Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs.

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Oxford nanopore sequencing can reveal changes in methylation patterns associated with obesity in minute quantities of cell-free DNA from serum.Bisulphite conversion and methylation-specific PCR can be used to validate differentially methylated regions in cell-free DNA.A differentially methylated region in an intronic region of the PPARGC1B gene was found associated with obesity.Differentially methylated regions in cell-free DNA could be useful as early risk markers of certain diseases and pathologies.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 Angiogenesis PET/MR in a Porcine Model of Chronic Myocardial Infarction.

Angiogenesis is crucial in tissue repair and prevents scar tissue formation following an ischemic event such as myocardial infarction. The ischemia induces formation of new capillaries, which have high expression of integrin αvß3. [68Ga]Ga-NODAGA-E[(cRGDyK)]2 ([68Ga]Ga-RGD) is a promising PET-radiotracer reflecting angiogenesis by binding to integrin αvß3. A Göttingen mini-pig underwent transient catheter-induced left anterior descending artery (LAD) occlusion for 120 min, and after 8 weeks was imaged on a Siemens mMR 3T PET/MR system. A large antero-septal infarction was evident by late gadolinium enhancement (LGE) on the short axis and 2-4 chamber views. The infarcted area corresponded to the area with high [68Ga]Ga-RGD uptake on the fused PET/MR images, with no uptake in the healthy myocardium. To support the hypothesis that [68Ga]Ga-RGD uptake reflects angiogenesis, biopsies were sampled from the infarct border and healthy myocardium. Expression of αvß3 was evaluated using immunohistochemistry. The staining showed higher αvß3 expression in the capillaries of the infarct border compared to those in the healthy myocardium. These initial data confirm in vivo detection of angiogenesis using [68Ga]Ga-RGD PET in a translational model, which overall support the method applicability when evaluating novel cardio-protective therapies.

Increased fibroblast accumulation in the equine heart following persistent atrial fibrillation.

BACKGROUND: Fibroblasts maintain the extracellular matrix homeostasis and may couple to cardiomyocytes through gap junctions and thereby increase the susceptibility to slow conduction and cardiac arrhythmias, such as atrial fibrillation (AF). In this study, we used an equine model of persistent AF to characterize structural changes and the role of fibroblasts in the development of an arrhythmogenic substrate for AF. MATERIAL AND METHODS: Eleven horses were subjected to atrial tachypacing until self-sustained AF developed and were kept in AF for six weeks. Horses in sinus rhythm (SR) served as control. In terminal open-chest experiments conduction velocity (CV) was measured. Tissue was harvested and stained from selected sites. Automated image analysis was performed to assess fibrosis, fibroblasts, capillaries and various cardiomyocyte characteristics. RESULTS: Horses in SR showed a rate-dependent slowing of CV, while in horses with persistent AF this rate-dependency was completely abolished (CV•basic cycle length relation p = 0.0295). Overall and interstitial amounts of fibrosis were unchanged, but an increased fibroblast count was found in left atrial appendage, Bachmann's bundle, intraatrial septum and pulmonary veins (p < 0.05 for all) in horses with persistent AF. The percentage of α-SMA expressing fibroblasts remained the same between the groups. CONCLUSION: Persistent AF resulted in fibroblast accumulation in several regions, particularly in the left atrial appendage. The increased number of fibroblasts could be a mediator of altered electrophysiology during AF. Targeting the fibroblast proliferation and differentiation could potentially serve as a novel therapeutic target slowing down the structural remodeling associated with AF.

Arrhythmogenic mechanisms of acute obstructive respiratory events in a porcine model of drug-induced long QT.

