H+ exchanger inhibition induced by Sildenafil

Author’s Accepted Manuscript p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil Romina G. Díaz, Daiana S. Escudero, María S. Brea, Patricio E. Morgan, Néstor G. Pérez www.elsevier.com/locate/ejphar

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S0014-2999(19)30053-6 https://doi.org/10.1016/j.ejphar.2019.01.070 EJP72211

To appear in: European Journal of Pharmacology Received date: 21 September 2018 Revised date: 11 January 2019 Accepted date: 21 January 2019 Cite this article as: Romina G. Díaz, Daiana S. Escudero, María S. Brea, Patricio E. Morgan and Néstor G. Pérez, p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.01.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil

Romina G. Díaz σ §, Daiana S. Escudero* §, María S. Brea*, Patricio E. Morganσ, Néstor G. Pérezσ

Centro de Investigaciones Cardiovasculares “Dr. Horacio E. Cingolani” Facultad de Ciencias Médicas, Universidad Nacional de La Plata Calle 60 y 120, 1900 La Plata ARGENTINA

§

Both authors contributed equally to this work.

Corresponding authors: Dr. NG Pérez and Dr. PE Morgan Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, UNLP Calle 60 y 120 (1900) La Plata, Argentina. Phone/FAX: (54-221) 483-4833 E-mail: [email protected], pemorgan@ med.unlp.edu.ar *Fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. σ Established Investigators of CONICET, Argentina.

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Abstract Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal–regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.

Keywords: NHE1; p38MAPK; protein kinase G; protein phosphatase 2A; Sildenafil

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1. Introduction Phosphodiesterase 5A (PDE5A) inhibitor Sildenafil, 1-((3-(6,7-dihydro-1-methyl-7-oxo-3propyl-1H-pyrazolo(4,3-d)pyrimidin-5-yl)-4-ethoxyphenyl)sulfonyl)-4-methylpiperazine citrate, was originally developed as an antianginal therapy because of its vasodilator activity (Jackson, Montorsi et al. 2006). Competitive inhibition of cyclic guanosine monophosphate (cGMP) breakdown promotes an increase of this second messenger, targeting protein kinase G (PKG), and vascular smooth muscle relaxation (Jackson, Montorsi et al. 2006). Due to its vasodilator outcomes, Sildenafil has been widely used as first-line therapy for erectile dysfunction (Burnett 2005), ranging to treatment of pulmonary hypertension (Ghofrani, Wiedemann et al. 2002), lung fibrosis (Ghofrani, Wiedemann et al. 2002) and pulmonary implications of cardiac heart failure (Behling, Rohde et al. 2008). Chronic administration of Sildenafil to rodents subjected to sustained pressure overload (Takimoto, Champion et al. 2005) or myocardial infarction (Perez, Piaggio et al. 2007) improved cardiac function and inhibited heart remodeling. Those effects came from a direct activation of cGMP/PKG-1 route (Perez, Piaggio et al. 2007). Cardioprotective actions of PKG-1 activation in ischemia reperfusion injury attenuated hypercontracture and reduced infarct size (Garcia-Dorado, Agullo et al. 2009), also reduced acceleration of cytosolic Ca2+ recovery and attenuation of Ca2+ oscillations (Abdallah, Gkatzoflia et al. 2005). Antihypertrophic effects of PKG-1 activation involved a negative regulation of the calcineurin-Nuclear Factor of activated T-cells hypertrophy pathway (Fiedler, Lohmann et al. 2002). Cardiac ischemia/reperfusion injury, hypertrophy development and progression to heart failure have been associated to Na+/H+ exchanger (NHE1) hyperactivity (Karmazyn, Gan et al. 1999, Odunewu-Aderibigbe and Fliegel 2014). Cardiac NHE1 is a main important housekeeping acid extrusion system (Leem, Lagadic-Gossmann et al. 1999) whose activity is

4 tightly controlled by intracellular H+ on its allosteric site (Wakabayashi, Fafournoux et al. 1992), phosphorylation (Karmazyn, Gan et al. 1999, Haworth, McCann et al. 2003, Coccaro, Karki et al. 2009) and other mechanisms (Wakabayashi, Bertrand et al. 1994, Wu and Vaughan-Jones 1994). During myocardial ischemia, NHE1 activity is stimulated not only by H+-triggered activation but also by an extracellular signal–regulated kinases (ERK)/p90 ribosomal s6 kinase (p90RSK)-dependent phosphorylation on the Ser703 residue of its cytosolic tail (Haworth, McCann et al. 2003). Chronic (Perez, Piaggio et al. 2007) or acute (Garciarena, Fantinelli et al. 2011) Sildenafil treatment of infarcted rat hearts reduced NHE1 hyperactivity and improved cardiac function. Acute Sildenafil effect on cardiac NHE1 hyperactivity was associated to a reduction in Ser703 phosphorylation (Diaz, Nolly et al. 2010, Garciarena, Fantinelli et al. 2011), through a mechanism that could be attributable to protein phosphatase 2A (PP2A) activation by PKG (Diaz, Nolly et al. 2010). Mitogen-activated protein kinases, as ERK and p38MAPK, mediate intracellular signaling pathways initiated by stress. Stress induced by ischemia (Garciarena, Fantinelli et al. 2011) or by H2O2 (Wei, Rothstein et al. 2001) activates ERK triggering NHE1 activation. The same stress stimuli produces p38MAPK activation (Ma, Kumar et al. 1999, Wei, Rothstein et al. 2001) without affecting NHE1 phosphorylation (Wei, Rothstein et al. 2001). Cardiac p38MAPK actions were initially described as pro-apoptotic (Ma, Kumar et al. 1999), however, new evidence revealed that p38MAPK activation participates in cell-survival mechanisms prompted by a-adrenergic stimulation (Tsang and Rabkin 2009), and cardioprotection against ischemia/reperfusion injury mediated by the mammalian target of rapamycin (Hernandez, Lal et al. 2011). In non-cardiac tissues, p38MAPK regulation of NHE1 activity was already described. In this regard, Angiotensin II stimulates NHE1 in vascular smooth muscles cells culture, where a concomitant activation of p38MAPKwas shown to negatively regulate H+ efflux directly by phosphorylation or indirectly by ERK

