Keywords: animal model, cardiac arrhythmias, diabetes, hypoglycemia, vitamin E
Abstract
In people with type 1 diabetes, hypoglycemia can induce cardiac arrhythmias. In rodent experiments, severe hypoglycemia can induce fatal cardiac arrhythmias, especially so in diabetic models. Increased oxidative stress associated with insulin-deficient diabetes was hypothesized to increase susceptibility to severe hypoglycemia-induced fatal cardiac arrhythmias. To test this hypothesis, Sprague–Dawley rats were made insulin deficient with streptozotocin and randomized into two groups: 1) control (n = 22) or 2) vitamin E treated (four doses of α-tocopherol, 400 mg/kg, n = 20). Following 1 week of treatment, rats were either tested for cardiac oxidative stress or underwent a hyperinsulinemic-severe hypoglycemic (10–15 mg/dL) clamp with electrocardiogram recording. As compared with controls, vitamin E-treated rats had threefold less cardiac oxidative stress, sixfold less mortality due to severe hypoglycemia, and sevenfold less incidence of heart block. In summary, vitamin E treatment and the associated reduction of cardiac oxidative stress in diabetic rats reduced severe hypoglycemia-induced fatal cardiac arrhythmias. These results indicate that in the setting of diabetes, pharmacological treatments that reduce oxidative stress may be an effective strategy to reduce the risk of severe hypoglycemia-induced fatal cardiac arrhythmias.
NEW & NOTEWORTHY For people with type 1 diabetes, severe hypoglycemia can be fatal. We show in our animal model that insulin-deficient diabetic rats have fatal cardiac arrhythmias during severe hypoglycemia that are associated with increased cardiac oxidative stress. Importantly, treatment with vitamin E, to reduce oxidative stress, decreased fatal cardiac arrhythmias during severe hypoglycemia.
INTRODUCTION
For people with type 1 diabetes, mild-to-moderate hypoglycemia is a common occurrence; and unfortunately, severe hypoglycemia is not an uncommon occurrence. Indeed, severe hypoglycemia occurs roughly 1.3 times per adult per year and accounts for 4%–10% of deaths in type 1 diabetes (1, 2). It remains unclear how severe hypoglycemia causes death, but cardiac arrhythmias have been implicated (3).
As compared with people without diabetes, people with type 1 diabetes have increased electrocardiogram (ECG) abnormalities, including atrioventricular heart block (4, 5), though this number is still relatively low. The incidence of heart block that occurs during severe hypoglycemia has not been well established because it is difficult to obtain this data given that 1) episodes of severe hypoglycemia are unpredictable, 2) unless someone is already connected to an ECG, heart rhythms cannot be analyzed in the time it takes to recover back to normal glucose levels, and 3) clinical studies are unethical due to the degree of hypoglycemia. However, there is some evidence of ECG abnormalities during hypoglycemia from studies whereby “real-world” analyses were performed in people with diabetes who were sent home with ambulatory ECG monitors and continuous glucose monitors. In these studies, bradycardia and atrial and ventricular ectopic beats were noted (6, 7).
Thus, animal models have the advantage of studying severe hypoglycemia in a controlled laboratory setting. We have described fatal cardiac arrhythmias during severe hypoglycemia (3). In this model, it was discovered that rats with insulin-deficient diabetes had an increased incidence of fatal cardiac arrhythmias during severe hypoglycemia compared with nondiabetic rats (8), but the mechanisms are currently unknown. Insulin-deficient diabetes can lead to inflammation and oxidative stress, which can increase the risk of cardiac events (9). Specifically, mitochondrial oxidative stress can alter the activities of many ion channels in the heart, which can increase the risk for cardiac arrhythmias (9, 10). In addition, in the setting of poorly controlled diabetes, increased free fatty acid utilization by heart tissue increases cardiac lipase activity, production of reactive oxidative species (ROS), and subsequent cardiac damage (11). Recent studies demonstrate that insulin-deficient diabetic rats show significant levels of oxidative stress biomarkers in cardiac tissue such as depletion of reduced glutathione and increased activity of superoxide dismutase, catalase, and malondialdehyde (12).
