QT Interval Instability and Variability in Dogs with Hyperadrenocorticism

Hyperadrenocorticism is one of the most common endocrine diseases in dogs. In humans, it is clearly associated with a higher risk of cardiovascular events, but studies in dogs are scarce. To investigate the arrhythmogenic risk of dogs with hyperadrenocorticism, indices of variability and instability of the QT interval were studied in 38 dogs with hyperadrenocorticism and in 12 healthy dogs: variance (QTv), total instability (TI), short-term (STI) and long-term (LTI), and mean (QTm). Except for QTm, all parameters studied were higher in the hyperadrenocorticism group than in the control group. In addition, STI and QTv showed moderate positive correlation with left ventricle wall thickness. To a better understanding on the effect of the hypothalamus-pituitary-adrenal axis on ventricular repolarization, the hyperadrenocorticism group was subdivided according to the percentage of variation in plasma cortisol concentration (<30.1%; 30.1-60%; >60%) 8 hours after low-dose administration of dexamethasone. There was statistical difference in QTv, TI and LTI indices between the control group and the <30.1% and >60% groups, and in STI index between the control group and the >60% group. There was no statistical difference between sex groups in any of the electrocardiographic parameters studied. This result may indicate that the etiology of hyperadrenocorticism, and its consequent influence on hypothalamus-pituitary-adrenal axis could interfere on the heterogeneity of ventricular repolarization parameters in different ways, especially in the short-term stability; however further studies are necessary to understand the role of cortisol on electrical instability in dogs.


Introduction
Hyperadrenocorticism is one of the most common endocrine disorders in dogs, with prevalence ranging from 0.2 to 1.46% (O'Neill et al. 2016;Carotenuto et al. 2019). In people, it is clear that chronic excessive secretion of cortisol can lead to several complications such as diabetes mellitus, central obesity, hyperlipidemia, hypercoagulability, and systemic arterial hypertension (Mancini et al. 2004). These abnormalities promote a higher risk of cardiovascular events, such as coronary artery disease, acute myocardial infarction, stroke and heart failure, which may remain high five years after successful treatment of the disease (Colao et al. 1999;Dekkers et al. 2013;Terzolo et al. 2014;Di Dalmazi et al. 2014;Bancos et al. 2016) .
In addition to the majority (80%) of people with hyperadrenocorticism having 'high' or 'very high' risk of cardiovascular complications (Mancini et al. 2004), the risk of cardiovascular disease in these patients is five times higher than the rest of the population, and the risk of death from cardiovascular complications doubles with each 20 mmHg increase in systolic blood pressure and 10 mmHg of diastolic blood pressure (Nieman 2019). Veterinary literature lacks information related to cardiovascular risk in dogs with hyperadrenocorticism. Despite no association with atherosclerosis (Hess et al. 2003), dogs with hyperadrenocorticism are at risk for thromboembolic disease, with a prevalence of 6.1% in that population (Hoffman et al. 2018). Also, they are prone to developing systemic arterial hypertension, left ventricular hypertrophy, diastolic dysfunction, left atrial enlargement and congestive heart failure (De Caterina et al. 2010;Nieman 2019).
In addition to functional and structural complications in the cardiovascular system secondary to hyperadrenocorticism, there is evidence of a higher arrhythmogenic risk. In people, several studies have already demonstrated the association between hypercortisolism and atrial fibrillation (Huerta et al. 2005;van der Hooft et al. 2006;De Caterina et al. 2010;Koracevic et al. 2020), but studies evaluating the risk for arrhythmias in dogs with such condition are lacking. Nonetheless, owing to the clear evidences in people and the high incidence of hyperadrenocorticism in dogs, investigation of surrogates for cardiovascular events in this species are indeed warranted.
The electrocardiogram is a low-cost exam widely used in veterinary practice, which can provide precious information in these situations. QT interval corresponds to the duration of the ventricular action potential, being one of the most used electrocardiographic parameters for the diagnosis of cardiac abnormalities (Niemeijer et al. 2015). The prolongation of QT interval has been historically associated with a higher frequency of arrhythmic events (Boulaksil et al. 2011;Itoh et al. 2016). Of note, some studies have demonstrated the inaccuracy of the isolated QT interval for stratification of arrhythmogenic risk (Hondeghem 2008a, b). However, parameters derived from QT, such as the instability and variability of QT, have been further studied, and proved more sensitive and specific for predicting the development of arrhythmias (Limprasutr et al. 2018).
In dogs with myxomatous mitral valve disease, our research group recently demonstrated that QT interval instability increases with disease progression and have predictive value for the development of ventricular arrhythmias (Brüler et al. 2018). While in people with hyperadrenocorticism the prolongation of QT interval corrected by heart rate (QTc) has been confirmed (Pecori Giraldi et al. 2011), to the best of our knowledge no study has investigated the influence played by high serum cortisol levels on myocardial repolarization in dogs. Thus, this study aimed at evaluating QT interval indices in dogs with hyperadrenocorticism. We hypothesized that an increased prolongation and instability of QT interval would be associated with a higher risk of developing arrhythmias.