5 inhibition (Kusuhara, Takahashi et al. 1998). On the contrary, in lymphocyte T-cells p38MAPK directly phosphorylate NHE1 leading to an increased exchanger activity (Khaled, Moor et al. 2001). Finally, PKG-dependent p38MAPK activation was also demonstrated as part of the integrin activation pathway of platelets (Li, Zhang et al. 2006), as well as of the presynaptic plasma membrane serotonin transporters regulation (Zhu, Carneiro et al. 2005). Considering the potential beneficial effects of cardiac p38MAPK activation, and the reported cross relationship between PKG and p38MAPK in different tissues, we hypothesized that activation of the latter would be involved in the PKG-1-mediated inhibitory action of Sildenafil on cardiac NHE1. Herein we will provide evidence that p38MAPK activation is an intermediate step in the signaling pathway triggered by PDE5A inhibition that leads to NHE1 dephosphorylation/inhibition.

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2. Materials and Methods 2.1. Animals All procedures followed during this investigation were done conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and to the guidelines laid down by the Animal Welfare Committee of La Plata School of Medicine (CICUAL). Four-month-old male Wistar rats (of approximately 300gr.) were anesthetized via intraperitoneal with ketamine plus diazepam (75mg/Kg; 5mg/Kg) until reached deep anesthesia, then were killed by extracting the heart. 2.2. Reagents To intracellular pH (pHi) measurements, acetoxymethyl ester form of the pH-sensitive fluorescent dye 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF-AM, Invitrogen, Molecular Probes) was used (final concentration of 10 μmol/l) in HEPES solution. HEPES buffer solution was composed by (in mmol/l) NaCl 146.2, KCl 4.5, CaCl2 1.35, MgSO4 1.05, glucose 11.0, HEPES 10.0, pH 7.40. Possible participation of catecholamines released by nerve endings was prevented by adding adrenergic receptors blockers (1.0 μmol/l prazosin plus 1.0 μmol/l atenolol) to HEPES buffer. To homogenize samples, lysis buffer containing (in mmol/l) sucrose 300; DTT 1; EGTA 4; Tris-HCl 20, and protease inhibitors cocktail (Complete Mini Roche), pH 7.4, was used. For immunoprecipitation assays, samples were homogenized with IP buffer containing Tris-HCl 10 mmol/l, EDTA 5 mmol/l, NaCl 150 mmol/l, NP40 1%, Sodium Deoxycholate 0.5%, and protease inhibitors cocktail (Complete Mini Roche), pH 7.5. Pharmacological intervention doses: Sildenafil citrate (kindly donated by Roemmers laboratories) was used at 1 μmol/l; SB202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4pyridyl)-1H-imidazole, Sigma-Aldrich) at 10 μmol/l; Okadaic acid (Sigma-Aldrich) at 1 nmol/l; Sodium Arsenite (Sigma-Aldrich) at 5 μmol/l, and Anisomycin ((2R,3S,4S)-2-(4-

7 Methoxybenzyl)-3,4-pyrrolidinediol-3-acetate, 2-[(4-Methoxyphenyl)methyl]-3,4pyrrolidinediol 3-acetate, Sigma-Aldrich) at 20 μmol/l. For p38MAPK activators Sodium Arsenite and Anisomycin, the micromolar range was selected to avoid activation of other kinases (like JNK) at higher doses of both compounds (Khaled, Moor et al. 2001, Duyndam, Hulscher et al. 2003), and to minimize possible proapoptotic effects of Arsenite (Trouba and Germolec 2004). 2.3. Measurement of pHi, determination of NHE1 activity and intrinsic buffer capacity (bi) pHi was measured in isolated left ventricular papillary muscles from rats following a previously described BCECF-epifluorescence technique (Perez, Piaggio et al. 2007). Once extracted the heart, muscles were carefully dissected, mounted in a perfusion chamber placed on the stage of an inverted microscope (Olympus), paced at 0.2 Hz at a voltage 10% over threshold and maintained at 30◦C. Muscles were incubated with BCECF-AM for 1 h in HEPES solution, and then washout extracellular medium with dye-free solution. pHi determinations were performed 30-60 min after washout, allowing uniform distribution of BCECF fluorescence throughout the muscle. To measure fluorescence emission, excitation light from a 75-W Xenon lamp was band-pass–filtered alternatively at 440 and 495 nm and was then transmitted to muscles by a dichroic mirror (reflecting wavelengths, <510 nm) located beneath the microscope. Fluorescence emission was collected by the microscope objective (10x), and transmitted through a band-pass filter at 535±5 nm to a photomultiplier (model R2693, Hamamatsu). NHE1 activity after sustained acidosis was assessed by the Na+dependent pHi recovery from an ammonium prepulse-induced acute acid load. Papillary muscles were acid loaded by brief (10 min) exposure to 20.0 mmol/l NH4Cl followed by washout with Na+-free HEPES buffer (NaCl was equimolar replaced with N-methyl-Dglucamine). Sustained acidosis lasting depends on Na+-free HEPES buffer permanence, SIL

