In the previous study [22], we detected a missense heterozygous variant A195V in one of 103 SUNDS cases, which was absent in 163 healthy controls as well as in Southern Chinese Han population, African population and Latin population in the Ensembl database (http://asia.ensembl.org/). The carrier of A195V was a 26-year-old male, who was found dead during night sleep. Comprehensive histopathological analysis, toxicological analysis and on-site investigation failed to explain the cause of the carrier’s death. In silico protein function prediction software Polyphen2 and Condel both predicted that the A195V was likely to be harmful or likely to be pathogenic. Previous studies also suggested that A195V was associated with sudden infant death syndrome (SIDS) [23], which further supports the pathological role of A195V in sudden death cases. The fact that A195V was discovered independently in SUNDS and SIDS strongly suggests A195V is closely related to sudden cardiac death. The present study was the first to confirm the pathogenicity of A195V through the molecular mechanism and cellular electrophysiological mechanism. Together with previous studies, it was the first time to more completely prove that HCN4 is the molecular cause and pathogenesis of SUNDS.
The sinus node pacing current in mammals is mainly generated by HCN4 ion channels. The variation of HCN4 gene leading to the decrease of HCN4 protein expression or channel dysfunction is the main cause of hereditary sinus arrhythmia. The caveolin (Cav) binding domains (CBD, Y259 and F262) locating in the intracellular amino terminus of HCN4 protein and closely to the S1 transmembrane domain, may regulate the membrane localization and expression of HCN4 protein by binding with Cav1 and Cav3 [24, 25]. On the contrary, loss of function variation on Cav3 or Cav3 knockout also hindered the binding of HCN4 and Cav 3, thus resulted in a shift to more negative values in HCN4 activation curve and a significant decrease in whole-cell current density [26, 27], which further supports the role of interaction between HCN4 and Cav3 in the sinus rhythm regulation and hereditary arrhythmias.
There are also β2-adrenergic receptors (β2-Ars) binding sites that selectively bind to β2-ARs and form HCN4 ion channel- β2-ARs complex, which binds with β2 receptor agonists, etc. isoproterenol, and promotes phosphorylation of gating related tyrosine on HCN4 channel, increasing the permeability of HCN4 channel to K+ and Na+ ions, thus increasing HCN4 channel current and sinus pacing rhythm [28–30]. Besides, the secondary α helix structure close to S1 transmembrane domain has an important effect on the movement of the S4 transmembrane voltage sensor domain of HCN4 channel and the replacement of amino acid residues in this domain may change the sensing sensitivity of the voltage sensor thus affects the gating process of HCN4 channel [31, 32]. It is reported that, an S4 mutation, K381E, retains S4-gate coupling and amplifies the sensitivity of HCN4 channel deactivation to cAMP with an activation mode shift to a more positive state [33]. Moreover, multiple protein kinase A (PKA) phosphorylation sites such as S14, S99, S110 and S117 at the amino terminus of HCN4 protein can be phosphorylated by PKA thus change the sensitivity of HCN4 channel to the regulation of β2 receptor agonists and cAMP [34, 35].
Yet, Zhang H, et al showed that inhibiting the expression and nuclear metastasis of HDAC4 by traditional Chinese medicine Shenxian-Shengmai Oral Liquid and Yixin-Fumai granules significantly promoted the expression of HCN4 in the SAN [36, 37]. And Yang B, et al proved that Loss of Trx2 reduces HCN4 expression via a mitochondrial ROS-HDAC4-MEF2C pathway, while mitochondria-specific ROS scavenger MitoTEMPO suppressed HDAC4 elevation and maintained HCN4 expression [38]. These studies suggested that HDAC4 and Trx2 might be the therapeutic targets for sudden death associated with loss-of-function variation of HCN4.
Here we analyzed the influence of A195V on mRNA and protein expression of HCN4. qPCR showed A195V decreased the HCN4 mRNA expression, while western blot analysis and confocal microscope analysis showed A195V decreased HCN4 protein expression on the membrane. The activation current density of HEK293 cells expressing A195V also decreased significantly compared to the WT group.
The site of A195V is close to the intracellular amino terminal CBD domain of HCN4 protein. A195V may decrease HCN4 expression and membrane localization through affecting the binding of CBD domain and Cav1 or Cav3. The decreased activation current density of A195V group could be induced by the reduction of HCN4 channel expression.
Previous study [39] demonstrated that the gain of function variation of HCN4 gene caused an increase in the expression of HCN4 protein channels and the funny current (If), resulting in a decrease in sodium calcium ion exchange by sodium/calcium ion exchanger (NCX1), and an increase in intracellular calcium ions. This was followed by a decrease in the expression level of cardiac sarcoplasmic reticulum calcium ATPase (SERCA2) and a decrease in the pumping of calcium ions from the cell into the sarcoplasmic reticulum, resulting in a significant increase in calcium ions during systole and an imbalance in intracellular calcium homeostasis. The administration of ivabradine, a HCN4 channel antagonist, can reverse the calcium homeostasis imbalance and myocardial remodeling caused by the increase of HCN4 protein expression and If augmentation. The treatment of orm10103, an NCX1 channel antagonist, can also reverse the calcium homeostasis imbalance caused by HCN4 overexpression. Since A195V altered the activation current density of HCN4, A195V might also change the intracellular sodium ion concentration and the intracellular calcium ion concentration through NCX1, thus induce intracellular calcium homeostasis imbalance, resulting in myocardial remodeling and related arrhythmias.
The significantly reduction of activation current density and activation at more negative voltages caused by A195V might induce decreased If and slow down the fourth-phase automatic depolarization process of sinoatrial node cells and reduce the resting heart rate, thus causing bradycardia and inducing related sudden death. The reverse potential calculated by analyzing the I-V curve according to the tail current were significantly more negative in A195V group than in the control group, which means that the A195V HCN4 channel reached a balance of internal and external current at a more negative potential than the wildtype HCN4 channel, and this indicates that the A195V HCN4 channel has a decreased sensitivity to the intracellular and extracellular voltage difference in tail current recording compared with the wildtype HCN4 channel, which, together with the slightly decreased tail current density of A195V HCN4 channel, suggests that the shutting down of the A195V HCN4 channel is delayed and the pacing automaticity is affected. The delayed pacing automaticity together with decreased If of sinoatrial node cells might allow the occurrence of atrial fibrillation and induce related sudden death.
In addition, A195V can influence the function of β2-ARs binding sites and PKA phosphorylation sites, thus affect the gating regulation of β2 receptor agonists and cAMP. In the present study, the activation I-V curve of A195V mutant HCN4 channel without cAMP stimulation was shifted to the right and less negative potential than the wildtype HCN4 channel, and was almost coincident with the activation I-V curve of the wildtype HCN4 channel as well as the A195V mutant channel under cAMP stimulation. This suggests that the A195V mutant HCN4 channel has a decreased sensitivity to cAMP regulation as it reached the cAMP gating stimulated state of wildtype HCN4 channel without cAMP stimulation and has no reserved space for further cAMP gating regulation. The loss of sensitivity to cAMP gating regulation together with the significantly decreased activation current density would severely impair the autonomic function of pacemaker cells, which is another mechanism for A195V causing atrial or ventricular arrythmias and related sudden death.
However, further studies using cardiomyocytes and mouse models are needed to more clearly clarify the A195V’s effect on myocardial action potential and its pathogenic effect in vivo.