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Nicotinamide-rich diet improves physical endurance by up-regulating SUR2A in the heart

Abstract SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K+ (KATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled KATP channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca2+ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal KATP channels and steady Ca2+ levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca2+. Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking KATP channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.


Introduction
Improving physical endurance is crucial for improving quality and duration of life in people suffering from different conditions including old age, heart failure, chronic obstructive pulmonary disease, cystic fibrosis, anticancer treatment and many others. At the moment there is no well-established strategy that can improve physical endurance, apart on physical exercise (reviewed in [1]). However, it is acknowledged that physical exercise is labour intensive with compliance being an important problem. In addition, some population of patients cannot perform the intensity of physical exercise required to achieve a desirable effect (reviewed in [2]).
Therefore, a pill that would improve physical endurance and produce similar effect of those produced physical exercise would be welcomed in context of many conditions and diseases.
The ATP-sensitive K ϩ (KATP) channel was originally discovered in membrane patches excised from ventricular cardiomyocytes [3]. Structurally, sarcolemmal KATP channels are heteromultimers composed of, at least, two distinct subunits. The pore-forming inwardly rectifying K ϩ channel core, Kir6.2, is primarily responsible for K ϩ permeance, whereas the regulatory subunit, also known as the sulfonylurea receptor, or SUR2A, has been implicated in ligand-dependent channel gating [4]. More recently, it has been suggested that the sarcolemmal KATP channel protein complex may be composed of more proteins than just Kir6.2 and SUR2A, including Kir6.1 and enzymes regulating intracellular ATP levels and glycolysis [5][6][7][8][9][10]. Sarcolemmal KATP channels have been shown to play a crucial role in ischaemic preconditioning (a phenomenon when brief episodes of ischaemia/reperfusion protect the heart against myocardial infarction, [11]) and myocardial resistance to ischaemia (reviewed in [12]). More recently, it has been suggested that these channels might play a central role in response to acute-exercise stress where they seem to contribute to the adaptation of cardiac function to match physical demand. It has been shown that mice lacking these channels are characterized by decreased physical endurance, which was manifested by their inability to sustain increased physical activity [13].
If a lack of sarcolemmal KATP channels is associated with decreased physical endurance, does it mean that an increase in number of sarcolemmal KATP channels would be associated with increased physical endurance? One of the problems in testing such strategy was finding a way to increase the numbers of sarcolemmal KATP channels in vivo. This was particularly complex as KATP channels are heteromultimers composed of more than one protein. It was recently proposed that SUR2A, a KATP channel regulatory subunit, is a rate-limiting factor in forming fully assembled KATP channels (reviewed in [14]). We have recently found that nicotinamide-rich diet up-regulates SUR2A [15].
If nicotinamide-rich diet can up-regulate SUR2A then it could also improve physical endurance. Testing this hypothesis would be clinically important, as it could lead to a safe and efficient strategy of improving physical performance in many different conditions. Mice are experimental models successfully used in preclinical studies of nicotinamide and KATP channels and findings on this species have been successfully interpolated in human beings many times [16][17][18]

SUR2A and Kir6.2 knockout mice
Generation, breeding and genotyping of these mice have previously been described in detail [13,19] , where m is animal mass, v is running velocity, g is acceleration due to gravity, t is time elapsed at a given protocol level and is the angle of incline [13].

Isolation of single cardiomyocytes
Ventricular cardiomyocytes were dissociated from the mouse using an established enzymatic procedure [23]. In brief, hearts were retrogradely perfused (at 37ЊC) with medium 199, followed by Ca 2ϩ -ethylene glycol-bis

Measurement of action membrane potential
Sarcolemmal membrane potential was monitored in cells in the absence and presence of isoprenaline (500 nM)/glybenclamide (10 M). Cells were loaded with di-8-ANEPPS according to the manufacturer's instruction (Invitrogen) and the sarcolemma imaged using laser confocal microscopy in line-scan mode (LSM-510, Zeiss; [25]). Cells were stimulated as described in above section and scanned under control conditions, then exposed to drugs. Cells were scanned at 5 min. intervals in the absence and presence of isoprenaline or isoprenaline/glybenclamide. Fluorescence was detected/imaged at 488 nM excitation wavelength and emission was captured at Ͼ505 nM.