8 effect on pHi recovery was determined at different times duration. Times tested were zero, 3, 5, 10 and 15 minutes. Experiments were performed in the nominal absence of bicarbonate (HEPES buffer, O2 100%) to assure that pHi recovery after the acidic load was entirely due to NHE1 activation. Intracellular buffer capacity (bi) was determined as previously described (Perez, Piaggio et al. 2007). For a better comparison of NHE1 activity among groups, raw data from each experiment were adjusted to an exponential function and then H+ efflux (JH+= maxdpHi/dt

.bi) was calculated at a common pHi of ~6.75. Since other authors (Leem, Lagadic-

Gossmann et al. 1999) demonstrated a greater increase in NHE1 activity at lower pHi, Table 1 shows that maximal intracellular acidification after the acidic load (measured just before Na+ re-addition) as well as bi were of similar magnitude in all experimental groups, validating comparison among them. At the end of each experiment, fluorescence emission was calibrated by exposing the muscles to a high-KCl solution containing 10 μmol/l Nigericin. Buffer pH was adjusted with KOH or HCl to four different values ranging from 7.5 to 6.5. Such a calibration revealed a linear relation between pH and the fluorescence ratio (F495/F440), which was used to correct experimental fluorescence ratios. All pharmacological interventions were started 20 minutes before the ammonium prepulse and kept for the entire protocol. Sample sizes are indicated in respective bars. For those comparative experiments, 10 minutes of sustained acidosis were implemented. 2.4. Protein determination-western blot Left ventricle cardiac tissue slices were superfused with bicarbonate free buffer (HEPES buffer bubbled with 100% O2). Sustained acidosis protocol consisted of 10 minutes of incubation with 20mM NH4Cl, followed by 10 min. washout with Na+-free HEPES buffer, after which samples were quickly frozen. For drug-treated muscles, drugs were added to the superfusate 20 minutes before ammonium prepulse, and maintained until the end of

9 experiment. Non-acidic controls (NAC) were time-matched by keeping samples in HEPES buffer during equivalent time to the entire acidic protocol (~40 min). Frozen samples were homogenized in lysis buffer. After a brief centrifugation the supernatant was kept and protein concentration determined by the Bradford method (BioRad Protein Assay). Samples were denatured and equal amounts of protein were subjected to PAGE and electrotransferred to PVDF membranes. To estimate kinases activation/phosphorylation, membranes were then blocked with non-fat-dry milk and incubated overnight either with antiphospho-ERK1/2 or anti-phospho-p38MAPK or anti-phospho-MAPKAP-2 or anti-phospho Akt1/2/3 (Santa Cruz Biotechnology: SC16982, SC166182, SC13746 or SC514032 respectively). Total ERK, p38MAPK, and GAPDH (Santa Cruz Biotechnology: SC1647, SC7972, SC47724) were assayed as loading controls. Akt expression was also assayed (SC5298). Peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) were used as secondary antibody and bands were visualized using the ECL-Plus chemiluminescence detection system (Amersham). Autoradiograms were analyzed by densitometric analysis (Scion Image). Signal obtained for the phosphor-proteins were each normalized by that obtained with respective loading control. Those normalized values were then expressed as a percentage of control ones (acidotic or non-acidotic, as respective figure shows). To estimate total expression of NHE1 same procedure was performed but signal of total protein was normalized with corresponding of GAPDH. 2.5. Immunoprecipitation and determination of NHE1 phosphorylation For NHE1 phosphorylation determination, samples processed as previous section details were immunoprecipitated using a NHE1 polyclonal antibody (Santa Cruz Biotechnology: SC28758) and Protein A/G plus-agarose (Santa Cruz Biotechnology, sc-2003) and then subjected to PAGE. Incubation was performed with an anti-14-3-3 binding motif (BM) antibody (Cell Signaling 9601). Previous reports have shown that the regulatory Ser703 of the

10 NHE1 lies within a sequence, which creates upon phosphorylation a binding motif for 14-3-3 proteins (Maekawa, Abe et al. 2006). Thus, the anti-14-3-3 BM antibody when probed with immunoprecipitated NHE1 represents a useful tool to estimate NHE1 phosphorylation at Ser703 (Snabaitis, D'Mello et al. 2006). Signals obtained for 14-3-3 BM were normalized by total NHE1 as loading control. 2.6. Statistics Results are presented as mean ± S.E.M. Statistical analysis was performed using one-way ANOVA followed by the Student-Newman-Keuls test. p<0.05 was considered significant.

