stands for the number of analyzed proband for each variant on LMNA gene.

Variant classification has been made applying the ACMG/AMP guidelines [24] (Table 2). Among 23 heterozygous patients, 18 (15 males and 3 females) (Table 3) referred symptoms and/or signs of DCM, while 5 subjects were asymptomatic with apparently no signs of the disease, most likely due to their young age (from 11 to 27 years old).


All variants were located within the coil domain (Figure 1), except for W467X, a non-sense variant within the tail domain. Its clinical features are very aggressive: early onset (3rd decade), LV dilation, severe reduced systolic function, large scar in the IVS and inferior wall, and complex ventricular arrhythmias. We also characterized a proband compound heterozygous (R216H/R331L) presenting worse clinical and instrumental findings. The patient (female) with onset of symptoms in the 6th decade, and severe LV systolic dysfunction (LVEF 30%), experienced supraventricular and ventricular arrhythmias, with multiple appropriate ICD shocks.

The mean age of signs/symptoms onset in 18 symptomatic patients was 51.3 ± 12.9 years. At echocardiographic examination, mean LVEDDi was 29.2 ± 4.3 mm/m2 with LVEF 42.6 ± 10.2%, LA dilation was present in 15 of them (83.3%). Three patients (16.6%) had right heart involvement with RV dilation and dysfunction (CG11, CG12, and CG14). Cardiac MR performed in 15 phenotype-positive patients showed a LVEF of 46 ± 12% and generally a mild-to-moderate LV enlargement with mean LVEDVi 86 ± 32.8 mL/m2. A late gadolinium enhancement (LGE) as a sign of fibrosis was present in 13 of 15 affected subjects that underwent cardiac MR (86.6%), with interventricular septum (IVS) involvement in nine of them (69.2%). Analysis of basal ECG showed AV delay in 10 of 17 patients in sinus rhythm (58.8%) and IV delay in 9 of 18 patients (50%) (LBBB in 66.6%), a mean cQT of 420 ± 28 ms. The first clinical manifestation was ventricular arrhythmias (VA) in eight (44.5%) patients, advanced atrioventricular block in four (22.2%), and left ventricular dysfunction in six (33.3%). Twelve (66.6%) patients underwent ICD implantation: nine patients received ICD implantation before diagnosis of lamin cardiomyopathy was made because of the occurrence of ventricular arrhythmias in eight (CG02, CG03, CG05, CG06, CG07, CG09, CG10, and CG11) as secondary prevention and because of severe LV dysfunction in one (CG12) as primary prevention; two patients (CG8A and CG14A) whose onset was characterized by moderate LVEF dysfunction underwent ICD implantation once LMNA mutation was diagnosed, while in one patient (CG13), first clinical presentation was an AVB, and so a PMK was implanted, and, only after diagnosis of LMNA, an upgrade to ICD was performed. As regards patients CG8A, CG14A, and CG13, they received ICD implantation after genetic diagnosis of a LMNA variant according to the European Guidelines that suggest ICD implantation in lamin DCM if two of the following conditions are met: male sex, non-sustained VT, non-missense LMNA variant, and LVEF < 45% (patient CG8A: male sex, non-missense variant, LVEF 44%; patient CG14A: male sex, LVEF 45%; patient CG13: male sex, evidence of VA at PMK interrogation).



arrhythmias;

 AF: atrial fibrillstion; VT/VF: ventricular

tachycardia/ventricular

 fibrillation; ICD: implantable

cardioverter–defibrillator.

**Figure 1.** Schematic representation of lamin A transcript and localization of exonic variants identified in this study. \* stands for nonsense mutations while = stands for synonymous mutations.

In eight patients (CG02, CG03, CG05, CG06, CG07, CG09, CG10, and CG11), whose clinical onset was characterized by ventricular arrhythmias, ICD was implanted as secondary prevention before diagnosis of lamin cardiomyopathy, as well as in another patient (CG12) that presented as the first phenotypic sign a severe reduction of the LVEF (20%); ICD was implanted as primary prevention before diagnosis of the LMNA variant.

Two patients (CG8A and CG14A) had a moderate LV dysfunction (LVEF 44% and 45%) and underwent ICD implantation after diagnosis of laminopathy because they both met the criteria for ICD implantation (as reported in the discussion, the European Guidelines recommend ICD implantation in lamin DCM if two of the following risk factors are present: male sex, non-sustained VT, non-missense variant, and LVEF < 45%):



One patient (CG13) had already undergone PMK implantation for AVB, and once diagnosis of LMNA variant was made, an upgrade to ICD was performed (male sex, evidence of VA at PMK interrogation). A mean follow-up of 31.5 months was achieved, during which seven (38.8%) patients experienced AF; two patients (14%) developed new AVB. Eleven (61.1%) patients experienced VA. At ICD interrogation, 8 of 12 patients (66.6%) received an appropriate ICD therapy (shock in 62.5%) (Table 3).

One patient (CG12) received heart transplantation for end-stage heart failure. Regarding the remaining five patients without a DCM clinical phenotype, all clinical and instrumental assessments were normal except for the presence of LA dilatation in two of them (two young brothers, IV7 and IV9, belonging to Family 14, with a left atrium volume of, respectively, 36.1 and 39.6 mL/m2).

#### *3.2. In Vitro Characterization of Two Novel LMNA Variants*

One patient belonging to Family 6 (III-2) and two brothers belonging to Family 14 (IV-7 and IV-9) underwent dermal biopsy in order to obtain in vitro fibroblasts (HDFs) (Figure 2). The missense R189Q variant of unknown significance (VUS) segregates in the daughter (IV-1) but not in the sibling (III-3 and III-4) of Family 6 (Figure 2A). In Family 14 (Figure 2B), the missense E317K is classified on ClinVar as likely pathogenic for DCM and reported in an Italian family with DCM and atrioventricular block [4].

