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Salbutamol‐induced electrophysiological changes show no correlation with electrophysiological changes during hyperinsulinaemic–hypoglycaemic clamp in young people with Type 1 diabetes

Abstract Aims Hypoglycaemia causes QT‐interval prolongation and appears pro‐arrhythmogenic. Salbutamol, a β2‐adrenoreceptor agonist also causes QT‐interval prolongation. We hypothesized that the magnitude of electrophysiological changes induced by salbutamol and hypoglycaemia might relate to each other and that salbutamol could be used as a non‐invasive screening tool for predicting an individual's electrophysiological response to hypoglycaemia. Methods Eighteen individuals with Type 1 diabetes were administered 2.5 mg of nebulized salbutamol. Participants then underwent a hyperinsulinaemic–hypoglycaemic clamp (2.5 mmol/l for 1 h). During both experiments, heart rate and serum potassium (and catecholamines during the clamp) were measured and a high‐resolution electrocardiogram (ECG) was recorded at pre‐set time points. Cardiac repolarization was measured by QT‐interval duration adjusted for heart rate (QT c), T‐wave amplitude (Tamp), T‐peak to T‐end interval duration (TpTend) and T‐wave area symmetry (Tsym). The maximum changes vs. baseline in both experiments were assessed for their linear dependence. Results Salbutamol administration caused QT c and TpTend prolongation and a decrease in Tamp and Tsym. Hypoglycaemia caused increased plasma catecholamines, hypokalaemia, QT c and TpTend prolongation, and a decrease in Tamp and Tsym. No significant correlations were found between maximum changes in QT c [r = 0.15, 95% confidence interval (95% CI) −0.341 to 0.576; P = 0.553), TpTend (r = 0.075, 95% CI −0.406 to 0.524; P = 0.767), Tsym (r = 0.355, 95% CI −0.132 to 0.706; P = 0.149) or Tamp (r = 0.148, 95% CI −0.347 to 0.572; P = 0.558) in either experiment. Conclusions Both hypoglycaemia and salbutamol caused pro‐arrhythmogenic electrophysiological changes in people with Type 1 diabetes but were not related in any given individual. Salbutamol does not appear useful in assessing an individual's electrophysiological response to hypoglycaemia.


Introduction
Considerable evidence indicates that hypoglycaemia is proarrhythmogenic during both experimental [1,2] and clinical conditions [3,4]. Hypoglycaemia causes QT c prolongation together with pro-arrhythmogenic changes in rate-independent T-wave characteristics (T p T end , T sym ) via catecholamine release, hypokalaemia and inhibition of rapid delayed rectifier potassium channels (I Kr ) [5]. Cases of bradycardia, atrial fibrillation, atrial flutter or ventricular ectopic beats linked with hypoglycaemia have been reported previously [3,4,6,7]. Although hypoglycaemia continues to be very common in people with Type 1 diabetes [8], sudden nocturnal deaths ('dead-in-bed' syndrome) [9], in which hypoglycaemia-induced malignant arrhythmias have been implicated, are fortunately very rare [10]. This indicates that the risk of arrhythmias is confined to only a few individuals although whether increased susceptibility is related to genetic factors, autonomic dysfunction other unrecognized influences remains unclear. If those at greatest risk of QT c lengthening and potentially hypoglycaemia-induced arrhythmias could be identified with a simple screening test, it might then be possible to use therapeutic or technological interventions to prevent the development of malignant cardiac arrhythmias.
Salbutamol is a b-adrenoreceptor agonist with predominant b 2 activity commonly used for the treatment of asthma and chronic obstructive pulmonary disease (COPD) [11]. Salbutamol's positive chronotropic effect has been classically linked with b 1 -adrenoreceptor stimulation, whereas QT c -prolonging and hypokalaemic effects are mediated by b 2 -adrenoreceptor activity [12]. We have previously shown that elevated catecholamine levels are the predominant cause of QT c prolongation during hypoglycaemia [13]. Furthermore, pretreatment with a selective b 1 -adrenoreceptor blocker atenolol [11] resulted in significant blunting of the QT c prolongation during experimental hypoglycaemia in people with Type 1 diabetes [14]. We therefore hypothesized that establishing electrophysiological changes induced by salbutamol inhalation might offer a practical, non-invasive approach to predicting an individual's electrophysiological responses induced by hypoglycaemia. This raised the possibility of identifying high-risk candidates who might be suitable for potential prophylactic treatment with b-adrenoreceptor blockers or an implantable cardioverter defibrillator. The aim of this study was to examine the potential use of inhaled salbutamol as a screening test for those at greatest risk of abnormal cardiac repolarization during hypoglycaemia.

