in the study by rinne et al., a heterozygous nonsense variant in popdc2 ( p. trp188ter; rs144241265, allele frequency of 1. 70 × 10 among european individuals in gnomad ) was identified in the heterozygous state in a monozygotic twin pair presenting with av block and in an unrelated family with a mother and son both diag nosed with first - degree av block ( i. e., prolongation of the pr - interval on the ecg ). the twin pair inherited the variant from the unaffected mother, indicating that it does not fully explain the phenotype observed in the two siblings. in support of this, single - variant analysis that we conducted in 162 carriers of the p. trp188ter variant in more than 1 million individuals from the general population did not show an association with sinus node or av node conduction disease in heterozygous state. thus, while the findings from rinne et al. are interesting and support our findings, they do not establish pathogenic genetic variation in popdc2 as a ( recessive or dominant ) mendelian cause of cardiac conduction disease in human. based on our findings, we recommend the inclusion of popdc2 in clinical genetic - testing panels for affected individuals presenting with unexplained sinus node dysfunction, av conduction defects with or without hcm. using population - level genetic data of more than 1 million individuals, we showed that none of the variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow - up. bi - allelic variants in popdc1 and popdc3 have been associated with muscular dystrophy, with and without ccd, respectively, while the affected individuals reported here presented with isolated cardiac disease. specifically, none of the affected individuals reported muscular weakness, atrophy, or cramps. furthermore, muscle biopsy in the proband from family a did not show evident signs of muscle disease. also, in contrast to popdc1 - and podpc3 - affected individuals, normal serum creatine kinase levels were found in the affected individuals reported here. while we did not detect a muscular phenotype in the affected individuals, currently aged 22 – 50 years, we cannot exclude subtle or age - dependent expression of muscular defects in popdc2 - related disease. although all three popdc proteins are expressed in both skeletal and cardiac muscle, differences in levels of expression between the popdc proteins might, in part, determine phenotypic expression. indeed, popdc2 is predominantly expressed in cardiac tissue, whereas popdc3, which presents with isolated muscular dystrophy, has a predominant expression in skeletal muscle. western blot analysis and immunostaining of popdc1 and popdc2 in muscle biopsies obtained in the proband from family a showed significant reduction of the expression of both popdc1 and popdc2. these findings are in line with previous studies that suggested that stability and / or membrane trafficking of the popdc1 - popdc2 complex is impaired by variants in each of the two proteins. using spatial transcriptomics and scrna - seq from human hearts, we showed that co - expression of popdc1 and 2 was most prevalent in av node, av node pacemaker, and av bundle cells. on the other hand, in the sinus node, popdc2 was abundantly expressed, but expression of popdc1 was sparse. while these results support the observed predisposition to av node disease in affected individuals with popdc2 lof variants, proper statistical testing using pseudo - bulk counts was not possible due to a low number of donors with conduction - system data. therefore, these results should be treated as hypothesis generating. together, these results concur with predisposition to av node disease in humans with lof variants in popdc1 and popdc2 and presence of sinus node disease in popdc2 - but not in popdc1 - related disease in human. popdc proteins are established interacting partners of the potassium channel trek - 1, which is known to underlie a background potassium current and is highly expressed in the sinus node. co - expression of trek - 1 with any of the three popdc proteins leads to an increase in trek - 1 current, a process modulated by the level of camp. furthermore, cardiac - specific trek - 1 - deficient mice display a sinus node phenotype characterized by bradycardia with frequent episodes of sinus pauses, partially resembling the phenotype in the affected individuals with popdc2 variants presented here. in an effort to shed light on the electrophysiological mechanism by which the variants in popdc2 lead to bradycardia, we therefore conducted co - expression studies of wt and mutant popdc2 with trek - 1. in vitro, both popdc2 variants tested failed to increase trek - 1 current and, by virtue of observations of bradycardia in trek - 1 - deficient mice, these variants would be expected to be associated with bradycardia. notwithstanding the clear observation of bradycardia in trek - 1 - deficient mice, how loss of background potassium current, causing an increase in diastolic net inward current, leads to bradycardia and sinus pauses is unclear. our in silico modeling studies showed that a 41 % reduction of trek - 1 current, simulating the effect of the variants in popdc2, leads to an increase in diastolic depolarization rate and spontaneous firing. these findings are in agreement with experiments using isolated sinus node cells of trek - 1 - deficient mice and computer simulations using a rabbit sinus node cell model. in a murine cardiac muscle cell line ( hl - 1 ), a stop variant in popdc2 associated with trek - 1 reduction, the maximum diastolic potential ( mdp ) was depolarized and the action potential upstroke velocity was reduced. in addition, a slower spontaneous firing rate was observed. in contrast, another study that examined loss of trek - 1 channel function in hl - 1 cells showed an increase in spontaneous firing rate. bradycardia may also be induced via changes in excitability of atrial cardio myoc ytes surrounding the sinus node. in our simulated human atrial cell, reduction in trek - 1 current density slightly depolarized the mdp and increased the apd, consistent with findings in rat ventricular myoc ytes, but the excitability was hardly affected. thus, while collectively these data support a role for trek - 1 in cardiac pacing and a causal effect of popdc2 variants through modulation of trek - 1 current, the exact cellular electrophysiological mechanism remains unclear. differences in the cellular models used cannot be excluded. furthermore, the differences in effects of trek - 1 deficiency observed in vivo and in vitro in trek - 1 - deficient mice suggest that other factors ( such as altered sympathetic or parasympathetic stimulation in vivo ) also contribute to the bradycardia observed at baseline in these mice. while an effect through modulation of sodium channel function could be postulated, no such effect was observed in our patch - clamp studies. although cardiac arrhythmias in affected individuals with recessive popdc2 variants are consistent with observations in mice and zebrafish, the role of popdc2 in cardiac hypertrophy remains unexplained. one of the potential mechanisms underlying cardiac hypertrophy in affected individuals with recessive variants in popdc2 is modulation of trek - 1. since trek - 1 is activated by biomechanical stretch, the role of trek - 1 in cardiac responses to chronic pressure was recently studied using transverse aortic constriction. notably, while no clear structural cardiac differences were seen at baseline, mice lacking trek - 1 exhibited an exaggerated pressure - overload - induced concentric hypertrophy with preserved systolic and diastolic cardiac function compared to wt mice.