Electroanatomical adaptations in the guinea pig heart from neonatal to adulthood.

AIMS: Electroanatomical adaptations during the neonatal to adult phase have not been comprehensively studied in preclinical animal models. To explore the impact of age as a biological variable on cardiac electrophysiology, we employed neonatal and adult guinea pigs, which are a recognized animal model for developmental research. METHODS AND RESULTS: Electrocardiogram recordings were collected in vivo from anaesthetized animals. A Langendorff-perfusion system was employed for the optical assessment of action potentials and calcium transients. Optical data sets were analysed using Kairosight 3.0 software. The allometric relationship between heart weight and body weight diminishes with age, it is strongest at the neonatal stage (R2 = 0.84) and abolished in older adults (R2 = 1E-06). Neonatal hearts exhibit circular activation, while adults show prototypical elliptical shapes. Neonatal conduction velocity (40.6 ± 4.0 cm/s) is slower than adults (younger: 61.6 ± 9.3 cm/s; older: 53.6 ± 9.2 cm/s). Neonatal hearts have a longer action potential duration (APD) and exhibit regional heterogeneity (left apex; APD30: 68.6 ± 5.6 ms, left basal; APD30: 62.8 ± 3.6), which was absent in adults. With dynamic pacing, neonatal hearts exhibit a flatter APD restitution slope (APD70: 0.29 ± 0.04) compared with older adults (0.49 ± 0.04). Similar restitution characteristics are observed with extrasystolic pacing, with a flatter slope in neonates (APD70: 0.54 ± 0.1) compared with adults (younger: 0.85 ± 0.4; older: 0.95 ± 0.7). Neonatal hearts display unidirectional excitation-contraction coupling, while adults exhibit bidirectionality. CONCLUSION: Postnatal development is characterized by transient changes in electroanatomical properties. Age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. Understanding heart development is crucial to evaluating therapeutic eligibility, safety, and efficacy.

The effect of sex and age on ex vivo cardiac electrophysiology: insight from a guinea pig model.

Highlighting the importance of sex as a biological variable, we recently reported sex differences in guinea pig in vivo electrocardiogram (ECG) measurements. However, substantial inconsistencies exist in this animal model, with conflicting reports of sex-specific differences in cardiac electrophysiology observed in vivo and in vitro. Herein, we evaluated whether sexual dimorphism persists in ex vivo preparations, using an isolated intact heart preparation. Pseudo-ECG recordings were collected in conjunction with dual optical mapping of transmembrane voltage and intracellular calcium from Langendorff-perfused hearts. In contrast to our in vivo results, we did not observe sex-specific differences in ECG parameters collected from isolated hearts. Furthermore, we observed significant age-specific differences in action potential duration (APD) and Ca2+ transient duration (CaD) during both normal sinus rhythm (NSR) and in response to dynamic pacing but only a modest sex-specific difference in CaD30. Similarly, the alternans fluctuation coefficient, conduction velocity during sinus rhythm or in response to pacing, and electrophysiology parameters (atrioventricular nodal effective refractory period, Wenckebach cycle length) were comparable between males and females. Results of our study suggest that the observed sex-specific differences in in vivo ECG parameters from guinea pigs are diminished in ex vivo isolated heart preparations, although age-specific patterns are prevalent. To assess sex as a biological variable in cardiac electrophysiology, a comprehensive approach may be necessary using both in vitro measurements from cardiomyocyte or intact heart preparations with secondary follow-up in vivo studies.NEW & NOTEWORTHY We evaluated whether the guinea pig heart has intrinsic sex-specific differences in cardiac electrophysiology. Although we observed sex-specific differences in in vivo ECGs, these differences did not persist ex vivo. Using a whole heart model, we observed similar APD, CaD, conduction velocity, and alternans susceptibility in males and females. We conclude that sex-specific differences in guinea pig cardiac electrophysiology are likely influenced by the in vivo environment and less dependent on the intrinsic electrical properties of the heart.

An electrophysiological substrate of COVID-19.

