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.

Despite the molecular mechanism involved in LMNA cardiomyopathies, the deterioration in electrical and contractile function correlates 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 instability and mechanical impairment in cardiac laminopathies 13 typically 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 resident macrophages that proliferate following myocardial damage such as in myocardial infarction, which in turn recruit other monocytes to the heart, contributing to myocardial interstitial fibrosis and adverse cardiac remodelling. 15 Moreover, it also well established that proinflammatory cytokines are produced in typical inflammatory cardiomyopathies 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 dysfunction and cardiac remodelling have not been fully investigated. Therefore, in this study, we aim at investigating the specific profile of 30

| Clinical and instrumental analysis
The patients who were referred to the Cardiomyopathy Unit, Cardiology 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 mutationnegative 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 study. The project conformed to the principles of the Declaration of Helsinki (World Medical Association) and was approved by the Ethics Committee of the University Hospital Consortium, Policlinico of Bari, Italy.

| Cytokine/chemokine assay
Plasma samples from the patients included in the study were prepared 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 Factorβ (TGFβ) 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 cytokines analysed and for detailed procedure, see Appendix S1.

| Serum exosome preparation and analysis
Serum exosomes were isolated from 250 μl of each patients' serum with the exosome isolation kit EXOQ50A-1 (System Biosciences) according to manufacturer's instructions. Serum exosomes were analysed by Western blotting for the expression of Hsp70 and exosome markers. For details on serum exosomes preparation and analysis, see Appendix S1.

| Cell culture and western blotting
HEK293 cells were transiently transfected with the previously de-

| 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 software version 14 (Stata). Student's t test for unpaired data was used to analyse differences in cytokines levels between each patient group and controls. Receiver operating characteristic (ROC) curves were used to estimate the diagnostic potential of the quantified individual cytokines to discriminate between groups. GraphPad Prism software (version 8) was used for statistical and graphical arts.
In all cases, significance was considered at p < 0.05.

| Molecular analysis of the patients' population
The lmna mutations included in this study were identified in members of Italian families with cardiac phenotypes screened in our Clinical Unit dedicated to cardiomyopathies.
The lmna mutations are as follows:
p-values ≤ 0.05 were considered statistically significant and reported in bold.
detected in three of them ( Figure 2D). Finally, a patient of this group underwent heart transplant (after unsuccessful VT trans-catheter ablation) at the age of 45 due to sustained VT recurrences in storm and subsequent LV function deterioration (LEVF 30%). 17

| Family 2
The patients of this family, with the p. Arg110Leufs*7 LMNA pathogenic variant, showed a less aggressive clinical phenotype in comparison with members of the previously described family. In these subjects, ventricular dimensions were in the normal range (LVEDD LVEDVi 90 ± 7 ml/m 2 ) and systolic dysfunction (LVEF 41 ± 15%).
Three patients also displayed conduction disorders: 2 of them presented first-degree AV-blocks with very long PR intervals (the longest reported was 520 ms), while the third patient developed high-degree AV block and received a dual-chamber pacemaker subsequently upgraded to a biventricular defibrillator. Atrial fibrillation and NSVT were found in 50% of these patients, while 2 patients also presented with SVT (one of them occurring during exercise testing, and the other was detected on defibrillator arrhythmia registry). One patient received an ICD in primary prevention due to severe left ventricular dysfunction (LVEF 20%), which subsequently progressively improved (last reported LVEF was 47%) after optimized drug treatment for heart failure, and the occurrence of several appropriate defibrillator interventions by ATP on SVT was also reported.

| Family 4
The p. Arg190Gln LMNA variant was related to neuromuscular involvement consisting of muscle cramps, reduced strength to stress and inappropriate muscular hypertrophy, associated with 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 during 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 abnormalities (Table 1).

| 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 undetectable (~1 pg/ml). 22 The levels of several pro-inflammatory cytokines resulted upregulated 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 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 variants and controls enrolled in our study with high degree of accuracy.
Interestingly, the levels of both IL-6 and G-CSF were significantly increased in all patients compared with controls ( Figure 4A, B,

| Analysis of Hsp70 in serum exosomes of LMNA mutant carriers
It has been reported that extracellular Hsp70 may act through surface receptors stimulating release of pro-inflammatory cytokines 23 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 phenotype in LMNA mutant carriers, we analysed the expression of Hsp70 in serum exosomes of both patients and controls involved in the study.
TA B L E 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

(Continues)
We found an increase in the Hsp70 expression in serum exosomes from LMNA-p. Leu140_Ala146dup carriers compared with controls ( Figure 6A). The same exosomes were tested for the expression 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 between the elevated serum levels of Hsp70 in serum exosomes from LMNA-p. Leu140_Ala146dup carriers and the specific LMNA mutant, 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.

| DISCUSS ION
In this work, we found that specific pro-inflammatory cytokines resulted upregulated in a cohort of patients affected by cardiomyopathy due to different mutations in lmna gene.
Carriers expressing the pathogenic LMNA variant are characterized by bradycardia, AF, a significant 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 controls, respectively ( Table 1).
p-values ≤ 0.05 were considered statistically significant and reported in bold.

TA B L E 2 (Continued)
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 inflammatory conditions since IL-1α and IL-1β lack the secretory peptide signal, and thus, they are not readily and by promulgating cardiomyocyte apoptosis. 29 In agreement with these experimental evidence, our data support the above pathogenetic mechanisms because we found high levels of IL-1β in Families 1 and 3. In particular, these patients displayed significantly lower LVEF values and higher prevalence of serious arrhythmic events in comparison with patients from other families (Table 1) To gain more insights on the molecular events involved in the inflammatory response in our patients, 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 carriers and a significant increase in Hsp70 upregulation in LMNA-p.
Leu140_Ala146dup-expressing cells. It has been reported that Hsp70 is released from cardiomyocytes undergoing lysis, necrosis and apoptosis, as well as via active secretion in response to a variety 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 circulating Hps70 have been detected in patients with acute myocardial infarction correlating with the extent of myocardial damage. 49 Moreover, GM-CSF has been reported to significantly increases the expression of Hsp70 in infarcted myocardium in mice. 50  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 TNFα, IL-1β and IL-6. 52 In the clinical practice, an anti-IL6 receptor antibody (tocilizumab) is widely used to treat the abnormal inflammatory 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 phenotype, 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

| CON CLUS IONS
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 therapeutic management might come from very early treatment with drugs hopefully already used in clinical practice. In this scenario, we believe 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 cardiomyopathy in lmna mutation carriers and correlate with the severity of the cardiac phenotype. Indeed, early identification of dysregulated proinflammatory 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.

ACK N OWLED G EM ENT
This work was supported by funding from 'Carmosino19LAMINOPATIE' and 'Carmosino20RIL' to Monica Carmosino and from the CLUSTER TECNOLOGICO REGIONALE 'DICLIMAX' (project # MTJU9H8) to Maria Svelto.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.