ (project #
MTJU9H8) to Maria Svelto
Abstract
Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies
strictly associated with significant deterioration of the electrical and contractile
function of the heart. Despite the continuous flow of biomedical evidence, linking
cardiac inflammation to heart remodelling in patients harbouring lmna mutations is
puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belong-
ing to four different families carr ying pathogenic lmna mutations segregating with
cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n =
11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony-
stimulating factor (G- CSF) and interleukin 6 (IL- 6) were found in all affected patients’
sera. In addition, elevated levels of Interleukins (IL) IL- 1Ra, IL- 1β IL- 4, IL- 5 and IL- 8
and the granulocyte- macrophage colony- stimulating factor (GM- CSF) were measured
in a large subset of patients associated with more aggressive clinical manifestations.
Finally, the expression of the pro- inflammatory 70 kDa heat shock protein (Hsp70)
was significantly increased in serum exosomes of patients harbouring the lmna muta-
tion associated with the more severe phenotype. Overall, the identification of patient
subsets with overactive or dysregulated myocardial inflammator y responses could
represent an innovative diagnostic, prognostic and therapeutic tool against Lamin
A/C cardiomyopathies.
KEYWORDS
cardiolaminopathies, cytokines, inflammation

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number of cardiac phenotypes reflects an even larger number of
specific mutations (about 500) identified in the lmna gene and col-
lected in the UMD- lmna mutations database.
The molecular mechanisms underlying the origin and develop-
ment of such a wide spectrum of cardiac pathologies are still in-
completely defined. Despite under the spotlight of many research
groups for quite a long time, a complete understanding of the cellular
functions mediated by LMNA is missing. Thus, mutations in the lmna
gene can either affect a specific function of LMNA or broadly impact
the whole repertoire of molecular mechanisms controlled by these
intermediate filaments such as mechanotransduction, gene regula-
tion and signal transduction.3,4 Yet, we still do not know whether
these different pathogenic mechanisms represent different pieces
of the same puzzle or each one of them can be correlated to a spe-
cific clinical entity.
For instance, alterations in several cellular pathways, including
WNT/β- catenin, mitogen- activated protein kinases (MAPKs) and pro-
tein kinas e B (AKT)/mammal ian target of rapamy cin (mTOR) signall ing,
have been identified in LMNAH222P/H222P mice, a mouse model of the
human LMNA- associated DCM- CD.5,6 Interestingly, lmna mutations
may also impinge the machinery involved in C a2+ handling into the
ER7 – 9 and the connexin 43 (CX43) expression/activity at the plasma
membrane in cardiomyopathies.10,11 More recently, Salvarini et al.
demonstrated that the epigenetic inhibition of the sodium voltage-
gated channel alpha subunit 5 (SCN5A) in cardiomyocytes differen-
tiated from IPS derived from patients with K219T- LMNA pathogenic
variant12 can account for the conduction defects reported in the clin-
ical history of these arrhythmogenic patients.
Despite the molecular mechanism involved in LMNA cardiomy-
opathies, the deterioration in electrical and contractile function cor-
relates with exacerbated cardiomyocytes damage or death, which,
in turn, may trigger myocardial inflammation, further aggravating
the progression of the cardiomyopathy. Myocardial fibrosis is also
thought to be responsible for the development of both electrical in-
stability and mechanical impairment in cardiac laminopathies13 typi-
cally developing within the interventricular septum, near the region of
the conduction system thus accounting for the conduction disease.14
It is known that the mammalian heart contains a population of resi-
dent macrophages that proliferate following myocardial damage such
as in myocardial infarction, which in turn recruit other monocytes to
the hear t, contributing to myocardial interstitial fibrosis and adverse
cardiac remodelling.15 Moreover, it also well established that pro-
inflammatory cytokines are produced in typical inflammatory cardio-
myopathies as consequence of pathogen infection and a wide variety
of toxic substances, drugs and systemic immune- mediated diseases.
These cardiomyopathies evolve also in DCM and heart failure.16
However, in inherited cardiomyopathies, cardiac inflammation
and its correlation to contractile dysfunc tion and cardiac remodel-
ling have not been fully investigated. Therefore, in this study, we aim
at investigating the specific profile of 30 cytokines and chemokines
in the serum of four dif ferent groups of patients harbouring differ-
ent pathogenic mutations in lmna gene and their family members
not carr ying the mutation. The clinical phenotype associated with
the lmna mutations under investigation was mainly characterized by
left ventricular dilation, left ventricular systolic dysfunction and con-
duction defects despite at different range of severity, even amongst
members of the same family. The profile of inflammator y cytokines
measured in our experiments did not show any specificity for the
location of each mut ation analysed. However, the severity of the
clinical manifestations associated with each patient correlates with
the degree of inflammation in terms of numbers of pro- inflammatory
cytokines upregulated. Since no specific pharmacological treatment
is available and the ICD implantation is currently the only ef fective
clinical approach for patients affected by cardiac laminopathies,
the identification of patient subsets affected by LMNA cardiomy-
opathies with overactive or dysregulated myocardial inflammatory
responses could be crucial for clarifying the pathogenesis of these
cardiomyopathies and for evaluating immediate successful thera-
peutic approaches.
2 | METHODS
2.1 | Clinical and instrumental analysis
The patients who were referred to the Cardiomyopathy
Unit, Cardiolog y Unit, Department of Emergency and Organ
Transplantation, University of Bari Aldo Moro, Bari (Italy), between
January 2020 and August 2020, and who fulfilled the inclusion and
exclusion criteria, were involved in the study. A total of 30 Italian
patients (19 patients with lmna mutation e and 11 lmna mutation-
negative family members) were enrolled in the study and subjected
to blood sampling for serum cytokine assay. To avoid variations in
serum levels of cytokines, no subject had exercised physical activity
prior to blood sampling and did not have any ongoing infections or
immunodeficiency conditions at the time of enrolment. For further
details on inclusion/exclusion criteria, see Appendix S1. All recruited
subjects provided their written informed consent to participate in
this stu dy. The proj ect conformed to th e principles of the D eclaration
of Helsinki (World Medical Association) and was approved by the
Ethics Committee of the University Hospital Consortium, Policlinico
of Bari, Italy.
2.2 | Cytokine/chemokine assay
Plasma samples from the patients included in the study were pre-
pared by centrifugation at 500 g for 12 min and stored at −80 °C.
