I. Morpho-functional ventricular abnormalities

Such as in 2010 ITF criteria, echocardiography, CMR and angiography were indicated as possible tools for assessing morpho-functional ventricular abnormalities. However, in attempt to identify the disease earlier, any degree of RV dilation or dysfunction in association with regional wall motion abnormalities was considered a major RV criterion while the presence of regional wall motion abnormalities without RV dilatation or dysfunction was introduced as minor criterion. This finding is due to the regional nature of ACM that can affect the segmental contractility because of local fibro-fatty replacement, without compromising the global hemodynamics of the RV [14, 21]. At the same time, it has been classified as a minor criterion because of the misinterpretation of some normal variants of the RV wall motion [18].

The two morpho-functional criteria introduced for the LV are both minor because of the low specificity for diagnosing left-sided ACM variants. They include the detection of global LV systolic dysfunction with or without dilatation, and regional LV hypokinesia or akinesia. The use of strain echocardiography is recommended, because of the ability to detect subtle changes, especially in the early stages of the disease [22, 23].

Instead of using fixed cut-off values, the Padua criteria recommend the use of current reference values for cardiac chamber size and function, normalized for sex, age and body surface area, recommended by international societies of cardiovascular imaging [24, 25], and proper reference values for athletes, especially if engaged in sports associated with the greatest RV remodeling, such as canoeing and rowing [26].

II. Structural myocardial abnormalities

Fibrous or fibro-fatty replacement of myocardium is the pathological manifestation of ACM, and its detection through EMB has been indicated since 1994 [7]. However, because of its invasive nature with potential serious complications, the 2020 criteria have limited EMB indication to selected cases of non-familial ACM with negative genotyping to exclude phenocopies, such as myocarditis, sarcoidosis or DCM [15]. The demonstration of fibrous replacement of the RV myocardium in at least 1 sample, with or without fatty tissue, represents a major criterion. EMB is particularly helpful in ARVC, because of the peculiar topographic and histological features of the disease with fibro-fatty replacement reaching the subendocardium. A negative EMB do not exclude the diagnosis of ACM because of the possibility of sampling error. Moreover, the LV EMB is not indicated at present because the risk/benefit ratio is not yet known [27].

The introduction of non-invasive tissue characterization with CMR is one of the most important innovations of the 2020 criteria. The major CMR criterion is the presence of transmural LGE in at least 1 RV segment, confirmed in 2 orthogonal views. Currently, the diagnostic specificity of RV LGE is considered high, instead the sensitivity is low. This is due to the CMR technology characterized by a poor spectral resolution and suboptimal contrast/noise ratio in quantifying the thin RV wall [17, 28, 29, 30]. The highest specificity is reached when wall motion abnormalities and pre/post contrast signal alterations are considered together [29] (Fig. 3, Ref. [31]).

In the LV, the presence of a stria of LGE with a non-ischemic distribution (subepicardial and/or midmyocardial, most affecting the inferolateral region) in at least 1 LV Bull’s Eye segment, confirmed in 2 orthogonal views (excluding junctional LGE, that is considered non pathologic) is a major criterion. Moreover, the circumferential involvement of septum and LV free wall in short axis view is called “ring-like” pattern, and it is considered as highly specific for ALVC [32]. Nonetheless, at present there is no gold standard for differentiating non-ischemic LGE secondary to ACM or to other diseases such as myocarditis: for this reason, in the absence of concomitant RV involvement, the diagnosis of left-dominant ACM in a proband requires positive genetic testing (Fig. 1). Fatty tissue infiltration is often observed on dedicated CMR sequences, but it is not considered a diagnostic criterion in isolation because of its low specificity.

In the early stages of LV involvement, the typical non-ischemic distribution of fibro-fatty replacement sparing the subendocardial layer can explain the absence of wall motion abnormalities, dilatation, or dysfunction of the LV. Thereby, the absence of LV functional abnormalities on echo, cine-CMR or angiography cannot rule out LV involvement, and CE-CMR characterization plays a key role in detection of left-sided ACM [14, 16, 33, 34, 35, 36] (Fig. 4, Ref. [14]).