Both lamin A/C and prelamin expression have been investigated, comparing them to healthy controls (WT). Lamin A/C localization, mainly situated at the nuclear peripheric rim, was comparable between WT and DCM-derived HDFs (99.8% positive cells), after immunofluorescence staining (Figure 3A). About 4.3% of WT nuclei were positive for prelamin A, as well as the IV-7 and IV-9 nuclei (5% and 4.98%, respectively), while III-2 nuclei (Family 6) were 12.2%. In these cells, a more punctate localization pattern relative to prelamin A was also revealed. These intra-nuclear aggregates differ significantly in size and number between cells within the same culture, and they are distributed next to a typical nuclear rim (Figure 3A, III-2). The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei was found to be the most discriminating parameter between patient and control cells. In fact, 6% of IV-7, 7.7% of IV-9, and 6.68% of III-2 nuclei showed an altered shape with nuclear invaginations and

blebs, considered typical markers associated with LMNA variants (Figure 3A,B). Results have evidenced statistically significant differences in circularity, roundness, and nuclear elongation (Figure 3C, \* *p* < 0.05). The percentage of DCM irregular nuclei strongly increases compared to WT cells, in which these alterations are present only in 1%, at the same age and number of passages (p3) (Figure 3B,C).

Successively, transcript and protein expression have been evaluated (Figure 3D), comparing quantitatively lamin A and C in DCMs with respect to WTs. In all patients, both lamin isoforms were decreased compared to WT in a statistically significant manner, except for the IV-7 patient, in whom lamin A expression slightly increased (Figure 3D). Protein quantification performed by Western blot confirmed a marked reduction of both lamin A and C isoforms (Figure 3E,F), except in IV-7, as expected, who did not show any significant protein reduction (Figure 3E).

**Figure 2.** (**A**) Segregation of R189Q in Family 6. (**B**) Segregation of E317K in Family 14. Arrows indicate the proband. Wt: wild type. \* stands for positive patients to Lamin A/C gene (LMNA) variants.

**Figure 3.** (**A**) Representative immunolabeling for lamin A/C (red) and Prelamin A (red) of WT and DCM HDFs (IV-7, IV-9, and III-2). Nuclei are counterstained with Hoechst 33,342 (blue). Scale bar 20 μm, 100 μm. (**B**) Percentage of WT and DCM cells with abnormal nuclear irregularities revealed in HDFs patients. Results represent three independent experiments with significant differences between WT and DCM HDFs (\*\* *p* < 0.01). (**C**) Bar graphs represent the four parameters relative to nuclear shape (area; circularity; elongation; roundness); they are reported as mean values ± SD (fold DCM vs. WT) of three independent experiments. Significant differences are denoted by the *p*-value (Student's two-tailed *t*-test; \* *p* < 0.05. (**D**) RT-qPCR of lamin A and C transcripts in WT and DCM HDFs; GAPDH was used as reference gene. Data are from three independent experiments and represented as mean ± SD (\* *p* < 0.05). (**E**) Densitometric analysis of Western blot performed on WT and DCM HDFs, showing the intensity of the band corresponding to lamin A and C normalized versus β-actin levels. WT densitometric value is the average between two different controls (\* *p* < 0.05; \*\* *p* < 0.01). Data are presented as mean ± SD. (**F**) Representative Western blot of lamin A and C; β-actin is used as housekeeping gene.

#### **4. Discussion**

LMNA-related DCMs have a more aggressive clinical course compared to other forms of dilated cardiomyopathies with higher rates of potentially fatal arrhythmias and end-stage heart failure [25]. The prevalence of LMNA mutations in familial DCM is about 5–10% [26]; however, only in 30–35% of familial cases, a Mendelian inheritance has been evidenced, suggesting a prevalent complex multi-variant or oligogenic basis of inheritance [27]. Moreover, a significant clinical heterogeneity has been reported within the same family in terms of onset, severity, and progression of the disease [28]. Both the genetic and phenotypic heterogeneities together with variable penetrance of LMNA variants make the pathogenic classification of the variants difficult.

LMNA variants occur in the head and rod domains, which comprise more than half of lamin A and two thirds of lamin C, but rarely in the tail domain [28]. The clinical heterogeneity observed in LMNA-DCM might be also explained by the different functional consequences of the variants. Most carriers exhibit an age-dependent penetrance: 7% under the age of 20 years to 100% above the age of 60 years [17].

Actually, a targeted therapy is not available for the early treatment of LMNA associated cardiac disease. However, the knowledge of the genotype of DCM patients allows a better prevention with ICD because of the high risk of SCD associated with LMNA variants.

The introduction of the NGS method in the daily practice of molecular diagnosis laboratories has allowed considerably reducing response times. The possibility to identify genetic variations in DCM patients allows early diagnosis before clinical manifestation, prognosis, genetic counselling, and preventive management of heterozygous subjects and their relatives.

In our cohort of patients with DCM, LMNA variants are present in about 19.5%. This finding is not in line with previous reports describing a 5–8% prevalence [29], probably because all patients come from a clinical center specialized in arrhythmology. Affected individuals frequently suffer from a progressive conduction system disease such as atrioventricular block, bradyarrhythmias, and tachyarrhythmias and have a high chance of developing thromboembolic disorder. It has been shown that men have a worse prognosis than women [30]; however, in our study, we have only three symptomatic female patients vs. 15 male patients, and we cannot draw any conclusions about this. Three variants described in our cohort belong to Class V, seven to Class IV, and one to Class III. Interestingly, the variant E317K was reported in Gnomad with only one allele count (frequency 3.19e-5), while in our study, it is present in six proband, allowing us to hypothesize a founder effect in the Italian population, as already reported for R331Q, a founder variant in autosomal dominant cardiac laminopathy with late-onset and mild cardiac phenotypes [31]. A specific study of haplotypes associated with this variant is necessary to confirm our hypothesis. Segregation analysis performed within each family lets us identify five members in the presymptomatic stage.

