The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the structural pore-forming subunits governing this current, with four HCN isoforms known (HCN1-4), among which HCN4 is the most highly expressed in the sinoatrial and atrioventricular nodes (Figure 1A).

D’Souza et al. ^[35][63] reported a direct biochemical interaction between miR-423-5p and HCN4 and they further demonstrated that miR-423-5p contributes to training-induced bradycardia by targeting HCN4. Thus miR-423-5p modulates the I_f current and the heart rate in mice. Yanni et al. ^[36][64] reported direct interaction between miR-370-3p and HCN4 (Figure 1B).

Indirect evidence on the role of miR-1 and miR-133 regulating HCN isoforms have been also reported. Inversed expression patterns of HCN2 and HCN4 (upregulated) and miR-1 and miR-133 (downregulated) have been reported in age-associated atrial fibrillation ^[38][66] myocardial infarction (MI) ^[39][40][67,68], and exercise training ^[41][69], yet it remains to be established if these microRNAs can direct target HCN isoforms (Figure 1B).

Similarly, no evidence has been reported to date on the direct functional role of microRNAs regulating HCN4 in arrhythmogenic syndromes, supporting the notion that additional studies are required in this context. A summary of the microRNA interaction with the HCN channels is provided on Table 1, while their links to distinct cardiac diseases is provided on Table 2.

2.3. ncRNAs in Sodium Channel Interacting Proteins

Calmodulin has been extensively reported to directly interact with Na_V1.5 (SCN5A) sodium channel and thus to modulate its function ^{[94][95][96][97][98][99][100][101]}[128,129,130,131,132,133,134,135]. Although distinct microRNAs such as miR-1 ^[102][136], let-7a ^[103][137], miR-625-5p ^[104][138], miR-525-5p ^[105][139], miR-338-5p ^[106][140], miR-185 ^[107][141], miR-145 ^[108][142], miR-30b-5p ^[109][143], and miR-675 ^[110][144] have been reported to modulate calmodulin expression, these reports exclusively describe their functional role in cardiac hypertrophy and failed to provide a direct link to sodium channel regulation. To date, the only report linking microRNAs, i.e., miR-26a, and cardiac arrhythmias, i.e., atrial fibrillation, is reported by Qi et al. ^[111][145]. Thus, the plausible contribution to sodium channel function by calmodulin interactive protein remains elusive.

3

3.1. ncRNAs in the Early Repolarization (ITO Transient Outward K+ Current)

After cardiac depolarization, the early repolarization process is governed by cardiac transient outward potassium current (I_TO). I_TO is rapidly activated after a fast increase of the membrane potential, where a short-lived, hyperpolarizing outward K+ current (I_TO) makes K+ ions from inside the cells to flow to the extracellular space, causing the transmembrane voltage to decrease. I_TO is then quickly deactivated, stopping the repolarization and ending the first phase of the action potential ^[112][155].

Several microRNAs have been described to be involved in the regulation of these channels. In particular, in 2007, Zhao and co-workers ^[42][70], demonstrated that Kcnd2 is positively regulated by miR-1, through Irx5 inhibition in mice, thus altering the endocardial to epicardial transmural gradient controlled by Kcnd2 within the ventricular cardiomyocytes and thus resulting in ventricular repolarization abnormalities. Kcnd2 is also regulated by miR-223-3p, a microRNA that is remarkably upregulated in a rat model of acute MI and consequently, K_v4.2 protein levels and I_TO density were significantly decreased ^[43][71] (Figure 1B). Such impaired modulation of K_v4.2 protein expression and thus of I_TO current can cause prolongation of the action potential duration and thus promote arrhythmias.

3.2. ncRNAs in the Plateau Phase and Terminal Repolarization (IKr, IKs, IKur K+ Current)

I_Kr currents are governed by hERG channels, also known as K_v11.1 ^[115][161]. As an homolog of the Drosophila “ether-a-go-go” (EAG) potassium channel, hERG was first cloned in the brain ^[116][162]. hERG channels are encoded by KCNH2 and mutations in KCNH2 have been associated to long QT syndrome (type 2; LQTS2) ^[87][113]. Ancillary MiRP1 (or KCNE2) subunits, that constitute single transmembrane protein homologous to KCNE1, was shown to associate with HERG channels and modulate I_Kr biophysical properties ^[117][163]. Mutations in KCNE2 have also been associated to long QT syndrome (type 6; LQTS6) ^[117][163].

3.5. ncRNAs Modulating the Slow Delayed Rectifier K + Current (IKs)

To date, scarce evidence is available regarding the functional impact of ncRNAs in I_Ks current modulation. Li et al. ^[48][76] examine miR-1/miR-133 levels, the potassium channel KCNE1 and KCNQ1 levels and I_Ks current in cardiac progenitor cells (CPCs) of normal human hearts. These authors observed that human CPCs expressed KCNE1 and KCNQ1 and possessed functional I_Ks currents (Figure 1B).

3.6. ncRNAs in the Resting Membrane Potential (IK1 Current and Na,K ATPase)

Several microRNAs have been reported to modulate I_K1 current in distinct biological contexts. miR-1 levels are increased in patients with coronary artery diseases (CAD) and also in an experimental rat model of MI. In this context, miR-1 silences KCNJ2 protein expression, and also GJA1, by directly targeting their 3´UTRs, respectively ^[51][78] (Figure 1B). On the contrary hand, miR-1 levels are greatly reduced in human AF as well as in AF experimental models, contributing to upregulation of Kir2.1 subunit, leading thus to increased I_K1, being this upregulation of inward-rectifier currents important for AF maintenance ^[50][52][49,79]. Additionally, miR-16 overexpression suppress KCNJ2/Kir2.1 expression in a rat experimental model of MI ^[53][80].

A summary of the microRNA interaction with the potassium channels is provided on Table 1, while their links to distinct cardiac diseases is provided on Table 2.

4

4.1. ncRNAs in Calcium Currents (ICa,L Current)

Transgenic mice overexpressing miR-1 resulted in severe electrophysiological defects, causing atrioventricular block ^[118][184]. Molecular analysis demonstrates that several key components contributing to the electrical wiring of the heart were altered, such as Cx43 and Kir2.1. Electrophysiological studies revealed that I_Ca and I_K1 currents were decreased. Knockdown of miR-1 overexpression using LNA-anti-miR-1 administration reversed such electrophysiological alteration, demonstrating a pivotal role for miR-1 in cardiac electrophysiology and particularly in calcium homeostasis. More recently, Zhang et al. (2019) demonstrated that transgenic mice overexpressing miR-1976 directly targeted two key calcium channels (i.e., Cav1.2 and Cav1.3, encoded by CACNA1C and CACNA1D, respectively) resulting in SAN dysfunction and thus lower heart rates, a phenotype reminiscent of sick sinus node syndrome in humans ^[119][185].

4.2. The Role of ncRNAs in Calcium-Induced Calcium Release