Cholecystokinin Octapeptide Promotes ANP Secretion through Activation of NOX4–PGC-1α–PPARα/PPARγ Signaling in Isolated Beating Rat Atria

Atrial natriuretic peptide (ANP), a canonical cardiac hormone, is mainly secreted from atrial myocytes and is involved in the regulation of body fluid, blood pressure homeostasis, and antioxidants. Cholecystokinin (CCK) is also found in cardiomyocytes as a novel cardiac hormone and induces multiple cardiovascular regulations. However, the direct role of CCK on the atrial mechanical dynamics and ANP secretion is unclear. The current study was to investigate the effect of CCK octapeptide (CCK-8) on the regulation of atrial dynamics and ANP secretion. Experiments were performed in isolated perfused beating rat atria. ANP was measured using radioimmunoassay. The levels of hydrogen peroxide (H2O2) and arachidonic acid (AA) were determined using ELISA Kits. The levels of relative proteins and mRNA were detected by Western blot and RT-qPCR. The results showed that sulfated CCK-8 (CCK-8s) rather than desulfated CCK-8 increased the levels of phosphorylated cytosolic phospholipase A2 and AA release through activation of CCK receptors. This led to the upregulation of NADPH oxidase 4 (NOX4) expression levels and H2O2 production and played a negative inotropic effect on atrial mechanical dynamics via activation of ATP-sensitive potassium channels and large-conductance calcium-activated potassium channels. In addition, CCK-8s-induced NOX4 subsequently upregulated peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) expression levels through activation of p38 mitogen-activated protein kinase as well as the serine/threonine kinase signaling, ultimately promoting the secretion of ANP via activation of PPARα and PPARγ. In the presence of the ANP receptor inhibitor, the CCK-8-induced increase of AA release, H2O2 production, and the upregulation of NOX4 and CAT expressions was augmented but the SOD expression induced by CCK-8s was repealed. These findings indicate that CCK-8s promotes the secretion of ANP through activation of NOX4–PGC-1α–PPARα/PPARγ signaling, in which ANP is involved in resistance for NOX4 expression and ROS production and regulation of SOD expression.