In total, 11 LMNA variants were identified in 15 families, and, as expected, phenotypepositive patients showed a complex cardiac phenotype ranging from a predominantly structural heart disease to a highly arrhythmic profile in an apparently normal heart. VA were the first relief in 44.5% of patients. This finding is of particular interest since VA generally do not appear as first clinical manifestation [32], with prevalence of any and all forms of sustained VA increasing from presentation to last follow-up [33].

Moreover, 62.5% of patients who experienced VT/VF as a first manifestation had a LVEF ≥50%. At follow-up, VA occurred in up to 61.1% of patients, and 66.6% of ICDimplanted patients received appropriate ICD therapy.

These data highlight once again the highly arrhythmic burden of LMNA-related DCM and the limits of systolic function in risk stratification, emphasizing instead the importance of a gene-based diagnosis. Systolic function evaluation is an important prognostic factor in DCM, but our data suggest its marginal role in predicting the risk of VA in patients with LMNA variants. Probably the early appearance of interstitial fibrosis together with ion channels anomalies [34] could represent the cause of ventricular arrhythmias even before systolic dysfunction occurs. In fact, as already recognized [35], the presence of a scar at CMR, generally located in the IVS, is extremely frequent in asymptomatic patients, and it represents a potential trigger of VA, explaining why VA often represents the first clinical manifestation in asymptomatic patients. In our study, MRI evidenced a scar in 87.5% of the patients who experienced VA. Moreover, EF represents only one of the four independent risk factors for malignant VA identified in lamin DCM: male sex, non-sustained VT, nonmissense variant, and LVEF < 45% [32]. According to the actual European Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death, ICD implantation in patient carriers of lamin variants should be considered when two of the above mentioned criteria are met [36]. The genetic analyses would be useful not only for diagnosing the laminopathy, but also for stratifying the prognosis of carriers [37].

Patients with LMNA-related DCM frequently face supraventricular arrhythmias including atrial fibrillation, atrial flutter, and focal atrial tachycardia, as expression of atrial disease [38]. In our study, the prevalence of supraventricular arrhythmias was 38.8% at follow-up, in line with other studies [33,39]. A common relief in our population was left atrial enlargement, found in 83.3% of symptomatic patients. Left atrial size is a wellknown predisposal factor for the occurrence of supraventricular arrhythmias, especially AF [40–42], commonly proposed as a barometer of diastolic burden and a predictor of common cardiovascular outcomes and cardiovascular death [43,44]. In our study, left atrial enlargement was the only abnormal finding evidenced in the two older male patients, aged 22 and 27 years, of the five total genotype positive asymptomatic patients. A third patient, female biological sex aged 26 years, showed an upper limit LA volume. Although this evidence comes from a low number of patients, we could speculate about atrial enlargement as an early marker of the disease in LMNA cardiomyopathies, rather than a mere consequence of pressure and/or volume overload due to LV dysfunction and worse LV compliance. The LA enlargement due to a "primitive LMNA induced atrial myopathy", if confirmed in further studies, could represent an important relief to look for in genotype-positive phenotype-negative subjects, as initial sign of structural heart disease. Moreover, defining molecular and cellular mechanism causing the "primitive LMNA induced atrial myopathy" could lead to identify novel pathogenic mechanisms involved in supraventricular arrhythmias' occurrence.

Successively, lamin expression and distribution were evaluated in vitro on HDFs carrying two novel LMNA variants: R189Q and E317K. Moreover, nuclear abnormalities and irregularities in lamin staining were assessed in order to correlate them to variant nature and disease phenotype [45–49]. The increased percentage of abnormal nuclei, irrespective of the type of nuclear malformation, is the most discriminating parameter between normal and lamin-defective cells [50], correlating with nuclear architecture instability [51]. Lamins are components of the nuclear lamina providing mechanical stability to the nucleus [52–57]. HDFs carrying R189Q displayed an abnormal prelamin accumulation, which usually correlates with senescent cells and premature aging. Accumulated prelamin A causes the captures of the transcription factor Sp1, resulting in altered extracellular matrix gene expression [58]. Nuclear dysmorphisms and nuclear envelope disorganization appear as a hallmark of human cultured laminopathic cells, independently of the associated clinical presentation [49,59,60].

Additionally, the transcription levels of both Lamin A and Lamin C have been evaluated. They are usually incorporated in the nuclear lamina in equivalent amounts and play distinct functions: any expression ratio variations may be due to altered splicing or mRNA stability [52].

In all patients, lamin C expression is statistically significantly reduced if compared to WT ones. In the III-2 patient, lamin A was also significantly reduced. Overall, lamin A/C ratio is modified from 1:1 value, especially in IV-7, in whom lamin A expression increased with respect to the younger brother. This unbalance, due to an aberrant splicing process, may lead to altered interaction with other important structural proteins and transcription factors, as well as altered chromatin interaction [61]. These data are confirmed at protein level in III-2 and IV-9 patients, expressing lower quantities of both protein isoforms, as evidenced by densitometric analysis of Western blot, suggesting a possible defective mechanotransduction and an enhanced nuclear fragility.