BACKGROUND: Obstructive sleep apnea is associated with increased risk of sudden cardiac death. OBJECTIVE: The purpose of this study was to elucidate changes in ventricular repolarization and electromechanical interaction during obstructive respiratory events simulated by intermittent negative upper airway pressure (INAP) in pigs. We also investigated the effect of a reduced repolarization reserve in drug-induced long QT (LQT) following INAP-induced changes in ventricular repolarization. METHODS: In sedated spontaneously breathing pigs, 75 seconds of INAP was applied by a negative pressure device connected to the endotracheal tube. Ventricular electromechanical coupling was determined by the electromechanical window (EMW) before (pre-INAP), during (INAP), and after INAP (post-INAP). Incidence rates of premature ventricular contractions (PVCs) were measured respectively. A drug-induced LQT was modeled by treating the pigs with the hERG1 blocker dofetilide (DOF). RESULTS: Whereas QT interval increased during and decreased after INAP (pre-INAP: 273 ± 5 ms; INAP 281 ± 6 ms; post-INAP 254 ± 9 ms), EMW shortened progressively throughout INAP and post-INAP periods (pre-INAP 81 ± 4 ms; post-INAP 44 ± 7 ms). DOF shortened EMW at baseline. Throughout INAP, EMW decreased in a comparable fashion as before DOF (pre-INAP/+DOF 61 ± 7 ms; post-INAP/+DOF 14 ± 9 ms) but resulted in shorter absolute EMW levels. Short EMW levels were associated with increased occurrence of PVCs (pre-INAP 7 ± 2 ms vs post-INAP 26 ± 6 ms; P = .02), which were potentiated in DOF pigs (pre-INAP/+DOF 5 ± 2 ms vs post-INAP/+DOF 40 ± 8 ms; P = .006). Administration of atenolol prevented post-INAP EMW shortening and decreased occurrence of PVCs. CONCLUSION: Transient dissociation of ventricular electromechanical coupling during simulated obstructive respiratory events creates a dynamic ventricular arrhythmogenic substrate, which is sympathetically mediated and aggravated by drug-induced LQT.

TL1A regulates adipose-resident innate lymphoid immune responses and enables diet-induced obesity in mice.

BACKGROUND/OBJECTIVES: TL1A is a pro-inflammatory cytokine that is homologous to TNFα and connected with the development of several chronic inflammatory disorders. The preliminary results of this study indicated reduced fat accumulation in 9-month-old TL1A-deficient mice at steady state. Thus, the objective was to investigate whether TL1A-deficient mice are resistant to the development of high-fat (HF) diet-induced obesity and to investigate the impact on lymphocyte infiltration in adipose tissue. METHODS: TL1A-deficient and TL1A-sufficient male BALB/cJ littermate mice were fed a 60% HF diet or a 10% low-fat control diet for 22 weeks. Mouse body composition and weight were monitored, and tissues were processed and evaluated by flow cytometry, qPCR, and histology. RESULTS: In this study, the TL1A-deficient HF-diet-fed mice had reduced whole-body weight gain, which was directly explained by a corresponding fat mass reduction (average 37.2%), compared with that of their TL1A-sufficient littermates. Despite previous data showing marked changes in the gut microbial community, TL1A-deficient GF mice also displayed reduced adiposity. Furthermore, the TL1A-deficient mice were resistant to hepatic steatosis and were shown to have improved glucose tolerance, as determined by oral glucose tolerance test (OGTT), and greater insulin sensitivity. In the epididymal white adipose tissue (eWAT), TL1A deficiency in HF-diet-fed mice resulted in a reduced abundance of IL-18Ra+ type-1 ILCs and γδT cells as well as markedly reduced expression of the mitochondria-regulating genes Ucp1, Ucp2, Ucp3, and Prdm16. Finally, to investigate the link of TL1A to obesity in humans, we identified a noncoding polymorphism (rs4979453) close to the TL1A locus that is associated with waist circumference in men (p = 0.00096, n = 60586). CONCLUSIONS: These findings indicate that TL1A plays an important role in regulating adipose tissue mass and that this role is independent of the gut microbiota. Furthermore, we show that TL1A regulates adipose-resident innate lymphocytes and mitochondria-mediated oxidative stress in eWAT.

Comparison of hemodynamics, cardiac electrophysiology, and ventricular arrhythmia in an open- and a closed-chest porcine model of acute myocardial infarction.