III. Repolarization abnormalities

As regards to repolarization abnormalities, the detection of T wave inversion (TWI) in right precordial leads (V1–V3) or beyond, or in V1–V2 only, remain major and minor criteria respectively. These findings require the absence of a complete right bundle branch block (RBBB). Otherwise, in the presence of RBBB, TWI in V1–V4 is a minor criterion. These criteria are valid in patients with complete pubertal development. A remarkable consideration is that TWI extension from right precordial leads (V1–V3) to left ones (V4–V6) is the expression of a more severe RV dilatation, with its displacement toward the axilla, rather than of LV involvement [34]. LV involvement can be only predicted with TWI in left precordial leads (V4–V6) in absence of complete LBBB, but it is a minor criterion because of its low specificity [15, 33, 34].

IV. Depolarization abnormalities

Signal averaged ECG is no more included among Padua criteria based on the experts’ opinion that they lacked specificity and showed low diagnostic accuracy [16]. Moreover, the epsilon wave in right precordial leads has been downgraded to minor criterion, because its identification and interpretation are highly influenced by ECG filtering and sampling rate, with a consequently large interobserver variability [37]. The ECG pattern in right precordial leads of a terminal activation duration (TAD) of the QRS $\ge$55 msec from the nadir of S wave to the end of QRS without a complete RBBB remains minor criterion, particularly if followed by TWI.

The presence of low QRS voltages in limb leads (peak-to-peak QRS amplitude 0.5 mV) is a predictor of LV involvement [14, 32, 35, 38]. The mechanism could be the reduction in generating of the electrical activity due to the fibro-fatty replacement of LV myocardial mass. It is a minor criterion in absence of other potential causes of low QRS voltages, such as emphysema, obesity, pericardial effusion, or inappropriate setting of low band-pass filters (100 Hz) (Fig. 5, Ref. [38]).

V. Ventricular arrhythmias

ACM is characterized by premature ventricular beats (PVBs) with origin from or around the fibro-fatty tissue. PVBs are considered in terms of absolute number ($>$500 PVBs/24 h), complexity (sustained or non-sustained VT) and morphology on 12-ECG leads 24 h Holter monitoring or 12-ECG leads exercise test [39]. According to the 2020 Padua criteria, PVBs or VT with LBBB morphology originating from RV regions other than RVOT are more specific for ACM, so it is a major criterion. Instead, the LBBB/inferior axis morphology is less specific for ACM, because PVBs originating from RVOT are often idiopathic (minor criterion). The detection of PVBs with RBBB morphology suggests the origin from the LV, excluding the fascicular pattern (QRS 130 msec). RBBB/wide QRS/superior axis is the most common PVBs morphology in patient with LV scar involving the lateral or the infero-lateral wall, in biventricular ACM and ALVC [33, 34].

Electrophysiology study (EPS) is not considered a diagnostic criterion; however, it can be useful in selected patients for differential diagnosis. In particular, response to programmed ventricular stimulation and isoproterenol may allow to differentiate between idiopathic right-ventricular outflow tract (RVOT) VT or Brugada syndrome and the scar re-entrant VT of ACM. Addition of RV endocardial voltage mapping may be of incremental diagnostic value for differential diagnosis with idiopathic RVOT VT, as demonstration of low-voltage areas suggest the presence of fibro-fatty scars [16].

VI. Family history and molecular genetics

Compared to the 2010 ITF criteria, in the Padua criteria the category family history and molecular genetics has not changed. Moreover, the category is shared between RV and LV criteria. This is because the manifestation of ACM and the predominant ventricular involvement may vary among members of the same family and with the same gene mutation. However, more restricted indications for genotyping have been proposed to the new criteria, trying to avoid misinterpretation of molecular genetic results and misdiagnosis. The genetic test is recommended in probands with a definite biventricular or ARVC diagnosis, in order to screen family members [40]; it may be considered in borderline forms to reach the definite diagnosis, provided that the results of genetic test are interpreted by experts in ACM; it is mandatory to reach the diagnosis of non-familial ALVC to exclude phenocopies [16].