Bio- Plex Pro Human Cytokine 27- plex Assay (#M500KCAF0Y; Bio-
Rad Laboratories) and Bio- Plex Pro Transforming Growth Factor- β
(TGF- β) 3- plex Assay (#171W4001 M; Bio- Rad Laboratories) were
used by following the manufacturer's instructions. Each sample was
analysed in triplicate in BioPlexMagpix Multiplex Reader (Bio- Rad
Laboratories), and the data automatically analysed using Bio- Plex
Manager 6.0 software (Bio- Rad Laboratories). For the list of cy-
tokines analysed and for detailed procedure, see Appendix S1.

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2.3 | Serum exosome preparation and analysis
Serum exosomes were isolated from 250 μl of each patient s’ serum
with the exosome isolation kit EXOQ50A- 1 (System Biosciences) ac-
cording to manufacturer's instructions. Serum exosomes were ana-
lysed by Western blotting for the expression of Hsp70 and exosome
markers. For details on serum exosomes preparation and analysis,
see Appendix S1.
2.4 | Cell culture and western blotting
HEK293 cells were transiently transfected with the previously de-
scribed17 vectors for the expression of either LMNA WT or LMNA- p.
Leu140_Ala146dup variant, using Lipofec tamine® 2000. After 72 h,
transfection cells were lysed in RIPA buf fer and lysate were analysed
by Western blotting for the expression of Hsp70. For details, see
Appendix S1.
2.5 | Statistical analysis
Continuous variables are expressed as mean values ± standard
deviation and compared between groups by using Student 's t test
(equal or unequal variance as appropriate). Categorical variables are
expressed as absolute frequency or percentage. Associations were
tested with Fisher's exact test. Analyses were performed using
STATA sof tware version 14 (Stata). Student's t test for unpaired data
was used to analyse dif ferences in cytokines levels bet ween each
patient group and controls. Receiver operating characteristic (ROC)
curves were used to estimate the diagnostic potential of the quanti-
fied individual cytokines to discriminate between groups. GraphPad
Prism software (version 8) was used for st atistical and graphical art s.
In all cases, significance was considered at p < 0.05.
3 | RESULTS
3.1 | Molecular analysis of the patients’ population
The lmna mutations included in this study were identified in mem-
bers of Italian families with cardiac phenotypes screened in our
Clinical Unit dedicated to cardiomyopathies.
The lmna mutations are as follows:
1. The heteroz ygous variant c.418_438dup consists of a duplication
of 21 nucleotides (CTGC TGAACTCCA AGGAGGCC) in the exon
2 of the lmna gene, located in the coil 1B of the central α-
helical rod domain of the LMNA protein. The resulting LMNA
variant is predicted to result in the duplication of seven amino
acids (LLNSKEA) in LMNA protein, from Leucine at position
140 to Alanine at position 146, without a frame shift in the
open reading frame. This LMNA variant hereinafter referred
to as p.Leu140_Ala146dup has been previously identified and
characterized in vitro by our group17 and classified as pathogenic.
2. The heterozygous variant c.329delG consists of a G deletion at
position 329 in the exon 2 of the lmna gene. This deletion causes
a shift in the reading frame starting at Arginine 110, changing
it to a Leucine and creating a premature stop codon at position
7 of the new reading frame. This LMNA variant, denoted as
p.Arg110Leufs*7, is present in the ClinVar data base and ranked
as likely pathogenic.
3. The heterozygous variant c.949G>A consists of a G to A substitu-
tion at position 949 in the exon 6 of the lmna gene leading to glu-
tamate to lysine exchange at the position 317 in the coil2 domain.
This variant (p.Glu317Lys) is ranked as pathogenic and has been
already described in patients with atrioventricular block (AVB)
and DCM.18,19
4. The heterozygous variant c.569G>A, located in coding exon 3 of
the lmna gene, results from a G to A substitution at nucleotide po-
sition 569. The arginine at codon 190 is replaced by glutamine, an
amino acid with highly similar properties. This variant is reported
as p.Arg190Gln and ranked as pathogenic. This alteration has
been already reported in individuals with DCM and reported as
LMNA R190Q variant.20,21
3.2 | Clinical data
Table 1 summarizes the most relevant clinical manifestations of all
the subjects evaluated in this study. Carriers of pathogenic LMNA
variants belonging to four different families (patient s) were com-
pared with family members not carr ying the mutation (controls).
Clinical data, electrical and mechanical abnormalities documented
by ECG recordings and cardiac imaging manifested during their clini-
cal history were reported.
3.2.1 | Family 1
Patients harbouring p. Leu140_Ala146dup LMNA variant mostly
exhibited (4 out of 6) a phenotype charac terized by left ventricular
dilation (LVEDD 52 ± 6 mm; LVEDVi 74 ± 16 ml/m2), left ventricular
systolic dysfunction (LVEF 45 ± 13%) (Figure 1A– D) and numerous
arrhythmic disorders. Indeed, frequent (up to 1000 per day) polymor-
phic premature ventricular complexes (PVCs), and non- sustained ven-
tricular tachycardia (NSVT) episodes were recorded on ECG Holter
monitoring in all patients of this group (Figure 2A); three patients also
showed sustained ventricular tachycardia (SVT) or ventricular fibrilla-
tion (VF) events, detected on telemetry or implantable cardioverter-
defibrillator (ICD) arrhythmia registry. Conduction disturbances (first- ,
second- and third- degree atrioventricular blocks, Figure 2B) and atrial
fibrillation (Figure 2C) were obser ved in about 80% of the patients.