Ventricular fibrillation (VF) during acute myocardial infarction (AMI) is an important contributor to sudden cardiac death. Large animal models are widely used to study AMI-induced arrhythmia, but the mode of AMI induction ranges from thoracotomy and surgical ligation of a coronary vessel (open chest) to minimally invasive techniques, including balloon occlusion (closed chest). How the choice of induction affects arrhythmia development is unclear. The aim of this study was to compare an open-chest and a closed-chest model with regard to hemodynamics, electrophysiology, and arrhythmia development. Forty-two female Danish Landrace pigs (20 open chest, 22 closed chest) were anesthetized, and occlusion of the mid-left anterior descending coronary artery was performed for 60 min. Opening the chest reduced blood pressure and cardiac output (Δ -22 mmHg, Δ -1.5 L/min from baseline, both P < 0.001 intragroup). Heart rate decreased with opening of the chest but increased with balloon placement (P < 0.001). AMI-induced ST elevation was lower in the open-chest group (P < 0.001). Premature ventricular contractions occurred in two distinct phases (0-15 and 15-40 min), the latter of which was delayed in the open-chest group (P = 0.005). VF occurred in 7 out of 20 and 12 out of 22 pigs in the open-chest and closed-chest groups, respectively (P = 0.337), with longer time-to-VF in the open-chest group (23.4 ± 1.2 min in open chest and 17.8 ± 1.4 min in closed chest; P = 0.007). In summary, opening the chest altered hemodynamic parameters and delayed the onset of ventricular arrhythmias. Hence, in the search for mechanisms and novel treatments of AMI-induced arrhythmia, caution should be taken when choosing between or comparing the results from these two models.NEW & NOTEWORTHY We demonstrated pronounced differences in hemodynamic parameters and time course of ventricular arrhythmias in regard to mode of infarct induction. Inducing myocardial infarction by thoracotomy and subsequent ligation decreased blood pressure and cardiac output and delayed the onset of ventricular arrhythmia, whereas balloon occlusion resulted in higher heart rates during infarct.

Longitudinal study of electrical, functional and structural remodelling in an equine model of atrial fibrillation.

BACKGROUND: Large animal models are important in atrial fibrillation (AF) research, as they can be used to study the pathophysiology of AF and new therapeutic approaches. Unlike other animal models, horses spontaneously develop AF and could therefore serve as a bona fide model in AF research. We therefore aimed to study the electrical, functional and structural remodelling caused by chronic AF in a horse model. METHOD: Nine female horses were included in the study, with six horses tachypaced into self-sustained AF and three that served as a time-matched sham-operated control group. Acceleration in atrial fibrillatory rate (AFR), changes in electrocardiographic and echocardiographic variables and response to medical treatment (flecainide 2 mg/kg) were recorded over a period of 2 months. At the end of the study, changes in ion channel expression and fibrosis were measured and compared between the two groups. RESULTS: AFR increased from 299 ± 33 fibrillations per minute (fpm) to 376 ± 12 fpm (p < 0.05) and atrial function (active left atrial fractional area change) decreased significantly during the study (p < 0.05). No changes were observed in heart rate or ventricular function. The AF group had more atrial fibrosis compared to the control group (p < 0.05). No differences in ion channel expression were observed. CONCLUSION: Horses with induced AF show signs of atrial remodelling that are similar to humans and other animal models.

Effects of dofetilide and ranolazine on atrial fibrillatory rate in a horse model of acutely induced atrial fibrillation.

