The Modulation of Cardiac Contractile Function by the ...

TOXICOLOGICAL SCIENCES, 148(2), 2015, 581–593 doi: 10.1093/toxsci/kfv202 Advance Access Publication Date: September 4, 2015 Research Article

The Modulation of Cardiac Contractile Function by the Pharmacological and Toxicological Effects of Urocortin2 Si Chen†, Zhenhua Wang*, Bo Xu*,‡, Xiangquan Mi§, Wanqing Sun¶,k, Nanhu Quan¶,k, Lin Wang¶,k, Xingchi Chen¶, Quan Liuk, Yang Zhengk, Jiyan Lengk, and Ji Li¶,1 *College of Life Sciences, Yantai University, Yantai, Shandong 264005, †State University of New York at Buffalo, Buffalo, New York 14214, ‡Key Laboratory of Pharmacology and Molecular Drug Evaluation, School of Pharmacy, Yantai University, Yantai, Shandong 264005, §School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, ¶Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216, and k The First Affiliated Hospital, Jilin University, Changchun, Jilin 130000 1

To whom correspondence should be addressed at Guyton Research Building, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State Street, Jackson, MS 39216. Fax: 601-984-1817. E-mail: [email protected]

ABSTRACT Urocortin2 (Ucn2) has been revealed to enhance cardiac function in heart failure. However, the pharmacological and toxicological effects of Ucn2 on cardiomyocytes are incompletely understood. In this study, we investigated the possible mechanisms of Ucn2 on mediating the contractility of cardiomyocytes. Mechanical properties and intracellular Ca2þ properties were measured in isolated cardiomyocytes from different treatment groups. The stress signaling was evaluated using Western blot. The results demonstrated that Ucn2 induced maximal velocity of shortening (þdL/dt), peak height, peak shortening (PS) amplitude, maximal velocity of relengthening (dL/dt), accompanied by a significant rise in intracellular Ca2þ level and a fall of the mean time constant of Ca2þ transient decay (Tau) in WT cardiomyocytes. However, these effects were abolished by preincubation of type 2 CRF receptors (CRFR2) antagonist anti-sauvagine 30 (a-SVG-30). We also found that Ucn2 treatment activated the AMPK pathway in isolated cardiomyocytes via CRFR2. Furthermore, Ucn2 induced protein kinase A (PKA) and phospholamban (PLN) phosphorylation. Pretreatment of PKA inhibitor H89 reduced the inotropic and lusitropic effects of Ucn2 as well as decreased the intracellular Ca2þ load and slowed down the Ca2þ transient decay. We also showed that preincubation of Compound C, an inhibitor of AMPK, inhibited the phosphorylation of PKA and the intracellular Ca2þ level in cardiomyocytes without affecting the contractile function and the Tau of cardiomyocytes. Taken together, it suggests that Ucn2 facilitate the contractility of cardiomyocytes via activating both AMPK and PKA. Key words: Ucn2; AMPK; PKA; cardiac toxicology; contractile function

American Heart Association reported that 8.1% of Americans self-reported have some type of cardiovascular disease (Roger et al., 2012). Among these, heart failure is one of the leading causes of morbidity and mortality, which can cause to left ventricular dysfunction; including systolic and diastolic dysfunction, and sudden cardiac death (Boonprasert et al., 2008; Roger et al., 2011). The contractile and metabolic proprieties of cardiomyocytes are critical factors in the regulation of heart performance and the susceptibility to cardiac dysfunction (Hill and

Olson, 2008). Corticotrophin releasing factor (CRF), a 41 aminoacid peptide hormone, is one of a family of peptides that includes urocortin (Ucn) 1, Ucn2, and Ucn3 (Salminen et al., 2011a,b). Study reported that centrally administered Ucn played a role in suppressing appetite, but it showed less effect on generating acute anxiety-like effects and generalized behavioral activation compared with CRF (Spina et al., 1996). Ucns have great cardiovascular actions in animals and men (Parkes et al., 2001; Weisinger et al., 2004). Several studies revealed that Ucn