Importantly, the different behavior between two brothers (IV-7 and IV-9) in lamin expression can explain the worse DCM phenotype evidenced in the youngest one. These data confirm the key role played by lamin in conferring a greater susceptibility to physical stress, especially in tissues exposed to mechanical strain, such as cardiac muscle [62].

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10215075/s1, Table S1: Genes sequenced for SCD.

**Author Contributions:** Conceptualization, V.F., J.C., F.S. and R.M.; methodology, V.F., J.C., P.S., M.M. and C.D.M.; validation, F.S., G.N., L.C., F.F. and R.M.; formal analysis, F.R., S.M., G.P., F.D.L. and A.M.; data curation, J.C. and V.F.; writing—original draft preparation, J.C., V.F., M.M. and P.S.; writing review and editing, F.S. and R.M.; supervision, F.S., R.M., G.N. and L.C.; project administration, F.S. and R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by Committees on Health Research Ethics of Tor Vergata Hospital n.2932/2017.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Variant submitted to ClinVar, Submission ID: SUB9594435.

**Acknowledgments:** Authors thank clinicians and patients for their collaboration.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Continuous Electrical Monitoring in Patients with Arrhythmic Myocarditis: Insights from a Referral Center**

**Giovanni Peretto 1,2,3,\*, Patrizio Mazzone 1, Gabriele Paglino 1, Alessandra Marzi 1, Georgios Tsitsinakis 1, Stefania Rizzo 4, Cristina Basso 4, Paolo Della Bella <sup>1</sup> and Simone Sala 1,2**


**Abstract:** Background. The incidence and burden of arrhythmias in myocarditis are under-reported. Objective. We aimed to assess the diagnostic yield and clinical impact of continuous arrhythmia monitoring (CAM) in patients with arrhythmic myocarditis. Methods. We enrolled consecutive adult patients (*n* = 104; 71% males, age 47 ± 11 year, mean LVEF 50 ± 13%) with biopsy-proven active myocarditis and de novo ventricular arrhythmias (VAs). All patients underwent prospective monitoring by both sequential 24-h Holter ECGs and CAM, including either ICD (*n* = 62; 60%) or loop recorder (*n* = 42; 40%). Results. By 3.7 ± 1.6 year follow up, 45 patients (43%) had VT, 67 (64%) NSVT and 102 (98%) premature ventricular complexes (PVC). As compared to the Holter ECG (average 9.5 exams per patient), CAM identified more patients with VA (VT: 45 vs. 4; NSVT: 64 vs. 45; both *p* < 0.001), more VA episodes (VT: 100 vs. 4%; NSVT: 91 vs. 12%) and earlier NSVT timing (median 6 vs. 24 months, *p* < 0.001). The extensive ICD implantation strategy was proven beneficial in 80% of the population. Histological signs of chronically active myocarditis (*n* = 73, 70%) and anteroseptal late gadolinium enhancement (*n* = 26, 25%) were significantly associated with the occurrence of VTs during follow up, even in the primary prevention subgroup. Conclusion. In patients with arrhythmic myocarditis, CAM allowed accurate arrhythmia detection and showed a considerable clinical impact.

**Keywords:** myocarditis; arrhythmias; telemonitoring; implantable cardioverter defibrillator; implantable loop recorder; Holter ECG

#### **1. Introduction**

Continuous arrhythmia monitoring (CAM) via implantable devices represents the gold standard for the detection of arrhythmias under many medical conditions [1,2]. In fact, in contrast to non-continuous monitoring by either Holter ECGs or short-term external devices [3], CAM allows the continuous and potentially life-long evaluation of cardiac electrical activity. In myocarditis, CAM may be useful to fill in relevant knowledge gaps on the incidence, type and burden of arrhythmias [4,5]. This is clinically important since ventricular arrhythmias (VAs) and bradyarrhythmias (BAs) constitute life-threatening complications of myocarditis [6,7]. Furthermore, the incidence of atrial fibrillation (AF) and other supraventricular arrhythmias (SVAs) is unknown in this setting. To date, no studies have investigated the benefits of CAM application in patients with myocarditis. In fact, indications for implantable cardioverter defibrillators (ICDs) are restricted in this population [5,6] and there is currently no experience about the use of implantable loop recorders (ILRs) as long-term monitoring devices. Because of the episodic nature of arrhythmias, we

**Citation:** Peretto, G.; Mazzone, P.; Paglino, G.; Marzi, A.; Tsitsinakis, G.; Rizzo, S.; Basso, C.; Della Bella, P.; Sala, S. Continuous Electrical Monitoring in Patients with Arrhythmic Myocarditis: Insights from a Referral Center. *J. Clin. Med.* **2021**, *10*, 5142. https://doi.org/10.3390/ jcm10215142

Academic Editor: Michael Henein

Received: 28 September 2021 Accepted: 29 October 2021 Published: 1 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

hypothesized that, even in the myocarditis population, CAM had a superior diagnostic yield compared to even regularly repeated Holter ECGs. In addition, we aimed to assess the appropriateness of the ICD implantation strategy in patients presenting with clinically defined acute myocarditis but heterogeneous histopathological findings.

#### **2. Methods**

#### *2.1. Study Design*

This was a single-center observational study with a prospective follow up reflecting the experience of a referral center. This study was in compliance with the Declaration of Helsinki and underwent Institutional Review Board approval. The study flowchart is presented in Figure 1. Between January 2013 and January 2019, consecutive patients with arrhythmic myocarditis were enrolled. The following inclusion criteria were applied: (1) age ≥ 18 year; (2) EMB-proven diagnosis of active myocarditis [5]; (3) evidence of previously unknown (or de novo) arrhythmias at index hospitalization; and (4) a CAM strategy started within 30 days from myocarditis diagnosis.