Antioxidants work as antagonists to reactive oxidative species, thus preventing potential damage from oxidizing molecules on bodily tissues. Previous studies have shown that antioxidant supplementation in rats can prevent oxidative stress (10). One such antioxidant, vitamin E, is a clinically relevant supplement with low levels of toxicity in high quantities (13). Studies have shown that vitamin E treatment via diet supplementation, intramuscular injection, and peritoneal injection can reduce oxidative stress in rat models (10, 14). In this study, it is hypothesized that increased oxidative stress in insulin-deficient diabetic rats mediate the previously noted increased susceptibility to severe hypoglycemia-induced fatal cardiac arrhythmias.
RESEARCH DESIGN AND METHODS
Animals
Adult, male Sprague–Dawley rats (Charles River) were investigated at ∼300 g, 7–9 wk of age. Rats were housed individually in 12:12 h light:dark cycles and received standard rat chow-fed ad libitum. All studies were done in accordance with and approved by the Animal Studies Committee at the University of Utah School of Medicine.
Experimental Protocol
All rats were injected with streptozotocin (STZ; 65 mg/kg) to induce insulin-deficient diabetes. Diabetic rats were randomly assigned to the control (n = 28) or vitamin E-treated (α-tocopherol, 400 mg/kg, n = 24) groups. Vitamin E was dissolved in a saline/ethanol mixture (4:1) and, due to its prolonged biological half-life (15), was injected subcutaneously on days 6, 8, 11, and 13 (Fig. 1). Control rats were injected with a vehicle solution containing saline and ethanol. Body weight and glucose were measured 3×/wk. On day 14, rats were either euthanized for testing of cardiac oxidative stress (experiment 1) or underwent hyperinsulinemic/hypoglycemic clamps (experiment 2).
Figure 1.
Experimental protocol. On day 1, rats for experiment 2 underwent surgery for vessel cannulation and EKG lead placement. On day 3, all rats (experiments 1 and 2) were injected with streptozotocin (STZ) to induce insulin-deficient diabetes. Rats were divided into two groups: 1) vitamin E treatment or 2) control. On days 6, 8, 11, and 13, rats were given their respective subcutaneous injection once each day. On day 14, one cohort of rats were euthanized and hearts were tested for oxidative stress. A second cohort of rats underwent hyperinsulinemic/severe hypoglycemic clamps.
Experiment 1: Oxidative Stress Testing
To test the effectiveness of vitamin E to reduce cardiac oxidative stress, control (n = 6) and vitamin E-treated (n = 4) rats were euthanized on day 14 without undergoing hypoglycemia. Left ventricles from the rat hearts were assayed for oxidative stress using the thiobarbituric acid reactive substances (TBARS) assay (Cayman Chemical). The TBARS assay is an accurate measure of lipid peroxidation, which is one of the most common indicators for evidence of oxidative stress (16).
Experiment 2: Hyperinsulinemic/Hypoglycemic Clamp
Rats first underwent surgery for the left carotid artery and right jugular vein cannulation and electrocardiogram (ECG) lead placement subcutaneously as done previously in our laboratory (3). Two days after surgery, rats were induced with diabetes with STZ injection as described in Experimental Protocol and separated into control (n = 22) or vitamin E treatment (n = 20). On day 14, overnight fasted rats underwent hyperinsulinemic (20 0mU/kg/min) (Humulin R; Eli Lilly, Indianapolis, IN) severe hypoglycemic (10–15 mg/dl) clamps for 3 h with continuous ECG recording (PowerLab 26 T; LabChart, ADInstruments, Colorado Springs, CO) (3). Blood samples were obtained through the cannulated carotid artery throughout the clamp for measurements of glucose (glucometer; Ascensia Contour BG monitors; Bayer Healthcare, Mishawaka, IN), epinephrine (ELISA; Abnova, Taiwan), and glucagon (ELISA; Mercodia, Salem, NC). Cardiac arrhythmias were monitored throughout the clamp duration and counted manually for the entire 3 h of severe hypoglycemia. QTc (Bazett’s formula) and heart rate were measured and analyzed every 15 min throughout the clamp. Respiration was obtained by counting breaths. Seizures were noted by visual observations of twitching, body rolls, and stiffening of the body. The time spent having a seizure was recorded as per hour to account for mortality before the end of 3 h of severe hypoglycemia. Observers during the clamp and analyses were blinded to the treatment groups.