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FIG. 1. Contractile properties of cardiomyocytes isolated from WT mice with or without Ucn2 and CRFR2 antagonist a-SVG-30. Ucn2 facilitates the contractile function of cardiomyocytes, while a-SVG-30 abolishes the contractility of cardiomyocytes. Freshly isolated cardiomyocytes from WT mice were incubated with or without Ucn2 (10 nM) and a-SVG-30 (10 nM) prior to mechanical assessment. A, Resting sarcomere length; B, Maximal velocity of relengthening (þdL/dt); C, Peak height; D, PS (normalized to the resting sarcomere length); E, Maximal velocity of shortening (dL/dt); F, Representative sarcomeric shortening traces obtained from isolated cardiomyocytes. Means 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus vehicle control group; †P < .05 versus Ucn2 alone group.

enhanced cardiovascular function in experimental heart failure (Rademaker et al., 2006). Ucns signal through 2 G protein-coupled receptors, CRF receptors (CRFR) 1 and 2 (Eckart et al., 2002). CRFR1 is mainly expressed in the central nervous system while CRFR2 is abundant in the periphery, including the heart and

systemic vasculature (Kishimoto et al., 1995; Lewis et al., 2001; Reyes et al., 2001; Stenzel et al., 1995). Although Ucn1 binds strongly to both receptor subtypes, Ucn2 is 40-fold more effective at competing for binding of CRFR2 (Dutton et al., 1999; Kishimoto et al., 1995; Reyes et al., 2001).

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FIG. 2. Ucn2 treatment induces phosphorylation of AMPK, ACC, and cTnI (Ser149) of isolated cardiomyocytes. A, Immunoblots of phosphorylated (p) AMPK, ACC, and cTnI (Ser149) of isolated cardiomyocytes with a-SVG-30 (10 nM), Ucn2 (10 nM), or the combination of a-SVG-30 and Ucn2 treatment. B, Bars represent the relative levels of phosphorylated AMPK, ACC, cTnI (Ser149). Values are expressed as means 6 SEM, n ¼ 3–4 per group. *P < .05 versus the corresponding control group, respectively. †

P < .05 versus corresponding Ucn2 alone group, respectively.

Recent studies demonstrated that Ucn2 treatment induced AMP-activated protein kinase (AMPK) activation and downstream ACC phosphorylation via CRFR2 in isolated heart muscle and in the intact heart in vivo (Dutton et al., 1999). AMPK senses metabolic stress and modulates myocardial signaling in the heart (Morrison and Li, 2011; Moussa and Li, 2012). Previous studies have revealed that AMPK stimulates glucose uptake and glycolysis during stress situations such as exercise, starvation, hypoxia and ischemia (Costa et al., 2012; Ma et al., 2010; Morrison et al., 2011; Wang et al., 2013). Activation of AMPK triggered troponin I (cTnI) phosphorylation at the Ser149 site which is related to the contractility of cardiomyocytes (Chen et al., 2014; Oliveira et al.,

2012). During ischemia reperfusion, AMPK plays a role in reducing ischemic injury (Young, 2008). It has been reported that Ucn2 treatment could activate AMPK in order to prevent injury during ischemia by decreasing the extent of myocardial necrosis and helping recovery of cardiac contractile function in the mouse model (Dutton et al., 1999). This activity is blunted by treatment with anti-sauvagine 30 (a-SVG-30), an antagonist to the cell surface CRFR2 receptor during hypoxia (Dutton et al., 1999). It has also been demonstrated that Ucn2 binds to CRFR2 to stimulate protein kinase A (PKA) activity (Turnley and Bartlett, 1999). Activation of PKA is responsible for enhancing Ca2þ influx and loading the sarcoplasmic reticulum (SR) with Ca2þ (Ullrich