Introduction
Atrial natriuretic peptide (ANP), as a cardiac hormone, is synthesized and secreted mainly from atrial myocytes in response to stretch and other stimuli; it is primarily involved in the regulation of body fluid volume and blood pressure [1][2][3]. In addition, ANP has anti-ischemic [4], anti-inflammatory [5], antihypertrophic [6], and anticancer [7] properties. Although very little is known about the relationship between ANP and reactive oxygen species (ROS) production under physiological conditions or during the development of cardiovascular disease, ANP is associated with important antioxidant defense in cardiomyocytes and vascular cells [5].
Cholecystokinin (CCK) is a classical gut hormone and a potent stimulator of gallbladder contraction that was found in extracts of small intestinal mucosa in the 1920s [8,9]. Subsequently, CCK has also been found in neurons [10], immune cells [11], kidney cells, and lung cells [12]. Pro-CCK is processed into several molecular forms such as CCK-58, CCK-33, CCK-22, CCK-8, and CCK-4; however, sulfated carboxyl-terminal CCK octapeptide (CCK-8) is the major biological active fragment of CCK, which retains most of the bioactivities of CCK. Two types of receptors for CCK (CCK 1 receptor (CCK 1 R) and CCK 2 receptor (CCK 2 R)) have been classified as belonging to the G protein-coupled receptor superfamily (GPCRs) and its distribution is tissue-dependent [12][13][14]. Recently, it has been demonstrated that pro-CCK, CCK, and its receptors are expressed in mammalian cardiomyocytes [15,16]. Studies have shown that CCK has physiological roles in regulating blood pressure [17] and heart rate [15] and can enhance cardiac contractility [18]. In addition, CCK can alleviate fibrosis in the noninfarcted regions and delay the left ventricular remodeling and the progress of heart failure in left coronary artery ligation induced myocardial infarction of rats [19]. Nevertheless, the development of postinfarction heart failure is associated with the upregulation of CCK levels in relation to cardiac functional parameters and brain natriuretic peptide levels [20]. Furthermore, mRNA and protein levels of CCK are increased in the hypertrophic myocardial tissue of rats, which was positively correlated with changes in the left ventricular wall thickness [12]. In clinical studies of heart failure patients, an increase in plasma CCK is associated with mortality in elderly females [21]. Thus, CCK signaling induces multiple effects on cardiovascular physiology and pathophysiology. Still, the direct roles of CCK on the regulation of atrial mechanical dynamics and ANP secretion are not yet identified.
Nicotinamide adenine dinucleotide phosphate oxidases (NOXs), a major enzymatic source of ROS in the cardiovascular system, are activated in specific cardiovascular diseases [22]. Previously, we have found that NOX4, controlled by phospholipase A2 (PLA2), is involved in the regulation of ANP secretion in isolated beating rat atria under hypoxic conditions [23]. Moreover, it has been confirmed that the insensitivity of the CCK 1 R inositol phosphate signaling to pertussis toxin (PTX) indicates that it couples through the Gq family of the G proteins, thereby linking with the PLA2-arachidonic acid (AA) pathway to mediate calcium oscillation and amylase secretion [24,25]. In contrast, CCK 2 R is coupled to two pathways through PTX-sensitive and PTX-insensitive G proteins, resulting in the regulation of AA release by PTX-sensitive G proteins [24,26] and mitogen-activated protein kinase (MAPK) signaling pathways [24,27]. Owing to the effect of PLA2-AA signaling on NOX4 activity and its role in the regulation of atrial ANP secretion [23,28], we hypothesize that CCK regulates ANP secretion through activation of NOX4 via PLA2-AA signaling. This study is to verify the hypothesis using CCK-8 in isolated perfused beating rat atria. This study shows that sulfated CCK-8 (CCK-8s) rather than desulfated CCK-8 promotes ANP secretion through activation of NOX4-peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α)-PPARα/PPARγ signaling, in which CCK-8s-induced ANP is involved in the resistance for NOX4 expression and ROS production and regulation of SOD expression.

Preparation of a Perfused Beating Rat Atrium Model In
Vitro. All animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Yanbian University and were in accordance with the National Institutes of Health Laboratory Animal Care Guidelines. Specific pathogen-free (SPF) Sprague-Dawley (SD) rats (250~300 g, 18 weeks, male) were obtained from the Laboratory Animal Center of Yanbian University (laboratory animal use license number: SYXK (Ji) 2020-0009) for preparation for the beating rat atrial perfusion model. The environmental temperature for the rats was about 18°C~26°C, the relative humidity was 40%~70%, the light cycle was 12 h light/dark, and water was available ad libitum alongside a standard diet. Sterile normal saline was used to prepare the pentobarbital sodium solution, and SD rats were intraperitoneally anesthetized at a dose of 90 mg/ kg. Immediate thoracotomy was performed, and the left atrium of the rat was harvested and fixed on a self-made beating rat atrial perfusion device. Each rat left atrium was subjected to atrial electrical stimulation, and a peristaltic pump was used to infuse HEPES buffer and the reagents required for the experiment into the atria at a constant rate of 1.0 mL/min. A continuous oxygen supply was maintained along with an atrial temperature of 36°C. HEPES buffer contained (in mM) 118.0 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.2 MgCl 2 , 25.0 NaHCO 3 , 10.0 glucose, 10.0 HEPES (pH 7.4 with NaOH), and 0.1% BSA.

