**5. DCM Caused by Disrupted NaV1.5 Interaction with Partner Proteins**

On the other hand, it is possible that the interaction of the defective NaV1.5 with its partner proteins of the cytoskeleton and intercalated disc, is responsible for the structural phenotype [28]. Interestingly, pathogenic *SCN5A* variants have been described in rare forms of left-ventricular non-compaction with a high arrhythmic burden [29]. Studies on induced pluripotent stem cell-derived cardiomyocytes have shown that the ARVC-associated missense variant *SCN5A-*R1898H leads to a significant reduction in peak *I*Na current, and of the abundance of NaV1.5 and N-cadherin clusters at the intercalated disc [13]. Other studies have shown that *SCN5A*-positive Brugada syndrome patients have significant cardiomyopathic changes, primarily in the right ventricular outflow tract, such as fatty wall replacement, fibrosis, and reduced expression of connexin 43 [30–32]. Moreover, missense *PKP2* variants identified in *SCN5A-*negative Brugada syndrome patients were shown to cause a loss of expression of desmosomal protein plakophilin-2, which was associated with decreased *I*Na, reduced number of NaV1.5 channels at the intercalated disc, and increased separation of microtubules from the cell end [33]. These interactions between the cardiac sodium channel complex and the intercalated disc likely underline mechanisms relevant to *SCN5A*-medicated ARVC and *PKP2*-mediated Brugada syndrome. Could an abnormal NaV1.5 channel result in reduced ventricular contractility through disrupting the function of its interacting proteins and/or those already implicated in DCM, such as proteins of the sarcomere, cytoskeleton, or the dystrophin-associated glycoprotein complex?

To date, several proteins interacting with the NaV1.5 channel have been shown to contribute to the *SCN5A*-mediated phenotypes through their alteration of the sodium channel availability or biophysical properties. NaV1.5 regulatory proteins caveolin 3 (*CAV3*), alpha 1 syntrophin (*SNTA1*), and cardiac sodium channel beta subunit 4 (*SCN4B*) have been reported in association with rare subtypes of congenital long QT syndrome. Pathogenic variants in cardiac sodium channel beta subunit 3 (*SCN3B*) and glycerol 3 phosphate dehydrogenase 1-like protein have been identified in patients with Brugada syndrome. Interestingly, cardiac sodium channel beta subunit 1 (*SCN1B*) has been linked to Brugada syndrome, atrial fibrillation (also *SCN2B*), and cardiac conduction disease. A number of other proteins have been shown to interact with and regulate the NaV1.5 channel. These include anchoring adaptor proteins ankyrin-G, syntrophins, MOG1, plakophilin-2, enzymes, such as nedd4-like enzymes, calmodulin kinase II δc, and protein tyrosine phosphatase H1, and other proteins that modulate the channel biophysical properties, such as 14-3-3η, calmodulin, telethonin, GPD1L, and FHF1B [34]. However, strong associations between pathogenic variants in these protein genes and development of DCM have not been reported.

The sarcolemmal membrane-associated protein (SLMAP) is localized at T-tubules and sarcoplasmic reticulum. Pathogenic variants in *SLMAP* have been shown to cause Brugada syndrome via modulating the intracellular trafficking of the NaV1.5 channel [35]. A recent report showed that transgenic mice with cardiac-specific expression of the SLMAP isoform 3 (SLMAP3) develop a significant decrease in fractional shortening and in cardiac output without notable hypertrophy, fibrosis, or fetal gene activation [36]. Electrocardiography identified increased PR interval and a decreased R amplitude. Western blot analysis revealed a decreased protein levels of NaV1.5 and calcium transport system of the sarcoplasmic reticulum (SERCA2a/PLN), suggesting a selective regulatory role of SLMPA3 in ion transport proteins at the level of gene expression. It is, however, unclear whether SLMAP3 is a contributor of DCM phenotype in humans or whether any of the reported DCM-associated *SCN5A* variants disrupts an interacting domain of its partner proteins or other DCM-associated genes/proteins. The list of proteins interacting with NaV1.5 is also not conclusive, and many aspects require further research. It is expected that these patterns will become clearer with further experimental evidence and with more genotype-phenotype analyses on DCM and related disorders. As a first step, *SCN5A* disruption has been demonstrated to result in TGF-β1-mediated fibrosis in a murine model of sinus node dysfunction. It is therefore possible that *SCN5A* variants can influence the pro-fibrotic milieu associated with other protein variants, and thereby contribute to the development of DCM [37].

#### **6. DCM Resulting from Long-Standing and Frequent Arrhythmias**

Alternatively, high arrhythmic burden may lead to ventricular dysfunction over time. This theory was primarily based on the finding that in several cases of MEPPC and DCM due to gain-of-function *SCN5A-*R222Q variant, therapy with hydroquinidine, flecainide, or amiodarone (in addition to standard treatment of heart failure) rapidly and effectively reduced the number of multifocal premature ventricular contractions and reversed the LV remodeling [10,16,38]. Following the initial report of R222Q, similar phenotypes have been reported for R222P, I141V, and G213D [39–41]. Nevertheless, whether the recovery of ventricular function relates to the premature ventricular contraction burden reduction or to intracellular mechanisms secondary to pharmacological blockade of the defective sodium channel, remain to be elucidated. Furthermore, some *SCN5A*–DCM patients, such as those carrying the D1275N variant, lack a history of long-lasting ventricular arrhythmias [14], and thus the LV dysfunction in these patients is unlikely to be a consequence of ventricular arrhythmias. It is therefore more likely that this is a contributing mechanism rather than a primary cause.