INTRODUCTION: The atrial fibrillatory rate is a potential biomarker in the study of antiarrhythmic drug effects on atrial fibrillation (AF). The purpose of this study was to evaluate whether dose-dependent changes in the atrial fibrillatory rate can be monitored on surface electrocardiography (ECG) following treatment with dofetilide, ranolazine, and a combination of the two in an acute model of AF in horses. METHODS AND RESULTS: Eight horses were subjected to pacing-induced AF on 4 separate days. Saline (control), dofetilide, ranolazine, or a combination of dofetilide and ranolazine was administered in four incremental doses. Atrial fibrillatory activity was extracted from surface ECGs using spatiotemporal QRST cancellation. The mean atrial fibrillatory rate before drug infusion was 297 ± 27 fpm. Dofetilide reduced the atrial fibrillatory rate following the infusion of low doses (0.89 µg/kg, P < 0.05) and within 5 minutes preceding cardioversion (P < 0.05). Cardioversion with ranolazine was preceded by a reduction in the atrial fibrillatory rate in the last minute (P < 0.05). The combination of drugs reduced the atrial fibrillatory rate in a similar manner to dofetilide used alone. A trend toward a lower atrial fibrillatory rate before drug infusion was found among horses cardioverting on low doses of the drugs. CONCLUSION: The atrial fibrillatory rate derived from surface ECGs showed a difference in the mode of action on AF between dofetilide and ranolazine. Dofetilide reduced the atrial fibrillatory rate, whereas ranolazine displayed a cardioverting mechanism that was distinct from a slowing of the fibrillatory process.

Time-dependent antiarrhythmic effects of flecainide on induced atrial fibrillation in horses.

BACKGROUND: Pharmacological treatment of atrial fibrillation (AF) in horses can be challenging because of low efficacy and adverse effects. Flecainide has been tested with variable efficacy. OBJECTIVE: To test whether the efficacy of flecainide is dependent on AF duration. ANIMALS: Nine Standardbred mares. METHODS: Factorial study design. All horses were instrumented with a pacemaker and assigned to a control or an AF group. On day 0, all horses were in sinus rhythm and received 2 mg/kg flecainide IV. Atrial fibrillation subsequently was induced in the AF group by pacemaker stimulation. On days 3, 9, 27, and 55, flecainide was administered to all horses, regardless of heart rhythm. RESULTS: All horses in AF cardioverted to sinus rhythm on days 3 and 9. On day 27, 5/6 horses cardioverted, whereas only 2/6 cardioverted on day 55. The time from the start of flecainide infusion to cardioversion (range, 3-185 min, log transformed) showed linear correlation with the cumulative duration of AF (r2 = .80, P < .0001). Flecainide induced abnormal QRS complexes in 4/6 AF horses and 1/3 controls. A positive correlation was found between heart rate before flecainide infusion and number of abnormal QRS complexes (0.14, P < .05). One horse suffered from cardiac arrest and died after flecainide infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Flecainide is effective for cardioversion of short-term induced AF, but the effect decreases with AF duration. Controlling heart rate may minimize adverse effects caused by flecainide, but the drug should be used with great caution.

Antiarrhythmic Effects of Combining Dofetilide and Ranolazine in a Model of Acutely Induced Atrial Fibrillation in Horses.

BACKGROUND: Antiarrhythmic compounds against atrial fibrillation (AF) often have reduced efficacy and may display cardiac and/or noncardiac toxicity. Efficacy can be improved by combining 2 compounds with distinct mechanisms, and it may be possible to use lower doses of each compound, thereby reducing the likelihood of adverse side effects. The purpose of this study was to investigate whether the effective doses of dofetilide and ranolazine can be reduced if the drugs are combined. METHODS: Dofetilide, ranolazine, and a combination of these were administered in 4 incremental dosing regimens to horses with acutely pacing-induced AF. Time to cardioversion, atrial effective refractory period, and AF vulnerability and duration were assessed. RESULTS: Of 8 horses, 6 cardioverted to sinus rhythm after infusion with a combination of 0.889 µg/kg dofetilide and 0.104 mg/kg ranolazine. Two horses cardioverted with 0.104 mg/kg ranolazine alone, and 3 cardioverted with 0.889 µg/kg dofetilide alone. The combination therapy decreased AF vulnerability (P < 0.05) and AF duration (P < 0.05). No change in atrial effective refractory period was detected with any of the drugs. CONCLUSIONS: The combination of dofetilide and ranolazine showed increased antiarrhythmic effects on acutely induced AF in horses, affecting time to cardioversion, AF vulnerability, and AF duration.