SARS-CoV-2 infection is associated with an increased risk of late cardiovascular (CV) outcomes. However, more data is needed to describe the electrophysiologic (EP) manifestation of post-acute CV sequelae of COVID-19. We compared two cohorts of adult patients with SARS-CoV-2 polymerase chain reaction (PCR) test and an electrocardiogram (ECG) performed between March 1, 2020, and September 13, 2020, in a retrospective double-cohort study, "Cardiovascular Risk Stratification in Covid-19" (CaVaR-Co19; NCT04555187). Patients with positive PCR comprised a COVID-19(+) cohort (n = 41; 61% women; 80% symptomatic), whereas patients with negative tests formed the COVID-19(-) cohort (n = 155; 56% women). In longitudinal analysis, comparing 3 ECGs recorded before, during, and on average 40 days after index COVID-19 episode, after adjustment for demographic and socioeconomic characteristics, baseline CV risk factors and comorbidities, use of prescription medications (including QT-prolonging drugs) before and during index COVID-19 episode, and the longitudinal changes in RR' intervals, heart rhythm, and ventricular conduction type, only in the COVID-19(+) cohort QTc increased by +30.2(95% confidence interval [CI] 0.1-60.3) ms and the spatial ventricular gradient (SVG) elevation increased by +13.5(95%CI 1.2-25.9)°. In contrast, much smaller, statistically nonsignificant changes were observed in the COVID-19(-) cohort. In conclusion, post-acute CV sequelae of SARS-CoV-2 infection manifested on ECG by QTc prolongation and rotation of the SVG vector upward.

Reproducibility of global electrical heterogeneity measurements on 12-lead ECG: The Multi-Ethnic Study of Atherosclerosis.

OBJECTIVE: Vectorcardiographic (VCG) global electrical heterogeneity (GEH) metrics showed clinical usefulness. We aimed to assess the reproducibility of GEH metrics. METHODS: GEH was measured on two 10-s 12­lead ECGs recorded on the same day in 4316 participants of the Multi-Ethnic Study of Atherosclerosis (age 69.4 ± 9.4 y; 2317(54%) female, 1728 (40%) white, 1138(26%) African-American, 519(12%) Asian-American, 931(22%) Hispanic-American). GEH was measured on a median beat, comprised of the normal sinus (N), atrial fibrillation/flutter (S), and ventricular-paced (VP) beats. Spatial ventricular gradient's (SVG's) scalar was measured as sum absolute QRST integral (SAIQRST) and vector magnitude QT integral (VMQTi). RESULTS: Two N ECGs with heart rate (HR) bias of -0.64 (95% limits of agreement [LOA] -5.68 to 5.21) showed spatial area QRS-T angle (aQRST) bias of -0.12 (95%LOA -14.8 to 14.5). Two S ECGs with HR bias of 0.20 (95%LOA -15.8 to 16.2) showed aQRST bias of 1.37 (95%LOA -33.2 to 35.9). Two VP ECGs with HR bias of 0.25 (95%LOA -3.0 to 3.5) showed aQRST bias of -1.03 (95%LOA -11.9 to 9.9). After excluding premature atrial or ventricular beat and two additional beats (before and after extrasystole), the number of cardiac beats included in a median beat did not affect the GEH reproducibility. Mean-centered log-transformed values of SAIQRST and VMQTi demonstrated perfect agreement (Bias 0; 95%LOA -0.092 to 0.092). CONCLUSION: GEH measurements on N, S, and VP median beats are reproducible. SVG's scalar can be measured as either SAIQRST or VMQTi. SIGNIFICANCE: Satisfactory reproducibility of GEH metrics supports their implementation.

Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart.

BACKGROUND: Three distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels. METHODS: Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity. RESULTS: RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3µM) and xestospongin C (XeC; 50µM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5-10µm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10µM). The velocity of wavelets was two times larger (86±16µm/s; n=14) compared to CWWs' (46±10µm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150µm/s (147±34µm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24µm/s; n=5) in the central region wherein predominant RyR2 expression was detected. CONCLUSION: We identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Electroanatomical Adaptations in the Guinea Pig Heart from Neonatal to Adulthood.