Five patients received an ICD in primary prevention, and several de-
vice appropriate interventions by antit achycardia pacing (ATP) or DC
shock on SVT or fast SVT recognized in VF zone, respectively, were

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TABLE 1 Clinical characteristics of study subjects according to lmna mutation carrier status
lmna mutation- positive subjects according to different genotypes (n = 19)
All lmna mutation-
positive subjects
(n = 19)
lmna mutation-
negative subjects
(n = 11)
p valuep. Leu140_Ala146dup (n = 6) p. Arg110Leuf s*7 (n = 4) p. Glu317Lys (n = 6) p. Arg190Gln (n = 3) Patients (n = 19) Controls (n = 11)
Age at time of blood
collection (years)
46 ± 10 42 ± 13 54 ± 6 35 ± 16 46 ± 12 41 ± 18 0.399
Gender (F/M) 4/2 3/1 2/4 0/3 9/10 7/4 0.466
Cardiac phenotype DCM, left ventricular systolic
dysfunction, AF, AVB,
PVCs, NSVT, SVT
Left ventricular systolic
dysfunction, AF, AVB,
PVCs, NSVT
DCM, lef t ventricular
systolic dysfunction, AF,
AVB, PVCs, NS VT, SVT
DCM, Lef t ventricle
systolic
dysfunction, AF
Healthy
Clinical myopathy 0 0 0 3 3 0
CPK levels (U/L) 163 ± 53 112 ± 68 59 ± 31 1274 ± 1255 339 ± 655 NA
SBP (mm Hg) 104 ± 13 106 ± 11 126 ± 20 153 ± 24 119 ± 24 118 ± 9 0.822
DBP (mm Hg) 65 ± 5 66 ± 8 83 ± 9 90 ± 13 75 ± 13 71 ± 7 0.320
BMI (K g/m2)24.9 ± 3.3 27. 1 ± 6.9 23.7 ± 3.4 27. 5 ± 4.3 25.4 ± 4.3 24. 3 ± 2.3 0.427
Heart rate (bpm) 60 ± 7 61 ± 11 62 ± 6 57 ± 2 60 ±773 ± 13 0.010
PR interval (ms) 250 ± 62 210 ± 68 277 ± 178 167± 23 237 ± 111 147± 29 0.003
QRS duration (ms) 93 ± 8 89 ± 10 97 ± 10 107 ± 12 96 ±11 87 ± 10 0.026
QTc interval (ms) 417 ± 24 398 ± 17 421 ± 22 410 ± 47 413 ± 26 415 ± 16 0.816
AF, n (%) 5 (83%) 2 (50%) 3 (50%) 1 (33%) 11 (58%) 0 (0%) 0.0 02
AV block, n (%) 5 (83%) 2 (50%) 3 (50%) 0 (0%) 10 (53%) 0 (0%) 0.004
PVCs >50 0/24h , n (%) 6 (100%) 3 (75%) 2 (33.3%) 0 (0%) 11 (58%) 0 (0%) 0.002
PVCs >1000/24h, n (%) 6 (100%) 3 (75%) 1 (16.6%) 0 (0%) 10 (53%) 0 (0%) 0.004
NS V T, n (%) 6 (100%) 2 (50%) 3 (50%) 0 (0%) 11 (58%) 0 (0%) 0.002
SV T/V F, n (%) 3 (50%) 0 (0%) 2 (33%) 0 (0%) 5 (26%) 0 (0%) 0.129
Indexed LVEDV (ml/m2)74 ± 16 66 ± 12 90 ± 7 86 ± 26 78 ± 17
LVEDD (mm) 52 ± 6 48± 4 53 ±953 ± 5 52 ± 6 44 ± 3 <0.001
LVEF (%) 45 ± 13 53 ± 6 41 ± 15 56 ± 5 47 ± 12 59 ± 3 0.001
CMRI, n (%) 5 (83%) 2 (50%) 3 (50%) 2 (66%) 12 (63%) /
LGE on CMRI, n (%) 4 (80%) 0 (0%) 1 (33%) 0 (0%) 5 (42%) /
PM/ICD implantation, n (%) 5 (83%) 2 (50%) 2 (33%) 1 (33%) 9 (47%) /
Heart transplant, n (%) 1 (17%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) /
Note: Mean ± Standard Deviation.
Abbreviations: AF, atrial fibrillation; AVB, atrioventricular block; BMI, body mass index; CMRI , cardiac magnetic resonance imaging; DBP, diastolic blood pressure; DCM, dilated cardiomyopathy; ICD,
implantable cardioverter- defibrillator; LGE, late gadolinium enhancement; LVEDD, left ventricular end- diastolic diameter; LVEDV, left ventricular end- diastolic volume; LVEF, left ventricular ejection fraction;
NSVT, non- sustained ventricular tachycardia; PM, pacemaker; PVCs, premature ventricular complexes; SBP, systolic blood pressure; SVT, sust ained ventricular tachyc ardia; VF, ventricular fibrillation.
p- values ≤ 0.05 were considered statistically significant and repor ted in bold.

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detected in three of them (Figure 2D). Finally, a patient of this group
underwent heart transplant (after unsuccessful VT trans- catheter ab-
lation) at the age of 45 due to sustained VT recurrences in storm and
subsequent LV function deterioration (LEVF 30%).17
3.2.2 | Family 2
The patients of this family, with the p. Arg110Leufs*7 LMNA patho-
genic variant, showed a less aggressive clinical phenotype in com-
parison with members of the previously described family. In these
subjects, ventricular dimensions were in the normal range (LVEDD
48 ± 4 mm; LVEDVi 66 ± 12 ml/m2) and only mild left ventricular
systolic dysfunction was observed (LVEF 53 ± 6%). Half of the car-
riers developed conduction disturbances (first- degree AV block),
atrial fibrillation and ventricular arrhythmias as NSVT episodes,
and many PVCs were recorded at the 24- hour Holter monitoring.
Two subjects underwent ICD implantation in primar y prevention,
and one of them, with moderate left ventricular systolic dysfunc-
tion (LVEF 45%), underwent up- grading of dual- chamber pacemaker
to biventricular ICD with subsequent recovery of the left ventricu-
lar systolic function. To date, no members of this family presented
life- threatening ventricular tachyarrhythmias. Furthermore, two
patients carrying the above variant showed neuromuscular involve-
ment (radiculopathy associated with motor block, waddling gait and
girdle hypertrophy) still undergoing diagnostic definition.