Participants and methods
Eighteen adults with Type 1 diabetes mellitus (age ≤ 50 years) were recruited from local diabetes outpatient clinics. Exclusion criteria were presence of ischaemic heart disease, cerebrovascular or peripheral vascular disease, asthma or COPD, presence of severe microvascular diabetic complications (diabetic maculopathy with severe visual impairment or eGFR CKD-EPI < 30 ml/min/1.73 m 2 ) and medication with b-blocking or QT-interval prolonging agents. Written informed consent was obtained from all participants and the study received local ethics approval.

Baseline assessment and salbutamol challenge
Each potential participant had a 12-lead electrocardiogram (ECG) performed and individuals with branch block or atrial fibrillation were excluded. Arterial BP, BMI, HbA 1c , urea and electrolytes, total cholesterol and triglyceride levels were obtained during the initial study visit. Cardiovascular autonomic tests were performed [15,16] to establish cardiovascular autonomic neuropathy status [17]. Participants were instructed to avoid vigorous exercise, caffeine and smoking 24 h prior to morning testing. Capillary blood glucose (BG) was measured immediately prior to administration of salbutamol to ensure participants were not hypoglycaemic (BG ≤ 3.5 mmol/l). Subsequently, 2.5 mg salbutamol was administered via a face mask. BP, heart rate, serum potassium and 5-min high-resolution three-lead ECG were measured at baseline and 10, 20, 40 and 60 min after salbutamol administration.

Hyperinsulinaemic-hypoglycaemic clamp
Following the salbutamol challenge, participants underwent a hyperinsulinaemic-hypoglycaemic clamp. Both study visits took place at least 4 weeks apart. Participants were instructed to measure BG before meals, on going to bed, at 03:00 and on waking on the day of the clamp to ensure no hypoglycaemia (BG ≤ 3.5 mmol/l) occurred 24 h prior to the clamp. Upon arrival at 08:30, an intravenous cannula was inserted into the antecubital fossa of the dominant arm initially for insulin and NaCl, and later for insulin and dextrose infusions. An infusion of 0.9% NaCl with KCl (20 mmol KCl for each 500ml of 0.9% NaCl) was initiated at a rate of 125 ml/h. For the following 4 h, BG was stabilized in the euglycaemic range (5-10 mmol/l) using a variable intravenous insulin infusion (Actrapid, Novo Nordisk Ltd, Crawley, UK) adjusted based on BG readings every 30 min. At 12:00 a retrograde cannula was inserted in the non-dominant hand in a warming chamber at 55°C for arterialized BG, potassium and catecholamine sampling. A hypoglycaemic clamp procedure began at 13:00. A primed continuous intravenous insulin infusion was administered at 60 mU/m 2 /min along with 20% dextrose commenced 4 min later at a variable rate, adjusted according to arterialized BG concentrations measured every 5 min (Analox GM9D Glucose Direct Analyser, Analox Instruments Ltd., Stourbridge, UK) for the whole duration of the clamp. Participants were blinded to BG values. From the start of the clamp, BG was What's new?
• We explored b 2 -agonist salbutamol inhalation to identify those at risk of abnormal cardiac repolarization during hypoglycaemia. We describe the electrophysiological effects of nebulized salbutamol and a hypoglycaemic clamp in people with Type 1 diabetes.
• We confirm that both salbutamol and hypoglycaemia have pro-arrhythmogenic electrophysiological effects.
• The magnitude of electrophysiological changes induced by these two stimuli did not show any relationship as measured by a statistically significant correlation in any of the examined variables.
• An individual's electrophysiological response to inhaled salbutamol does not appear useful in predicting their electrophysiological response to hypoglycaemia. To measure catecholamines, 6 ml of whole blood was collected into chilled lithium heparin tubes containing 50 ll EGTA/glutathione preservative and centrifuged at 4°C, 3000 rpm for 10 min. The resulting supernatant was stored at À80°C until assayed by highperformance liquid chromatography.

