Oxytocin Downregulates the Cav1.2 L-Type Ca Channel Via Gi/Camp/PKA/CREB Signaling Pathway in Cardiomyocytes

membranes

Article 2+ Oxytocin Downregulates the CaV1.2 L-Type Ca Channel via Gi/cAMP/PKA/CREB Signaling Pathway in Cardiomyocytes

Masaki Morishima 1,2, Shintaro Tahara 1, Yan Wang 1 and Katsushige Ono 1,*

1 Department of Pathophysiology, Oita University School of Medicine, Oita 879-5593, Japan; [email protected] (M.M.); [email protected] (S.T.); [email protected] (Y.W.) 2 Department of Food Science and Nutrition, Kindai University Faculty of Agriculture, Nara 631-8505, Japan * Correspondence: [email protected]; Tel.: +81-97-586-5650

Abstract: Oxytocin (OT) and its receptor (OTR) are expressed in the heart and are involved in the physiological cardiovascular functional system. Although it is known that OT/OTR signaling is cardioprotective by reducing the inflammatory response and improving cardiovascular function, the role of OT in the cardiac electrical excitation modulation has not been clarified. This study investigates the molecular mechanism of the action of OT on cardiomyocyte membrane excitation focusing on the L-type Ca2+ channel. Our methodology uses molecular biological methods and a patch-clamp technique on rat cardiomyocytes with OT, combined with several signal inhibitors and/or activators. Our results show that long-term treatment of OT significantly decreases the 2+ expression of Cav1.2 mRNA, and reduces the L-type Ca channel current (ICa.L) in cardiomyocytes. OT downregulates the phosphorylated component of a transcription factor adenosine-30,50-cyclic

monophosphate (cAMP) response element binding protein (CREB), whose action is blocked by OTR antagonist and pertussis toxin, a speciﬁc inhibitor of the inhibitory GTP-binding regulators

Citation: Morishima, M.; Tahara, S.; of adenylate cyclase, Gi. On the other hand, the upregulation of Cav1.2 mRNA expression by Wang, Y.; Ono, K. Oxytocin isoproterenol is halted by OT. Furthermore, inhibition of phospholipase C (PLC) was without effect

Downregulates the CaV1.2 L-Type on the OT action to downregulate Cav1.2 mRNA—which suggests a signal pathway of Gi/protein Ca2+ Channel via Gi/cAMP/PKA kinase A (PKA)/CREB mediated by OT/OTR. These ﬁndings indicate novel signaling pathways of /CREB Signaling Pathway in OT contributing to a downregulation of the Cav1.2-L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes. Membranes 2021, 11, 234. https://doi.org/10.3390/ Keywords: oxytocin; L-type Ca2+ channel; gene transcription; CREB membranes11040234

Academic Editor: Terry Hébert 1. Introduction Received: 25 February 2021 Oxytocin (OT) is a neurohypophyseal hormone secreted from both central and pe- Accepted: 24 March 2021 Published: 25 March 2021 ripheral tissues, resulting in reproduction, brain neuromodulation, and central regulation of blood pressure [1]. Although OT is commonly known as a female hormone, besides

Publisher’s Note: MDPI stays neutral reproductive properties, there is growing evidence that OT plays a role in regulating car- with regard to jurisdictional claims in diovascular function. OT acts upon the OT receptor (OTR), which belongs to the family published maps and institutional affil- of G-protein-coupled receptor (GPCRs). These receptors are abundantly expressed in the iations. heart, and local OT action in the heart is important for blood pressure regulation [2], as well as cardioprotection associated with secretion of atrial natriuretic peptide (ANP) and production of nitric oxide (NO) [3,4]. Similarly, OT is associated with a direct, centrally mediated regulation of autonomic outflow to the heart leading to negative inotropic and chronotropic effects [5]. Under the pathophysiological condition, OT has anti-inflammatory Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and anti-fibrotic effects that could explain the observed myocardial protection in in vivo This article is an open access article studies [6]. Recent evidence indicates that OT stimulates the proliferation and differen- distributed under the terms and tiation of embryonic stem cells to cardiomyocytes [7]. Although many of these studies conditions of the Creative Commons demonstrated that OT reduced the heart rate and the force of atrial contractions [1], the po- Attribution (CC BY) license (https:// tential role of oxytocin in cardiac electrical excitation has not been intensively investigated. creativecommons.org/licenses/by/ Changes in ion channel expression modulation are hallmarks of heart failure-related 4.0/). electrical remodeling. Heart failure is defined as the end-stage of various cardiac diseases,