FIGURE 1 Cardiac phenotype
evaluated by Echocardiographic imaging
in LMNA mutants’ carriers. The upper
half of the image shows LMNA DCM
phenotype: (A) and (B) panels show apical
four chamber (4C) views with chamber
dilatation (LVEDVi 82 ml/m2 and LVESVi
52 ml/m2) and reduced left ventricular
systolic function (LVEF 35%) due to
global hypocontractility. (C) PLA X view:
LVEDD (55 mm). (D) PSAX view: mid- LV
at the level of papillar y muscles. The
lower part (panels E– H) of the picture
shows a hypokinetic non- dilated LMNA
cardiomyopathy characterized by normal
ventricular volumes both in end- diastole
(E) (LVEDVi 64 ml/m2) and end- systole
(F) (LVESVi 38.8 ml/m2) with moderate
systolic left ventricular dysfunction
(LVEF 36%). (G) In PLAX view: LVEDD
(51 mm). (H) PSAX view: mid- LV at the
level of papillary muscles. DCM, dilated
cardiomyopathy; LVEDD, left ventricular
end- diastolic diameter; LVEDV, left
ventricular end- diastolic volume index;
LVEF, left ventricular ejection fraction;
LVESVi, left ventricular end- systolic
volume index; PLAX view, parasternal
long- axis view; PSAX, parasternal short-
axis view

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FIGURE 2 Abnormal ECG findings in LMNA mutants’ carriers. (A) Episode of non- sustained VT with LBBB morphology and inferior
axis on 12- lead Holter monitoring. (B) ECG showing sinus rhythm, first- degree atrioventricular block (upper trace), type 1 s- degree
atrioventricular block (middle trace) and 2:1 atrioventricular block (lower trace) recorded in the same patient during 24- h ECG recording. (C)
12- lead ECG displaying atrial fibrillation and ventricular rhythm induced by pacemaker with VVI pacing mode. (D) ICD remote monitoring
report showing PVCs which trigger an episode of sustained TV, terminated, after ineffective ATP, by internal ICD shock. ATP, antitachycardia
pacing; ICD, implantable cardioverter- defibrillator; LBBB, left bundle branch block; PVCs, premature ventricular complexes; VT, ventricular
tachycardia

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3.2.3 | Family 3
Cardiac phenotype of p. Glu317Lys LMNA variant carriers was
characterized by left ventricular dilatation (LVEDD 53 ± 9 mm;
LVEDVi 90 ± 7 ml/m2) and systolic dysfunction (LVEF 41 ± 15%).
Three patients also displayed conduction disorders: 2 of them
presented first- degree AV- blocks with very long PR inter vals (the
longest reported was 520 ms), while the third patient developed
high- degree AV block and received a dual- chamber pacemaker sub-
sequently upgraded to a biventricular defibrillator. Atrial fibrillation
and NSV T were found in 50% of these patients, while 2 patients
also presented with SV T (one of them occurring during exercise
testing, and the other was detected on defibrillator arrhythmia
registr y). One patient received an ICD in primar y prevention due
to severe left ventricular dysfunction (LVEF 20%), which subse-
quently progre ssively imp roved (last reported LVEF was 47%) af ter
optimized drug treatment for heart failure, and the occurrence of
several appropriate defibrillator inter ventions by ATP on SVT was
also reported.
3.2.4 | Family 4
The p. Arg190Gln LMNA variant was related to neuromuscu-
lar involvement consisting of muscle cramps, reduced strength to
stress and inappropriate muscular hypertrophy, associated with
significantly increased CPK values (1274 ± 1255 U/L), in addition
to cardiac abnormalities. One of the variant carriers had left ven-
tricular dilatation with normal ventricular function, while his brother
showed a hypokinetic non- dilated cardiomyopathy phenotype with
mildly impaired left ventricular systolic function (LVEF 50%) and nor-
mal ventricular volumes (Figure 1E– H); thus far, none of these two
patients has developed ventricular and supraventricular arrhyth-
mias or conduction disturbances. The third member of this family,
the father, having normal left ventricular size and systolic function,
showed paroxysmal atrial fibrillation with low ventricular rate and
underwent ICD implantation in primary prevention due to sinus
node disease and a wide QRS tachycardia episode occurrence dur-
ing exercise testing.
All lmna mutation- negative family members were clinically
asymptomatic, and none of them showed LMNA- linked cardiac
phenotypes either in terms of electrical disorders or mechanical ab-
normalities (Table 1).
3.3 | Cytokine and chemokine levels in the
sera of the LMNA patients and comparison with
those of healthy controls
Sera from the patients and controls described in the Table 2 were
screened for the circulating levels of 30 cytokines/chemokines
(Table 2). The levels of IL- 15 are under the lower limit of the assay
sensitivity, thus resulted undetectable in our cohort of patients. This
is in line with the fact that it has been reported that in humans, the
levels of circulating IL- 15 under normal conditions are low or unde-
tectable (~1 pg/ml ). 22
The levels of several pro- inflammatory cy tokines resulted up-
regulated in the sera of patients compared with controls. A more
detailed analysis of these cytokines was performed.
As shown in Figure 3A and Table 2, IL- 1ra resulted signifi-
cantly upregulated in the serum of both p. Leu140_Ala146dup, p.
Arg110Leufs*7 and p. Glu317Lys carriers compared with controls
(p = 0.0001, p = 0.0016 and p = 0.0002 respectively). The area
under the ROC curve (AUC) for IL- 1ra was 1 (Figure 3A’, p = 0.0003),
suggesting that the measurement of the levels of this cytokine
might allow us to discriminate between patients carrying either p.
Leu140_Ala146dup, p. Arg110Leufs*7 or p. Glu317Lys LMNA vari-
ants and controls enrolled in our study with high degree of accuracy.
Moreover, levels of IL- 8 resulted significantly increased in the serum
of p. Arg110Leufs*7, p. Glu317Lys and p. Arg190Gln LMNA carri-
ers compared with controls (Figure 3B, Table 2). The AUC values are
0.93 ± 0.04 (Figure 3B’, p = 0.0 004), indicating that IL- 8 levels may
be also considered as accurate biomarkers for the cardiomyopathy in
p. Arg110Leufs*7, p. Glu317Lys and p. Arg190Gln carriers.
Interestingly, the levels of both IL- 6 and G- CSF were signifi-
cantly increased in all patients compared with controls (Figure 4A , B,
Table 2) with the AUC value of 0.94 ± 0.04 (Figure 4A’, p = 0.0003)
and 1 (Figure 4B’, p < 0.00 01), respectively, indicating the levels of
these cytokines as valuable parameters to distinguish between pa-
tients af fected by LMNA- associated cardiomyopathy and controls at
least in our cohort of 30 individuals analysed.
Serum levels of IL- 1β, IL- 4, IL- 5 and GM- CSF were also signifi-
cantly upregulated in some of the families enrolled in the study
compared with controls (Figure 5). Interestingly, these cytokines
were more significantly upregulated in p. Leu140_Ala146dup car-
riers sug gesting a more complex pattern of inflammation in these
patients. Interestingly, amongst the cohort of p. Leu140_ Ala146dup
carriers, we identified a 57- year- old patient who underwent heart
transplantation in 2017 and asymptomatic at the time of the analy-
sis. In this carrier, the serum levels of cytokines were comparable to
that of control healthy subject s, thus suggesting a clear correlation
between the clinical manifestation of the cardiomyopathy and the
increased levels of pro- inflammatory cytokines (not shown).