**Figure 1.** Study flowchart: study design with inclusion criteria is shown. AF = atrial fibrillation; AFlu = atrial flutter; AT = atrial tachycardia; AVB = atrioventricular blocks; CAM = continuous arrhythmia monitoring; CMR = cardiac magnetic resonance; EMB = endomyocardial biopsy; FU = follow up; NSVT = nonsustained ventricular tachycardia; PVC = premature ventricular complexes; VA = ventricular arrhythmia; VF = ventricular fibrillation; VT = ventricular tachycardia.

As part of the baseline diagnostic work-up, all patients underwent complete blood exams, continuous 12-lead ECG telemonitoring, transthoracic echocardiogram and cardiac magnetic resonance (CMR).

#### *2.2. Definitions*

Arrhythmias were defined based on updated standards [8–10] and classified into VA, SVA and BA. In detail, VA included ventricular fibrillation (VF), tachycardia (VT), nonsustained VT (NSVT) and grade ≥2 premature ventricular complexes (PVCs) according to Lown's classification (i.e., >1 PVC/min or >30 PVC/h) [11]; SVA included AF, atrial flutter and atrial tachycardia; BA included 2nd degree type II, 2:1, or 3rd degree atrioventricular blocks (AVBs) and pauses >3 s. Further definitions, including details concerning VA characterization, are reported in the Supplementary Materials.

Histological signs of fibrosis, cardiac myocyte hypertrophy and nuclear atypia were used to define "chronically active" rather than true "acute" myocarditis [12,13].

#### *2.3. CAM Selection*

In the absence of clear guideline recommendations for patients with chronically active myocarditis [5–7], the choice between ICD and ILR was patient-tailored and guided the experience of a referral center for arrhythmia management [14]. In detail, the following putative risk factors were identified a priori as markers of arrhythmic risk: (1) left ventricular ejection fraction (LVEF) < 35% at baseline echocardiogram; (2) non-lymphocytic histotypes, namely cardiac sarcoidosis and giant cell myocarditis; (3) 2nd or 3rd degree AVB; (4) fast (>180 bpm for at least 10 beats) or recurrent (>3 episodes at telemonitoring) NSVT despite antiarrhythmic therapy; (5) induction of VT or VF at baseline programmed ventricular stimulation (PVS) when applicable; (6) extensive areas of either late gadolinium enhancement (LGE) at CMR (>1 LV wall, or >5 of 17 LV segments) or replacement fibrosis at histology (>50% of tissue samples).

For secondary prevention, the ICD implant was indicated following either VT or VF onset. Otherwise, CAM was proposed to all patients: the decision between the primary prevention ICD and ILR implant was personalized, and guided by the above defined risk factors. Details about CAM programming are reported in the Supplementary Materials.

#### *2.4. Follow-Up*

All patients underwent prospective follow-up (FU) reassessment [15] through both CAM and 12-lead 24 h Holter ECGs, according to a defined schedule (4/year in the first year; 2/year in years 2–5; and then 1/year). Both in-person and remote monitoring were allowed for CAM, and the arrhythmia timeline was defined by the real event date. The association with symptoms was assessed both by the analysis of manually activated device alerts, and by direct patient interrogation.

#### *2.5. Endpoints*

VA occurrence, burden and timing—as detected by CAM vs. Holter ECG monitoring were analyzed as the primary study endpoint. During FU, appropriate ICD interventions (anti-tachycardia pacing or shock) also constituted VT events. The occurrence of other arrhythmias (SVA, BA) constituted the secondary endpoints. In addition, the appropriateness of the ICD implantation strategy was retrospectively evaluated.

#### *2.6. Statistical Analysis*

SPSS Version 20 (IBM Corp., Armonk, NY, USA) was used for the analysis, and Prism Version 6 (GraphPad Software Inc., La Jolla, CA, USA) was used for graphic presentations. Continuous variables were expressed as the mean and standard deviation, or as median and IQR of 25th to 75th percentiles, depending on the distribution of data. Accordingly, continuous variables were compared by Student's *t*-test or by Mann–Whitney U-test. Categorical variables, reported as counts and percentages, were compared by the Fisher

exact test. Cox regression and Kaplan–Meier curves were used for event rate analyses. Where relevant, 2-sided *p*-values < 0.05 were set as statistically significant. Confidence intervals were set at 95%.

#### **3. Results**

#### *3.1. Baseline Characteristics of the Population*

Overall, 104 patients (71% males, mean age 47 ± 11 year) were enrolled, including those with arrhythmic presentation (*n* = 70) and those with arrhythmias detected during in-hospital telemonitoring (*n* = 34). Patients' complete characteristics are shown in Table 1. Arrhythmias included VAs, SVAs and BAs in 104 (100%), 11 (11%), and 9 patients (9%), respectively. Overall, 19 patients (18%) had LVEF < 35% at presentation. EMB identified 73 cases of chronically active myocarditis (70%) and CMR showed anteroseptal LGE in 26 cases (25%).

**Table 1.** Baseline characteristics of the population.




Baseline characteristics of the population are shown. ACS = acute coronary syndrome; AF = atrial fibrillation; AVB = atrioventricular block; BA = bradyarrhythmia; CMP = cardiomyopathy; CRP = C-reactive protein; EDD = end-diastolic diameter; EDVi = end-diastolic volume (indexed); EF = ejection fraction; EGE = early gadolinium enhancement; HF = heart failure; HR = heart rate; IQR = interquartile range; LGE = late gadolinium enhancement; LV = left ventricle; n.v. = normal value; NSAT = nonsustained atrial tachyarrhythmia; NSVT = nonsustained ventricular tachycardia; PVC = premature ventricular complexes; RV = right ventricle; SCD = sudden cardiac death; SD = standard deviation; SVA = supraventricular arrhythmias; T-Tn = T troponin; TAPSE = triscupid annular plane systolic excursion; VA = ventricular arrhythmias; VF = ventricular fibrillation; VT = ventricular tachycardia; WMA = wall motion abnormality.