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including ischemic or dilated cardiomyopathy [8]. Chronic stimulation of β-adrenergic receptors by catecholamines leading to activation of cAMP-dependent signal transduction pathway plays a central role in the pathogenesis in heart failure [9]. Accordingly, it is postulated that transcription factors that are related to catecholamine-dependent cardiac compli- cations, including CREB, play an important mechanism for catecholamine-dependent gene controls and altered-gene regulation in heart failure. The voltage-gated L-type Ca2+ channel is one of the ion channels responsible for heart failure-associated electrical remodeling in cardiomyocytes. Previous studies have demonstrated that the positive inotropic effect 2+ 2+ of noradrenaline to increase Ca inﬂux through the L-type Ca channel current (ICa.L) was accelerated under the prolonged intensive stimulation of the β-adrenergic receptor in the heart [10]. Although it is well known that cardiac L-type Ca2+ channels function is modulated by a cAMP-dependent protein kinase, little is known about the molecular mechanisms regulating L-type Ca2+ channel gene expression in cardiomyocytes. Although we have previously demonstrated that OT downregulated the Cav1.2-L-type Ca2+ channel expression accompanied by a reduction of ICa.L in cardiomyocytes [11], the signal pathways underlying cardiac OT actions are poorly understood. In the present study, we investigated the intracellular molecular mechanism for L- type Ca2+ channel expression regulation by OT. We revealed a novel function of OT/OTR signals which act in concert to regulate the transcription of the Cav1.2 channel via the reduction of distinct downstream effectors, Gi/PKA/CREB pathway. These pathways in cardiomyocytes will likely be related to the rescue of the heart in pathological conditions, such as heart failure.

2. Results 2.1. OT and OTR in Cardiomyocytes We ﬁrst examined whether OT and OTR could be detected in the adult rat heart and neonatal rat ventricular cardiomyocytes by using quantitative real-time PCR. Figure1 clearly demonstrates that OT and OTR are detectably expressed in adult and neonatal rat hearts, in accordance with previous studies [1]. Of note, there was an approximate 3.5-fold difference of OT mRNA expression between the atrium and the ventricles in the adult heart (Figure1A,B). Interestingly, the levels of OTR mRNA expression were signiﬁcantly higher in the atrium than in the ventricle approximately by 4.5 times in the adult heart, which is nearly identical to the difference of OT mRNA between the atrium and the ventricle (Figure1A,C). The expression levels of OT and OTR in neonatal ventricular cardiomyocytes were nearly identical to those in adult atrial tissue. These data indicate that the expression Membranes 2021, 11, x FOR PEER REVIEWof OT and OTR is appreciably high in neonatal cardiomyocytes. It is reasonable to evaluate 3 of 13

the action of OT by use of neonatal cardiomyocytes, which also strengthens the rationale to utilize neonatal rat ventricular cardiomyocytes to access the effect of OT for this purpose.

Figure 1. Cont.

Figure 1. Detection of oxytocin (OT) and OT receptor (OTR) mRNA in adult and neonatal rat hearts. (A) The photograph presents OT and OTR RT-PCR products after electrophoresis on 2% agarose in ethidium bromide from the following targets: Neonatal ventricle (left), adult atrium (middle), and adult ventricle (right). (B,C) Relative quantification of OT (B) and OTR (C) mRNA in rat neonatal ventricle and adult heart, atrium, and ventricle, by real-time PCR. Changes in expression of OT and OTR normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were evaluated. Data are expressed as mean  S.E. (n = 8). * p < 0.05 between groups indicated.

2.2. Long-Term Effects of OT on ICa.L We decided to evaluate the long-term effects of OT on the L-type Ca2+ channel. A long-term treatment (24 h) of neonatal rat ventricular cardiomyocytes with OT (10−7 M) significantly decreased expression of Cav1.2 mRNA by 41%, which was blocked by an OTR antagonist (OTRA). These results clearly indicate that OT exerted its effect on the L- type Ca2+ channel via the OTR signaling pathway (Figure 2A). Importantly, L-type Ca2+ channel current (ICa.L) was also reduced by OT. The maximum inward ICa.L was decreased by 20.8% (Figure 2B), and the maximum chord conductance (GCa.L) was reduced by 18.9% by long-term treatment with OT (Figure 2C). These electrophysiological results verified the reduction of Cav1.2 by OT (Figure 2A) and our previous observations [11].