ECG and cardiac repolarization
Three orthogonal ECG leads (X, Y and Z) were low-pass filtered (40 Hz) and the isoelectric line was subtracted. For each lead, an average beat was calculated from a 5-min recording. A composite wave was then calculated from averaged beats as the square root of X 2 + Y 2 + Z 2 and was used for further analysis of cardiac repolarization. Semiautomatic custom-built software was used to detect the fiducial points and to calculate the parameters of T-wave morphology. Measurement of the QT-interval was based on the tangent method and Bazett correction for heart rate was applied (QT c ). T-wave morphology was characterized by the T-wave amplitude (T amp ), normalized to the baseline [18], the T-peak to T-end interval duration (T p T end ), T p T end corrected for heart rate (T p T end ) and T-wave area symmetry ratio (T sym ), which was defined as the area under the T-wave from T-wave onset to T-peak divided by the area under the T wave between T-peak to T-end [19]. The onset and offset of the T-wave were characterized by tangents at the ascending and descending sections of the T-wave.

Statistical analysis
Statistical analysis was performed with SPSS (version 23; IBM, Chicago, IL, USA). Graphing was completed using GraphPad Prism (version 7.03, GraphPad Software, Inc., San Diego, CA, USA). Repeated measures analysis of variance (ANOVA) was applied to both salbutamol and clamp studies. The Greenhouse-Geisser correction was used to test the effect of time where the sphericity condition was violated according to Mauchly's test. Where a significant effect of time was identified, contrasts to baseline were used to compare means of variables at multiple time points during salbutamol challenge (T10, T20, T40 and T60), and euglycaemic time point (60 EU1) and hypoglycaemic time point (150 HYPO2) of the hypoglycaemic clamp procedure vs. their corresponding baseline. For all parameters, normality of residuals was confirmed by revision of QQ plots. Linear dependence between variables was examined using Pearson correlation coefficient (r). P < 0.05 was deemed statistically significant and correlations of > 0.6 were considered clinically relevant.

Baseline characteristics and salbutamol challenge
Baseline participant characteristics are shown in Table 1.
Following administration of 2.5 mg nebulized salbutamol, systolic and diastolic BP readings did not change significantly ( Table 2). Serum potassium concentrations showed a nonsignificant trend towards lower values but remained within the normal range (Fig. 1a). A trend towards higher heart rates compared with baseline was observed from T20 onwards but did not increase significantly (P = 0.170 for the group) (Fig. 1b). Both QT c interval and T p T end significantly prolonged following salbutamol inhalation (P < 0.001 for the group). QT c interval was significantly prolonged at all time points vs. baseline with a maximum mean difference of 18.0 ms at T20 [95% confidence interval (95% CI) 12.5 to 23.4] (Fig. 1c). Similarly, T p T end interval duration was significantly prolonged at all time points vs. baseline with a maximum mean difference of 7.4 ms at T20 (95% CI 4.5 to 10.3) (Fig. 1d). T-wave area symmetry (T sym ) decreased (P = 0.010 for the group) after salbutamol administration. This difference was statistically significant at all time points with a maximum mean difference vs. baseline À0.082 at T20 (95% CI À0.127 to À0.038) (Fig. 1e). The normalized Twave amplitude (T amp ) dropped during the salbutamol challenge (P < 0.001 for the group). The difference was statistically significant at all time points with a maximum mean difference vs. baseline À0.127 at T40 (95% CI À0.177 to À0.078) (Fig. 1f). Numerical values for all observed variables during salbutamol challenge are listed in Table 2.
Correlations between responses to salbutamol inhalation and to hyperinsulinaemic-hypoglycaemic clamp We examined whether the magnitude of salbutamol-induced changes in cardiac repolarization correlated with changes induced during the hyperinsulinaemic-hypoglycaemic clamp. For this purpose, the maximum change in each parameter vs. baseline was calculated for the salbutamol challenge and for the hypoglycaemic part of the clamp. The maximum differences in both challenges were then assessed for their linear dependence. There was no statistically significant correlation between the magnitude of the QT c interval change during salbutamol challenge and during hyperinsulinaemic-hypoglycaemic clamp (r = 0.15, 95% CI À0.341 to 0.576;

