sinus node and av node / his scrna - seq data from mice were obtained from a previously published study. using these data, t - distributed stochastic neighbor embedding ( t - sne ) maps with a perplexity of 50 were generated on the r2 environment genomics analysis and visualization platform ( http: / / r2. amc. nl ). the cells were subsequently clustered into different populations using the t - sne dbscan tool. sentinel gene expression was used to characterize the different clusters ( e. g., sinus node cells, expressing higher levels of tbx3, isl1, and hcn4 ). thereafter, popdc1 - 3 expression intensities were plotted on the t - sne maps to identify their expression profiles across the present tissue clusters. htrek - 1a cloned in pires2 - egfp was obtained from drs. delphine bichet and florian lesage ( universite nice sophia antipolis, france ). full - length popdc2 cdna sequences ( nm _ 001308333 - hg19; wild type [ wt ], c. 516 _ 527del: p. arg263his ) were synthesized, cloned into pbluescript iisk + ( genecust, boynes, france ), and subsequently subcloned into pires - gfp ( pcgi ). details on cell preparation and expression can be found in the supplemental information. i na and trek - 1 currents were measured with ruptured and amphotericin - perforated patch - clamp technique, respectively, using an axopatch 200b amplifier ( molecular devices corporation, sunnyvale, ca, usa ). voltage control, data acquisition, and analysis were accomplished using custom software. i na recordings were low - pass filtered with a cutoff frequency of 5 khz and digitized at 20 khz, while this was 2 and 4 khz, respectively, for trek - 1 current measurements. series resistance was compensated by ≥80 %, and potentials were corrected for the calculated liquid junction potential. cell membrane capacitance ( c m ) was calculated by dividing the time constant of the decay of the capacitive transient after a −5 - mv voltage step from −40 mv by the series resistance. patch pipettes were pulled from borosilicate glass ( harvard apparatus, uk ) and had resistances of 2. 5 – 3. 5 mω after filling with the solutions as indicated below. measurements from a minimum of nine cells from three independent transfections were acquired for each condition. trek - 1 currents were recorded at 36 ± 0. 2°c. cells were superfused with solution containing ( in mm ) nacl 140, kcl 5. 4, cacl 2 1. 8, mgcl 2 1, glucose 5. 5, and hepes 5 at ph 7. 4 ( naoh ). pipette solution contained ( in mm ) k - gluc 125, kcl 20, nacl 5, amphotericin - b 0. 88, and hepes 10 at ph 7. 2 ( koh ). trek - 1 currents were measured using 500 - ms voltage - clamp steps to test potentials ranging from −100 to + 50 mv from a holding potential of −80 mv. the trek - 1 current was measured at the end of the voltage - clamp step and current densities were calculated by dividing current amplitude by c m. i na was measured at room temperature using a bath solution containing ( in mm ) nacl 20, cscl 120, cacl 2 1. 8, mgcl 2 1. 0, glucose 5. 5, and hepes 5. 0 at ph 7. 4 ( csoh ). pipettes were filled with solution containing ( in mm ) naf 10, cscl 10, csf 110, egta 11, cacl 2 1. 0, mgcl 2 1. 0, na 2 atp 2. 0, and 10 hepes at ph 7. 2 ( csoh ). the i na density and voltage dependence of activation were determined by 50 - ms depolarizing pulses to test potentials ranging from −80 to + 40 mv from a holding potential of −120 mv. voltage - dependent inactivation was obtained by measuring the peak currents during a 50 - ms test step to −20 mv, which followed a 500 - ms prepulse to membrane potentials between −140 and 0 mv to allow inactivation. the holding potential was −120 mv. all voltage - clamp steps were applied with a 5 - s cycle length. peak i na was defined as the difference between peak and steady-state current. current density was calculated by dividing the measured currents by c m. to determine the activation characteristics of i na, current - voltage curves were corrected for differences in driving force and normalized to maximum peak current ( i max ). steady-state activation and inactivation curves were fitted using the boltzmann equation i / i max = a / to determine the membrane potential for half - maximal ( in ) activation ( v 1 / 2 ) and the slope factor k. source paper: pmc12256823 data are presented as mean ± standard error of the mean ( sem ). statistical analysis was carried out with sigmastat 3. 5 software. normality and equal variance assumptions were tested with the kolmogorov - smirnov and the levene median test, respectively. groups were compared with one - way anova. p < 0. 05 defines statistical significance. the spontaneous electrical activity of a single human sinus nodal pacemaker cell was simulated using the comprehensive mathematical model developed by fabbri et al. for simulations of a single human atrial cell, we used the model by maleckar et al. the cellml code of both models, as available from the cellml model repository at https: / / www. cellml. org /, was edited and run in version 0. 9. 31. 1409 of the windows based cellular open resource ( cor ) environment. trek - 1 currents are not included in both original models. to study whether the homozygous loss lof variants in popdc2 contribute to bradycardia via trek - 1 current changes, we fitted our experimental data of the trek - 1 current - voltage relationship ( figure 3 a ) and implemented the thus - obtained trek - 1 current in both models as a control over a range of trek - 1 current densities. subsequently, the trek - 1 density was reduced to 59 % of the trek - 1 + popdc2 - wt current according to the effects induced by the popdc2 variants ( figure 3 a ) to assess the functional effects of the variants. all simulations were run for a period of 200 s, which appeared a sufficiently long time to reach steady-state behavior. the analyzed data are from the final 10 s of the 200 - s period. the recent availability of population - level cohorts with both clinical as well as whole - genome sequencing and well - imputed array genotyping data now provide the opportunity for orthogonal validation of genetic findings through complementary population - level analysis. samples were included from four large population biobanks ( total n = 1, 089, 031 ) with genetic data, namely decode genetics in iceland ( n = 173, 025 ), uk biobank ( n = 428, 503 ), copenhagen hospital biobank and the danish blood donor study in denmark ( n = 487, 356 ), and intermountain in utah, usa ( n = 138, 006 ). disease status was obtained from electronic health records and ascertained using the following international classification of diseases 10th revision codes: atrioventricular block ( i44. 1 and i44. 2 ), bradycardia ( r00. 1 ), cardiac arrest ( i46 ), hypertrophic cardiomyopathy ( i42. 1 and i42. 2 ), muscular dystrophy ( g71. 0 ), myoc arditis ( i40, i41 and i51. 4 ), and sinus node dysfunction ( i49. 5 ). pacemaker implantation was defined based on procedure codes ( decode: nomesco classification of surgical procedures [ ncsp ] codes fpe / fpse and fpf / fpsf. copenhagen hospital biobank: ncsp codes fpe / fpse and fpf / fpsf. uk biobank: national clinical coding standards opcs code k60 ). heart rate was available only in the uk biobank and decode. in the uk biobank, heart rate was obtained during blood - pressure measurement at assessment. both measurements were taken twice, and multiple measurements for one individual were averaged. in decode, heart - rate measurements were sourced from electro cardiogram s ( ecgs ) from landspitali university hospital in iceland between 1998 and 2015. mean values from sinus - rhythm ecgs were obtained for each individual, which were subsequently standar diz ed and adjusted for age and sex. details on each biobank and dna genotyping and sequencing methods for each biobank can be found in the supplemental information.