Appropriate threshold levels of cardiac beat-to-beat variation in semi-automatic analysis of equine ECG recordings.

BACKGROUND: Although premature beats are a matter of concern in horses, the interpretation of equine ECG recordings is complicated by a lack of standardized analysis criteria and a limited knowledge of the normal beat-to-beat variation of equine cardiac rhythm. The purpose of this study was to determine the appropriate threshold levels of maximum acceptable deviation of RR intervals in equine ECG analysis, and to evaluate a novel two-step timing algorithm by quantifying the frequency of arrhythmias in a cohort of healthy adult endurance horses. RESULTS: Beat-to-beat variation differed considerably with heart rate (HR), and an adaptable model consisting of three different HR ranges with separate threshold levels of maximum acceptable RR deviation was consequently defined. For resting HRs <60 beats/min (bpm) the threshold level of RR deviation was set at 20%, for HRs in the intermediate range between 60 and 100 bpm the threshold was 10%, and for exercising HRs >100 bpm, the threshold level was 4%. Supraventricular premature beats represented the most prevalent arrhythmia category with varying frequencies in seven horses at rest (median 7, range 2-86) and six horses during exercise (median 2, range 1-24). CONCLUSIONS: Beat-to-beat variation of equine cardiac rhythm varies according to HR, and threshold levels in equine ECG analysis should be adjusted accordingly. Standardization of the analysis criteria will enable comparisons of studies and follow-up examinations of patients. A small number of supraventricular premature beats appears to be a normal finding in endurance horses. Further studies are required to validate the findings and determine the clinical significance of premature beats in horses.

Changes in heart rate, arrhythmia frequency, and cardiac biomarker values in horses during recovery after a long-distance endurance ride.

OBJECTIVE To evaluate heart rate, heart rate variability, and arrhythmia frequency as well as changes in cardiac biomarker values and their association with heart rate in horses before and after an endurance ride. DESIGN Cross-sectional study. ANIMALS 28 Arabian horses competing in a 120- or 160-km endurance ride. PROCEDURES ECG recordings were obtained from each horse before (preride) and after (recovery) an endurance ride to evaluate changes in heart rate and the SD of normal R-R intervals (SDNN) during the initial 12 hours of recovery. Frequencies of supraventricular and ventricular premature complexes before and after the ride were evaluated. Blood samples were obtained before the ride and twice during recovery. Hematologic analyses included measurement of serum cardiac troponin I concentration and creatine kinase isoenzyme MB activity. RESULTS Heart rate was significantly increased and SDNN was decreased during the recovery versus preride period. Frequency of ventricular premature complexes increased during recovery, albeit not significantly, whereas frequency of supraventricular premature complexes was not significantly different between preride and recovery periods. Serum cardiac troponin I concentration and creatine kinase isoenzyme MB activity were significantly increased in the recovery versus preride period. No associations were identified between cardiac biomarkers and velocity, distance, or mean heart rate. CONCLUSIONS AND CLINICAL RELEVANCE Heart rate increased and SDNN decreased in horses after completion of an endurance ride. These and other cardiac changes suggested that prolonged exercise such as endurance riding might have cardiac effects in horses. Additional studies are needed to clarify the clinical relevance of the findings.

Pharmacologic inhibition of small-conductance calcium-activated potassium (SK) channels by NS8593 reveals atrial antiarrhythmic potential in horses.