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FIG. 3. Intracellular Ca2þ transient properties of isolated cardiomyocytes with a-SVG-30 (10 nM), Ucn2 (10 nM) or the combination of a-SVG-30 and Ucn2 treatment. Ucn2 increased the intracellular Ca2þ and decreased the mean time constant of Ca2 þ transient decay (Tau) significantly. A, Histograms showing the resting intracellular calcium level; B, Histograms showing mean Ca2þ transient amplitude; C, Histograms showing the mean time constant of Ca2þ transient decay (Tau); D, Representative Ca2þ transient traces obtained from isolated cardiomyocytes of vehicle and Ucn2 groups. Mean 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus vehicle control group; †P < .05 versus Ucn2 alone group.

et al., 2012). Phosphorylation of phospholamban (PLN) enables its dissociation from sarco/endoplasmic reticulum Ca2þ-ATPase (SERCA) then leads to the increase of SERCA activity (Ullrich et al., 2012). These changes would result in the elevation of SR Ca2þ load and systolic Ca2þ release, then stimulate cardiomyocyte contractility (Turnley et al., 1999; Ullrich et al., 2012). Our earlier studies demonstrated that activation AMPK by AMPK activator facilitate the contractile function of cardiomyocytes without changing the intracellular Ca2þ transient (Chen et al., 2014). We also reported that Ucn2 treatment stimulated the AMPK pathway in isolated heart muscles and in the intact heart in vivo (Li et al., 2013). However, the cellular actions of Ucn2 on mouse cardiomyocytes still remain elusive. In this study, we identified the effect of Ucn2 pharmacologically on isolated cardiomyocytes by measuring mechanical properties and intracellular Ca2þ levels, try to test the hypothesis that the positive contractile function upon Ucn2 treatment was mediated by both AMPK and PKA signaling.

MATERIALS AND METHODS Experimental Animals Male wild-type (WT) mice were used in the experiments. All of these mice were of a C57BL/6 background and 4–6 months of age. All of the animal procedures conducted in this study were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee.

Isolation of Cardiomyocytes and Cell Mechanics Mice were given 100 U of heparin i.p. (Sagent Pharmaceuticals, Schaumburg, Illinois) for anticoagulation before anesthetized with 100 mg/kg sodium pentobarbital i.p. (Sigma, St Louis, Missouri). The heart was excised and fastened onto the cardiomyocyte perfusion apparatus (Radnoti, Monrovia, California) and perfusion was initiated in the Langendorff mode. Hearts were perfused at 37 C with a Ca2þ-free Krebs-Henseleit based buffer (pH 7.3) containing: 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 10 mM HEPES, 14.7 mM KCl, 1.7 mM MgSO4, 120.3 mM NaCl, 4.6 mM NaHCO3, 30 mM taurine, 10 mM glucose, and 10 mM 2,3butanedione monoxime that was bubbled with 95% O2/5% CO2. After a few minutes of stabilization, the heart was then digested with the same perfusion buffer containing 0.067 mg/ml Liberase Blendzyme 4 (Roche, Indianapolis, Indiana). After digestion, the heart was removed and minced. Extracellular Ca2þ was added back to the cells to reach a final concentration of 1 mM. Cardiomyocytes were then subjected to pharmacological drug treatment with either vehicle dimethyl sulfoxide (DMSO), 10 mM Compound C (Enzo Life Sciences, Farmingdale, New York) or 10 nM a-SVG-30 (Sigma, St Louis, Missouri), 10 nM H89 (Enzo Life Sciences, Farmingdale, New York) and 10 nM Ucn2 (Sigma, St Louis, Missouri) for 10 min at room temperature (20–25 C).

