Increase in cardioprotective SUR2A does not alter heart rate and heart rate regulation by physical activity and diurnal rhythm

Abstract

Objectives

SUR2A is an ABC protein serving as a regulatory subunit of ATP-sensitive (K_ATP) channels. An increase in SUR2A levels is cardioprotective and it is a potential therapeutic strategy against ischaemic heart disease, heart failure and other diseases. However, whether overexpression of this protein has any adverse effects is yet to be fully understood. Here, we examined the heart rate and the heart rate diurnal variation in mice overexpressing SUR2A (SUR2A+) and their littermate controls (WT) using ECG telemetry that was continuously recorded for 14 days (days 8–23 post-radiotransmitter implantation).

Methods

Using SigmaPlot 14.0 and Microsoft Excel, Area Under the Curve (AUC) for each parameter was calculated and plotted in a graph.

Results

Both WT and SUR2A+ mice were more physically active during nights and there were no significant differences between two phenotypes. Physical activity was associated with increased heart rate in both phenotypes, but there were no differences in heart rate between phenotypes irrespective of physical activity or time of the day. A diurnal heart rate variation was preserved in the SUR2A+ mice. As area under the curve (AUC) analysis has the potential to reveal differences that are invisible with other statistical methods, we compared AUC of heart rate in SUR2A+ and WT mice. This analysis did not yield anything different from traditional analysis.

Conclusions

We conclude that increased SUR2A levels are not associated with changes in physical activity, heart rate and/or circadian rhythm influence on the heart rate. This lack of adverse effects supports a notion that manipulation with SUR2A levels is a promising cardioprotective strategy.

Introduction

The heart rate is determined by the internal excitation caused by the autonomous pacemaker cells and the external heart rate modulation by the autonomic nervous system (ANS). The sinoatrial node is the main pacemaker of the heart, generating the fastest firing rate of 60–100 impulses/min as compared to the atrioventricular node and other pacemaker cells. There is a multitude of factors that extrinsically influence the heart rate by modifying the sympathetic and parasympathetic stimulation. The sympathetic nervous system increases the heart rate via having a positive effect on chronotropy and dromotropy, while the parasympathetic nervous system has a negative chronotropic and dromotropic effect. During physical activity and exercise, the sympathetic heart innervation predominates and produces an increase in heart rate to meet the adequate oxygenation demands of the muscles, while at rest, the parasympathetic nervous system takes over and slows down the heart rate [1].

The circadian rhythms are endogenous homeostatic mechanisms that regulate the sleep–wake cycle and create a 24-h oscillatory rhythm for many physiological parameters, including heart rate. Circadian rhythms are regulated by the suprachiasmatic nucleus and the pineal gland that produce melatonin, a hormone which has high levels in darkness and low levels in light. Melatonin induces an increase in parasympathetic drive which peaks around the latter part of the habitual sleep time, around 05:00–06:30, which leads to a decrease in the heart rate [2, 3]. It was noted that strokes and heart attacks tend to occur more commonly during the early morning, which is attributed to the sudden increase in heart rate following rising from supine to an upright position during maximal parasympathetic drive [3]. Physical activity/exercise is suggested to be associated with the circadian influence on the heart rate. On the other hand, the circadian rhythms prevent oxidative stress on cardiac muscle and other tissue [4, 5].

Sulfonylurea receptor (SUR) is a regulatory subunit of the hetero-octameric sarcolemmal ATP-sensitive K⁺ channel (K_ATP). Two SUR genes were identified, SUR1 which codes for the high affinity SUR1 regulatory protein present in the K_ATP channels of the pancreatic islets; SUR2 produces two low-affinity proteins via alternate splicing which are: SUR2A and SUR2B [6]. An increase in SUR2A levels increases myocardial resistance to different types of metabolic stresses. It has been suggested that regulating SUR2A expression is a potential therapeutic strategy against conditions where increased myocardial resistance to stress is desirable such as ischaemic heart disease, heart failure and others [7], [8], [9].

Whether overexpression of this protein has any adverse effects is yet to be fully understood. Our recent study demonstrated that SUR2A overexpression leads to changes in the pattern of Q–T interval [10]. Whether heart rate is affected as well is yet to be determined. In this study, we have examined the heart rate and the heart rate diurnal variation SUR2A mice and their littermate controls.