3. Results 3.1. NHE1 activity during sustained acidosis. Role of p38MAPK in the signaling pathway triggered by Sildenafil. Sustained acidosis (10 min) was induced by washing out ammonium chloride with a Na+ free buffer. Fig. 1A shows that p38MAPK specific inhibitor, SB202190, did not affect the characteristic rapid recovery of pHi upon Na+ restoration into the superfusing buffer. To analyze whether the inhibitory effect of Sildenafil was mediated by p38MAPK, SB202190 was added to the Sildenafil containing buffer before inducing acidosis. Fig. 1B shows that Sildenafil induced reduction of the NHE1-mediated pHi recovery was prevented in the presence SB202190, restoring pHi to control levels. Fig. 1C represents statistical analysis of the proton efflux (JH+), calculated immediately after Na+ re-addition. These results indicate that although activation of NHE1 during sustained acidosis appears to be independent of p38MAPK, this kinase participates in the inhibition pathway induced by Sildenafil. In order to determine whether Sildenafil-triggered NHE1 inhibition could be affected by the time-duration of acidosis, new series of experiments were performed in which time-extension

11 of the ammonium prepulse washout under Na+-free buffer was varied. Supplemental Fig. 1 shows averaged JH+ results obtained immediately after Na+ re-addition under all experimental conditions. Sustained acidosis promoted a greater NHE1 activation than the transient one at any time-duration tested. Exchanger activity was maximal after three-five min of acidosis. Further prolongation of acidosis showed a significant slowing down of exchanger activity that reached an apparent stable condition around 10 to 15 min. Sildenafil promoted NHE1 inhibition only after sustained acidosis as was previously shown (Diaz, Nolly et al. 2010). Interestingly, the remaining NHE1 activity observed after PDE5A inhibition was similar under all experimental conditions and of similar magnitude to that observed after transient acidosis. This finding would suggest that Sildenafil treatment only targeted the described phosphorylation-mediated activation of the exchanger (Haworth, McCann et al. 2003). A prolonged condition of acidosis was associated to an enhanced phosphorylation on NHE1 at Ser703 (Haworth, McCann et al. 2003). In the present study, cardiac left ventricle samples were subjected to acidosis for 10 min, immediately frozen and afterwards processed for NHE1 expression and phosphorylation analysis. Fig. 2A, shows that pharmacological treatment either with Sildenafil or SB202190 did not affect NHE1 expression compared to non-treated control samples. Fig. 2B shows that increased Ser703 phosphorylation during sustained acidosis was prevented by Sildenafil, effect that was avoided in the presence of SB202190. The sole presence of SB202190 under same experimental condition did not modify the magnitude of NHE1 phosphorylation. These results indicate that Sildenafil inhibition of NHE1 hyperactivity, and the potential intervention of p38MAPK in this route, would be due to modulation of its Ser703 phosphorylation. To further analyze the role of p38MAPK as a signaling protein in the Sildenafil dependentNHE1 inhibitory pathway during acidosis, we directly stimulate p38MAPK. After 10 min of acidosis, papillary muscles pretreated with p38MAPK activator Sodium Arsenite showed a

12 significantly reduction in NHE1 activity (Fig. 3A and C). In order to confirm that NHE1 inhibition was specifically due activation of p38MAPK and not any other kinase (Duyndam, Hulscher et al. 2003), the same protocol was repeated in the presence of SB202190. As shown in Fig. 3A and C, addition of SB202190 to the superfusate completely reverted the Arsenitemediated inhibitory effect on pHi recovery after acidosis, suggesting that at the concentration of Arsenite used (5mmol/L) the effect on NHE1 activity was entirely mediated by p38MAPK activation. These results were confirmed using another activator of p38MAPK, Anisomycin, which was capable of mimicking the effect of Sildenafil on NHE1 activity, preventing the characteristic fast recovery seeing in control experiments (Fig. 3B and C). Since comparison of pHi after 20 min of incubation with every pharmacological intervention showed no changes in basal pHi (Supplemental Table 1), the results observed on NHE1 activity under each experimental condition, would be exclusively consequence of the acidic challenge. These results lead us to conclude that p38MAPK activation effectively promotes NHE1 inhibition under sustained acidosis. Since p38MAPK inhibition reversed Sildenafil inhibitory effect on NHE1 activity and conversely, p38MAPK activation mimicked the inhibitory effect of Sildenafil on the exchanger, we explored the possible p38MAPK activation by Sildenafil and/or sustained acidosis. We determined p38MAPK phosphorylation (as activation index) in cardiac samples after sustained acidosis with or without Sildenafil stimulus and compared to non-acidotic controls. Fig. 4A shows that p38MAPK phosphorylation was not significantly affected by sustained acidosis itself, but was increased when Sildenafil was present in the superfusate. In order to establish a correlation between phosphorylation and kinase activity, we performed western blots assessing phosphorylation state of MAPKAP-2, a target protein for p38MAPK. Since for 10 min of acidosis, total protein expression for p38MAPK (% of Control: 100 ± 6 Control, n=4; 96 ± 12 Acidosis, n=4; 107 ± 11 Sildenafil, n=4), ERK (% of Control: 100 ± 4