#### *3.2. CAM Types, Indications and Complications*

ICDs were implanted in 62 patients (60%; *n* = 47 for secondary prevention), including dual-chamber (*n* = 48), single-chamber (*n* = 5) and subcutaneous devices (S-ICD, *n* = 9). The remaining 42 patients (40%) underwent ILR implant. The mean number of risk factors was two in ICD carriers and <1 in ILR cases (Table S1). Among the 61 patients undergoing PVS, 25 had VT or VF inducibility and underwent ICD implant (Table S2). Complications were documented in 9/62 ICD carriers (15%) including infection (*n* = 3), catheter dislocation or

malfunctioning (*n* = 3), hematoma (*n* = 2) and pneumothorax (*n* = 1). No complications occurred following ILR implants.

#### *3.3. Treatment and Follow Up*

All patients were discharged on medical treatment, including RAAS-inhibitors (*n* = 87), betablockers (*n* = 96), and either single (*n* = 47) or combined (*n* = 23) antiarrhythmic drug (AAD) therapy (Table S3).

The study FU was 3.7 ± 1.6 year. There were no patients lost to FU. The average number of Holter ECGs per patient was 9.5, and the proportion of missed exams was 3.6% (maximum one exam per patient). Three patients died (end-stage heart failure, *n* = 1; infectious complications of cardiac transplantation, *n* = 1; malignancy, *n* = 1), all of which were ICD carriers (guideline-driven implant in two of them). No patients experienced systemic embolism or hemorrhagic complications.

During FU, CMR was repeated in 40 cases (38%), and its interpretation was limited by susceptibility artifacts in all ICD (*n* = 5) and no ILR carriers (*n* = 35, 28 of whom were proven healed from myocarditis). All devices were replaced following the end-of-life status. No quality-of-life issues were reported by 91% of the device carriers (Table S4).

#### *3.4. VA Detection, Burden and Timing*

During FU, 45 patients (43%) underwent VT episodes including *n* = 3 incessant VTs, *n* = 10 electrical storms (≥3 shocks/24 h) and *n* = 32 paroxysmal VTs only. In 10/45 cases (22%), there was no prior history of VT. In addition, 67 patients (64%) had NSVT and 102 (98%) PVC. Complete data are reported in Table 2. As compared to Holter ECG, CAM identified more patients either with VT (45 vs. 4, *p* < 0.001) or NSVT (64 vs. 45, *p* < 0.001). Kaplan–Meier curves are shown in Figure 2. All VT episodes and most of the NSVT ones were only detected by CAM (Table 2); in addition, CAM allowed earlier NSVT detection (median 6, IQR 3–24 vs. median 24, IQR 9–36 months, respectively, *p* < 0.001). Event rates are shown in Figure S1.

**Figure 2.** Detection of ventricular arrhythmias by CAM vs. sequential 24 h Holter ECGs. Kaplan–Meier curves are shown for the endpoint of VT (panel **A**) and NSVT (panel **B**). CAM = continuous arrhythmia monitoring (red); ER = event rate; FU = follow up; Holter = 24 h Holter ECG (blue); NSVT = nonsustained ventricular tachycardia; VT = ventricular tachycardia.


**Table 2.** Arrhythmia detection during follow up.

Arrhythmia types documented during follow up are shown as detected by Holter ECG vs. CAM. Both the number of episodes and the number of patients are reported: <sup>1</sup> VT includes sustained VT and appropriate ICD therapy (either ATP or shock); <sup>2</sup> AF and AT only include episodes lasting > 30 s; <sup>3</sup> NSAT includes supraventricular arrhythmia episodes lasting ≤ 30 s; <sup>4</sup> BA includes 2nd type II, 2:1 or 3rd degree atrioventricular blocks and pauses > 3 s. AF = atrial fibrillation (paroxysmal); AT = atrial tachycardia; ATP = anti-tachycardia pacing; BA = bradyarrhythmia; CAM = continuous arrhythmia monitoring; ICD = implantable cardioverter defibrillator; NSAT = nonsustained atrial tachyarrhythmia; NSVT = nonsustained ventricular tachycardia; PVC = premature ventricular complex; VT = ventricular tachycardia.

> Although an alert for clustered PVC was reported by CAM in 21 cases (21%), PVCs were documented by Holter ECG in 102/102 patients (*p* < 0.001). During FU, CAM showed a significant reduction in VT/NSVT cycle length variability, whereas the Holter ECG documented a progressive prevalence of monomorphic PVC (Figure S2).

#### *3.5. Other Arrhythmias*

During FU, SVA episodes were documented in 27 patients (26%) including AF in 19 cases (18%). In addition, six patients had BA, mainly second- and third-degree AVB. Complete data are shown in Table 2. Overall, CAM identified more patients either with SVA lasting > 24 h (9 vs. 1, *p* < 0.001), or BA (6 vs. 1, *p* = 0.015) and only missed pauses in the range of 2–3 s. SVA detection by CAM was earlier than that by Holter ECG (22 ± 8 months in 27 patients vs. 36 ± 12 months in 7 patients, respectively, *p* = 0.001).