Nicotinamide-rich diet increases SUR2A mRNA levels in heart ventricles
It has been previously shown that the level of SUR2A mRNA correlates well with the number of fully assembled sarcolemmal KATP channels and that this parameter could be used as a proxy measurement of the level of sarcolemmal KATP channels [19,[26][27][28][29]. We have analysed levels of SUR2A mRNA as well as mRNAs of two other KATP channel subunits, Kir6.2 and Kir6.1, in mice fed with control and nicotinamide-rich diet. Real time RT-PCR revealed that nicotinamide-rich diet significantly increased the levels of SUR2A mRNA in the heart as the threshold cycle for mice on control and nicotinamide-rich diet was 32.0 Ϯ 0.4 and 22.0 Ϯ 0.9, respectively (n ϭ 6 for each; P Ͻ 0.01; Fig. 1 Fig. 1). Also, no significant difference was found in glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA levels ( Fig. 1; threshold Fig. 2A). We have also elucidated possibility that nicotinamide-rich diet affects expression of other subtypes of sulfonylurea receptors (SUR1 and SUR2B) in ventricular tissue as well as expression of SUR1, SUR2A and SUR2B in some non-ventricular tissues. Real time RT-PCR did not reveal any statistically significant differences in level of expression of these subunits between mice on different diets in tested tissues (Fig. 2B).

Nicotinamide-rich diet increases physical endurance
To test physical endurance of mice on control and nicotinamide-rich diet, we have used treadmill as described previously [13]. The tolerated workload, a parameter that incorporates time of effort with speed and incline of the treadmill, was significantly higher in mice on nicotinamide-rich diet compared with the mice on control diet (tolerated workload was 54.7 Ϯ 5.1 J in mice on control and 104 Ϯ 8.2 J in mice on nicotinamide-rich diet, n ϭ 6 for each, P Ͻ 0.01; Fig. 3). Mice on control diet were able to stay on treadmill for 67.3 Ϯ 6.2 min. (n ϭ 6), while this time was significantly increased in mice on nicotinamide-rich diet (to 144.0 Ϯ 14.9 min.; Fig. 3; P Ͻ 0.01).

A sole increase in SUR2A mimics the effect of nicotinamide-rich diet on physical endurance
To determine whether a sole increase in SUR2A would have similar effect on physical endurance as a nicotinamide-rich diet, we

Fig. 1 Expression of KATP channel subunits in hearts of mice on control and nicotinamide-rich diet. Representative progress curves for the realtime PCR amplification of SUR2A (A and C) and GAPDH (B and D) cDNA from mice on control or nicotinamide-rich diet (as labelled in the figure) and a corresponding bar graphs (E and F) as well as bar graphs depicting cycle threshold values for Kir6.2 and Kir6.1. Each bar represents mean Ϯ S.E. of the mean (n ϭ 4-6). *P Ͻ 0.05.
have taken advantage of transgenic phenotype overexpressing SUR2A alone [19]. Under the exercise-stress test, mice in which the SUR2A was under control of cytomegalovirus promoter performed at a significantly increased level than the littemate controls (Fig. 4). The tolerated workload was ~2-fold higher in transgenic mice compared with the wild-type animals (tolerated workload was 45.6 Ϯ 2.2 J in wild-type mice and 91.6 Ϯ 13.4 J in SUR2A mice, n ϭ 5-7, P ϭ 0.01; Fig. 4). Time spent on treadmill was significantly increased in SUR2A mice when compared to the wildtype (Fig. 4).

A shortening in action membrane potential in response to ␤-adrenergic stress in cardiomyocytes from SUR2A mice
It is generally accepted that the activation of ␤-adrenergic receptors and increased cardiac output and blood/oxygen supply underlies general adaptation to stress and ability of mammalians to sustain increased physical activity [30]. The activation of KATP channels induces shortening of the action membrane potential. Therefore,