Materials and methods

SUR2A mice

All experiments have been done on four male mice overexpressing SUR2A (SUR2A+ mice) and their four littermate controls (WT). Generation and breeding of these mice have been described in detail in our previous reports [7, 11]. Briefly, linearized CMV promoter-SUR2A construct was injected into fertilized eggs from superovulated female derived from CBAxBalb/c crosses (male and female F1′s). Founder mice were checked for successful integration of the transgene CMV-SUR2A by PCR using CMV-specific primers. Mouse genomic DNA as template was extracted from mouse ears using the Wizard^® SV Genomic DNA Purification System (Promega). Founder mice were bred between themselves and with C57BL/6J mice. Every mouse was geno-typed using the CMV-specific primers. All wild-type mice used in the study as controls were littermates. A moderate increase in SUR2A mRNA and protein levels (4–10 times) was demonstrated by real-time RT-PCR and Western blotting, respectively [7, 11]. The experiments in this study have been done under authority of Project Licenses 60/3152 and 60/3925 approved by the Home Office (UK) (this statement is equivalent to statement that the research complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving animals).

Telemetry

Telemetry radiotransmitters (ETA-F20, Data Sciences International, St. Paul, MN, USA) were implanted in the peritoneum as described in Refs. [10, 12]. Thus, mice were anesthetized using a constant flow of oxygen and isoflurane into a whole body chamber (Harvard Apparatus). The peritoneal cavity was exposed and the transmitter was placed down in the abdominal cavity (ETA‐F20, Data Sciences International, St. Paul, MN). A needle was used to feed the leads of the transmitter from the abdominal cavity into the peritoneal cavity. The transmitter was then anchored in place by incorporating the grooves present on the transmitter body into the stitch line that sealed the abdominal cavity. The electrodes were then positioned on the body, with the positive electrode being positioned on the left xiphoid process and the negative electrode being positioned on the right shoulder of the mouse. The incision was closed with surgical staples. The system was set to record the locomotor activity (spontaneous physical activity/PA) and the ECG over a 10‐s interval and calculate the average values of these parameters within this period. Physical activity was measured by the horizontal displacement of the mouse in relation to two antennas in the receiver situated under the respective cages. Activity 0 indicated inactive state while any other number indicated active state. In each of the mice, the data recordings were initiated immediately postoperatively at 4 pm following reintroduction to cage. The recording room was maintained at 21–22 °C with a 12:12 h light–dark cycle (6 pm–6 am night and 6 am–6 pm day with 5.30–6 am dawn).

Data analysis and statistics

The data in this study were collected from day 8 to day 23, covering 15 days and giving the mice a week to habituate to the environment post-intervention. Furthermore, two periods of time were assigned, from 12:00 to 16:00 (Day) and from 00:00 to 04:00 (Night), to assess the diurnal effect on the heart rate. Using SigmaPlot 14.0 and Microsoft Excel, area under the curve (AUC) for each parameter was calculated and plotted in a graph. Data are presented in mean ± standard error of the mean (SEM) which was obtained as a function of average in addition to the sample size. When doing the comparisons, α=0.05 and CI=95%. The p-value was obtained using repeated measures two-way ANOVA and n value refers to the number of analysed animals; Paired, two-tailed Student’s t-test; and two-sample, two-tailed t-test depending on what the comparison entailed. A value of p<0.05 indicated a statistically significant comparison while 0.1>p>0.05 indicated trend.

Results

Physical activity in SUR2A+ and WT mice

When looking into the time spent in active state from day 8 to day 23, no statistically significant difference was found between WT and SUR2A+ mice (18.3% ± 2.2 vs. 17.6% ± 0.8% respectively, n=4 for each, p=0.846; Figure 1). WT mice were significantly more active during nights than during days (14.3% ± 1.1% vs. 21.2% ± 1.1%, n=4 for each, p=0.012; Figure 1). Similar pattern was observed in SUR2A+ mice (13.8% ± 2.1% vs. 23.0% ± 4.3%, n=4 for each; Figure 1), but the difference was not statistically significant (p=0.168). All mice spent significantly more time in inactive than in active state (Figure 1).

Figure 1:

Physical activity of SUR2A+ and WT mice. Bar graph showing time spent in inactive and active states in depicted conditions and line graphs showing physical activity of a wild type (WT) mouse (upper line graph) and physical activity of SUR2A+ (SUR2A) mouse (lower line graph) from day 3 to day 28. Each bar represents mean ± SEM (n=4).