Background: Electroanatomical adaptations during the neonatal to adult phase have not been comprehensively studied in preclinical animal models. To explore the impact of age as a biological variable on cardiac electrophysiology, we employed neonatal and adult guinea pigs, which are a recognized animal model for developmental research. Methods: Healthy guinea pigs were categorized into three age groups (neonates, n=10; younger adults, n=13; and older adults, n=26). Electrocardiogram (ECG) recordings were collected in vivo from anesthetized animals (2-3% isoflurane). A Langendorff-perfusion system was employed for optical assessment of epicardial action potentials and calcium transients, using intact excised heart preparations. Optical data sets were analyzed and metric maps were constructed using Kairosight 3.0. Results: The allometric relationship between heart weight and body weight diminishes with age, as it is strongest at the neonatal stage (R 2 = 0.84) and completely abolished in older adults (R 2 = 1E-06). Neonatal hearts exhibit circular activation waveforms, while adults show prototypical elliptical shapes. Neonatal conduction velocity (40.6±4.0 cm/s) is slower than adults (younger adults: 61.6±9.3 cm/s; older adults: 53.6±9.2 cm/s). Neonatal hearts have a longer action potential duration (APD) and exhibit regional heterogeneity (left apex; APD30: 68.6±5.6 ms, left basal; APD30: 62.8±3.6), which was absent in adult epicardium. With dynamic pacing, neonatal hearts exhibit a flatter APD restitution slope (APD70: 0.29±0.04) compared to older adults (0.49±0.04). Similar restitution characteristics are observed with extrasystolic pacing, with a flatter slope in neonatal hearts (APD70: 0.54±0.1) compared to adults (Younger adults: 0.85±0.4; Older adults: 0.95±0.7). Finally, neonatal hearts display unidirectional excitation-contraction coupling, while adults exhibit bidirectionality. Conclusion: The transition from neonatal to adulthood in guinea pig hearts is characterized by transient changes in electroanatomic properties. Age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. Understanding postnatal heart development is crucial to evaluating therapeutic eligibility, safety, and efficacy. What is Known: Age-specific cardiac electroanatomical characteristics have been documented in humans and some preclinical animal models. These age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. What the Study Adds: Cardiac electroanatomical characteristics are age-specific in guinea pigs, a well-known preclinical model for developmental studies. Age-dependent adaptations in cardiac electrophysiology are readily observed in the electrocardiogram recordings and via optical mapping of epicardial action potentials and calcium transients. Our findings reveal unique activation and repolarization characteristics between neonatal and adult animals.

Evoked centripetal Ca(2+) mobilization in cardiac Purkinje cells: insight from a model of three Ca(2+) release regions.

Despite strong suspicion that abnormal Ca(2+) handling in Purkinje cells (P-cells) is implicated in life-threatening forms of ventricular tachycardias, the mechanism underlying the Ca(2+) cycling of these cells under normal conditions is still unclear. There is mounting evidence that P-cells have a unique Ca(2+) handling system. Notably complex spontaneous Ca(2+) activity was previously recorded in canine P-cells and was explained by a mechanistic hypothesis involving a triple layered system of Ca(2+) release channels. Here we examined the validity of this hypothesis for the electrically evoked Ca(2+) transient which was shown, in the dog and rabbit, to occur progressively from the periphery to the interior of the cell. To do so, the hypothesis was incorporated in a model of intracellular Ca(2+) dynamics which was then used to reproduce numerically the Ca(2+) activity of P-cells under stimulated conditions. The modelling was thus performed through a 2D computational array that encompassed three distinct Ca(2+) release nodes arranged, respectively, into three consecutive adjacent regions. A system of partial differential equations (PDEs) expressed numerically the principal cellular functions that modulate the local cytosolic Ca(2+) concentration (Cai). The apparent node-to-node progression of elevated Cai was obtained by combining Ca(2+) diffusion and 'Ca(2+)-induced Ca(2+) release'. To provide the modelling with a reliable experimental reference, we first re-examined the Ca(2+) mobilization in swine stimulated P-cells by 2D confocal microscopy. As reported earlier for the dog and rabbit, a centripetal Ca(2+) transient was readily visible in 22 stimulated P-cells from six adult Yucatan swine hearts (pacing rate: 0.1 Hz; pulse duration: 25 ms, pulse amplitude: 10% above threshold; 1 mm Ca(2+); 35°C; pH 7.3). An accurate replication of the observed centripetal Ca(2+) propagation was generated by the model for four representative cell examples and confirmed by statistical comparisons of simulations against cell data. Selective inactivation of Ca(2+) release regions of the computational array showed that an intermediate layer of Ca(2+) release nodes with an ~30-40% lower Ca(2+) activation threshold was required to reproduce the phenomenon. Our computational analysis was therefore fully consistent with the activation of a triple layered system of Ca(2+) release channels as a mechanism of centripetal Ca(2+) signalling in P-cells. Moreover, the model clearly indicated that the intermediate Ca(2+) release layer with increased sensitivity for Ca(2+) plays an important role in the specific intracellular Ca(2+) mobilization of Purkinje fibres and could therefore be a relevant determinant of cardiac conduction.