Fig. 2 Acute electrophysiological effect of nicotinamide on isolated cardiomyocytes and expression of different SUR types in cardiac and some extra-cardiac tissues. (A) Typical whole cell currents at 80 mV in a cardiomyocyte in the absence (control) and presence (nicotinamide) of nicotinamide (1 mM) and a current density graph corresponding to conditions (A1). Each bar represents mean Ϯ S.E. of the mean (n ϭ 4). (B) Bar graphs showing real time RT-PCR cycle threshold values for depicted tissues and genes. Each bar represents mean Ϯ S.E. of the mean (n ϭ 3-6).
we have assessed the duration of action membrane potential in cells from both phenotypes under control conditions and when stimulated with a ␤-adrenoceptor agonist, isoprenaline. Under control conditions, action membrane potential duration remained stable over time in both phenotypes, i.e. at the beginning of field stimulation duration of action membrane potential was 228 Ϯ 11 msec. in wild-type and 223 Ϯ 44 msec. in SUR2A mice ( Fig. 4; n ϭ 6-10; P ϭ 0.90) and these values were not significantly changed after 30 min. of stimulation (action membrane potential duration; wild-type: 234 Ϯ 17 msec., n ϭ 10, P ϭ 0.74; SUR2A: 218 Ϯ 22 msec., n ϭ 6, P ϭ 0.89; Fig. 4). When isoprenaline (500 nM), an agonist of ␤-adrenergic receptors used to induce stress, was added, no changes in action membrane potential duration was observed in wild-type (from 243 Ϯ 17 msec. in the absence to 254 Ϯ 24 msec. in the presence of isoprenaline, n ϭ 7, P ϭ 0.61; Fig. 5). In contrast to the wild-type, in SUR2A mice action membrane potential was significantly shortened after exposure to 500 nM isoprenaline (from 240 Ϯ 19 msec. in the absence to 163 Ϯ 13 msec. in the presence of isoprenaline, n ϭ 6, P ϭ 0.0007; Fig. 5). To test whether this shortening is induced by the activation of sarcolemmal KATP channels, we have used glybenclamide, a blocker of KATP channels. When applied alone, glybenclamide (10 M) did not alter duration of action membrane potential in SUR2A cells (data not shown), but glybenclamide (10 M) blocked the shortening of action membrane potential in SUR2A cell (from 238 Ϯ 15 msec. in the absence to 228 Ϯ 37 msec. in the presence of isoprenaline and glybenclamide, n ϭ 6, P ϭ 0.76; Fig. 5).

An improved Ca
2؉ homeostasis in SUR2A cardiomyocytes exposed to ␤-adrenergic stress It was suggested that KATP channels regulate Ca 2ϩ homeostasis during ␤-adrenergic stress and that this is crucial for regulation of physical endurance [13]. A shortening of cardiac action membrane potential is known to be associated with decreased Ca 2ϩ influx and prevention of Ca 2ϩ loading.  Nicotinamide-rich diet has no effect on physical endurance in Kir6.

knockout mice
In addition to up-regulating SUR2A expression, nicotinamide-rich diet could have other cardiac effects [31] Fig. 7). Time spent on treadmill was also not significantly different (Fig. 7).