3.4 | Analysis of Hsp70 in serum exosomes of
LMNA mutant carriers
It has been reported that extracellular Hsp70 may act through sur-
face receptors stimulating release of pro- inflammatory cytokines23
and that elevated serum levels of Hsp70 correlate with hypertrophy
and fibrosis in cardiovascular diseases.24
Indeed, to investigate more in deep the molecular mechanisms
involved in the establishment of the inflammatory phenot ype in
LMNA mutant carriers, we analysed the expression of Hsp70 in
serum exosomes of both patients and controls involved in the study.

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TABLE 2 Serum levels of chemokines/cytokines expressed in pg/ml. Only the p values of significative differences compared with controls
were indicated (in bold). OOR< indicates that cytokine levels are under the lower limit of the assay sensitivity
p. Leu140_Ala146dup (n = 6) p. Arg110Leufs*7 (n = 4) p. Glu317Lys (n = 6) p. A rg190Gln (n = 3) Controls (n = 11)
IL- 1β6. 530 ± 1.522
p = 0.0011
3.715 ± 1.937
p = 0.56
4.918 ± 1.3 61
p = 0.01
2.643 ± 0.6233
p = 0.5
1.849 ± 0.2817
IL- 1r a 398.4 ± 30.03
p = 0.001
279.5 ± 14.29
p = 0.0016
396.0 ± 39. 83
p = 0.00 02
213.9 ± 82. 23
p = 0.22
132.4 ± 23 .15
IL- 2 9. 259 ± 2.24 4
p = 0.5608
8.193 ± 2.0 69
p = 0.7
12.95 ± 4.312
p = 0.19
8.237 ± 1 .411
p = 0.74
7.6 98 ± 0. 8279
IL- 4 9.68 0 ± 1.680
p = 0.00 05
4.578 ± 1.091
p = 0.52
7.9 4 8 ± 3.129
p = 0.16
4.523 ± 0.8 520
p = 0.53
3.343 ± 0.4856
IL- 5 20.99 ± 4.10 0
p = 0.00 04
9.923 ± 1.691
p = 0.07
31.84 ± 11.3 2
p = 0.0064
18.76 ± 10 .69
p = 0.05
5.679 ± 1.150
I L - 6 5.82 ± 1.0
p = 0.00 4
7.195 ± 1.805
p = 0.003
11.79 ± 3.19
p = 0.001
6.657 ± 1.76 4
p = 0.00 4
2.743 ± 0.3996
I L - 7 3 9.71 ± 5.560
p = 0.1554
29.17 ± 4.482
p = 0.78
35.56 ± 3. 523
p = 0.31
30.71 ± 8.330
p = 0.99
30.68 ± 2 .903
IL- 8 75.39 ± 18.43
p = 0.7383
198.8 ± 35.50
p = 0.005
256.5 ± 41 .4 6
p = 0.00 03
216.5 ± 36.85
p = 0.003
83.73 ± 16. 44
IL- 9 140.8 ± 6.28 8
p = 0.4683
139.1 ± 6.032
p = 0.58
169. 2 ± 14.11
p = 0.44
141.0 ± 11. 05
p = 0.68
152.4 ± 13.69
IL- 10 2.684 ± 0.6863
p = 0.9671
1.677 ± 0.7888
p = 0.49
2.564 ± 1.309
p = 0.97
2.523 ± 0.658 4
p = 0.93
2.633 ± 1.020
I L - 1 2 ( p 7 0 ) 6.952 ± 0.9793
p = 0.5925
6.720 ± 1. 217
p = 0.7
9.2 6 3 ± 1.709
p = 0.07
11.0 6 ± 4.952
p = 0.10
6.298 ± 0.7212
I L - 1 3 4.295 ± 0.8268
p = 0.6949
3.093 ± 0.3060
p = 0.67
5.575 ± 0.9616
p = 0.24
3.083 ± 0.8539
p = 0.72
3.785 ± 0.9604
IL- 15 OOR<OOR<OOR<OOR<OOR<
I L - 1 7 A 2 7.16 ± 5 .176
p = 0.186 0
24.26 ± 6.430
p = 0.34
39. 71 ± 14. 88
p = 0.09
24.65 ± 3.60 0
p = 0.21
20.02 ± 1.597
Eotaxin 1 7 7. 8 ± 29. 02
p = 0.3068
138.6 ± 21.04
p = 0.94
178.0 ± 22.04
p = 0.26
130.9 ± 38.26
p = 0.81
141. 2 ± 20. 28
bFGF 54.50 ± 6.378
p = 0.3689
52.57 ± 7.433
p = 0.49
72.84 ± 16 .73
p = 0.08
56.65 ± 6.106
p = 0.19
48.02 ± 2 .953
G - C S F 2980 ± 523.8
p = 0.00 01
1486 ± 442 .6
p = 0.0035
1140 ± 220.4
p = 0.002
1398 ± 188.5
p = 0.00 01
340.6 ± 57. 89
G M - C S F 6.250 ± 1.060
p = 0.00 05
3.067 ± 1.011
p = 0.19
5.355 ± 0.9873
p = 0.05
5.473 ± 2.787
p = 0.06
1.978 ± 0.3146
IFN- γ14.97± 5.036 10.3 3±0.8 819 11. 35± 1.888 12.45± 5.019 11.47± 4.499
IP - 10 748.8 ± 52.22
p = 0.0650
610. 3 ± 105.9
p = 0.84
1030 ± 285.7
p = 0.08
402.4 ± 53 .65
p = 0.13
58 7.4 ± 63.58
MCP- 1 (MCAF) 37.97 ± 4.896
p = 0.1871
31.78 ± 3.089
p = 0.10
73.28 ± 15.05
p = 0.11
37. 91 ± 5.729
p = 0.35
48.88 ± 6.413
MIP- 1α21.52 ± 8.44 3
p = 0.4025
33. 59 ± 14. 43
p = 0.08
29. 0 8 ± 6 .912
p = 0.06
40.18 ± 1.161
p = 0.005
14.02 ± 3.919
PDGF- bb 3233 ± 360.7
p = 0.2717
3388 ± 1 77. 8
p = 0.52
38 51 ± 394.6
p = 0.96
4797 ± 332.4
p = 0.31
3883 ± 4 37.6
MIP- 1b 148.5 ± 35.34
p = 0. 3511
142. 2 ± 23. 39
p = 0.28
155.5 ± 22.78
p = 0.10
108. 4 ± 10. 58
p = 0.82
114.0 ± 12. 52
RANTES 11929 ± 556 .9
p = 0.1784
1244 6 ± 421.6
p = 0. 51
14188 ± 518 .9
p = 0.711
11606 ± 1082
p = 0.35
13630 ± 1043
TNF- γ99. 09 ± 2 9.64
p = 0.2234
80.49 ± 21.18
p = 0.23
95.85 ± 20.49
p = 0.05
64.04 ± 7.89 3
p = 0.92
63.16 ± 4.318
(Continues)

10910
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G ERBINO E t al.