Measurement of Cardiomyocyte Contractile Function The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, Massachusetts) (Li et al., 2008; Ma et al., 2010; Wang et al.,

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FIG. 4. Ucn2 treatment induces phosphorylation of PKA and PLN (Ser16/Thr17) of isolated cardiomyocytes. A, Immunoblots of phosphorylated (p) PKA and PLN (Ser16/ Thr17) of isolated cardiomyocytes with a-SVG-30 (10 nM), Ucn2 (10 nM), or the combination of a-SVG-30 and Ucn2 treatment. B, Bars represent the relative levels of phosphorylated PKA and PLN (Ser16/Thr17). Values are expressed as means 6 SEM, n ¼ 3–4 per group. *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

2011). Cardiomyocytes were placed in a chamber and stimulated with a suprathreshold voltage at a frequency of 0.5 Hz. IonOptix SoftEdge software was used to capture changes in sarcomere length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: PS, the amplitude myocytes shortened on electrical stimulation, which is indicative of peak ventricular contractility; time-to-90% relengthening, the duration of myocytes to reach 90% relengthening, an indicative of diastolic duration; and maximal velocities of shortening and relengthening. Intracellular Ca21 Transient Measurement Intracellular Ca2þ was measured using a dual-excitation, singleemission photomultiplier system (IonOptix) (Li et al., 2008; Wang et al., 2011; Zhao et al., 2009). Cardiomyocytes were loaded with fura 2-AM (2 lM) and were exposed to light emitted by a 75 W halogen lamp through either a 340- or 380-nm filter while being stimulated to contract at a frequency of 0.5 Hz. Fluorescence emissions were then detected. Immunoblotting Isolated cardiomyocytes were lysed in a lysis buffer containing: 50 mM b-glycerol phosphate, 2 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM NaF, 1 mM sodium orthovanadate, 20 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail tablet. Protein levels of AMPK, p-AMPK, ACC, p-ACC, pcTnI (Ser150), p-PLN and p-PKA were examined by standard western immunoblotting (Cui et al., 2013; Tong et al., 2013). Membranes were probed with anti-rabbit AMPK (Cell Signaling), anti-rabbit phosphor-AMPK (Thr172), anti-rabbit ACC (Cell Signaling), anti-rabbit phosphor-ACC (Ser79), anti-rabbit

phosphor-PLN, and anti-rabbit phosphor-PKA substrates (Cell Signaling) followed by incubation with horseradish peroxidasecoupled anti-rabbit secondary antibody. For detection of cTnI Ser150 phosphorylation, a polyclonal antibody was generated against the phosphopeptide LRRVRIS (phos)ADAMMQA and purified with affinity cross-absorption with the nonphosphorylated peptide (Sancho Solis et al., 2011). Blue x-ray film (Phenix, Candler, North Carolina) was used for photon detection and image development. Films were scanned with the Bio-Rad GS700 scanner in the core facility of the SMBS and the relative density of the bands on the film was determined by Image J software. Statistical Analysis Data are presented as the means 6 SEM. Differences between groups were assessed using analysis of variance (Zhu et al., 2008), followed by Newman-Keuls’ post hoc test. P < .05 was considered significant.

RESULTS Effect of Ucn2 on Cell Shortening and Relengthening in Cardiomyocytes To investigate the potential role of Ucn2 in regulating myocardial function, we tested the effects of Ucn2 and CRFR2 antagonist a-SVG-30 on contractile function in myocytes isolated from mouse hearts. The contractility was measured in the isolated cardiomyocytes by the IonOptix system (Ma et al., 2010; Wang et al., 2011). Figure 1F shows representative cell shortenings obtained after exposure to Ucn2. Average values are shown in

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FIG. 5. H89 abolished the phosphorylation of PKA, PLN (Ser16/Thr17), AMPK and ACC of isolated cardiomyocytes. A, Immunoblots of phosphorylated (p) PKA, PLN (Ser16/Thr17), AMPK, and ACC of isolated cardiomyocytes with H89 (10 nM), Ucn2 (10 nM), or the combination of H89 and Ucn2 treatment. B, Bars represent the relative levels of phosphorylated PKA, PLN (Ser16/Thr17), ACC and AMPK. Values are expressed as means 6 SEM, n ¼ 3–4 per group. *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