13 Control, n=8; 101 ± 10 Acidosis, n=6; 105 ± 14 Sildenafil, n=7), Akt (Fig. 5C) and NHE1 (Fig. 2A) remained unchanged, phospho-MAPKAP-2 was normalized to GAPDH. In accordance to the observed increase in phospho-p38MAPK, MAPKAP-2 phosphorylation was found increased during sustained acidosis only in presence of Sildenafil (Fig. 4B, middle bar), reinforcing the notion that sustained acidosis per se does not affect p38MAPK activation. In addition, SB202190 completely reverted the increase in phospho-MAPKAP-2 promoted by Sildenafil (Fig. 4B, fourth bar), giving further support to the evidence that Sildenafil leads to p38MAPK phosphorylation/activation. 3.2. Effect of Sildenafil on ERK 1/2 and Akt activation under sustained acidosis. NHE1 phosphorylation at Ser703 following a sustained acidosis was reported to be caused by activation of ERK1/2-p90RSK kinases cascade (Karmazyn, Gan et al. 1999, Haworth, McCann et al. 2003). Since Sildenafil-induced p38MAPK-mediated inhibitory effect on NHE1 also has Ser703 phosphorylation site as a target, it may result from modulation of either ERK1/2-p90RSK or an alternative pathway. As an initial approach to disclose these possibilities, we aimed to assess ERK1/2 phosphorylation (as kinase activation index) under our experimental conditions. Fig. 5A shows that sustained acidosis significantly increased ERK1/2 phosphorylation, and that this effect was not modified by Sildenafil treatment. Furthermore, addition of SB202190 to Sildenafil-treated muscles did not modify phosphorylation status of ERK1/2, suggesting that activation of p38MAPK by PDE5A inhibition does not affect the activation of these kinases upstream NHE1. Fig. 5A also shows that p38MAPK inhibition per se did not change the increase in ERK1/2 phosphorylation promoted by sustained acidosis. These findings provide evidence to propose that the effect of both Sildenafil and p38MAPK on NHE1 activity are independent of ERK1/2 activity. In a mouse model of pressure overload-triggered cardiac hypertrophy, Takimoto et al. (Takimoto, Champion et al. 2005) reported protein kinase B (Akt) inactivation after chronic

14 Sildenafil treatment. On the other hand, Mockridge el al. (Mockridge, Marber et al. 2000, Armstrong 2004) showed activation of Akt in neonatal rat cardiomyocytes during simulated ischemia/reperfusion, pathological condition that is associated to intracellular acidification, while Snabaitis el at. (Snabaitis, Cuello et al. 2008) reported direct inhibitory action of Akt on NHE1 activity. In this context, we sought to test whether Akt activation would play a role in the described mechanism. Fig. 5B shows a slight not significant increase in Akt phosphorylation/activation during acidosis in the presence of Sildenafil without changes in Akt expression (Fig. 5C), suggesting that Akt would not participate in the Sildenafil-triggered pathway leading to NHE1 inhibition. 3.3. Role of p38MAPK in NHE1 regulation under sustained acidosis. Participation of PP2A Dephosphorylation of NHE1induced by Sildenafil during sustained acidosis is mediated by PP2A phosphatase activation (Diaz, Nolly et al. 2010). Since we are currently providing evidence that p38MAPK activation participates in that Sildenafil-triggered mechanism, we next hypothesized that PP2A may conceivably be an intermediary step of this inhibitory pathway. In agreement, Fig. 6 shows that pretreatment of papillary muscles with Okadaic Acid, at a concentration that specifically inhibits PP2A (1 nmol.L-1) (Cohen 1989), completely reversed the inhibitory effect of p38MAPK activation (Arsenite promoted) on NHE1 activity. Taken together, these results allow suggesting that PDE5A inhibition by Sildenafil leads to a PKG-1-triggered p38MAPK-mediated PP2A activation leading to NHE1 dephosphorylation, with the consequent decrease in the exchanger activity.

15 4. Discussion Sustained acidosis causes a time dependent MAPK-mediated phosphorylation of the cytosolic tail of the cardiac NHE1 (Karmazyn, Gan et al. 1999, Haworth, McCann et al. 2003). This mechanism induces an increased exchanger activity that reaches a maximum after 3-5 min of acidosis, and that can be inhibited by preventing ERK-MAPK activation (Haworth, McCann et al. 2003) but also by inducing PKG activity (Perez, Piaggio et al. 2007, Diaz, Nolly et al. 2010). Current work demonstrates that activation of another MAPK, such as p38MAPK, is a key factor in the signaling pathway that links PDE5A inhibition and PKG-1 activation with NHE1 inactivation. Using an activator of p38MAPK as Sodium Arsenite, we also demonstrate that p38MAPK-triggered NHE1 inhibition is mediated by phosphatase PP2A. Our data constitute an evidence of a PKG-1-p38MAPK-PP2A sequential activation pathway in the myocardium following PDE5A inhibition targeting NHE1 phosphorylation. Cardiac tissue role of p38MAPK is not entirely defined. Whereas most evidence assigned it deleterious actions, growing evidence reveals p38MAPK protective effects. Association between p38MAPK and apoptosis has been demonstrated in ischemia/reperfusion injury, and its inhibition was proposed as a therapeutic target (Ma, Kumar et al. 1999). In fact, the p38MAPK inhibitors Losmapimod and SB-681323 when tested in clinical trials failed to show conclusive results (Sarov-Blat, Morgan et al. 2010, Aronow and Kaple 2016, O'Donoghue, Glaser et al. 2016). Conversely, p38MAPK activation was demonstrated to be crucial against cardiomyocyte apoptosis (Tsang and Rabkin 2009, Hernandez, Lal et al. 2011). Since results provided in Fig. 1 and 3 show a p38MAPK inhibitory effect on NHE1 hyperactivity, and cardiac NHE1 hyperactivity exerts a well-known detrimental role (Odunewu-Aderibigbe and Fliegel 2014), our results favor the p38MAPK protective premise (Javadov, Jang et al. 2014). In this regard, several cardiac pathological conditions (Karmazyn, Gan et al. 1999, Odunewu-Aderibigbe and Fliegel 2014) are associated with increased NHE1