We found an increase in the Hsp70 expression in serum exo-
somes from LMNA- p. Leu140_Ala146dup carriers compared with
controls (Figure 6A). The same exosomes were tested for the ex-
pression of the exosome’ markers, CD81 and CD9 (Figure 6B).
Densitometric analysis of Hsp70 band in the serum of all patients
and controls showed that only the circulating levels of Hsp70 were
significantly upregulated in LMNA- p. Leu140_Ala146dup carriers
compared with controls (Figure 6C).
To corroborate the relationship bet ween the elevated serum lev-
els of Hsp70 in serum exosomes from LMNA- p. Leu140_ Ala146dup
carriers and the specific LMNA mut ant, we analysed the expression
levels of Hsp70 in HEK293 cells after LMNA- p. Leu140_Ala146dup
expression. As shown in Figure 6D and E, the expression levels of
Hsp70 increased by about twofold in LMNA- p. Leu140_Ala146dup
compared with LMNA- WT- expressing HEK293 cells.
4 | DISCUSSION
In this work, we found that specific pro- inflammatory cytokines re-
sulted upregulated in a cohort of patients affec ted by cardiomyopa-
thy due to different mutations in lmna gene.
Carriers expressing the pathogenic LMNA variant are character-
ized by bradycardia, AF, a signific ant increase in PR interval and QRS
duration, high frequency of AV block, PVCs and NSVT occurrence,
an increase in LVEDD and a decrease in LVEF compared with con-
trols, respectively (Table 1).
The PR inter val duration >200 ms and a QRS complex prolonga-
tion (Table 1) denote the presence of AV- conduction blocks and an
impaired electrical conduction within the ventricles in LMNA variant
ca rri ers . In ad d it ion , hig h fre que ncy of PVC s an d NSVT in LMNA mu-
tant carriers clearly demonstrates the presence of early depolariza-
tions originating in the ventricles of these patients due to increased
ventricular automaticity. Both PVCs and NSVT are associated with
an overall increased risk for clinically relevant heart failure and an in-
creased risk for death.25 Moreover, LMNA mutant carries recruited
in this study are charac terized by mean increase in LVEDD and a
decrease in LVEF compared with controls denoting a left ventricular
dilation and dysfunction.
We found high levels of circulating G- CSF and IL- 6 in all patient s,
and elevated levels of IL- 1ra and IL- 8 in a large subset of the patients
enrolled in the study.
In particular, high levels of both G- C SF and IL- 6 were obser ved in
all the families we examined, independently from the mutation t ype
and the phenotypes detected in each family. On the base of these
findings, it is possible to hypothesize that high levels of both G- CSF
and IL- 6 could be associated with cardiolaminopathies. On the con-
trary, high values of IL- 1ra are present in three (Families 1, 2 and 3) of
the investigated families whose members carrying the LMNA variant
displayed a high percent age of AV- blocks, NSVT and PVCs, which
are completely absent from patients of the fourth family and from
control subjects (Table 1). In addition to this, the observed PR in-
tervals were significantly longer in patients from Families 1, 2 and 3
when compared either to patients from the fourth family or subjec ts
not carr ying LMNA variants (Table 1). Interestingly, the members of
Family 4 were characterized by a different laminopathic phenotype
than those belonging to the other three families, because they dis-
played a clear neuromuscular involvement associated with high CPK
values and less severe electrical and mechanical cardiac damage.
This suggests that IL- 1ra could be a biomarker associated with con-
duction defects and arrhythmic manifestations in LMNA patients
carrying an over t cardiac phenotype.
G- CSF is produced by bone marrow stromal cells, endothelial
cells, macrophages and fibroblasts. Its production is induced by in-
flammatory stimuli such as pro- inflammatory cytokines (TNF- α, I L - 6
and IL- 1), and it may enhance the pro- inflammatory responses by
controlling neutrophil numbers and their activity during inflamma-
tion. Although we did not find any signific ant increase in TNF- α in
LMNA mutant carriers, we did measure a significant increase in the
serum levels of IL- 1ra in p. Leu140_Ala146dup, p. Arg110Leufs*7
and p. Glu317 Lys ca rr iers an d in th e se rum level s of IL- 6 in all LMNA
mutants carriers compared with controls. IL- 1ra is a receptor an-
tagonist of IL- 1 activity, and it is released rapidly in the circulation
under the same inflammator y conditions that stimulate IL- 1α and
p. Leu140_Ala146dup (n = 6) p. Arg110Leufs*7 (n = 4) p. Glu317Lys (n = 6) p. A rg190Gln (n = 3) Controls (n = 11)
VEGF 108.2 ± 47.78
p = 0.555 4
20 9.5 ± 86 .52
p = 0.15
133.4 ± 47.72
p = 0.34
161. 0 ± 43.49
p = 0.14
72.98 ± 29. 99
TGF- β149020 ± 2220
p = 0.6832
54484 ± 476 .7
p = 0.9888
51638 ± 4105
p = 0.9959
63726 ± 3358
p = 0.1202
52788 ± 2577
TGF- β23169 ± 75. 88
p = 0.9999
3282 ± 65.51
p = 0.8934
3421 ± 172.1
p = 0.196 8
3244 ± 186.3
p = 0.9827
3173 ± 35 .17
TGF- β31627 ± 23.58
p = 0.6128
1666 ± 57.1 2
p = 0.9790
1548 ± 105,3
p = 0.153 4
1770 ± 83.44
p = 0.920 4
1705 ± 38.60
Abbreviations: IL, interleukin; bFGF, basic fibroblas t growth factor; G- CSF, granulocyte colony- stimulating f actor; GM- CSF, granuloc yte- macrophage
colony- stimulating factor; IFN- γ, γ- interferon; IP- 10, interferon gamma- induced protein- 10; MCP- 1, monocyte chemoattractant protein- 1; MIP- 1α,
MIP- 1β, macrophage inflammatory protein- 1α and 1β; PDGF- BB, platelet- derived grow th fac tor- BB; TNF- α, tumour necrosis factor- α; VEGF, vascular
endothelial growth factor; TGF- β, transforming growth factor- β.
p- values ≤ 0.05 were considered statistically significant and repor ted in bold.