Figure 1, indicating that Ucn2 alone significantly increased maximal velocity of shortening (þdL/dt) (Fig. 1B), peak height (Fig. 1C), PS amplitude (Fig. 1D) and maximal velocity of relenthening (dL/dt) (Fig. 1E) without changing the resting sarcomere length (Fig. 1A) in WT cardiomyocytes. In the presence of a-SVG-30 however, the effects of Ucn2 were abolished, similar to the rundown observed in untreated control cardiomyocytes (Fig. 1B–E), confirming the lack of effect of Ucn2 in cardiomyocytes pretreated with a-SVG-30. Effect of Ucn2 on AMPK Signaling As activating AMPK modulates contractile functions of cardiomyocytes via phosphorylation of troponin I (Chen et al., 2014)

and previous studies demonstrated that Ucn2 treatment increased AMPK activation in isolated cardiomyocytes (Dutton et al., 1999), the question was asked as to whether the facilitation of cardiomyocytes contractile function seen with Ucn2 treatment was due to induced AMPK phosphorylation. To investigate this, isolated cardiomyocytes from WT mice were treated with vehicle (saline), CRFR2 antagonist a-SVG-30, Ucn2 and the combination of a-SVG-30 and Ucn2 for 10 min. Protein expression and phosphorylation of AMPK, its downstream signaling target acetyl-CoA carboxylase (ACC) and cTnI (Ser149) was examined (Fig. 2). Results show that cardiomyocytes responded to Ucn2 with the phosphorylation of AMPK, ACC, and cTnI (Ser149).

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FIG. 6. Contractile properties of isolated cardiomyocytes with H89 (10 nM), Ucn2 (10 nM), or the combination of H89 and Ucn2 treatment. H89 down-regulated the contractile function of cardiomyocytes. A, Resting sarcomere length; B, Maximal velocity of relengthening (þdL/dt); C, Peak height; D, PS (normalized to the resting sarcomere length); E, Maximal velocity of shortening (dL/dt); F, TPS90. Means 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

Effect of Ucn2 on Intracellular Ca21 Level To explore the underlying mechanism involved in the role of Ucn2 in inducing the contractility of cardiomyocytes, we evaluated the intracellular Ca2þ transients using the fura-2 fluorescence technique (Wang et al., 2011). Fig. 3D shows representative Ca2þ transients with or without treatment of Ucn2. Average values shown in Figure 3 indicate that Ucn2 elevated intracellular Ca2þ levels (Fig. 3B) and decreased the mean time constant of Ca2þ transient decay (Tau) (Fig. 3C) without changing the baseline of Ca2þ transient (Fig. 3A). However,

intracellular Ca2þ levels were blocked in myocytes pretreated with a-SVG-30 (Fig. 3B), consistent with the change of cell shortening. This data suggest that Ucn2 stimulates intracellular Ca2þ levels in cardiomyocytes. Signaling Pathway Involved in the Ucn2-Induced Increase in Cardiomyocytes Contractility Previous study in our lab showed that activation of AMPK by A769662 or AMPK deficiency did not change the intracellular Ca2þ level (Chen et al., 2014). Therefore, we investigated whether

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pharmacologically via applying PKA inhibitor H89 to isolated myocytes. Immunoblots of cardiomyocytes demonstrated that H89 blocked the phosphorylation of p-PKA with or without the presence of Ucn2 (Fig. 5). Activation of PLN was also inhibited after H89 treatment (Fig. 5). Besides, we found that pretreatment of H89 abolished the phosphorylation of both AMPK and ACC (Fig. 5). The contractility was measured in the isolated cardiomyocytes by the IonOptix system (Ma et al., 2010; Wang et al., 2011). Average values are shown in Figure 6, indicating that H89 significantly increased time to 90% PS (TPS90) and down-regulated maximal velocity of relenthening (þdL/dt) (Fig. 6B), peak height (Fig. 6C), PS amplitude (Fig. 6D) and maximal velocity of shortening (dL/dt) (Fig. 6E) without changing the resting sarcomere length (Fig. 6A) in WT cardiomyocytes. Besides, we observed that the effects of Ucn2 were abolished after H89 pretreatment, similar to untreated control cardiomyocytes (Fig. 6B–E). Ca2þ dynamics were measured in cardiomyocytes loaded with fura-2 (Fig. 7D). Cardiomyocytes treated with H89 showed significant prolongation of mean time constant of Ca2þ transient decay (Tau) (Fig. 7C) without changing the amplitude of Ca2þ transients (Fig. 7B). However, the elevated intracellular Ca2þ levels by Ucn2 were blocked in myocytes pretreated with H89 (Fig. 7B), consistent with the change of cell shortening. Taken together, these results suggest that Ucn2 increases phosphorylation of p-PLN in a PKA-dependent manner, suppression of PKA signaling by H89 abolishes the contractile function as well as Ca2þ dynamics of cardiomyocytes, confirming that Ucn2 also modulates the contractility of cardiomyocytes through PKA signaling pathway.