Experimental Protocols.
Each left atrium was first stably perfused for 60 min, followed by six cycles of reperfusion according to the experimental group, each cycle for 12 min, as shown in Table 1. To evaluate the effects of CCK-8 on atrial pulse pressure and ANP secretion, two cycles of control period were followed by infusion of CCK-8s or CCK-8d for four cycles. In another series of experiments, one cycle of the control period was followed by one cycle of treatment reagents and then CCK-8s for four cycles in the continuous presence of the treatment reagents. To test the effects of GLX and A71 on the atrial pulse pressure and ANP secretion, the control period was followed by five cycles of GLX or A71. All atria were harvested immediately after the end of perfusion and cryopreserved at -80°C for subsequent experiments. Beijing, China). Denatured samples were electrophoresed in 8% or 15% SDS-PAGE gels at room temperature. After electrophoresis, the separated proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using the wet transfer method and transferred for 60 min or 90 min at 4°C. The blotted membranes were blocked with 5% nonfat dry milk in PBS for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. The next day, after incubation with secondary antibodies for 2 h, the membranes were washed for 30 min and developed using a sensitive ECL chemiluminescence detection kit (PK10002, Proteintech; Wuhan, China). The bands were analyzed grayscale using Ima-geJ (National Institutes of Health; Bethesda, USA) software, and the results of the analysis were normalized.
2.6. RT-qPCR. In brief, total RNA was extracted from left atrial tissues using an RNApure Total RNA Kit (RN03, Aidlab; Beijing, China). The first strand of cDNA was then synthesized by high-performance reverse transcription using a SweScript RT I First Strand cDNA Synthesis Kit (G3330, Servicebio; Wuhan, China). Finally, the real-time quantitative polymerase chain reaction (RT-qPCR) was performed with 2× SYBR Green qPCR Master Mix (High ROX) (G3322, Servicebio; Wuhan, China). Results were calculated using the 2 -ΔΔCT relative quantification method and were normalized. Primer sequences are shown in Table 2.
2.7. Statistical Analysis. Statistical analysis was performed by one-way ANOVA and two-way ANOVA with Tukey's multiple comparison test using GraphPad Prism 9.0 (GraphPad

Oxidative Medicine and Cellular Longevity
Software; San Diego, USA). All data were normally distributed (Kolmogorov-Smirnov test) and given as mean ± standard error of the mean (SEM). P < 0:05 was considered statistically significant.

Effects of CCK-8 on the ANP Secretion and Atrial
Dynamics. To determine the effects of CCK-8 on atrial dynamics and ANP secretion, CCK-8s and CCK-8d were used in the experiments. In isolated beating rat atria, the infusion of CCK-8s significantly increased ANP secretion and inhibition of atrial pulse pressure (P < 0:001 vs. control period; Figures 1(a) and 1(b)) in a time-dependent manner. Meanwhile, CCK-8d obviously inhibited the atrial pulse pressure (P < 0:001 vs. control period; Figure 1(d)) but did not affect the ANP secretion ( Figure 1(c)). In the presence of CCK 1 R and CCK 2 R antagonists, Loxiglumid and YM022, the CCK-8s-induced increase of ANP secretion was abolished completely (P < 0:001 vs. CCK-8s alone period; Figures 1(e) and 1(g)). The CCK-8s-induced inhibition of atrial pulse pressure was almost completely blocked by Loxiglumid (P < 0:001 vs. CCK-8s alone period; Figure 1(f)) and notably attenuated by YM022 (P < 0:001 vs. CCK-8s alone period; Figure 1(h)). In addition, the expressions of CCK 1 R and CCK 2 R were significantly upregulated in atrial tissue after infusion of CCK-8s rather than CCK-8d (P < 0:001 vs. control group; Figures 1(i)-1(k)). The results indicated that sulfated rather than desulfated CCK-8 was able to promote ANP secretion through CCK receptors and has a negative inotropic effect on the mechanical dynamics of isolated perfused beating rat atria.
The data suggested that PLC and cPLA2 mediate the process of CCK-8s-induced increase of atrial ANP secretion.
3.3. Effects of CCK-8s on NOX4 Expression and Its Role in ANP Secretion. According to the role of CCK-8s on the levels of p-cPLA2 and the effect of cPLA2 on AA release and NOX4 activation [29], the levels of AA, NOX4 expression, and H 2 O 2 production induced by CCK-8s were observed. Results revealed that the levels of AA were remarkably increased in atrial tissue after infusion of CCK-8s (P < 0:001 vs. control group; Figure 3(a)), which were blocked by pretreatment with antagonists of CCK receptors and U73122 (P < 0:001 and P < 0:01 vs. CCK-8s group; Figure 3(a)). In addition, the expression of NOX4 was dramatically upregulated in CCK-8s-infused atrial tissue (P < 0:001 vs. control group; Figures 3(b)-3(d)); this effect was blocked by antagonists of CCK receptors and CAY10650 pretreatment (P < 0:001 vs. CCK-8s group; ). The levels of H 2 O 2 were also markedly increased in atrial tissue after infusion of CCK-8s (P < 0:001 vs. control group; Figure 3(e)), which were abolished by pretreatment with antagonists of CCK receptors (P < 0:001 vs. CCK-8s group; Figure 3(e)). Furthermore, in the presence of GLX351322, an inhibitor of NOX4, CCK-8s-induced increase of ANP secretion was not observed (P < 0:001 vs. CCK-8s alone period; Figure 3(f)), and the inhibition of atrial pulse pressure was also removed (P < 0:001 vs. control period and CCK-8s alone period; P > 0:05 vs. GLX351322 alone period; Figure 3(g)). In the presence of Glibenclamide, an inhibitor of K ATP , the effects of CCK-8s on atrial ANP secretion and mechanical dynamics were removed (P < 0:001 vs. CCK-8s alone period, respectively; Figures 3(h) and 3(i)), and a blocker of BK Ca , GAL-021, mimicked the role of Glibenclamide (P < 0:001 vs. CCK-8s alone period, respectively; Figures 3(j) and 3(k)). These results suggested that CCK-8s upregulated the expression of NOX4 via cPLA2-AA signaling, thereby increasing the secretion of ANP and exerting a negative inotropic effect on atrial mechanical dynamics, in which K ATP and BK Ca were involved.