Discussion
In this study we sought to determine whether nebulized b 2 -adrenoreceptor agonist salbutamol induced electrophysiological changes were related in character and magnitude to hypoglycaemia-induced electrophysiological changes in young people with Type 1 diabetes.  We showed that administration of 2.5 mg nebulized salbutamol led to significant prolongation of QT c interval duration with concomitant T p T end interval prolongation, flattening of the T-wave and a change towards a more symmetric T-wave. These findings are largely in keeping with previously published data indicating that salbutamol causes QT c interval prolongation, T-wave flattening and, in some participants, ST-segment depression and emergence of U-waves [20]. To our knowledge, we are the first to report that salbutamol inhalation results in significant T p T end interval prolongation and a change in T-wave area symmetry. T p T end and T sym are measures of dispersion of cardiac repolarization, an important factor in the initiation of ventricular arrhythmias [19,21] and T p T end prolongation has been shown to be associated with increased risk of sudden cardiac death [22]. In our study, we observed non-significant trends towards increased heart rate and decreased serum potassium levels. Given the previously reported significant changes in these characteristics in similar settings [23], this probably reflects the relatively small number of participants. Since the introduction of b 2 -adrenoreceptor agonists in the treatment of obstructive airway disease in the 1960s, concerns have been raised regarding their short-and long-term cardiovascular safety profile and this issue has remained controversial. Case reports suggesting increased risk of cardiac arrhythmias linked with the use of salbutamol have been published [24]; however, data from larger studies and meta-analyses have been conflicting. Some studies have reported a significantly increased risk for adverse cardiovascular events [25], whereas others have not [26]. Careful use of b 2 -adrenoreceptor agonists, especially in specific patient populations such as in those with underlying cardiac conditions, is therefore warranted.
During the hyperinsulinaemic euglycaemic part of the clamp we observed a decrease in plasma potassium levels with concomitant QT c and T p T end interval prolongation and decreased T-wave amplitude (T amp ) and more symmetric Twaves (decreased T-wave area symmetry ratio T sym ). Significant increases in plasma catecholamine levels were detected during the subsequent hyperinsulinaemic-hypoglycaemic part of the clamp, which resulted in an additional decrease in plasma potassium levels, QT c and T p T end prolongation, and decreased T amp and T sym . These biochemical and electrophysiological findings confirm our previous studies indicating that hypoglycaemia causes an acquired long-QT syndrome with pro-arrhythmogenic changes in T-wave morphology and that these changes are caused mainly by a combination of insulin-induced hypokalaemia and hypoglycaemia-stimulated catecholamine release [1,13]. Importantly, similar electrophysiological changes are present during both experimental [1] and clinical hypoglycaemia in various groups of individualsin children and adolescents [27], in young and otherwise healthy people with type 1 diabetes [4] or in middle-aged people with Type 2 diabetes with cardiovascular risk factors [3]. We also detected a nonsignificant trend towards increased heart rate during the hypoglycaemic part of the clamp in keeping with changes observed in our previously published studies [13,14].
Lastly, we examined whether the character and magnitude of salbutamol-induced changes in cardiac repolarization correlated with changes induced by a hyperinsulinaemichypoglycaemic clamp. We did not find any significant correlation in any of the examined electrophysiological variables, which, at first sight and given the current knowledge about the mechanisms causing hypoglycaemia and salbutamol-induced QT c interval prolongation, appears surprising. Clearly, one possible explanation is that with this number of participants our study lacked sufficient statistical power to demonstrate a statistically significant association. Eighteen is a relatively large number of participants for studies inducing experimental hypoglycaemia using a hyperinsulinaemic clamp and this number provided 80% power to identify a statistically significant r-value of 0.6. We reasoned that it would be necessary to identify this strength of association if we were to justify inhaled salbutamol as a screening test. The upper limits of the 95% CI of correlations for QT c, T p T end and T amp in our study do not include values of > 0.6, suggesting that these results are neither statistically significant nor clinically relevant. The rather wide 95% CI for T sym includes the clinically relevant correlation however, which suggests that this result is not statistically significant but might be potentially clinically relevant and further studies on larger number of participants will be required.