Statistics
GraphPad Prism (v. 6.07) was used to calculate statistical differences between the groups. Student’s t test was used for differences between the two groups. Fisher exact test with a Freeman–Halton extension was used for differences in mortality, time to onset of heart block, and incidence of heart block. One-way ANOVA was used for the comparison of three or more groups. Post hoc analysis was performed with Tukey’s test. Statistical significance was determined at P < 0.05. Power analysis was performed using G Power 3.1.
RESULTS
STZ injection resulted in glucose levels that increased to an average of 407 ± 25 and 463 ± 20 mg/dL in control and vitamin E-treated rats, respectively (Fig. 2A). During the treatment protocol, glucose levels and body weight did not differ between vitamin E-treated rats and control rats (Fig. 2, A and B).
Figure 2.
Glucose and body weight during the experiments. A: glucose levels in control (open circle) and vitamin E-treated (black triangle) rats were not different between the groups. After STZ injection, all rats had glucose levels rise to around 400 mg/dL. n = 20–22/group. B: body weight was not different between the two groups. n = 20–22/group. C: In experiment 1, rats were euthanized and ventricles were measured for oxidative stress. Vitamin E treatment (square dots) successfully decreased the amount of ventricular MDA (lipid peroxidation) compared with control rats (circle dots). n = 4-6/group. *P < 0.05, t test. Data are means ± SE. STZ, streptozotocin.
Experiment 1
To confirm that vitamin E treatment reduced oxidative stress in diabetic rats, hearts were excised and analyzed for lipid peroxidation via the TBARS assay. Vitamin E-treated rats had significantly reduced (0.89 ± 0.46 nmol/mg protein; P < 0.02) cardiac oxidative stress compared with control rats (2.79 ± 0.54 nmol/mg protein; Fig. 2C).
Experiment 2
The second cohort of rats underwent hyperinsulinemic/hypoglycemic clamps following control or vitamin E treatment. Glucose levels were evenly matched during the clamp (Con: 12.5 ± 0.4; VitE: 12.6 ± 0.4 mg/dL; Fig. 3A). The average glucose infusion rate required to maintain severe hypoglycemia during the 3 h was increased in vitamin E (0.83 ± 0.14 mg/kg/min; P < 0.04) compared with control (0.45 ± 0.12 mg/kg/min) rats (Fig. 3B). Epinephrine levels were not different between the groups (Fig. 3C). Glucagon levels trended to be decreased in vitamin E-treated rats compared with control rats during severe hypoglycemia (Fig. 3D).
Figure 3.
Experiment 2. Hyperinsulinemic/severe hypoglycemic clamp. A: during the clamp, glucose levels were evenly matched between the two groups. Rats were maintained in the severe hypoglycemic range (10–15 mg/dL) for 3 h. B: the glucose infusion rate was similar between the two groups until the last timepoint where vitamin E-treated rats (black triangle) had a significantly higher glucose infusion rate compared with control rats (open circle). The average glucose infusion rate during severe hypoglycemia was also increased in vitamin E-treated rats. *P < 0.05, t test. C: epinephrine levels increased to a similar extent in both groups during severe hypoglycemia compared with basal conditions. D: glucagon increased during severe hypoglycemia in control rats (white bars) and vitamin E-treated rats (grey bars) with no statistical difference between the two groups. *P < 0.05, one-way ANOVA. Data are means ± SE. n = 20–22/group.
Mortality due to severe hypoglycemia was 37.5% in control rats but treatment with vitamin E significantly reduced mortality to just 6.25% (P < 0.04; Fig. 4A). Reduced mortality was associated with reduced cardiac arrhythmias in vitamin E-treated rats. Third-degree heart block was similarly reduced 7.4-fold with an occurrence in 46% of the control rats and just 6.25% of the vitamin E-treated rats (P < 0.02; Fig. 4B). Third-degree heart block was 100% sensitive and 96% specific at predicting mortality during severe hypoglycemia. Although vitamin E treatment reduced second-degree heart block 1.6-fold compared with control rats, this did not reach significance (Fig. 4C). However, the time to onset of second-degree heart block was delayed in vitamin E-treated rats compared with control rats (P < 0.05; Fig. 4D). Mean respiratory rate during severe hypoglycemia was similar between control (60 ± 2 breaths/min) and vitamin E-treated (61 ± 2 breaths/min) rats. In addition, seizure occurrence was similar between control (44 ± 9 s/h of severe hypoglycemia) and vitamin E-treated (41 ± 8 s/h of severe hypoglycemia) rats.
Figure 4.