Major criteria are the detection of a pathogenic or likely pathogenic ACM gene mutation in the patient under evaluation, the history of a first-degree relative with ACM confirmed pathologically at autopsy or surgery or who reached the criteria necessary for ACM diagnosis. The minor criteria are the suspicion of ACM without confirmation in a first-degree relative, the suspicion of ACM in a first-degree relative who suddenly died before the age of 35, the confirmed diagnosis of ACM in a second-degree relative.

The second step is the identification of the specific ACM phenotype according to the number of criteria for the RV and LV involvement that are fulfilled. According to the 2020 ITF criteria, any diagnosis of ACM requires that at least 1 criterion from category I (morpho-functional abnormalities) or II (structural abnormalities) must be reached, either major or minor, and only these two categories are taken into consideration to classify the phenotypic variant.

If these criteria are only fulfilled for the RV, the diagnosis is the classical right-dominant variant (ARVC). Whereas, if the criteria are fulfilled for both RV and LV, the diagnosis is “biventricular” form. Moreover, it is possible to define the likelihood of disease according to the number of major and minor criteria reached from all categories. So, the diagnosis can be “definite” if either 2 major criteria, or 1 major and 2 minor criteria or 4 minor criteria are fulfilled, “borderline” if either 1 major and 1 minor criterion or 3 minor criteria are reached, and “possible” if either 1 major criterion or 2 minor criteria are satisfied.

The diagnosis of ALVC is reached in patients with structural LV abnormalities (major criterion) and no RV involvement, when a pathogenic or likely pathogenic ACM-causing gene mutation is identified. In this case, the diagnosis of ALVC is always definite. The need for positive genotyping testing is due to the possible overlap of these morpho-functional and structural findings with phenocopies, such as DCM, cardiac sarcoidosis or myocarditis.

After the clinical diagnosis of ACM and the definition of the specific phenotype, the third step is to define the aetiology of ACM and to exclude phenocopies. This purpose can be reached thanks to molecular genetic testing and cascade family screening. Indeed, ACM is generally transmitted as an autosomal dominant trait, with variable expressivity and incomplete penetrance. So, the molecular genetic test can identify either desmosomal or non-desmosomal gene defects causing ACM.

Desmosomes are proteins forming the area composita of the intercalated disc that are structure crucial for electromechanical connection of cardiomyocytes and intracellular signaling cascades. These structures are also composed by adherens junctions, gap junctions and ion channels. Pathogenic mutations of gene encoding desmosomal proteins such as plakophilin (PKP2), desmoplakin (DSP), desmoglein (DSG2) and desmocollin (DSC2) are identified in $\approx$50% of patients with ARVC [40] and rarely (1%) of gene encoding adherens junctional proteins such as N-cadherin (CDH2) and $\alpha$-T-catenin (CTNNA3) [41, 42].

ACM left-sided variants are also associated with mutations in non-desmosomal genes encoding for ion channels and cytoskeletal components, such as lamin A/C (LMNA), filamin C (FLNC), transmembrane protein 43 (TMEM 43), desmin (DES), titin (TTN), phospholamban (PLN), the cardiac ryanodine receptor-2 (RYR2), sodium voltage-gated channel alpha subunit 5 (SCN5A) and transforming growth factor beta-3 (TGF$\beta$-3) [40].

In case of a negative molecular genetic testing but positive clinical family screening, ACM is defined familial but “gene elusive”. Also in this case, the presence of affected relatives allows to rule out non-hereditary conditions mimicking ACM.

If both the genetic testing and the cascade clinical family screening for ACM are negative, it is essential to perform further evaluations in order to exclude mimics, both acquired (sarcoidosis, DCM, pulmonary artery hypertension, myocarditis, Chagas disease) and congenital (left-to right shunt or Ebstein anomaly) phenocopies.