KairoSight-3.0 : A Validated Optical Mapping Software to Characterize Cardiac Electrophysiology, Excitation-Contraction Coupling, and Alternans.

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical mapping of voltage-sensitive and calcium-sensitive probes allow for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package ( KairoSight-3 . 0 ) with features to enhance characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3 . 0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80% repolarization), calcium transient duration (30 or 80% reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97% of manual and software values falling within 10 ms of each other and >75% within 5 ms for action potential duration and calcium transient duration measurements (n=1000-2000 pixels). Further, our software package includes additional cardiac metric measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3 . 0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, and the excitation-contraction coupling with satisfactory accuracy. Graphical Abstract Demonstrating Experimental and Data Analysis Workflow: Created with Biorender.com.

KairoSight-3.0: A validated optical mapping software to characterize cardiac electrophysiology, excitation-contraction coupling, and alternans.

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexylphthalate (DEHP).

Di-2-ethylhexylphthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell (RBC) units stored between 7-42 days (23-119 µg/mL). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 µg/mL), but sinus rate declined by 43% and sinus node recovery time prolonged by 56.5% following 30-minute exposure to 100 µg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length, and increased incidence of atrioventricular uncoupling. Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on atrioventricular conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using hiPSC-CM confirmed that DEHP slows electrical conduction in a time (15 min - 3 hours) and dose-dependent manner (10-100 µg/mL). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexyl phthalate (MEHP). This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 µg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 µg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 µg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 µg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

Digitizing ECG image: A new method and open-source software code.

BACKGROUND AND OBJECTIVE: We aimed to develop and validate an open-source code ECG-digitizing tool and assess agreements of ECG measurements across three types of median beats, comprised of digitally recorded simultaneous and asynchronous ECG leads and digitized asynchronous ECG leads. METHODS: We used the data of clinical studies participants (n = 230; mean age 30±15 y; 25% female; 52% had the cardiovascular disease) with available both digitally recorded and printed on paper and then scanned ECGs, split into development (n = 150) and validation (n = 80) datasets. The agreement between ECG and VCG measurements on the digitally recorded time-coherent median beat, representative asynchronous digitized, and digitally recorded beats was assessed by Bland-Altman analysis. RESULTS: The sample-per-sample comparison of digitally recorded and digitized signals showed a very high correlation (0.977), a small mean difference (9.3 µV), and root mean squared error (25.9 µV). Agreement between digitally recorded and digitized representative beat was high [area spatial ventricular gradient (SVG) elevation bias 2.5(95% limits of agreement [LOA] -7.9-13.0)°; precision 96.8%; inter-class correlation [ICC] 0.988; Lin's concordance coefficient ρc 0.97(95% confidence interval [CI] 0.95-0.98)]. Agreement between digitally recorded asynchronous and time-coherent median beats was moderate for area-based VCG metrics (spatial QRS-T angle bias 1.4(95%LOA -33.2-30.3)°; precision 94.8%; ICC 0.95; Lin's concordance coefficient ρc 0.90(95%CI 0.82-0.95)]. CONCLUSIONS: We developed and validated an open-source software tool for paper-ECG digitization. Asynchronous ECG leads are the primary source of disagreement in measurements on digitally recorded and digitized ECGs.