FIG. 7. Intracellular Ca2þ transient properties of isolated cardiomyocytes with H89 (10 nM), Ucn2 (10 nM) or the combination of H89 and Ucn2 treatment. A, Histograms showing the resting intracellular calcium level; B, Histograms showing mean Ca2þ transient amplitude; C, Histograms showing the mean time constant of Ca2þ transient decay (Tau). Mean 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

Ucn2 action is mediated by PKA and SERCA regulator PLN since both activation of PKA and phosphorylation of PLN are responsible for increasing SR Ca2þ load and systolic Ca2þ release (Turnley et al., 1999; Ullrich et al., 2012). Figure 4 shows immunoblots of phosphorylated PKA and PLN (Fig. 4A) of cardiomyocytes treated with Ucn2. This rise in PKA and PLN phosphorylation was suppressed by preincubation of the cardiomyocytes with a-SVG-30. These results indicate that Ucn2 stimulates PKA and PLN phosphorylation in cardiomyocytes.

Effect of PKA Inhibitor H89 on Mechanical Properties of Intracellular Ca21 To further confirm the contribution of PKA signaling on cardiomyocytes contractility. We abolished the activation of PKA

Effect of AMPK Inhibitor Compound C on Mechanical Properties of Intracellular Ca21 To clarify the cross talk between AMPK and PKA signaling upon Ucn2 treatment, we exclude the involvement of AMPK by applying AMPK inhibitor Compound C with or without the presence of Ucn2. Immunoblots in Figure 8 showed that Compound C alone didn’t change the phosphorylation of PKA as well as PLN. However, Compound C inhibited Ucn2-induced phosphorylation of PKA (Fig. 8A). The contractility was measured in the isolated cardiomyocytes by the IonOptix system (Ma et al., 2010; Wang et al., 2011). Average values are shown in Figure 9. We observed that Compound C didn’t change resting sarcomere length (Fig. 9A), maximal velocity of relenthening (þdL/dt) (Fig. 9B), peak height (Fig. 9C), PS amplitude (Fig. 9D) and maximal velocity of shortening (dL/dt) (Fig. 9E) with or without Ucn2 treatment. We also measured Ca2þ dynamics in cardiomyocytes (Fig. 10). Compound C failed to change the mean time constant of Ca2þ transient decay (Tau) (Fig. 10C) similar to the change of contractile function. However, the combination of Ucn2 and Compound C suppressed the intracellular Ca2þ level as compared with Ucn2 treatment group (Fig. 10B). These results indicate that inhibition of AMPK showed no effect on changing the contractile function of cardiomyocytes and the calcium decay with or without Ucn2 even though pretreating Compound C blocked Ucn2-induced PKA phosphorylation as well as intracellular Ca2þ level.