16 activity, leading to an augmentation in intracellular Na+ concentration, finally enhancing cytosolic Ca+2 that activates deleterious-signaling transcription factors (Nakamura, Iwata et al. 2008). Positive results reported with NHE1 inhibitors in experimental studies were not reflected on clinical outcomes (Theroux, Chaitman et al. 2000, Mentzer, Bartels et al. 2008) because of inconsistent results evidenced by the poor efficacy and serious side effects (Karmazyn 2013). However, NHE1 inhibition if applied properly, continues to represent one of the most effective approaches for myocardial rescue following ischemic insult (Karmazyn 2013). In contrast with classical pharmacological inhibitors, Sildenafil reduced NHE1 hyperactivity without affecting its basal housekeeping activity (Diaz, Nolly et al. 2010). Herein we demonstrated that this inhibitory effect was mediated by p38MAPK activation. p38MAPK inhibitory action on NHE1 activity was also shown in isolated vascular smooth muscle cells after stimulation with Angiotensin II (Kusuhara, Takahashi et al. 1998). Although, in that case p38MAPK effect could be consequence from a direct phosphorylation of the exchanger or a decreasing phosphorylation of its upstream kinases ERK1/2-p90RSK. Results presented in Fig. 5 propose another possibility, since either activation or inactivation of p38MAPK did not affect the acidosis-triggered increase in ERK1/2 phosphorylation. Complete reversion of p38MAPK effect by Okadaic acid, showed in Fig. 6, suggests that NHE1 dephosphorylation by PP2A would be responsible for p38MAPK inhibitory actions. In our experiments, NHE1 phosphorylation was evaluated by the ability of adaptor protein 143-3 to recognize and bind to phospho-Ser703 microenvironment. Fig. 2 shows that Sildenafilinduced dephosphorylation of that residue was cancelled by inhibiting p38MAPK. A limitation of our study is that we only assayed NHE1 phosphorylation at Ser703 and no other regulatory sites (Liu, Stupak et al. 2004, Snabaitis, Cuello et al. 2008, Coccaro, Karki et al. 2009). In this regard, a dichotomy in p38MAPK actions on NHE1 was presented by two different laboratory groups using GST-NHE1 fusion, p38MAPK resulted able to directly

17 phosphorylate the cytosolic tail of the exchanger, decreasing (Kusuhara, Takahashi et al. 1998) or increasing (Khaled, Moor et al. 2001) its activity. We cannot discard then an additional direct NHE1 phosphorylation by this kinase, but in any case, p38MAPK activation finally lead to NHE1 inhibition. The possibility of a direct effect may provide a potential explanation to the greater inhibitory effect of Arsenite on NHE1 activity (Fig. 3C), even more pronounced than that promoted by Sildenafil. Sildenafil, as other PKG-1 activator strategies, has an established cardioprotective role (Takimoto, Champion et al. 2005, Perez, Piaggio et al. 2007, Garcia-Dorado, Agullo et al. 2009). Present data suggests that p38MAPK activation would mediate some of those effects (Fig. 4). Regarding cross activation of these kinases, the available literature is rather controversial. Experiments in platelets provide evidence that p38MAPK could be activated by PKG (Li, Zhang et al. 2006). Additionally, p38MAPK activation decreased NHE1 activity in vascular smooth muscle cells (Kusuhara, Takahashi et al. 1998) supporting as a whole the proposal of p38MAPK activation to mediate PKG-1-triggered NHE1 inactivation. However, in apparent contradiction Fiedler et al. (Fiedler, Feil et al. 2006) showed inhibition of p38MAPK by PKG-1 as part of a cardioprotective pathway in experimental ischemia/reperfusion injury. In this regard, we should highlight that p38MAPK activation in cardiomyocytes during ischemia/reperfusion occurs independent of the upstream MKK3/6 kinase, being instead mediated by interaction of p38MAPK with the scaffold protein TAB1 (Ge, Gram et al. 2002, Tanno, Bassi et al. 2003). Fiedler`s study identified an inhibitory interaction between PKG-1 and TAB1-p38MAPK signaling pathway that protects against myocardial post-ischemic injury. Therefore, we may speculate that our findings could result from modulation of MKK3/6 signaling pathway during sustained acidosis. Results in Fig. 4 showing increased p38MAPK phosphorylation/activity in presence of Sildenafil support this possibility.