Demographic and Methodological Heterogeneity in Electrocardiogram Signals From Guinea Pigs.

Electrocardiograms (ECG) are universally used to measure the electrical activity of the heart; however, variations in recording techniques and/or subject demographics can affect ECG interpretation. In this study, we investigated variables that are likely to influence ECG metric measurements in cardiovascular research, including recording technique, use of anesthesia, and animal model characteristics. Awake limb lead ECG recordings were collected in vivo from adult guinea pigs using a platform ECG system, while recordings in anesthetized animals were performed using both a platform and needle ECG system. We report significant heterogeneities in ECG metric values that are attributed to methodological differences (e.g., ECG lead configuration, ECG recording platform, presence or absence of anesthesia) that persist even within the same cohort of animals. Further, we report that variability in animal demographics is preserved in vivo ECG recordings-with animal age serving as a significant contributor, while sex-specific influences were less pronounced. Methodological approaches and subject demographics should be fully considered when interpreting ECG values in animal models, comparing datasets between studies, or developing artificial intelligence algorithms that utilize an ECG database.

Risk of Cardiovascular Events After COVID-19.

We aimed to determine absolute and relative risks of either symptomatic or asymptomatic SARS-CoV-2 infection for late cardiovascular (CV) events and all-cause mortality. We conducted a retrospective double cohort study of patients with either symptomatic or asymptomatic SARS-CoV-2 infection (COVID-19+ cohort) and its documented absence (COVID-19- cohort). The study investigators drew a simple random sample of records from all patients under the Oregon Health & Science University Healthcare (n = 65,585), with available COVID-19 test results, performed March 1, 2020 to September 13, 2020. Exclusion criteria were age <18 years and no established Oregon Health & Science University care. The primary outcome was a composite of CV morbidity and mortality. All-cause mortality was the secondary outcome. The study population included 1,355 patients (mean age 48.7 ± 20.5 years; 770 women [57%], 977 White non-Hispanic [72%]; 1,072 ensured [79%]; 563 with CV disease history [42%]). During a median 6 months at risk, the primary composite outcome was observed in 38 of 319 patients who were COVID-19+ (12%) and 65 of 1,036 patients who were COVID-19- (6%). In the Cox regression, adjusted for demographics, health insurance, and reason for COVID-19 testing, SARS-CoV-2 infection was associated with the risk for primary composite outcome (hazard ratio 1.71, 95% confidence interval 1.06 to 2.78, p = 0.029). Inverse probability-weighted estimation, conditioned for 31 covariates, showed that for every patient who was COVID-19+, the average time to all-cause death was 65.5 days less than when all these patients were COVID-19-: average treatment effect on the treated -65.5 (95% confidence interval -125.4 to -5.61) days, p = 0.032. In conclusion, either symptomatic or asymptomatic SARS-CoV-2 infection is associated with an increased risk for late CV outcomes and has a causal effect on all-cause mortality in a late post-COVID-19 period.

Characteristics of Cardiac Memory in Patients with Implanted Cardioverter-defibrillators: The Cardiac Memory with Implantable Cardioverter-defibrillator (CAMI) Study.