DISCUSSION Pharmacotherapy of heart failure has shown major improvement over the years. Ucn2, a peptide of the CRF family, has gained lots of attention as a potential treatment of heart failure (Dutton et al., 1999). Ucn2 signal through CRFR2 caused a potent enhancement of myocardial inotrophy and lusitrophy (Bale et al., 2004). Studies have demonstrated that Ucn2 induced an

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FIG. 8. Compound C inhibited Ucn2-induced phosphorylation of PKA. A, Immunoblots of phosphorylated (p) PKA and PLN (Ser16/Thr17) of isolated cardiomyocytes with Compound C (10 mM), Ucn2 (10 nM), or the combination of Compound C and Ucn2 treatment. B, Bars represent the relative levels of phosphorylated PKA and PLN (Ser16/Thr17). Values are expressed as means 6 SEM, n ¼ 3–4 per group. *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

increase in cardiac output, heart rate, and left ventricular ejection fraction, while decreasing systemic vascular resistance and mean arterial pressure without hormone response (Davis et al., 2007a,b; Mackay et al., 2003; Ng et al., 2004). However, the cellular effects of Ucn2 on cardiomyocytes are not fully understood. The goal of this study is to investigate the effect of Ucn2 on mediating the contractility of cardiomyocytes. Here we show that Ucn2 stimulated both PKA and AMPK signaling, and that the 2 kinases combine to facilitate contractile function. In this study, Ucn2 exerted powerful positive inotropic and lusitropic effects in cardiomyocytes. As shown in Figure 1, we found that Ucn2 alone significantly stimulated maximal velocity of shortening (þdL/dt), peak height, PS amplitude, maximal velocity of relengthening (dL/dt) in WT cardiomyocytes. Previous research in isolated cardiomyocytes indicated CRFR2mediated improvement of intracellular calcium level by Ucn2 as one of the mechanism accounting for the stimulation of cardiomyocyte contractility (Meili-Butz et al., 2010). In this study, we found that the positive effects of cardiomyocytes after Ucn2 treatment were accompanied by a significant rise in intracellular Ca2þ level as well as a fall of the mean time constant of Ca2þ transient decay (Tau) (Fig. 3). To determine the mechanism by which Ucn2 treatment stimulates the contractility of cardiomyocytes, we used the CRFR2 antagonist a-SVG-30, an N-terminally truncated version of Ucn2 (Brar et al., 2004). Preincubation with a-SVG-30 abolished contractile function, accompany by a decrease in the peak level and a prolongation of the mean time constant of Ca2þ transient decay. Within the last decade, AMPK has emerged as a central metabolic regulator that balances energy supply (Chen et al., 2014; Young, 2008). Impaired cardiac function and susceptible cell death were found in transgenic mice which lack functional

AMPK (Russell et al., 2004). cTnI is recognized as the ‘inhibitory’ subunit of the troponin complex playing a critical regulatory role in cardiac muscle contraction and relaxation. ACC is a classic AMPK target and phosphorylation inhibits the action of ACC to synthesize malonyl-CoA, (Kudo et al., 1996). Previous study showed that pharmacologic treatment with Ucn2 activated AMPK and downstream ACC in isolated heart muscle (Li et al., 2013). Recent study in our lab also noted that activation of AMPK could regulate the phosphorylation status of cTnI (Ser150), leading to the result of increase Ca2þ sensitivity and contractility of cardiomycytes (Chen et al., 2014). In this study, we found that Ucn2 treatment activated the AMPK pathway in isolated cardiomyocytes via CRFR2 (Fig. 2). In addition, treatment with aSVG-30 partially inhibited the activation of AMPK after Ucn2 treatment. Because recent study in our lab demonstrated that activation of AMPK by AMPK activator A-769662 or AMPK deficiency did not change the intracellular Ca2þ level while Ucn2 significantly improved the handling of intracellular Ca2þ (Chen et al., 2014). Therefore, the activation of AMPK signaling and cTnI (Ser150) might not be the only mechanism involved in regulating the contractile function of cardiomyocytes. CRFR2 couples to Gs– cAMP–PKA signaling (Nishikimi et al., 2000). A previous study showed that Ucn2 stimulated positive inotropic and lusitropic effects and activated PKA signaling in rabbit ventricular myocytes (Yang et al., 2006). Stimulation of PKA is expected to reveal a positive effect on intracellular Ca2þ level via phosphorylation of key Ca2þ -regulating proteins including PLN, which will result in the increase of SR Ca2þ content and the acceleration of Ca2þ transient (Turnley and Bartlett, 1999). In our study, we found that cardiomyocytes respond to PKA phosphorylation via Ucn2. Furthermore, Ucn2 induced PLN phosphorylation at Ser16, the site phosphorylated by PKA (Fig. 4). Pretreatment of a-SVG-30