Experiment 2. Mortality and cardiac data. A: mortality due to severe hypoglycemia was decreased in vitamin E-treated rats (black bar) compared with control rats (white bar). *P < 0.05, Fisher exact test. B: incidence of third-degree heart block was decreased in vitamin E-treated rats compared with control. *P < 0.05, Fisher exact test. C: the amount of second-degree heart block during severe hypoglycemia was not different between the two groups. D: time to onset of second-degree heart block was delayed in rats with vitamin E treatment (black triangle) compared with control rats (open circle). *P < 0.05, Fisher exact test. E: heart rate decreased during severe hypoglycemia compared with basal in both groups, but to a greater extent in vitamin E-treated rats. *P < 0.05, one-way ANOVA. F: QTc prolongation was increased at basal in the vitamin E-treated rats compared with control and did not further increase during severe hypoglycemia. In control rats, QTc prolongation occurred during severe hypoglycemia. G: representative ECG tracings from control (left) and vitamin E-treated (right) rats during basal settings show normal sinus rhythm and during severe hypoglycemia show second-(arrows) and third-degree (p wave represents the atrium contraction) heart block. *P < 0.05, one-way ANOVA. Data are means ± SE. n = 20–22/group.
Heart rate was reduced in both groups during severe hypoglycemia but to a greater extent in vitamin E-treated rats (211 ± 5 beats/min; P < 0.02) versus control rats (230 ± 7 beats/min) (Fig. 4E). Vitamin E-treated rats had QTc prolongation at baseline (P < 0.05; Fig. 4F). However, during severe hypoglycemia, QTc length was similar between the vehicle (188 ± 4 ms)- and vitamin E (183 ± 4 ms)-treated rats (Fig. 4F). Representative ECG tracings are shown in Fig. 4G.
DISCUSSION
Type 1 diabetes is associated with increased oxidative stress (17), which can lead to cardiac arrhythmias (9, 10). We have previously shown in rat models that diabetes increases the incidence of severe hypoglycemia-induced fatal cardiac arrhythmias, but the mechanisms were unclear. Prior to this study, it was not known whether increased oxidative stress associated with diabetes increases susceptibility to fatal cardiac arrhythmias during a subsequent severe hypoglycemic episode. This current study reveals that 1) cardiac oxidative stress can be lowered with vitamin E and 2) vitamin E treatment in diabetic rats reduces severe hypoglycemia-induced mortality and cardiac arrhythmias, specifically heart block. This is the first study to show a direct link between cardiac oxidative stress and fatal cardiac arrhythmias during severe hypoglycemia.
Vitamin E treatment in diabetic rats led to a reduction in some specific cardiac arrhythmias during severe hypoglycemia but not all. Although second-degree heart block was not reduced by vitamin E treatment, the time to onset of these arrhythmias was delayed. This finding is consistent with other studies that showed vitamin E reduced and delayed the onset of epinephrine-induced cardiac arrhythmias in the absence of hypoglycemia (18). In the current study, third-degree heart block was reduced with vitamin E treatment. Third-degree heart block is highly sensitive and specific at predicting severe hypoglycemia-induced cardiac arrhythmias and mortality in our rodent studies (8). This suggests that vitamin E treatment has the potential to decrease this life-threatening cardiac rhythm in the setting of severe hypoglycemia.
Baseline QTc prolongation occurred in rats treated with vitamin E. QTc prolongation has been associated with hypoglycemia and is a marker of increased risk of cardiac arrhythmias (19). However, in the setting of severe hypoglycemia, in animal models, QTc prolongation is not always associated with fatal cardiac arrhythmias (8). Clinically, the potential for vitamin E to increase QTc length will need to be examined in more detail, especially in those with underlying Long QT syndrome.
Reactive oxygen species are known contributors to cardiac arrhythmias in the setting of diabetes, particularly due to the alteration of ion homeostasis (20). Alterations in calcium, sodium, and/or potassium currents are thought to be the major contributors of ROS to cardiac arrhythmias (21). Although these currents were not measured in this study, these may also be altered by vitamin E treatment indirectly by the reduction in oxidative stress, which is one possible mechanism of action for reduction in cardiac arrhythmias with vitamin E treatment in diabetic rats.