TABLE 2 (Continued)

|
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GERBIN O Et al.
IL- 1β. Of note, the measurement of IL- 1ra levels rather than IL- 1α
or IL- 1β is a more reliable parameter of an increase in production
of IL- 1 family members in inflammator y conditions since IL- 1α and
IL- 1β lack the secretory peptide signal, and thus, they are not readily
secreted into the systemic circulation.
IL- 1 family members can act on cardiac resident macrophages,
neutrophils and parenchymal cells to trigger production of IL- 6, G-
CSF and other inflammator y mediators. It has been reported that IL- 1
is consistently upregulated in experimental models of heart failure
due to a wide range of aetiologies, including myocardial infarc tion
and diabetic cardiomyopathy.26 Moreover, there are experimental
evidence supporting the role of IL- 1 signalling in the pathogenesis
of cardiac dysfunction and adverse remodelling associated with
heart failure. For instance, KO mice for IL- 1 receptors showed at-
tenuated adverse remodelling after myocardial infarction, exhibit-
ing suppressed inflammatory responses.27 IL- 1 suppresses systolic
FIGURE 3 Serum levels of IL1- ra
(A) and IL- 8 (B) in the family enrolled in
the study and in healthy controls. **p <
.01; ***p < .001. Areas under the curves
(AUC) obtained with the sensitivity and
specificity for the serum levels of IL1- ra
(A’) and IL- 8 (B’) in patients with LMNA
mutant shown in A and in B respectively
FIGURE 4 Serum levels of IL6 (A) and
G- CSF (B) in the family enrolled in the
study and in healthy controls. **p < .01;
***p < .001; ****p < .0001. Areas under
the curves (AUC) obtained with the
sensitivity and specificity for the serum
levels of IL6 (A’) and G- CSF (B’) in patients
with LMNA mutant shown in A and in B,
respectively

10912
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G ERBINO E t al.
cardiomyocyte function by the disruption of calcium handling28
and by promulgating cardiomyocyte apoptosis.29 In agreement with
these experimental evidence, our data support the above pathoge-
netic mechanisms because we found high levels of IL- 1β in Families 1
and 3. In particular, these patients displayed signific antly lower LVEF
values and higher prevalence of serious arrhythmic events in com-
parison with patients from other families (Table 1). Both left ventric-
ular dysfunction and arrhythmias seem to be associated with IL- 1β
increased levels in our patients, thus potentially confirming the role
of this cytokine as a biomarker related to more severe cardiac me-
chanica l and electrical abnormalities in LMNA patients. Interestingly,
IL- 1ra and G- CSF levels were found upregulated in patient cohort
affected by striated muscle laminopathies, including LMNA cardio-
myopathy,30 thus suggesting not only a link of these cytokines with
cardiac dysfunction but also laminopathies in general.
The role of IL- 6 in the pathogenesis of cardiac disease is well es-
tablished in experimental models and in humans. IL- 6 is consistently
upregulated in experimental models of cardiac injury and hear t
failure regardless of the underlying aetiology and is expressed by
cardiomyocytes, infiltrating mononuclear cells and fibroblasts.31,32 In
cardiomyocytes, IL- 6 is able to decrease intracellular Ca2+ transients
and depress cell contraction through a nitric oxide (NO)- cGMP-
mediated pathway,33,34 and in fibroblasts, IL- 6 promotes prolifera-
tion and stimulates extracellular matrix synthesis.34 Moreover, in in
vivo studies, infusion of IL- 6 caused hyper trophy and fibrosis, and
increased myocardial stiffness in mice.35
Importantly, high plasma levels of IL- 6 can provide prognostic in-
formation in patient s with chronic heart failure (CHF), independently
of ventricular dysfunction and of aetiology, suggesting an important
role for IL- 6 in the pathophysiology of HF.36,37 Regardless of specific
aetiology and organ localization, systemic inflammation, via IL- 6 eleva-
tion, rapidly induces atrial electrical remodelling and electrical instabil-
ity thus increasing susceptibility to atrial fibrillation, by downregulating
cardiac connexins.38 Emerging experimental evidence suggests that
IL- 6 may play a critical role in contributing to the modulation of ICa,
L and IK current s, and both factors are active contributors to cardiac
FIGURE 5 Serum levels of IL- 1β, IL- 4,
IL- 5 and GM- CSF in some of the family
enrolled in the study and in healthy
controls. **p < .01; ***p < .001

|
10913
GERBIN O Et al.
instabilities.39 In accordance with these experimental findings, atrial
fibrill ation occurrence w as documented in all e valuated LMNA fami lies.
IL- 8, which also resulted upregulated in most of the patients in-
volved in this study, has been shown to be induced in the failing myo-
cardium,40 and it was reported to predict the development of left
ventricular dysfunction and the following HF together with IL- 6.41
Interestingly, Family 4 with a neuromuscular phenotype shows
circulating levels MIP- 1α significantly higher than those from other
family patients or control subject s. The association between high ex-
pression of this biomarker and neuromuscular phenotype in LMNA
variant patients is consistent with data from Cappelletti C et al. who
examined the cytokine profile in patients with striated muscle lami-
nopathies.30 Thus, MIP- 1α could be associated with skeletal muscle
involvement in cardiac laminopathy patients.
In addition to the cy tokines discussed so far, IL- 4, IL- 5 and GM-
CSF resulted also significantly increased in the serum of some pa-
tients enrolled in the study.
A recent and elegant study demonstrated the cooperative role of
IL- 5 and IL- 4 in the development of inflammatory dilated cardiomy-
opathy (DCMi). Transgenic mice overexpressing IL- 5 developed eo-
sinophil infiltration and a severe spontaneous cardiac enlargement.
The role of IL- 5 in the pathogenesis of DCMi was to mobilize IL- 4-
producing eosinophils into the myocardium, which then in turn were
responsible for the dilated cardiomyopathy.42 Accordingly, several
studies have shown a positive correlation between systemic IL- 4 lev-
els and cardiac fibrotic remodelling and dilation in both patients and
experimental animals.43- 45
Interestingly, p. Leu140_Ala146dup carriers with the widest
spectrum of pro- inflammatory circulating cytokines dysregulated
show the most severe cardiac phenotype in our cohort of LMNA
mutant carriers. Notably, IL- 4 and GM- CSF resulted to be highly ex-
pressed only in patients belonging to the above family. Since this
family displayed the most severe cardiac phenotype, both in terms
of cardiac function impairment and in relationship with malignant
arrhythmic event s occurrence, this further supports the hypothesis
that the pro- inflammatory cy tokines we found upregulated in our
study, significantly contribute to pathogenesis of the LMNA cardio-
myopathy at least in our cohort of LMNA mutant carriers.