This study sought to determine factors associated with cardiac memory (CM) in patients with implantable cardioverter-defibrillators (ICDs). Patients with structural heart disease [n = 20; mean age: 72.6 ± 11.6 years; 80% male; mean left ventricular ejection fraction (LVEF): 31.7 ± 7.6%; history of myocardial infarction in 75% and nonsustained ventricular tachycardia (NSVT) in 85%] and preserved atrioventricular conduction received dual-chamber ICDs for primary (80%) or secondary (20%) prevention. Standard 12-lead electrocardiograms were recorded in AAI and DDD modes before and after seven days of right ventricular (RV) pacing in DDD mode with a short atrioventricular delay. The direction (azimuth and elevation) and magnitude of spatial QRS, T, and spatial ventricular gradient vectors were measured before and after seven days of RV pacing. CM was quantified as the degree of alignment between QRSDDD-7 and TAAI-7 vectors (QRSDDD-7 -TAAI-7 angle). Circular statistics and mixed models with a random slope and intercept were adjusted for changes in cardiac activation, LVEF, known risk factors, and the use of medications known to affect CM occurring on days 1 through 7. The QRSDDD-7-TAAI-7 angle strongly correlated (circular r = -0.972; p < 0.0001) with a TAAI-7-TDDD-7 angle. In the mixed models, CM-T azimuth changes [+132° (95% confidence interval (CI): 80°-184°); p < 0.0001] were counteracted by the history of MI [-180° (95% CI: -320° to -40°); p = 0.011] and female sex [-162° (95% CI: -268° to -55°); p = 0.003]. A CM-T area increase [+15 (95% CI: 6-24) mV*ms; p < 0.0001] was amplified by NSVT history [+27 (95% CI: 4-46) mV*ms; p = 0.007]. These findings suggest that preexistent electrical remodeling affects CM in response to RV pacing, that CM exhibits saturation behavior, and that women reach CM saturation more easily than men.

Detection and removal of pacing artifacts prior to automated analysis of 12-lead ECG.

BACKGROUND: Pacing artifacts must be excluded from the analysis of paced ECG waveform. This study aimed to develop and validate an algorithm to identify and remove the pacing artifacts on ECG and vectorcardiogram (VCG). METHODS: We developed a semi-automatic algorithm that identifies the onset and offset of a pacing artifact based on the VCG signal slope steepness and designed a graphical user interface that permits quality control and fine-tuning the constraining threshold values. We used 1054 ECGs from the retrospective, multicenter cohort study "Global Electrical Heterogeneity and Clinical Outcomes," including 3825 atrial and 10,031 ventricular pacing artifacts for the algorithm development and 22 ECGs including 108 atrial and 241 ventricular pacing artifacts for validation. Validation was performed per digital sample. We used the kappa-statistic of interrater agreement between manually labeled sample (ground-truth) and automated detection. RESULTS: The constraining parameter values were for onset threshold 13.06 ± 6.21 µV/ms, offset threshold 34.77 ± 17.80 µV/ms, and maximum window size 27.23 ± 3.53 ms. The automated algorithm detected a digital sample belonging to pacing artifact with a sensitivity of 74.5% and specificity of 99.6% and classified correctly 98.8% of digital samples (ROC AUC 0.871; 95%CI 0.853-0.878). The kappa-statistic was 0.785, indicating substantial agreement. The agreement was on 98.81% digital samples, significantly (P < 0.00001) larger than the random agreement on 94.43% of digital samples. CONCLUSIONS: The semi-automated algorithm can detect and remove ECG pacing artifacts with high accuracy and provide a user-friendly interface for quality control.

Electrophysiological ventricular substrate of stroke: a prospective cohort study in the Atherosclerosis Risk in Communities (ARIC) study.