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FIG. 9. Contractile properties of cardiomyocytes isolated from WT mice with Compound C (10 mM), Ucn2 (10 nM), or the combination of Compound C and Ucn2 treatment. Compound C didn’t affect the contractile function of cardiomyocytes with or without Ucn2 treatment. A, Resting sarcomere length; B, Maximal velocity of relengthening (þdL/dt); C, Peak height; D, PS (normalized to the resting sarcomere length); E, Maximal velocity of shortening (dL/dt); F, TPS90. Means 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus control group.

suppressed all of the effects of Ucn2. Besides, pretreatment of cardiomyocytes with H89, an inhibitor of PKA, largely reduced the inotropic and lusitropic effects of Ucn2 (Fig. 6). H89 also decreased the intracellular Ca2þ load and slowed down the Ca2þ transient decay (Fig. 7), consistent with previous findings (Yang et al., 2006, 2011). Given the observation that both AMPK and PKA contributed to the positive stimulation of cardiomyocytes contractility, we asked next whether the crosstalk between AMPK and PKA exists. A study demonstrated that PKAa and AMPKa1 physically interact in primary adipocytes and PKA phosphorylates AMPKa1 at Ser173 (Djouder et al., 2010). It has also been reported that phosphorylation of cTnI at Ser150 by

AMPK reduced the effects of cTnI Ser23/24 PKA phosphorylation at the myofilament (Nixon et al., 2012). However, the interaction and the differential regulation of the PKA- and AMPK-signaling pathways in the heart have not been thoroughly investigated yet. We found that preincubation of Compound C, an inhibitor of AMPK inhibited the intracellular Ca2þ level in cardiomyocytes due to the blunted phosphorylation of PKA while it did not affect the contractile function and the mean time constant of Ca2þ transient decay (Tau) of cardiomyocytes with or without Ucn2. Interesting, preincubation of H89 suppressed the phosphorylation of both PKA and AMPK. As a result, both calcium dynamics and sarcomeric shortening were impaired.

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FUNDING This work was supported by American Heart Association 14IRG18290014, American Diabetes Association Basic Sciences Grant 1-14-BS-131, NIH R21AG044820, and R01AG049835, National Natural Science Foundation of China 31171121, and 81500264.

REFERENCES

FIG. 10. Intracellular Ca2þ transient properties of isolated cardiomyocytes with Compound C (10 mM), Ucn2 (10 nM), or the combination of Compound C and Ucn2 treatment. Compound C suppressed the intracellular Ca2þ level. A, Histograms showing the resting intracellular calcium level; B, Histograms showing mean Ca2þ transient amplitude; C, Histograms showing the mean time constant of Ca2þ transient decay (Tau). Mean 6 SEM, n ¼ 50–120 cells from 3 mice per group, *P < .05 versus control group; †P < .05 versus Ucn2 alone group.

CONCLUSIONS In this study, we proposed that Ucn2 facilitates the contractility of cardiomyocytes via activating both AMPK and PKA. By activation of AMPK, cTnI (Ser150) was phosphorylated therefore help regulate the contractility of cardiomyocytes. PKA and Ca2þ -regulating proteins PLN would be phosphorylated upon Ucn2 treatment, which loading the SR with Ca2þ as well as accelerating Ca2þ decay, resulting in stimulating the contractility. Inhibition of AMPK by Compound C before Ucn2 treatment suppressed the activation of PKA without affecting the contractility of cardiomyocytes while inhibition of PKA by H89 before Ucn2 treatment abolished the activation of AMPK, leading to the down-regulation of calcium dynamics and sarcomeric shortening.

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