Effects of CCK-8s-Induced NOX4 on p38 MAPK and Akt
Expressions. In accordance with the relationship between the activation of the NOX4-dependent p38 MAPK axis and cell oxidative damage [30], the effects of CCK-8s-induced NOX4 on the p38 MAPK and Akt protein expression in atrial tissue after infusion of CCK-8s were observed. The data showed that CCK-8s notably increased the levels of phosphorylated p38 (p-p38) MAPK, (P < 0:05 vs. control group; Figures 4(a) and 4(b)) and phosphorylated Akt (p-Akt) (P < 0:01 vs. control group; Figures 4(a) and 4(c)), which      These results demonstrated that p38 MAPK and Akt controlled by NOX4 were involved in the regulation of CCK-8s-induced ANP secretion.

Effects of Endogenous ANP on NOX4, SOD, and CAT
Expressions under CCK-8s Action. To define the effects of endogens ANP on NOX4, SOD, and CAT protein expressions under CCK-8s action, another series of experiments were performed with an ANP receptor antagonist. Results showed that CCK-8s significantly increased SOD and CAT rather than GPX protein expression levels (P < 0:01 and P < 0:001 vs. control group; Figures 7(a)-7(d)); these effects were blocked by inhibitors of CCK receptors and PPARα as well as PPARγ (P < 0:001 vs. CCK-8s group; Figures 7(a)-7(c) and 7(e)-7(g)). In the presence of an antagonist of the ANP receptor, A71915, the CCK-8sinduced increase of AA release and H 2 O 2 levels were further augmented (P < 0:05 and P < 0:001 vs. CCK-8s group; Figures 8(a) and 8(b)). In addition, A71915 further enhanced the effects of CCK-8s-induced increase of NOX4 and CAT protein expression levels (P < 0:05 vs. CCK-8s group; Figures 8(c)-8(e)), but the CCK-8s-induced increase of SOD protein expression was abolished by A71915 (P < 0:05