To gain more insight s on the molecular events involved in the
inflammatory response in our patient s, we paid attention to the heat
shock proteins since some of them have been shown to be potent
activators of the innate immune system.46
Of note, we found significantly elevated expression levels of
Hsp70 in the serum exosomes of LMNA- p. Leu140_Ala146dup car-
riers and a significant increase in Hsp70 upregulation in LMNA- p.
Leu140_Ala146dup- expressing cells. It has been repor ted that
Hsp70 is released from cardiomyocytes undergoing lysis, necrosis
and apoptosis, as well as via active secretion in response to a vari-
ety of stress stimuli, including ischaemia and oxidative stress.47, 48
Accordingly, we previously reported that LMNA- p. Leu140_
Ala146dup- expressing cardiomyocytes have decreased nuclear
stability, resulting in a higher rate of apoptosis.17 High levels of cir-
culating Hps70 have been detec ted in patients with acute myocar-
dial infarction correlating with the extent of myoc ardial damage.49
Moreover, GM- CSF has been reported to significantly increases the
expression of Hsp70 in infarcted myocardium in mice.50 Indeed, the
most severe cardiac phenotype and the upregulated levels of GM-
CSF found only in LMNA- p. Leu140_Ala146dup carriers may ac-
count for the elevated levels of circulating Hsp70 in these patient s.
Of note, Hsp70 and IL- 6 are potential new therapeutic targets for
this subset of cardiolaminopathies.
In fact, a significant cardioprotective effect of the anti- Hsp70
blocking antibody has been demonstrated in an HF mouse model,
with resolution of myocardial inflammation, left ventricular dilation
FIGURE 6 Hsp70 expression in patients’ serum exosomes and in LMNA- p. Leu140_Ala146dup- expressing cells. (A) Western blot ting on
serum exosomes from LMNA- p. Leu140_Ala146dup- carrying patients (p. Leu140) and healthy controls (controls) with anti- Hsp70 antibodies.
Equal volumes of sera from each patient were loaded. (B) Western blotting on serum exosomes from LMNA- p. Leu140_Ala146dup- carrying
patients (p. Leu140) and healthy controls (controls) with anti- CD81, anti- CD- 9 antibodies used as markers of exosomes purity. GAPDH
absence is also index of exosome purity and was included in the panel. (C) Densitometric analysis of Hsp70- immunoreactive bands in serum
exosomes from all patients and controls included in the study. The data are means of 3 independent experiments. *p < .05. (D) Western
blotting of Hsp70 and LMNA in either LMNA WT or LMNA- p. Leu140_Ala146dup- expressing HEK293 cells. (E) Densitometric analysis
of Hsp70- immunoreactive bands in either LMNA W T or LMNA- p. Leu140_ Ala146dup- expressing HEK293 cells. The data are means of 3
independent experiments. **p < .01

10914
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G ERBINO E t al.
and dysfunction and a significant inhibition of cardiac fibrosis.51
Moreover, Hsp70 blocking improves cardiac functional recovery in
mice after global ischaemia reperfusion and reduces expression of
the pro- inflammatory cytokines TNF- α, IL- 1β and IL- 6.52 In the clin-
ical practice, an anti- IL6 receptor antibody (tocilizumab) is widely
used to treat the abnormal inflammator y response that occurs in
autoimmune diseases as rheumatoid arthritis and it is well tolerated
by patients.53 In addition, its in vivo administration to the mouse
model of progeria significantly ameliorates the progeroid pheno-
type, including cardiac histology in these mice.54 Of note, the anti-
IL- 6 receptor antibody (MR16- 1) prevents the development of LV
remodelling after MI in mice.55
5 | CONCLUSIONS
Clinical course of cardiac laminopathies is characterized by a poor
prognosis and a high rate of major cardiac events. So far, therapeutic
approaches are exclusively symptomatic. Improvement in therapeu-
tic management might come from ver y early treatment with drugs
hopefully already used in clinical practice. In this scenario, we be-
lieve that our data are of great interest on the translational point of
view. The main finding of our study is that inflammatory cytokines
could significantly contribute to the pathogenesis of the cardiomyo-
pathy in lmna mutation carriers and correlate with the severity of the
cardiac phenotype. Indeed, early identification of dysregulated pro-
inflammatory serum cytokines in LMNA- cardiomyopathy patients
could be crucial for therapeutic approaches able to counteract the
progression of the disease and to finally improve the prognosis of
this subset of severe cardiomyopathies.
ACKNOWLEDGEMENT
This work wa s sup por te d by fu ndi ng fro m ‘C arm osin o19LAMIN OPATIE’
and ‘Carmosino20RIL’ to Monica Carmosino and from the CLUSTER
TECNOLOGICO REGIONALE ‘DICLIMAX’ (project # MTJU9H8) to
Maria Svelto.
CONFLICT OF INTEREST
The authors have no conflict of interest to declare.
AUTHOR CONTRIBUTIONS
Andrea Gerbino: Conceptualization (equal); Data curation (equal);
Investigation (equal); Writing- original draft (equal). Cinzia Forleo:
Data curation (equal); Methodology (equal); Resources (equal);
Writing- original draft (equal). Serena Milano: Data curation (equal);
Formal analysis (supporting); Investigation (equal). Francesca
Piccapane: Data curation (equal); Methodology (equal). Giuseppe
Procino: Writing- review & editing (equal). Martino Pepe: Resources
(equal). Mara Piccolo: Resources (equal). Piero Guida: Data curation
(lead). Nicoletta Resta: Data curation; Investigation (equal). Stefano
Favale: Writing- review & editing (equal). Maria Svelto: Funding ac-
quisition (lead); Writing- review & editing (equal). Monica Carmosino:
Conceptualization (equal); Formal analysis (supporting); Funding ac-
quisition (supporting); Project administration (lead).
DATA AVAIL ABI LIT Y STAT EME NT
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Monica Carmosino https://orcid.org/0000-0001-7600-8816
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SUPPORTING INFORMATION
Additional supporting information may be found in the online ver-
sion of the ar ticle at the publisher’s website.
How to cite this article: Gerbino A, Forleo C, Milano S, et al.
Pro- inflammatory cytokines as emerging molecular
determinants in cardiolaminopathies. J Cell Mol Med.
2021;25:10902– 10915. https://doi. or g/10.1111/ jcmm.16975