Vitamin E, specifically α-tocopherol, works by reducing the formation of new free radicals and protecting cell membranes from oxidative damage (22). Vitamin E can also inhibit platelet aggregation and release prostacyclin from endothelial cells (23). This is important, especially during hypoglycemia which can lead to platelet aggregation which is associated with major cardiovascular events (24, 25). Therefore, the reduction in fatal heart block with vitamin E in insulin-deficient diabetic rats could be due to a combination of reduced oxidative stress and/or reduced platelet aggregation. Future studies are needed to determine the role of platelet aggregation in fatal cardiac arrhythmias during severe hypoglycemia.
Vitamin E was injected subcutaneously and distributed widely within the body. We infer that its effects are direct consequences of its action on the heart. Cardiac oxidative stress was reduced with vitamin E treatment suggesting that this treatment specifically led to a reduced risk of cardiac arrhythmias during the subsequent severe hypoglycemic episode. It is possible that extra-cardiac reductions in oxidative stress led to the reduction in cardiac arrhythmias. However, we did not measure oxidative stress in other tissues. The intergroup differences in glucose infusion rates and glucagon response suggest extra-cardiac sites of this drug’s effect.
Another limitation of the study is that we did not measure cardiac oxidative stress at the end of severe hypoglycemia. This was due to the fact that hypoglycemia itself is known to increase oxidative stress. Thus, our measurements of cardiac oxidative stress were done in a cohort of rats that did not undergo severe hypoglycemia so that we could test the ability of vitamin E to reduce cardiac oxidative stress in insulin-deficient diabetic rats without the added effect of hypoglycemia.
Although vitamin E supplementation in people with diabetes has been shown to reduce oxidative stress and cardiovascular events (26–28), the clinical efficacy of short-term antioxidant use in attempting to reverse years of oxidative stress and organ damage is still questionable (29, 30). In addition, vitamin E treatment does not work in all populations of people with diabetes perhaps due to the length of the study or the different genetic backgrounds of the individuals being tested (29). For instance, some studies have shown that people with diabetes who also have the specific haptoglobin 2-2 genotype, which leads to the production of a plasma glycoprotein to help reduce oxidative stress, are more responsive to vitamin E treatment to reduce cardiovascular disease than individuals with diabetes who have different haptoglobin genotypes (31). In addition to vitamin E, other antioxidants have also been shown to have beneficial cardiovascular effects in people with diabetes. Specifically, resveratrol reduced oxidative stress and improved cardiovascular function in people with diabetes that experienced a recent myocardial infarction (32). However, there are no known clinical studies that have evaluated antioxidants or specifically vitamin E’s effects on reducing cardiac arrhythmias during severe hypoglycemia in individuals with type 1 diabetes.
Together with our current data in insulin-deficient diabetic rats revealing that vitamin E treatment can reduce fatal heart block during severe hypoglycemia, people at risk for severe hypoglycemia might benefit from treatment with vitamin E. Studies that examine vitamin E treatment in people with type 1 diabetes at risk for severe hypoglycemia are needed to determine potential treatment benefits observed in these animal models.
GRANTS
This research was supported by funding from the Juvenile Diabetes Research Foundation 1-FAC-2020-984-A-N (to C.M.R.-B.), the division of Endocrinology, Metabolism, and Diabetes at the University of Utah (to C.M.R.-B.), the Native American summer research internship program at the University of Utah (I.H.), the Diabetes and Metabolism center at the University of Utah (to C.M.R.-B.), the National Institute of Diabetes and Digestive and Kidney Disease (NIH 5T32DK091317 to C.M.R.-B.; R01DK118082 to S.J.F.), and the Barnstable Brown Diabetes Center and University of Kentucky Diabetes and Obesity Research Priority Area (to S.J.F.).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
C.M.R.-B. and S.J.F. conceived and designed research; C.M.R.-B., M.O., J.B., E.Y.H., and I.H. performed experiments; C.M.R.-B., M.O., J.B., E.Y.H., and I.H. analyzed data; C.M.R.-B., M.O., and J.B. interpreted results of experiments; C.M.R.-B. prepared figures; C.M.R.-B. and M.O. drafted manuscript; C.M.R.-B., M.O., and S.J.F. edited and revised manuscript; C.M.R.-B., M.O., J.B., and S.J.F. approved final version of manuscript.
ACKNOWLEDGMENTS
C.M.R.-B. is the guarantor of this manuscript and takes responsibility for its content. This research was presented at the American Diabetes Association’s Scientific sessions in June 2020.
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