Discussion
NOX4 is one of the enzymatic sources of ROS generation in the cardiovascular system expressed in the heart [22]. In addition, the role of PLA2 and AA on the activation of NOXs has been demonstrated [28]. In our previous study, we have demonstrated that activated cPLA2 induced by endothelin-1 was involved in the regulation of hypoxiainduced ANP secretion through activation of NOX4 and H 2 O 2 production in isolated beating rats' atria [23]. The increased NOX4 activity in response to a high pacing frequency also promoted ANP secretion in rat atria [31]. These results suggest that NOX4 is one of the regulatory factors for the secretion of ANP in the atria.
In the current study, CCK-8s rather than CCK-8d significantly upregulated the expression of CCK 1 and CCK 2 receptors and increased the levels of p-cPLA2, AA release, NOX4 relative protein levels, and H 2 O 2 production, concomitant with an increase of ANP secretion and inhibition of mechanical dynamics in isolated beating rat atria. The CCK-8s-induced increases of p-cPLA2 expression and AA release were blocked by inhibitors of CCK receptors and PLC, respectively. Similarly, the CCK-8s-induced upregulation of NOX4 expression and H 2 O 2 production was abolished by antagonists of CCK receptors, and an inhibitor of cPLA2 also nullified the effect of CCK-8s on NOX4 expres-sion. Furthermore, CCK-8s-induced promotion of ANP secretion was blocked by antagonists of CCK receptors, PLC and cPLA2, respectively, without changes in the inhibition of atrial mechanical dynamics induced by CCK-8s. Nevertheless, CCK-8s-induced inhibition of atrial dynamics was abolished by inhibitors of NOX4, K ATP , and BK Ca , which was accompanied by blockage of CCK-8s-induced ANP secretion. These results demonstrated that CCK-8s (but not CCK-8d) triggered NOX4 activation and increased H 2 O 2 production through CCK receptor-mediated activation of cPLA2, leading to an increase in ANP secretion and a negative inotropic action, in which K ATP and BK Ca were involved. The results of the current study are similar to those of the previous studies mentioned above and support previous reports that K ATP and BK Ca are regulated by H 2 O 2 [32][33][34].
The PGC-1α is a member of the PGC-1 family that regulates adaptive thermogenesis and mitochondrial function [35]. PGC-1α can be directly activated by p38 MAPK [36] and participates in the elimination of ROS in the heart [37,38]. PPARα and PPARγ, as downstream signal molecules of PGC-1α, inhibit NOX and ROS generation by enhancing the activity of SOD as well as CAT, thereby resisting oxidative stress damage [39,40]. In addition, the promoter region of the human ANP gene located on the short arm of chromosome 1 contains binding sites for many transcription factors, including PPARα and PPARγ [41]. Furthermore, it has been demonstrated that PPARγ is involved in the regulation of ANP secretion in beating rat atria under normoxic or hypoxic conditions [42][43][44]. These results suggest that the changes in PPARα and PPARγ activity are closely related to the secretion of ANP.
the upregulation of PGC-1α expression. The CCK-8sinduced increase of p-p38 MAPK and p-Akt levels was blocked by an inhibitor of NOX4, and the CCK-8sinduced upregulation of PGC-1α expression was repealed by inhibitors of p38 MAPK and Akt, respectively. In addi-tion, CCK-8s also markedly increased PPARα as well as PPARγ mRNA levels and their phosphorylated protein expressions, concomitant with the promotion of ANP secretion. An inhibitor of PGC-1α abolished the CCK-8sinduced increase of phosphorylated PPARα as well as phosphorylated PPARγ levels by attenuation of PPARα as well as PPARγ mRNA levels; it also abrogated the CCK-8s-induced promotion of ANP secretion. Similarly, inhibitors of PPARα as well as PPARγ attenuated the CCK-8sinduced upregulation of NPPA mRNA levels and repealed the promotion of ANP secretion induced by CCK-8s. The current study data indicate that CCK-8s-induced NOX4 upregulates PGC-1α expression through phosphorylation of p38 MAPK and Akt, leading to the activation of PPARα as well as PPARγ, thereby promoting the secretion of ANP. The data is similar to those of the previous studies mentioned above. (a-f) n = 5. * * P < 0:01 and * * * P < 0:001 vs. control group; # P < 0:05, ## P < 0:01, and ### P < 0:001 vs. CCK-8s group; &&& P < 0:001 vs. A71+CCK-8s group.