Animals
Medical records of dogs diagnosed with hyperadrenocorticism between January 2013 and April 2016 at a veterinary teaching facility were retrospectively analyzed. The inclusion criteria for the hyperadrenocorticism group (HG) were the diagnosis of hyperadrenocorticism through the low-dose dexamethasone suppression test (LDDST) (0.01 mg/kg IV), in addition to the presence of an electrocardiographic tracing performed within seven days of confirmation of hyperadrenocorticism. Dogs that did not have electrocardiographic tracing or that were already receiving any hyperadrenocorticism drug therapy at the time of electrocardiographic recording were not admitted into this investigation. To access the presence of left ventricle (LV) concentric hypertrophy, echocardiographic data were also collected from dogs that had this information available in their records. Concentric hypertrophy was defined as diastolic LV free wall (LVFW) and/or interventricular septum (IVS) thickness above the normal range for body weight (Gonçalves et al. 2002). For the control group (CG), healthy dogs from a Beagle research colony were prospectively enrolled. Only dogs without clinical characteristics compatible with hyperadrenocorticism or any other systemic disease were included in this group. These dogs underwent a thorough clinical examination as well as electrocardiographic evaluation. In either group, demographic data (breed, sex, age, body weight) was recorded.

Electrocardiographic Analysis
In all animals, the electrocardiographic tracing was obtained using a computer-based equipment. Bipolar (I, II and III), and unipolar (aVL, aVR and aVF) leads were recorded, with paper velocity adjusted to 50 mm/s and sensitivity calibrated to 1 mV/1 cm. The analysis of the electrocardiographic traces was performed in lead II, and included duration of the QT interval, which corresponds to the beginning of the Q wave to the end of the T wave (Santilli et al. 2019). Fifty consecutive QT intervals were measured and recorded, allowing the calculation of the following parameters: QTm (mean QT intervals); QTv (variance of QT intervals); TI (total instability); LTI (long-term instability); and STI (short-term instability). The variables TI, LTI and STI were calculated according to the methodology proposed by Van der Linde and colleagues (Van Der Linde et al. 2005), which is based on Poincaré plots (Fig 1) constructed from the intersection between QT n (X axis) and QT n+1 (Y axis). TI was calculated by applying the equation described below: Where: : The width and length of the plot XY were measured after rotating it in -45 o () around its origin.
Thus, rotational centers of gravity were obtained, which were later used to calculate LTI and STI. Where: For the interpretation of the Spearman correlation magnitude, the following classification was adopted: correlation coefficients 0 to 0.1 (negligible), 0.1 to 0.39 (weak), 0.4 to 0.69 (moderate), 0.7 to 0.89 (strong), and 09 to 1 (very strong) (Schober et al. 2018).

Statistical analysis
In addition, Kruskal-Wallis test followed by Dunn's post hoc test was used to check for differences in accordance to the percentage of reduction of basal cortisol in response to LDDST. Lastly, Mann-Whitney test was used to evaluate differences attributed to sex in HG. All statistical analyses were performed using the software Graphpad Prism for Windows (v.5.02), considering p<0.05 as significant.

QT mean, variability and instability indices
With the exception of QTm, a significant difference was documented between CG and HG in all parameters studied. QTv, LTI, STI and TI were higher in HG than in CG (

Discussion
In this study, we investigated the influence of hypercortisolism in some QT interval variables in dogs. Although QT interval was not prolonged, the variability and instability of total ventricular electric activity was higher in dogs with hyperadrenocorticism compared to healthy dogs. In addition, the indices derived from the QT interval varied according to the intensity of hypothalamic pituitary-adrenal axis (HPAA) suppression during the LDDST.
QT interval reflects the duration of cardiac action potential and its prolongation is considered proarrhythmic. The association between QT prolongation and a higher risk of ventricular arrhythmias and sudden death has been discussed in the literature for more than 40 years (Schwartz and Wolf 1978;Straus et al. 2006;Ahmad and Dorian 2007;Hondeghem 2011;Niemeijer et al. 2015). In dogs, the prolongation of QTc has been demonstrated with the progression of several heart diseases (Koyama et al. 2004;Brüler et al. 2018), which might potentially result in higher arrhythmogenic risk. Nonetheless, in spite of the progressive increase of QT interval along the worsening of mitral valve degeneration in dogs, we recently showed that QTc failed to predict the development of ventricular arrhythmias in those animals (Vila et al.