OBJECTIVES: The goal of the study was to determine an association of cardiac ventricular substrate with thrombotic stroke (TS), cardioembolic stroke (ES) and intracerebral haemorrhage (ICH). DESIGN: Prospective cohort study. SETTING: The Atherosclerosis Risk in Communities (ARIC) study in 1987-1989 enrolled adults (45-64 years), selected as a probability sample from four US communities (Minneapolis, Minnesota; Washington, Maryland; Forsyth, North Carolina; Jackson, Mississippi). Visit 2 was in 1990-1992, visit 3 in 1993-1995, visit 4 in 1996-1998 and visit 5 in 2011-2013. PARTICIPANTS: ARIC participants with analysable ECGs and no history of stroke were included (n=14 479; age 54±6 y; 55% female; 24% black). Ventricular substrate was characterised by cardiac memory, spatial QRS-T angle (QRS-Ta), sum absolute QRST integral (SAIQRST), spatial ventricular gradient magnitude (SVGmag), premature ventricular contractions (PVCs) and tachycardia-dependent intermittent bundle branch block (TD-IBBB) on 12-lead ECG at visits 1-5. OUTCOME: Adjudicated TS included a first definite or probable thrombotic cerebral infarction, ES-a first definite or probable non-carotid cardioembolic brain infarction. Definite ICH was included if it was the only stroke event. RESULTS: Over a median 24.5 years follow-up, there were 899 TS, 400 ES and 120 ICH events. Cox proportional hazard risk models were adjusted for demographics, cardiovascular disease, risk factors, atrial fibrillation, atrial substrate and left ventricular hypertrophy. After adjustment, PVCs (HR 1.72; 95% CI 1.02 to 2.92), QRS-Ta (HR 1.15; 95% CI 1.03 to 1.28), SAIQRST (HR 1.20; 95% CI 1.07 to 1.34) and time-updated SVGmag (HR 1.19; 95% CI 1.08 to 1.32) associated with ES. Similarly, PVCs (HR 1.53; 95% CI 1.03 to 2.26), QRS-Ta (HR 1.08; 95% CI 1.01 to 1.16), SAIQRST (HR 1.07; 95% CI 1.01 to 1.14) and time-updated SVGmag (HR 1.11; 95% CI 1.04 to 1.19) associated with TS. TD-IBBB (HR 3.28; 95% CI 1.03 to 10.46) and time-updated SVGmag (HR 1.23; 95% CI 1.03 to 1.47) were associated with ICH. CONCLUSIONS: PVC burden (reflected by cardiac memory) is associated with ischaemic stroke. Transient cardiac memory (likely through TD-IBBB) precedes ICH.

Electrocardiogram machine learning for detection of cardiovascular disease in African Americans: the Jackson Heart Study.

AIMS: Almost half of African American (AA) men and women have cardiovascular disease (CVD). Detection of prevalent CVD in community settings would facilitate secondary prevention of CVD. We sought to develop a tool for automated CVD detection. METHODS AND RESULTS: Participants from the Jackson Heart Study (JHS) with analysable electrocardiograms (ECGs) (n=3679; age, 6212 years; 36% men) were included. Vectorcardiographic (VCG) metrics QRS, T, and spatial ventricular gradient vectors magnitude and direction, and traditional ECG metrics were measured on 12-lead ECG. Random forests, convolutional neural network (CNN), lasso, adaptive lasso, plugin lasso, elastic net, ridge, and logistic regression models were developed in 80% and validated in 20% samples. We compared models with demographic, clinical, and VCG input (43 predictors) and those after the addition of ECG metrics (695 predictors). Prevalent CVD was diagnosed in 411 out of 3679 participants (11.2%). Machine learning models detected CVD with the area under the receiver operator curve (ROC AUC) 0.690.74. There was no difference in CVD detection accuracy between models with VCG and VCG + ECG input. Models with VCG input were better calibrated than models with ECG input. Plugin-based lasso model consisting of only two predictors (age and peak QRS-T angle) detected CVD with AUC 0.687 [95% confidence interval (CI) 0.6250.749], which was similar (P=0.394) to the CNN (0.660; 95% CI 0.5970.722) and better (P<0.0001) than random forests (0.512; 95% CI 0.4930.530). CONCLUSIONS: Simple model (age and QRS-T angle) can be used for prevalent CVD detection in limited-resources community settings, which opens an avenue for secondary prevention of CVD in underserved communities.

Competing risks in patients with primary prevention implantable cardioverter-defibrillators: Global Electrical Heterogeneity and Clinical Outcomes study.