18 We would like to emphasize that we are providing first evidence to suggest a PDE5A inhibition/PKG-1-p38MAPK-PP2A sequential activation targeting NHE1 phosphorylation in the heart. p38MAPK was already proposed to be able to activate PP2A in rat myocardium (Liu and Hofmann 2003). Studies of the same group and others (Kusuhara, Takahashi et al. 1998, Westermarck, Li et al. 2001, Liu and Hofmann 2004) showed that p38MAPK activation decreases ERK activity, which differs from present findings showing no changes in acidosis triggered phosphorylation of ERK1/2 under p38MAPK inhibition (Fig. 5). However, the increased oxidative stress used by Liu et al. (Liu and Hofmann 2004) to study cardiac apoptosis, is a different setting than sustained acidosis. Sustained acidosis should be longer than 12 h to promote cardiomyocyte apoptosis (Kubasiak, Hernandez et al. 2002). Different from our cardiac model, the activation of p38MAPK by high Arsenite concentration by Westermarck et al. (Westermarck, Li et al. 2001) or Angiotensin II by Kusuhara et al. (Kusuhara, Takahashi et al. 1998) were studied in different cellular types like fibroblasts and smooth muscle cells. Under sustained acidosis, PP2A inhibition cancelled the inhibitory effect of p38MAPK on NHE1 transport activity (Fig. 6). Additionally p38MAPK inhibition reverted Sildenafil reduction on NHE1-Ser703 phosphorylation (Fig. 2). Thus, active PP2A must target Ser703 at NHE1 cytosolic tail recognizing 14-3-3 binding site. In same direction, it was described that PP2A removes phosphate groups of 14-3-3 binding sites on different proteins such as Ser287 of Cdc25 in Xenopus embryonic cells (Margolis, Perry et al. 2006), Ser392 from Kinase Suppressor of Ras complexes in brain tissue, and Ser259 from Raf-1 in NIH3T3 cells (Ory, Zhou et al. 2003). Moreover, the specific capability of PP2A to dephosphorylate NHE1 cytosolic tail was also showed in cultured adult rat ventricular myocytes (Snabaitis, D'Mello et al. 2006). Active PP2A heterotrimeric holoenzyme coexist as a dimeric core of a catalytic subunit (PP2Ac) and a scaffold A subunit that associate a regulatory B subunit.

19 Phosphorylation and methylation of PP2Ac trigger different B subunit selection, shaping the final effector of PP2A activity or its selectivity pathway (Eichhorn, Creyghton et al. 2009). Okadaic acid affect all possible heterotrimeric ensembles, hindering to dissect here which one was responsible for NHE1 dephosphorylation. Although it is generally accepted that PP2A activity can be inhibited by phosphorylation (Janssens and Goris 2001), it can also be directly activated by PKCδ (Zhang, Kanthasamy et al. 2007). In different contexts, different authors described a cardiac PP2A activation by cGMP/PKG (Shen and Pappano 2002, Xu, Lee et al. 2013), a p38MAPK-triggered PP2A traslocation (Liu and Hofmann 2003), and migration to sarcolemmal membrane to dephosphorylate proteins (Zuluaga, Alvarez-Barrientos et al. 2007). Since OKA reversed p38MAPK-mediated actions on NHE1 activity (Fig. 6), we may suggest that under sustained acidosis Sildenafil-triggered p38MAPK activation would lead to PP2A ensemble, activation, and interaction with NHE1. From a clinical perspective, these results validate the potential benefits of using PDE5A inhibitors like Sildenafil against cardiac pathologies characterized by exacerbated NHE1 activity (Karmazyn, Gan et al. 1999, Odunewu-Aderibigbe and Fliegel 2014). Sildenafil has the benefit of being widely clinically used for the treatment of diseases like erectile dysfunction (Jackson, Montorsi et al. 2006), stable angina or pulmonary hypertension (Ghofrani, Wiedemann et al. 2002). It is important to emphasize that Sildenafil was only effective during sustained acidosis, therefore preserving the crucial NHE1 housekeeping role, which constitutes a clear benefit compared to other NHE1 inhibitors. Finally, the proposal of involving p38MAPK-PP2A activation as a potential cardioprotective signaling route increases novel evidence assigning beneficial effects to p38MAPK activation in cardiac tissue (Javadov, Jang et al. 2014).

5. Conclusion

20 Current data provide evidence to propose an alternative modulation of NHE1 activity by PDE5A inhibition. The underlying mechanism involves p38MAPK-mediated PP2A activation leading to NHE1 dephosphorylation as schematized in Fig. 7. Furthermore, the specific reduction of NHE1 phosphorylation/activity by this mechanism does not affect its basal activity, establishing a difference with classical NHE1 inhibitors, which completely block transport activity.

Acknowledgments - Funding This work was supported in part by grants PICT 2012-2396 and PICT 2016-2289 from Agencia Nacional de Promoción Científica y Tecnológica of Argentina to Dr. Néstor Gustavo Pérez, and PIP0750 from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina to Dr. Néstor Gustavo Pérez.

Table 1

Experimental groups

pHi

Control (n=6)

6.75 ± 0.02 27.0 ± 0.7

Sildenafil (n=5)

6.77 ± 0.03 26.8 ± 1.2

Sildenafil +SB (n=4)

6.73 ± 0.01 28.6 ± 0.5

Ars (n=4)

6.71 ± 0.01 29.2 ± 0.3

Ars+SB (n=3)

6.68 ± 0.01 30.3 ± 0.4

SB (n=4)

6.71 ± 0.01 29.7 ± 1.4

bi (mmol/L)

Ars+OKA (1hM) (n=5) 6.70 ± 0.01 29.3 ± 0.3 Anisomycin (n=3)

6.73 ± 0.02 28.4 ± 0.8

21 Table 1. Maximal acidification and bi after 10 min of ammonium prepulse and 10 min of sustained acidosis in each experimental condition. No statistical differences were observed among groups (ANOVA).