BACKGROUND: Small-conductance calcium-activated potassium (SK) channels have been found to play an important role in atrial repolarization and atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to investigate the existence and functional role of SK channels in the equine heart. METHODS: Cardiac biopsies were analyzed to investigate the expression level of the most prominent cardiac ion channels, with special focus on SK channels, in the equine heart. Subcellular distribution of SK isoform 2 (SK2) was assessed by immunohistochemistry and confocal microscopy. The electrophysiologic and anti-AF effects of the relative selective SK channel inhibitor NS8593 (5 mg/kg IV) were evaluated in anesthetized horses, focusing on the potential of NS8593 to terminate acute pacing-induced AF, drug-induced changes in atrial effective refractory period, AF duration and vulnerability, and ventricular depolarization and repolarization times. RESULTS: Analysis revealed equivalent mRNA transcript levels of the 3 SK channel isoforms in atria compared to ventricles. Immunohistochemistry and confocal microscopy displayed a widespread distribution of SK2 in both atrial and ventricular cardiomyocytes. NS8593 terminated all induced AF episodes (duration ≥15 minutes), caused pronounced prolongation of atrial effective refractory period, and reduced AF duration and vulnerability. QRS duration and QTc interval were not affected by treatment. CONCLUSION: SK channels are widely distributed in atrial and ventricular cardiomyocytes and contribute to atrial repolarization. Inhibition by NS8593 terminates pacing-induced AF of short duration and decreases AF duration and vulnerability without affecting ventricular conduction and repolarization. Thus, inhibition by NS8593 demonstrates clear atrial antiarrhythmic properties in healthy horses.

Cardiac arrhythmias and electrolyte disturbances in colic horses.

BACKGROUND: Despite increased focus on cardiac arrhythmias in horses, the nature and prevalence is still poorly described. Case reports suggest that arrhythmias occurring secondary to systemic disease are seen more commonly in the clinic than arrhythmias caused by cardiac disease. The aim of this study was to investigate the prevalence of arrhythmias in colic horses referred for hospital treatment. Associations between electrolyte disturbances and arrhythmias were also investigated. RESULTS: Heart rate was 37.4 ± 3.7 bpm in the control group, and 51.6 ± 11.8 bpm, in the colic group, which was significantly different (P < 0.0001). AV blocks and SVPCs were found in both groups, however only colic horses showed VPCs. No significant difference between the two groups was found for AV blocks, SVPCs, and VPCs (P = 0.08 - 0.76). The mean levels of potassium, sodium, ionized calcium, and chloride were significantly lower in the colic group compared to the control group at admission. Mean levels of glucose and L-lactate were significantly elevated in the colic group (P < 0.05). CONCLUSIONS: This study describes prevalence of cardiac arrhythmias and electrolytes concentrations in colic horses compared to healthy controls. Although we only observed VPCs in the colic horses, no significant differences between colic horses and controls were found. Despite the colic horses having electrolyte changes at admission no correlation was found between the electrolyte disturbances and cardiac arrhythmias. Although no clear conclusions can be drawn from the present study, the results indicate that relatively mild colic per se is not pro-arrhythmogenic, whereas severe colic probably are more likely to result in ventricular arrhythmia.

Diurnal modulation and sources of variation affecting ventricular repolarization in Warmblood horses.

OBJECTIVES: Irregularities in cardiac repolarization are known to predispose for arrhythmias and sudden cardiac death in humans. The QT interval is a quantitative measurement of repolarization, and clinically, the QTc (QT interval corrected for heart rate) and Tpeak to Tend intervals (TpTe) are used as repolarization markers. To support the use of these markers in horses, we sought to describe the possible influence of the environment, time of day, day-to-day effects, T wave conformation, age, body weight (BW), and horse-to-horse variation on repolarization measurements. ANIMALS: 12 Warmblood geldings, age 10.8 ± 4.8 years. METHODS: Holter ECGs were performed on days 0, 7 and 14. Measures of RR, QT, QTp, QTc and TpTe intervals and T wave conformation were obtained each hour during the recordings. An ANCOVA analysis was performed to estimate diurnal variation and the sources of variation affecting these intervals. RESULTS: Differences between individual horses were the largest source of repolarization variability although the environment had a significant effect on repolarization as well. Diurnal variation affected both the RR interval and the repolarization markers. The QT, QTc and TpTe intervals were prolonged on day 0. Biphasic T waves shortened the TpTe interval approximately 10 ms. Age and BW did not appear to affect repolarization. CONCLUSIONS: Equine repolarization markers exhibit significant variation. Factors affecting repolarization measurements include horse-to-horse variation, diurnal variation, the environment, and T wave conformation. These factors must be considered if markers of equine repolarization are used diagnostically.