Our hypothesis was based on our previous work which showed that QT c interval prolongation induced by hyperinsulinaemic-hypoglycaemic clamp in healthy individuals was completely abolished by atenolol [13]. In the same individuals, keeping the serum potassium levels within the normal range (3.5-4.5 mmol/l) during a hyperinsulinaemic-hypoglycaemic clamp on a different occasion only partially diminished the magnitude of QT c prolongation [13]. Atenolol was subsequently shown to significantly reduce QT c prolongation during hyperinsulinaemic-hypoglycaemic clamps in individuals with Type 1 diabetes [14]. These data therefore implicated sympathoadrenal activation as a major driver of hypoglycaemia-induced QT c prolongation. Both sympathoadrenal response, independently of its trigger, and hypoglycaemia cause hypokalaemia. Catecholamines and salbutamol cause hypokalaemia via binding to b 2 -adrenoreceptors with resulting formation of cyclic adenosine monophosphate (cAMP) and subsequent stimulation of membrane-bound sodium potassium adenosine triphosphatase (Na + /K + -ATPase) [12]. This ion channel causes a shift of potassium from the extracellular to the intracellular compartment with resulting hypokalaemia. Hypoglycaemia causes hypokalaemia via two mechanisms: sympathoadrenal activation and via the effects of insulin, which stimulates potassium cellular uptake by elevation and increased sensitivity to intracellular sodium, translocation and activation of Na + /K + -ATPase and by inhibition of potassium cellular efflux [28]. Thus, mechanisms behind b 2 -adrenergic and hypoglycaemia-induced hypokalaemia are similar, but not identical. In addition, hypoglycaemia causes QT c prolongation by direct inhibition of I Kr channels in the cardiomyocyte membrane [29]. The resulting prolongation of action potential duration, together with intracellular calcium loading in the cardiomyocyte caused by increased b-adrenergic stimulation, represent the two major pro-arrhythmic pathomechanisms [5]. The one obvious difference between the salbutamol challenge and hyperinsulinaemic-hypoglycaemic clamp in our study was the presence of hypokalaemia (K + < 3.5 mmol/l) during the clamp. Salbutamol administration, in our hands, did not lead to hypokalaemia and potassium levels were relatively constant during the observed period of 60 min post administration. It is possible, that the effect of hyperinsulinaemia-induced sympathoadrenal activation with hypokalaemia and potential direct effect of hypoglycaemia on I Kr channels [29] influenced the resulting electrophysiological changes in a given individual. Consistent with our previous observations that sympathoadrenal activation appears to be the main driver of hypoglycaemia-induced electrophysiological changes, we wonder whether the response is also modulated by hypokalaemia and direct inhibitory effect of hypoglycaemia on I Kr channels.
There are other limitations to the current study. First, we could not control for the variable impairment in hypoglycaemia-induced sympathoadrenal responses among individuals with Type 1 diabetes during a given hypoglycaemic stimulus. This known heterogeneity may be an important reason for the limited correlation.
Second, we examined electrophysiological responses during experimental hypoglycaemia rather than clinical hypoglycaemia. Circulating insulin concentrations during experimental hypoglycaemia are greater than those generally observed in clinical hypoglycaemia with resulting greater electrophysiological effects due to more pronounced hypokalaemia [1,30].
Third, it is unclear to what extent variable degrees of autonomic dysfunction contributed to the lack of a relationship. Most of the participants did not have cardiovascular autonomic neuropathy (15/18), three had some degree of autonomic dysfunction as measured by cardiovascular autonomic function tests, but none had definite cardiovascular autonomic neuropathy.
In summary, we have described electrophysiological changes following a single dose of nebulized salbutamol and during a subsequent hyperinsulinaemic-hypoglycaemic clamp in 18 young people with Type 1 diabetes. Both stimuli caused prolongation of QT c and T p T end intervals and decreased Twave amplitude (T amp ) and T-wave area symmetry (T sym ). The magnitude of electrophysiological changes induced by these two stimuli does not correlate in a given individual, however. Salbutamol does not, therefore, appear to be a useful screening tool to assess an individual's electrophysiological response to hyperinsulinaemic hypoglycaemia. A further search for ways of identifying those with diabetes at greatest risk of QT c lengthening and potentially hypoglycaemiainduced arrhythmias is warranted. The current emergence of continuous glucose monitoring into clinical practice and the increasing sophistication of ambulatory Holter ECG monitoring may offer a more promising approach particularly as it involves direct measurement of arrhythmias. Screening for those exhibiting arrhythmias rather than identifying a surrogate measure of abnormal cardiac repolarization such as QT interval may be a better approach in management of this rare but devastating syndrome.
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