Figure Legends Fig. 1. Effect of p38MAPK inhibition on NHE1 activity during sustained acidosis. Cardiac papillary muscles were superfused with bicarbonate free buffer, and subjected to sustained acidosis protocol (section 2.3). After 10 min of acidosis, Sodium re-addition triggered the NHE1-mediated pHi recovery. A, Representative experiments of a muscle treated with SB202190 (SB, 10 μmol/l) or without any drug (Control). B, Representative traces for pHi recovery in the presence of Sildenafil (SIL, 1 μmol/l) or SIL+ SB. C, Average H+ flux mediated by NHE1 (JH+) was calculated for all groups at the same pHi of 6.75 as described in section 2.3. All drugs were added 20 min before ammonium pulse and remained present until the end of the experiment. ANOVA, Student-Newman-Keuls,* P<0.05 vs. control; # P<0.05 vs. SIL. Fig. 2. Effect of p38MAPK inhibition on NHE1 expression and phosphorylation during sustained acidosis. Left ventricle tissue slices were subjected to sustained acidosis, treated with Sildenafil (SIL), SB202190 (SB) (10 μmol/l), SB + SIL (1 μmol/l) or without drugs (Control, C) and frozen immediately after. All drugs were added 20 min before acidification and kept during the entire experiment. A, On top representative immunoblots for cardiac NHE1 expression. Bottom panel shows bar graph of the average protein expression normalized to the amount of GAPDH, as described in section 2.4. B, Top panel shows representative immunoblots of cardiac NHE1 and 14-3-3 binding motif expression on immunoprecipitated samples with specific NHE1 antibody (described in section 2.5).Bottom

22 panel shows a bar graph with average protein expression, normalized to the amount of NHE1. ANOVA, Student-Newman-Keuls, * P< 0.05. Fig. 3. Effect of p38MAPK activation on NHE1 activity. Cardiac papillary muscles were subjected to sustained acidosis protocol (section 2.3). A, Representative experiments of NHE1 dependent pHi recovery in p38MAPK activation condition with Sodium Arsenite (Ars, 5 μmol/l), in presence or absence of SB (10 μmol/l). B, Representative traces for pHi recovery in presence of another p38MAPK activator, Anisomycin (Aniso, 20 μmol/l). Control experiment with no drugs added allow comparison of p38MAPK effect on pHi recovery. C, Graph bar of average H+ flux mediated by NHE1 (JH+) calculated for all groups at the same pHi of 6.75. All drugs were added 20 min before ammonium and kept until the end of the experiment. ANOVA, Student-Newman-Keuls,* P<0.05 vs. control, # P<0.05 vs. Ars. Fig. 4. p38MAPK activation by Sildenafil. Cardiac left ventricle tissue slices were superfused with bicarbonate free buffer and, except control (NAC), subjected to sustained acidosis for 10 min and immediately homogenized. A, Top panel shows representative immunoblots of cardiac p-p38MAPK for control sample (NAC) or samples under sustained acidosis with or without Sildenafil (SIL). Bottom panel shows a bar graph with average protein expression, normalized to the total amount of p38MAPK. B, Top panel shows representative immunoblots of cardiac phospho-MAPKAP-2 (p-MK2) in non-acidotic control (NAC) or samples under sustained acidosis with SB202190 (SB), SIL, SB+SIL or without any drug. Bottom panel shows a bar graph with average protein expression, normalized to the amount of GAPDH (section 2.4). ANOVA, Student-Newman-Keuls, *P<0.05 vs. nonacidotic control. Fig. 5. Effect of Sildenafil on ERK 1/2 and Akt activation. Cardiac left ventricle tissue slices were superfused with bicarbonate free buffer and, except control, were subjected to sustained acidosis for 10 min and immediately homogenized. Drugs were added 20 min

23 before ammonium prepulse. A, Top panel shows representative immunoblots for cardiac pERK expression in samples treated with Sildenafil (SIL), SB20290 (SB), SB+ SIL or without drugs (C) during sustained acidosis and in control sample (NAC). Bottom panel shows a bar graph with average protein expression, quantified by densitometry and normalized to the amount of ERK. B, top panel shows representative immunoblots of cardiac p-Akt for control sample (NAC) or samples under sustained acidosis with (SIL) or without (C) Sildenafil. Bottom panel shows a bar graph with average protein expression, normalized to the amount of GAPDH. C, same as B but showing total Akt expression under the same experimental conditions. ANOVA, Student-Newman-Keuls,* P< 0.05 vs. non-acidotic sample. Fig. 6. Role of PP2A in NHE1 activity inhibition by p38MAPK during sustained acidosis. In cardiac papillary muscles, sustained to 10 min of acidosis, NHE1-dependent pHi recovery was assayed by sodium re-addition (section 2.3). Left panel shows representative traces of NHE1-dependent pHi recovery of muscle treated with Sodium Arsenite (Ars, 5 μmol/l) + Okadaic Acid (OKA, 1 nmol/l) or without drugs (Control). Right panel shows a bar graph with average H+ flux (JH+) calculated at the same pHi of 6.75 for all groups, comparative bar of Arsenite effect is shown to clarify Okadaic actions. All drugs were added 20 min before ammonium pulse and remained present until the end of the experiment. ANOVA, Student-Newman-Keuls * P<0.05 vs. control; # P<0.05 vs. Ars. Fig. 7. Proposed mechanism of SIL actions on NHE1 activity/phosphorylation. NHE1 activation after sustained acidosis in presence of SIL could result from the counterbalance between a stimulatory process induced by MEK-ERK-p90RSK activation and the inhibitory effect induced by PDE5A inhibition that involves p38MAPK activation and PP2A activation and migration to membrane.

24

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