BACKGROUND: Global electrical heterogeneity (GEH) is associated with sudden cardiac death in the general population. Its utility in patients with systolic heart failure who are candidates for primary prevention (PP) implantable cardioverter-defibrillators (ICDs) is unclear. OBJECTIVE: The purpose of this study was to investigate whether GEH is associated with sustained ventricular tachycardia/ventricular fibrillation leading to appropriate ICD therapies in patients with heart failure and PP ICDs. METHODS: We conducted a multicenter retrospective cohort study. GEH was measured by spatial ventricular gradient (SVG) direction (azimuth and elevation) and magnitude, QRS-T angle, and sum absolute QRST integral on preimplant 12-lead electrocardiograms. Survival analysis using cause-specific hazard functions compared the strength of associations with 2 competing outcomes: sustained ventricular tachycardia/ventricular fibrillation leading to appropriate ICD therapies and all-cause death without appropriate ICD therapies. RESULTS: We analyzed 2668 patients (mean age 63 ± 12 years; 624 (23%) female; 78% white; 43% nonischemic cardiomyopathy; left ventricular ejection fraction 28% ± 11% from 6 academic medical centers). After adjustment for demographic, clinical, device, and traditional electrocardiographic characteristics, SVG elevation (hazard ratio [HR] per 1SD 1.14; 95% confidence interval [CI] 1.04-1.25; P = .004), SVG azimuth (HR per 1SD 1.12; 95% CI 1.01-1.24; P = .039), SVG magnitude (HR per 1SD 0.75; 95% CI 0.66-0.85; P < .0001), and QRS-T angle (HR per 1SD 1.21; 95% CI 1.08-1.36; P = .001) were associated with appropriate ICD therapies. Sum absolute QRST integral had different associations in infarct-related cardiomyopathy (HR 1.29; 95% CI 1.04-1.60) and nonischemic cardiomyopathy (HR 0.78; 95% CI 0.62-0.96) (Pinteraction = .022). CONCLUSION: In patients with PP ICDs, GEH is independently associated with appropriate ICD therapies. The SVG vector points in distinctly different directions in patients with 2 competing outcomes.

Sex differences in vectorcardiogram of African-Americans with and without cardiovascular disease: a cross-sectional study in the Jackson Heart Study cohort.

OBJECTIVES: We hypothesised that (1) the prevalent cardiovascular disease (CVD) is associated with global electrical heterogeneity (GEH) after adjustment for demographic, anthropometric, socioeconomic and traditional cardiovascular risk factors, (2) there are sex differences in GEH and (3) sex modifies an association of prevalent CVD with GEH. DESIGN: Cross-sectional, cohort study. SETTING: Prospective African-American The Jackson Heart Study (JHS) with a nested family cohort in 2000-2004 enrolled residents of the Jackson, Mississippi metropolitan area. PARTICIPANTS: Participants from the JHS with analysable ECGs recorded in 2009-2013 (n=3679; 62±12 y; 36% men; 863 family units). QRS, T and spatial ventricular gradient (SVG) vectors' magnitude and direction, spatial QRS-T angle and sum absolute QRST integral (SAI QRST) were measured. OUTCOME: Prevalent CVD was defined as the history of (1) coronary heart disease defined as diagnosed/silent myocardial infarction, or (2) revascularisation procedure defined as prior coronary/peripheral arterial revascularisation, or (3) carotid angioplasty/carotid endarterectomy, or (4) stroke. RESULTS: In adjusted mixed linear models, women had a smaller spatial QRS-T angle (-12.2 (95% CI -19.4 to -5.1)°; p=0.001) and SAI QRST (-29.8 (-39.3 to -20.3) mV*ms; p<0.0001) than men, but larger SVG azimuth (+16.2(10.5-21.9)°; p<0.0001), with a significant random effect between families (+20.8 (8.2-33.5)°; p=0.001). SAI QRST was larger in women with CVD as compared with CVD-free women or men (+15.1 (3.8-26.4) mV*ms; p=0.009). Men with CVD had a smaller T area (by 5.1 (95% CI 1.2 to 9.0) mV*ms) and T peak magnitude (by 44 (95%CI 16 to 71) µV) than CVD-free men. T vectors pointed more posteriorly in women as compared with men (peak T azimuth + 17.2(8.9-25.6)°; p<0.0001), with larger sex differences in T azimuth in some families by +26.3(7.4-45.3)°; p=0.006. CONCLUSIONS: There are sex differences in the electrical signature of CVD in African-American men and women. There is a significant effect of unmeasured genetic and environmental factors on cardiac repolarisation.