leu37serfs 20 ) as they affect the same residue as the variant in family b ( p. arg263his ) or are expected to result in premature truncation of the protein and possibly nonsense - mediated decay, respectively. we co - transfected hek293 cells with wt and mutant popdc2 with trek - 1 containing plasmids. as expected, co - expression of wt popdc2 with trek - 1 increased trek - 1 current density ( figures 2 g – 2i ). however, when we co - expressed trek - 1 with a p. gln172 _ tyr176delinshis or p. arg263his popdc2 - containing plasmid, no increase in trek - 1 current density was observed, comparable to the co - expression of trek - 1 with an empty vector ( figures 2 g – 2i ). we then tested the effect of the popdc2 variants on sodium current ( i na ) as it was recently hypothesized that popdc2 may interact with na v 1. 5. however, expression of wt, p. gln172 _ tyr176delinshis, or p. arg263his popdc2 in an hek293 cell line stably expressing human na v 1. 5 channels ( encoded by scn5a ) showed an effect neither on i na density nor on i na gating properties by wt or mutant popdc2 ( figure s3 ). western blot analysis showed levels of expression of the mutant proteins that were comparable to the levels seen for wt popdc2 ( figure s4 ). to evaluate whether the observed decrease in trek - 1 current density, associated with the popdc2 variants, is responsible for the bradycardia observed in affected individuals, we conducted computer simulations using comprehensive mathematical models of both a human sinus nodal pacemaker cell and a human atrial cell. as shown in figure 3 a ( blue line ), the experimental data on the voltage dependence of the trek - 1 + popdc2 - wt current could be well fitted ( r > 0. 99 ) with the relationship i trek - 1 = −117. 1 + 284. 24 × exp ( v m / 90. 52 ), in which i trek - 1 and v m denote trek - 1 current density ( in pa / pf ) and membrane potential ( in mv ), respectively. the mutant data could be well fitted by scaling down i trek - 1 to 59 % of the trek - 1 + popdc2 - wt current over the entire volage range ( figure 3 a, red line ). we started our simulations with incorporating i trek - 1, which is not present in the original model cell, into the human sinus node model cell. because data on the density of i trek - 1 in human sinus node cells are lacking, we set its density to 1. 2 pa / pf ( at + 30 mv ), as observed in mouse sinus node cells by unudurthi et al. this, however, resulted in cessation of pacemaker activity in the human sinus node model cell. we then repeated our simulations with a 10 times lower density of i trek - 1. with this density, the cycle length of the simulated action potential amounted to 1, 160 ms ( figure 3 b, top panel, blue solid line ), as compared to 813 ms in the original model ( figure 3 b, top panel, gray dotted line ). cycle length was reduced by 16 % from 1, 160 to 973 ms upon reduction of i trek - 1 density to 59 %, thus simulating the effect of the variants in popdc2 ( figure 3 b, top panel, red solid line; figure 3 c, vertical arrow ). the decrease in cycle length was mainly due to an increase in diastolic depolarization rate ( figure 3 b, top panel ) as a result of the decrease in the small but effective i trek - 1 during this phase of the action potential ( figure 3 b, bottom panel ). qualitatively similar results were obtained with other i trek - 1 densities. the observed effects on cycle length are summarized in figure 3 c. source paper: pmc12256823 one may argue that the popdc2 variant - induced decrease in i trek - 1 affects pacemaker activity by changing the excitability of the atrial tissue surrounding the sinus node. this was assessed in simulations of a human atrial cardio myoc yte after incorporating i trek - 1, which was not represented in the original model cell. figure 3 d shows the results obtained with an i trek - 1 density of 4. 0 pa / pf ( at + 30 mv ). simulating the effect of the variants in popdc2 by lowering the density of i trek - 1 to 59 % led to a 0. 63 - mv depolarization of diastolic potential, a 5. 1 % decrease in threshold current ( figures 3 e and 3f, vertical arrows ), and a 23 - ms increase in action - potential duration ( apd ) at 90 % repolarization. the relatively small effects on diastolic potential and threshold current were obtained with a probably overestimated i trek - 1 density, given the large effect of i trek - 1 on the apd of the original model ( figure 3 d, top panel ). smaller, but qualitatively similar, effects on diastolic potential and threshold current were obtained with lower i trek - 1 densities ( figures 3 e and 3f ), associated with a less prominent effect of i trek - 1 on the apd of the original model. overall, despite the observation of bradycardia in mice lacking trek - 1, findings from our simulation studies do not provide an explanation for the clinically observed sinus bradycardia in individuals with popdc2 variants based on a decrease in i trek - 1 per se. individual ii - 3 from family a ( figure 1 a ) underwent muscle biopsy of the m. vastus lateralis ( supplemental information ). histological analysis disclosed mild fiber size variability and a slight increase of connective tissue. neither nuclear centralizations nor fiber splittings were observed. no necrotic or degenerated fibers were detected ( figures 4 a and 4b ). non - specific myopathic features and increased connective tissue were previously observed in muscle samples from individuals with pathogenic popdc1 variants, while affected individuals with popdc3 variants displayed typical features of muscle dystrophies, although the severity of the histopathological findings differed among affected individuals. in healthy skeletal muscle fibers, popdc1 and popdc2 were robustly localized in the sarcol emma. conversely, in the affected individuals ’ muscle, immunoflu orescence staining showed a significant reduction of popdc1 ( figure 4 c ) and popdc2 ( figure 4 d ) levels. on the other hand, the immunoflu orescent signal for cav eol in - 3, which also stains muscle membranes, showed normal distribution and intensity in the muscle sample of the affected individual ii - 3 from family a ( figure 1 a ) compared to control. the severe reduction of popdc1 and popdc2 levels was also documented by sds - page analysis of muscle protein lysates ( figure s5 ). these findings suggest that popdc2 p. gln172 _ tyr176delinshis affects the stability of popdc2, hampering its membrane localization and leading to the secondary reduction of popdc1. indeed, the stability and / or membrane trafficking of the popdc1 - popdc2 complex have been found to be impaired by genetic variants in each of the two proteins. ultrastructural examination detected the presence of tubular aggregates in few muscle fibers ( figure 4 e ). alterations in the structure of the sarcol emma, characterized by small microvilli - like projections, together with sub sarcol emmal vacuoles were also observed. basal lamina appeared unstructured and enlarged in some fibers ( figure 4 f, asterisks ). we also noted increased level of lipids, which in some cases were arranged to form rows of droplets ( figure 4 g ). heterogeneous transmission electron microscopy findings were previously observed in muscle samples from affected individuals harboring variants in popdc1 but not in those with popdc3 pathogenic variants ( mim: 605824 ). to explore the anatomical distribution of popdc1 - 3 expression ( popdc1 ’ s previous official gene name is bves ), we analyzed a previously published visium spatial and single-nucleus transcriptomic dataset. for the visium spatial dataset, annotation of histological tissue sections of the av node and the sinus node was used and expression of popdc1 - 3 was explored across those structures ( figure 5 a ). popdc2 showed higher expression than popdc1 / bves and popdc3.