2021).
Curiously, several studies also suggest that prolongation of the QT interval and QTc are not reliable predictors of ventricular arrhythmia (Spier et al. 2001;Hondeghem 2008a, b;Leonard et al. 2013). In fact, the prolongation of the duration of the action potential can be antiarrhythmic, since it is the mechanism of class III antiarrhythmic agents (Brendorp et al. 2002). Thus, other factors must coexist with QT interval duration abnormalities for the promotion of proarrhythmic effects, such as spatial or temporal heterogeneity of cardiac repolarization (Schneider et al. 2005). Our results showed no difference between the HG and the CG in the mean duration of QT interval (QTm), but significant difference was found in the parameters that assess repolarization temporal heterogeneity through QT variability (QTv) and instability (TI, LTI and STI).
QT interval exhibits spontaneous fluctuations with each beat, reflecting subtle time variations in ventricular depolarization and repolarization. Because ventricular depolarization is much more stable than repolarization, QT variability may be used as a tool to measure the variance of ventricular repolarization duration (Baumert et al. 2016). Parameters derived from QT variability have already been shown to be useful tools to investigate pro-arrhythmogenic and non-arrhythmogenic effect of drugs in dogs (Schneider et al. 2005), and to be a strong predictor of ventricular fibrillation in rabbits anesthetized with myocardial ischemia, presenting greater sensitivity and specificity than the QT and QTc intervals (Sarusi et al. 2014;Limprasutr et al. 2018). Therefore, the greater variability and instability of QT in dogs with hyperadrenocorticism as compared to healthy dogs (Table 2 and Figure 2) points to a higher risk for cardiovascular events in that population.
In our study, dogs with plasma cortisol reduction >60% (i.e., likely to have pituitary-dependent hyperadrenocorticism) and dogs with plasma cortisol reduction <30.1% (i.e., likely to present adrenal tumor and/or pituitary-dependent hyperadrenocorticism) showed similar results. QTv, TI and LTI were similar in these groups, and significantly higher than in CG. in. For STI, however differences existed only between the >60% group and the CG (Table 2). This result may indicate that the etiology of hyperadrenocorticism, and its consequent influence on HPAA, could interfere on the heterogeneity of ventricular repolarization parameters in different ways, especially in the short-term stability. At least in people with either adrenal tumor or pituitary-dependent hyperadrenocorticism, the risk of death and cardiovascular events was shown to be similar (Dekkers et al. 2013). Nevertheless, to the best of authors' knowledge, there are no studies which investigated such characteristic in dogs. It is not clear why the group of dogs with 30-60% of cortisol suppression did not differ from control dogs. Further studies are necessary to understand the role of hyperadrenocorticism etiology on electrical instability in dogs.
Lastly, while studies in people have reported that women have higher QTc than men (Linde et al. An important limitation of this study is its retrospective design, which limits the acquisition of some data, such as the presence of comorbidities. It is known that ventricular repolarization rates may change with heart diseases (Brüler et al. 2018;Vila et al. 2021), with non-cardiac diseases (Armstrong et al. 2017;Kim et al. 2021), as well as with antiarrhythmic drugs administration (Shantsila et al. 2007  QTn+1 (Y axis) in one of the healthy dogs (a), whose total calculated instability was 6.4, and in three dogs with hyperadrenocorticism, whose calculated total instability was 11.6 (b), 16.9 (c) and 25.8 (d). The black squares represent the projection of the center of gravity coordinates of the QT intervals on the X and Y axes Figure 1 Poincaré plot constructed from the intersection between QTn (X axis) and QTn+1 (Y axis) in one of the dogs with hyperadrenocorticism included in the study. The circles represent the 49 cartesian combinations formed between 50 sequenced QT intervals (QT1 to QT50), and the black square represents the projection of the gravity center coordinates of the QT intervals on the X and Y axes Poincaré plots showing the dispersion of the intersection points between QTn (X axis) and QTn+1 (Y axis) in one of the healthy dogs (a), whose total calculated instability was 6.4, and in three dogs with hyperadrenocorticism, whose calculated total instability was 11.6 (b), 16.9 (c) and 25.8 (d). The black squares represent the projection of the center of gravity coordinates of the QT intervals on the X and Y axes