#### *3.6. CAM Type and Indication*

Arrhythmia recordings in different CAM subgroups are shown in Table S5. Although most VA occurred in patients following secondary prevention ICD implant, VTs were also documented within primary prevention ICD (10 episodes in *n* = 8 patients) and ILR subgroups (two episodes in two patients).

A FU VT was found in 40/80 patients with putative risk factors vs. 5/24 without putative risk factors (HR 3.8, 95% CI 1.3–11.2, *p* = 0.015). However, there was no a single risk factor capable of predicting the occurrence of a de novo VT (Table 3). Instead, our post hoc analysis identified both anteroseptal LGE distribution pattern at CMR, and signs of chronically active myocarditis at EMB, as significantly associated with the first episode of VT during FU (respectively: 50 vs. 13% and 90 vs. 49%, both *p* < 0.05). Results were confirmed for the whole study cohort, where VT episodes were more common in the chronically active myocarditis and anteroseptal LGE subgroups (respectively: 40/73 vs. 5/31 acute cases, *p* < 0.001; and 16/26 vs. 29/78 inferolateral cases, *p* = 0.04).

**Table 3.** Characteristics of primary prevention CAM patients with follow-up VT vs. without follow-up VT.


Characteristics of the 10 patients experiencing their first VT episode (VT+) during follow up are shown. Significant differences are evidenced in bold. \* The definition includes extensive areas of LGE (>1 left ventricular wall, or >5 of 17 left ventricular segments) at cardiac magnetic resonance, or replacement fibrosis in >50% of endomyocardial samples undergoing histological analysis. AVB = atrioventricular blocks; CAM = continuous arrhythmia monitoring; ILR = implantable loop recorder; LGE = late gadolinium enhancement; LVEF = left ventricular ejection fraction; NSVT = nonsustained ventricular tachycardia; PVS = programmed ventricular stimulation; VT = ventricular tachycardia.

#### *3.7. Clinical Impact*

Guided by CAM for VT episodes and by Holter ECG for high-burden PVCs, 41 patients (39%) underwent transcatheter ablation during FU. Apart from the VT episodes, most FU arrhythmias were asymptomatic (Table S6). Significantly, de novo oral anticoagulants were started in eight SVA patients (8%) including six asymptomatic ILR carriers. An upgrade to dual-chamber ICD was performed in eight cases (8%) including ILR patients (*n* = 5; two for VT and three for NSVT associated with BA), and ICD cases experiencing inappropriate shocks for AF (*n* = 3; two single-chamber ICDs and one S-ICD).

Based on the current guideline recommendations [5,6], only the five patients with granulomatous myocarditis (5%) and VT/VF onset would have met the criteria for an early ICD implant. However, among the 99 candidates for an ICD-sparing strategy, 41 (41%) experienced at least one VT episode during FU. By the end of the study, the ICD implantation strategy was appropriate in 80% of the population instead of 60%, resulting from the strict application of the guidelines (Figure 3).

**Figure 3.** Events by myocarditis stage and implantation strategy. Panel **A**: VT events (VT+) in patients with true acute vs. chronically active myocarditis according to endomyocardial biopsy findings; Panel **B**: Appropriateness of the ICD implantation strategy by application of current guidelines for acute myocarditis (left panel) vs. by multiparametric approach as described in this study (right panel). FU = follow up; ICD = implantable cardioverter defibrillator; VF = ventricular fibrillation; VT = ventricular tachycardia.

#### **4. Discussion**

#### *4.1. Major Findings*

We described the first study aimed at exploring the advantages of CAM as compared to standard Holter ECG monitoring in patients with EMB-proven active myocarditis [5,13] and evidence of arrhythmias at index hospitalization. Remarkably, the comparison between techniques was unbiased since all patients underwent both CAM and Holter monitoring strategies. Despite the considerable number of Holter ECG exams per patient, we showed that CAM was more accurate in both detecting and quantifying most of the clinically impactful arrhythmias. In addition, we showed that despite a uniform clinical presentation with acute myocarditis [5,6], many patients had histopathological signs suggesting chronically active disease [4,14]: in light of the significant association with follow-up VT episodes, an earlier indication of the ICD implant could be considered for the latter ones.

#### *4.2. Diagnostic Accuracy for VA*

As shown in Table 2, all FU VT episodes were detected by CAM. Compared to Holter ECG, CAM was superior in both identifying patients with VA and detecting total VA episodes. Although more frequently detected by ICDs, VA episodes were also found in a relevant proportion of ILR carriers (Table S5). Conversely, the CAM accuracy in detecting PVCs was remarkably lower compared to Holter ECG, which allowed precise PVC quantification over time [10]. As a relevant guidance for the planning of catheter ablation strategies, the clinical VA morphology requires documentation by 12-lead ECG recording [10,16]. Recently, VA characterization by ECG has also been proposed as a tool to assess the myocardial inflammatory stage [17,18] and identify the suitable candidates for VT ablation [16]. In keeping with myocarditis healing, CAM recordings documented a progressive reduction in VA cycle length variability during follow-up, in parallel with a prevalence of monomorphic PVC by Holter ECG (Figure S2).

#### *4.3. Other Arrhythmias*

Table 2 shows that CAM was an accurate tool also for diagnosing SVA and BA. Remarkably, most of the long-lasting SVAs were those which were late onset (Figure S1) and asymptomatic (Table S6). In this setting, the CAM-guided anticoagulation strategy [19] was safe since no ischemic or hemorrhagic complications occurred. In turn, advanced AVBs, commonly reported in acute-phase cardiac sarcoidosis [4], were documented even later during FU. Although iatrogenic effects from betablockers and AADs were likely (Table S3), the documentation of both BA and NSVT constituted an indication to ICD upgrading in three ILR carriers (Table S6). Instead, the possible underdiagnosis of BA in transvenous ICD carriers constituted a clinically neglectable issue.