Heart rate in SUR2A and WT mice

In both phenotypes, heart rate was significantly higher in mice when they were active compared to inactive (p<0.001 for all, n=4 for each; Figure 2). Heart rate did not significantly differ between day and night at the same level of physical activity (p=0.592 for animals in active state and p=0.566 in inactive state for WT and p=0.695 for animals in active state and p=0.325 in active state for SUR2A+; n=4 for each; Figure 2). There was no statistically significant difference between WT and SUR2A+ mice irrespective of day/night-time or state of physical activity (p=0.573; n=4 for each; Figure 2). As AUC analysis has the potential to reveal differences that are invisible with other statistical methods, we compared AUC of heart rate in SUR2A+ and WT mice during daytime and night-time, respectively. Using this methodology, it was revealed that heart rate is significantly higher during the night than during the day in both phenotypes (p=0.026 for all; n=4 for each; Figure 3). There was no statistically significant difference between WT and SUR2A+ mice irrespective of day/night-time or state of physical activity, (p=0.420; n=4 for each; Figure 3).

Figure 2:

Heart rate in SUR2A+ and WT mice. Bar graph showing heart rate in depicted conditions and line graphs showing heart rate of a wild type (WT) mouse (upper line graph) and heart rate of SUR2A+ (SUR2A) mouse (lower line graph) from day 3 to day 28. Each bar represents mean ± SEM (n=4).

Figure 3:

Area under the curve (AUC) of the heart rate in SUR2A+ and WT mice. Bar graph showing AUC of heart rate in SUR2A+ and WT mice under conditions described in each graph. Each bar represent mean ± SEM (n=4). *p<0.05.

Discussion

In the present study, it was found that increased expression of SUR2A does not affect physical activity or heart rate. In addition, regulation of heart rate by physical activity and circadian rhythm remains intact in SUR2A+ mice. Taken all together, these findings showing a lack of SUR2A adverse effects further support the idea about clinical viability of SUR2A-based cardioprotective strategy.

SUR2A is an ABC protein servings as a regulatory subunit of sarcolemmal K_ATP channels [6]. It has been shown that a moderate increase in SUR2A increases resistance of cardiomyocytes to hypoxia/ischaemia/reperfusion [7, 13], protect the myocardium against ischaemia-reperfusion [7, 14], increase physical endurance [10, 11], counteract some of ageing-induced heart alterations [11] and prolong life-span [15]. SUR2A regulates levels of sarcolemmal K_ATP channels and an increase in SUR2A results in an increase in the number of fully-assembled sarcolemmal K_ATP channels [7]. In turn, this is associated with their earlier channels activation during metabolic challenge and prevention of subsarcolemmal ATP decline [13]. Sarcolemmal K_ATP channel subunits SUR2A and kir6.2 physically associate with creatine kinase and glycolytic enzymes that, by virtue of their catalytic activity, produce ATP [16], [17], [18], [19]. Thus, an increased presence of fully-assembled K_ATP channels in sarcolemmal results in increased presence of ATP-producing enzymes to provide ATP in subsarcolemmal space where it is most needed for maintaining ion homeostasis [13]. During ischaemia, subsarcolemmal ATP levels decrease and SUR2A overexpression counteract this decrease by increasing number of fully-assembled K_ATP channels, which is cardioprotective [13]. Increased myocardial resistance to stress seems to be responsible for the anti-ageing effect of SUR2A and increased physical endurance [10, 11, 20]. Recent study suggested that sarcolemmal K_ATP channels open not only in pathological conditions, but also under some physiological conditions such as increased physical activity [10]. Recent studies suggested that some drugs that are already on the market, such as nicotinamide and pyrazinamide, regulate levels of SUR2A [21, 22]. These drugs can be quickly introduced into clinical practice and it is important to reveal possible unexpected/adverse effects of SUR2A.

In this study, we have found that increase in SUR2A did not have any effect on physical activity and/or heart rate. Recently, it has been demonstrated that K_ATP channels are expressed in sinoatrial (SA) node where they regulate heart rate. It has been shown that mice lacking K_ATP channels in SA node have a slow heart rate, with episodes of sinus arrest [23]. However, an increase in SUR2A did not affect heart rate suggesting that SUR2A did not influence K_ATP channels in SA node.

The increased expression of SUR2A did not alter the diurnal variation of heart rate of SUR2A+ mice. Circadian rhythms are regulated by the suprachiasmatic nucleus in hypothalamus that regulates production of melatonin in the pineal gland. So far, no role of SUR2A or K_ATP channels has been ascribed in regulation of melatonin production [24]. However, it has been shown that, at least, some of the effects of melatonin are mediated via activation of K_ATP channels [25, 26]. The fact that SUR2 did not alter circadian rhythm of physical activity and heart rate suggests that SUR2A is most likely not a part of K_ATP channels mediating melatonin action.

Conclusions

In conclusion, we have shown that increased SUR2A levels are not associated with changes in physical activity, heart rate and/or circadian rhythm influence on the heart rate. This lack of adverse effects supports the notion that manipulation with SUR2A levels is a promising cardioprotective strategy.