Discussion
In the present study we have shown that nicotinamide-rich diet increases physical endurance by up-regulating SUR2A in the heart. It seems that this is sole mechanism of nicotinamide-rich diet-induced improvement of physical endurance. Nicotinamide is the amide of nicotinic acid and a member of water-soluble vitamin B group. In cells, nicotinamide is incorpo-rated into nicotinamide adenine dinucleotide (NAD) and NAD phosphate, coenzymes in various enzymatic oxidation-reduction reactions (reviewed in [28]). It has been reported that exposure of cardiac cells to mild hypoxia and consequent increase in intracellular NAD stimulate SUR2A expression by activating PI3-kinase signalling pathway that stimulates SUR2 promoter via c-jun transcription factor [29]. Most interestingly, this signalling pathway has solely regulated SUR2A expression without affecting any other genes, as revealed by gene array technology [29]. Further experiments have supported the idea that increase in intracellular NAD seem to triggers signalling pathway that up-regulate SUR2A. In turn, an increase in SUR2A expression leads to increase in number of sarcolemmal KATP channels and cardiac phenotype with increased resistance to ischaemia reperfusion [29]. Nicotinamide-rich diet  increases levels of NAD in many tissues, although this has not been specifically reported for the heart [32]. We have recently found that nicotinamide-rich diet up-regulates SUR2A and increases myocardial resistance to ischaemia reperfusion [15], which is in agreement with the effect of (1) nicotinamide-rich diet on intracellular NAD levels [32], (2) intracellular NAD on SUR2A expression [29] and (3) increased SUR2A on heart resistance to ischaemia reperfusion [19,33]. The effect of increased SUR2A and sarcolemmal KATP channels on physical endurance has never been studied before. In the present study, nicotinamide-rich diet has increased the expression of SUR2A and numbers of sarcolemmal KATP channels, which is in agreement with the effect of nicotinamide-rich diet described in Ref. [15]. It has been demonstrated that a lack of sarcolemmal KATP channels is associated with decreased physical endurance due to heart inability to respond to increased metabolic demand and to increase cardiac output. Here we have shown that nicotinamide-rich diet has increased physical endurance in mice and that would (1) confirm a crucial role of sarcolemmal KATP channels in regulating physical endurance, (2) show that by manipulating KATP channel numbers physical endurance can be increased considerably over the physiological level and (3) demonstrate a potential of nicotinamide-rich diet as a strategy against conditions associated with impaired physical performance.
It is appreciated that nicotinamide-rich diet might have other cardiac and non-cardiac effects and that SUR2 is not necessarily only gene regulated by nicotinamide-rich diet [31]. On the other hand, we have previously shown that there are conditions, like mild hypoxia is, that could regulate the expression of SUR2A alone [29]. Indeed, in the present study, SUR2A was the only gene regulated by nicotinamide-rich diet out of all genes we tested. If upregulation of SUR2A is the main mechanism of nicotinamide-rich diet increase in physical endurance, then the same phenomenon should be observed in transgenic mice with solely increased SUR2A (this phenotype is published in [19]). The treadmill endurance test has demonstrated that a sole increase in SUR2A levels in transgenic mice has mimicked the effect of nicotinamiderich diet in the wild-type suggesting that up-regulation of SUR2A could be indeed the mechanism by which nicotinamide-rich diet improves physical performance. In study by Zingman et al. [13], it has been shown that a lack of sarcolemmal KATP channels lead to impaired Ca 2ϩ homeostasis in cardiomyocytes during stimulation with 500 nM of isoprenaline, which is a ␤-adrenergic stress; a difficulty to maintain Ca 2ϩ homeostasis in the absence of sarcolemmal KATP channels seems to be the main factor leading to decreased physical endurance in Kir6.2 knockout phenotype [13]. Here, we have shown that 500 nM of isoprenaline was associated with shortening of action membrane potential in a glybenclamidesensitive manner in SUR2A, but not wild-type, cells. This indicates that there is opening of sarcolemmal KATP channels in cells with increased numbers of sarcolemmal KATP channels under the applied level of stress. It has been previously shown that increased number of sarcolemmal KATP channels is associated with earlier activation of KATP channels [19,21], which is in accord with our present findings. The activation of KATP channels prevents intracellular Ca 2ϩ loading, which is the main cause of impaired cellular function during adrenergic stress. Our results that Ca 2ϩ homeostasis was improved in SUR2A cells when compared to the wild-type ones is in agreement with a role of sarcolemmal KATP channels as regulators of intracellular Ca 2ϩ (reviewed in [12]). It also supports a theory that the regulation of Ca 2ϩ homeostasis in the heart is the cellular mechanism underlying KATP channelsmediated regulation of physical endurance [13]. The final test for our hypothesis that SUR2A up-regulation is the main/sole mechanism of nicotinamide-rich diet was in determining whether nicotinamide-rich diet can improve physical endurance of Kir6.2 knockout mice. These mice have disrupted Kir6.2 gene and they lack all components of KATP channels in sarcolemma as this subunit is crucial for SUR2A trafficking [34]. Consequently, no intervention that manipulates with the sarcolemmal KATP channels number would be successful in Kir6.2 knockout mice. Conversely, if there is a KATP channel-independent mechanism involved, it would be revealed in Kir6.2 knockout mice. The fact that nicotinamide-rich diet did not affect physical endurance in Kir6.2 knockout mice suggest that increase in sarcolemmal KATP channels is the main mechanism involved in nicotinamide-rich diet increase in physical endurance.
It has been previously shown that a small change in intracellular NAD might regulate the expression of SUR2A without affecting expression of other genes [29]. In the present study, a dose of nicotinamide per os used in mice was low and it is much lower than the dose that was required to protect the heart against ischaemia reperfusion [15]. The human equivalent dose of nicotinamide to increase physical endurance would be lower than any other dose of nicotinamide that is currently used in clinical practice for any other indication. This means that oral nicotinamide has a clear potential as a drug that can regulate SUR2A expression and improve physical endurance in conditions where this effect would be desirable.