#### *4.4. Arrhythmic Risk Estimation*

In our study, the indication of ICD was supported by a number of pre-selected risk factors, namely: LVEF < 35% [6,7]; malignant histotypes [4]; major BA [9]; fast/recurrent NSVT [10]; positive PVS [20]; and extensive LGE or myocardial fibrosis [21,22]. Although VT events more commonly occurred in patients with at least one of the above risk factors, none of the candidates were able to predict an adverse outcome in primary prevention. In keeping with prior studies, we identified anteroseptal LGE [23–26] and histological signs suggesting chronic myocarditis [12,13] as factors associated with adverse arrhythmic outcomes, both in the whole cohort and in patients without malignant VA onset. Results are consistent with recently published data [27]. As suggested by Table 3, mild systolic dysfunction (i.e., LVEF < 50%) may play an additional role for primary prevention risk stratification, as already suggested both in myocarditis and other cardiomyopathies [28,29].

#### *4.5. Device Indication and Choice*

Overall, our data challenge the uniform application of an ICD-sparing strategy in patients with VA onset and newly diagnosed active myocarditis [5,6]. Actually, our analysis revealed that, despite the clinically acute myocarditis onset, the majority of patients in our cohort had histological signs of chronic myocarditis, as supported by myocardial fibrosis and additional cellular abnormalities [12,13]. In contrast to the truly "acute" myocarditis cases, those with "chronically active" inflammation showed a significantly higher occurrence of VT during FU—even in the absence of granulomatous myocarditis (Figure 3). Our findings indicate that clinical guidelines may benefit from a clear distinction between the scenarios, and we suggest that a multiparametric assessment could be implemented in chronic setting to identify the most suitable candidates for an early ICD implant [14].

As for the device choice, in our experience, dual-chamber ICDs are advisable to minimize the risk of inappropriate shocks by single-lead devices. In turn, since scar-related VA may even occur during the post-inflammatory stage of myocarditis [16,17], the use of wearable cardioverter defibrillators could be undermined by the unpredictable optimal timing for device withdrawal: while life-vests are currently recommended as a bridge for decision making in acute myocarditis [5,30], S-ICDs may constitute a valuable alternative in the chronic setting. Finally, because of a combination of high diagnostic accuracy, general acceptance among patients (Table S4) and CMR feasibility [31,32], we suggest the widespread use of ILRs as optimal diagnostic tools for the remaining low-risk patients with arrhythmic myocarditis [33,34].

#### *4.6. Study Limitations*

Our study specifically focused on patients with myocarditis and the evidence of VA at the index of hospitalization. Although the arrhythmic population is underinvestigated and clinically demanding [4–7], results should not be inappropriately generalized to different clinical scenarios. Selection bias related to the center experience [14,33] as well as baseline arrhythmia overdetection due the use of continuous in-hospital telemonitoring should be taken into account. Importantly, CAM choice was conditioned by a number of risk factors that, although reasonable, were not supported by robust evidence—this introduces a bias by indication. Baseline PVS was not performed in all patients, and wearable devices were not hereby investigated. Finally, some differences in arrhythmia detection capability should be considered for ICDs (unable to detect BA unless permanent pacing is needed) and for single-chamber and subcutaneous devices (which may be less reliable in differentiating SVA and VA subtypes). Larger prospective multicenter studies are needed to validate our findings and improve patient selection for each device type at different inflammatory stages [16–18].

#### **5. Conclusions**

In patients with arrhythmic myocarditis, CAM was a clinically useful tool to detect arrhythmias and guide relevant therapeutical decisions. As compared to sequential Holter ECGs, CAM allowed an earlier detection and greater diagnostic yield for most arrhythmias. As a complementary tool, Holter ECG allowed PVC quantification and morphology characterization. Based on our findings, efforts are needed to identify patients with chronically active myocarditis, as well as those with anteroseptal LGE at CMR, who may benefit from an earlier ICD implant. In low-risk patients, ILR was a feasible and sensitive diagnostic tool, allowing also to monitor myocarditis evolution by informative CMR. Prospective controlled trials including appropriate myocarditis staging and a uniform implantation strategy are needed, to improve the arrhythmic risk stratification and patient selection for different device types.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390jcm10215142/s1, Supplementary Materials. Table S1: Indications to different CAM types; Table S2: Programmed ventricular stimulation; Table S3: Treatment; Table S4: Quality of life; Table S5: Arrhythmia detection according to CAM type and indication; Table S6: Clinical impact of arrhythmia detection; Figure S1: Arrhythmic event rates; Figure S2: VA modifications during follow-up.

**Author Contributions:** Conceptualization, G.P. (Giovanni Peretto) and S.S.; methodology, G.P. (Gabriele Paglino), P.M., A.M., G.T., S.R. and C.B.; validation, P.M. and P.D.B.; formal analysis, G.P. (Giovanni Peretto); investigation, G.P. (Giovanni Peretto) and S.S.; resources, P.D.B.; data curation, G.P. (Giovanni Peretto), G.T. and A.M.; writing—original draft preparation, G.P. (Giovanni Peretto); writing—review and editing, S.S.; visualization, G.P. (Giovanni Peretto), P.M., G.P. (Gabriele Paglino), A.M., G.T., S.R., C.B., P.D.B. and S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of San Raffaele Scientific Institute (MYOCAR, 24/01/2018).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data will be made available, upon reasonable request, by emailing the correspondent author.

**Conflicts of Interest:** The authors declare no conflict of interest regarding the publication of this manuscript.

#### **Abbreviations**



#### **References**