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Figure 1. Detection of oxytocin (OT) and OT receptor (OTR) mRNA in adult and neonatal rat hearts. Figure 1. Detection of oxytocin (OT) and OT receptor (OTR) mRNA in adult and neonatal rat (A) The photograph presents OT and OTR RT-PCR products after electrophoresis on 2% agarose in ethidiumhearts. ( bromideA) The fromphotograph the following presents targets: OT Neonatal and OTR ventricle RT-PCR (left), products adult atrium after (middle), electrophoresis and on 2% adultagarose ventricle in ethidium (right). (B bromide,C) Relative from quantification the following of OT (targets:B) and OTR Neonatal (C) mRNA ventricle in rat neonatal (left), adult atrium ventricle(middle), and and adult adult heart, ventricle atrium, and (right). ventricle, (B, byC)real-time Relative PCR. quantification Changes in expression of OT (B of) OTand and OTR (C) mRNA in OTRrat neonatal normalized ventricle to glyceraldehydes-3-phosphate and adult heart, atrium dehydrogenase, and ventricle, (GAPDH) by real were-time evaluated. PCR. Data Changes in expres- aresion expressed of OT and as mean OTR± normalizedS.E. (n = 8). * p to< 0.05glyceraldehydes between groups- indicated.3-phosphate dehydrogenase (GAPDH) were evaluated. Data are expressed as mean  S.E. (n = 8). * p < 0.05 between groups indicated. 2.2. Long-Term Effects of OT on ICa.L We decided to evaluate the long-term effects of OT on the L-type Ca2+ channel. A long-term2.2. Long treatment-Term Effects (24 h) of of OT neonatal on ICa.L rat ventricular cardiomyocytes with OT (10−7 M) significantlyWe decided decreased to expressionevaluate ofthe Cav1.2 long mRNA-term effects by 41%, of which OT wason blockedthe L-type by an Ca2+ channel. A OTRlong antagonist-term treatment (OTRA). (24 These h) resultsof neonatal clearly indicaterat ventricular that OT exerted cardiomyocytes its effect on thewith OT (10−7 M) L-type Ca2+ channel via the OTR signaling pathway (Figure2A). Importantly, L-type Ca 2+ significantly decreased expression of Cav1.2 mRNA by 41%, which was blocked by an channel current (ICa.L) was also reduced by OT. The maximum inward ICa.L was decreased byOTR 20.8% antagonist (Figure2B), (OTRA). and the maximum These results chord conductance clearly indicate (G Ca.L )that was reducedOT exerted by 18.9% its effect on the L- bytype long-term Ca2+ channel treatment via with the OT O (FigureTR signaling2C). These electrophysiologicalpathway (Figure results 2A). verifiedImportantly, the L-type Ca2+ reductionchannel ofcurrent Cav1.2 (I byCa.L OT) was (Figure also2A) reduced and our previous by OT. observations The maximum [11]. inward ICa.L was decreased by 20.8% (Figure 2B), and the maximum chord conductance (GCa.L) was reduced by 18.9% by long-term treatment with OT (Figure 2C). These electrophysiological results verified the reduction of Cav1.2 by OT (Figure 2A) and our previous observations [11].

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Figure 2. Long-term effects of OT on Cav1.2 expression and ICa.L.(A) Expression ratio of Cav1.2 mRNA after treatment with Figure 2. OTLong (100-term nM), OTReffects antagonist of OT OTRAon Cav1.2 (1 µM), expression and OT (100 nM)and with ICa.L OTRA. (A) (1ExpressionµM) for 24 h.ratio (B) Representative of Cav1.2 mRNA ICa.L traces after in treatment with OT (the100 vehicle nM), andOTR OT antagonist (inset) applied OTRA for 24 (1 h, andμM), their and group OT data (100 of nM) current with (I)-voltage OTRA (V) (1 relationship.μM) for 24 Current h. (B) tracesRepresentative were ICa.L traces in theobtained vehicle from and a holding OT (inset) potential applied of −40 for mV 24 to h, test and potentials their group up to 50 data mV withof current 10 mV increments.(I)-voltage (C(V)) Group relationship. data of Current traces werechord obtained conductance from (Ga Ca.Lholding)-voltage potential (V) relationship of −40 mV for vehicle to test and potentials OT constructed up to from50 mV I-V with curves 10 in mV panel increments. (B)* p < 0.05 (C) Group data of chordbetween conductance groups at the (G sameCa.L)- potentials.voltage (V) The relationship numbers of cells for are vehicle indicated and in OT parentheses. constructed from I-V curves in panel (B). * p < 0.05 between groups at the same potentials. The numbers of cells are indicated in parentheses. 2.3. OT Downregulates Cav1.2 mRNA and ICa.L via the Gi Protein

To investigate the signal pathway for the downregulation of Cav1.2 and ICa.L by OT, 2.3. OTwe examinedDownregulates the possible Cav1.2 roles mRNA of OTR and focusing ICa.L via on Gthe proteins, Gi Protein because OTR differentially signals via Gq and Gi proteins [12]. Interestingly, OT was unable to reduce Cav1.2 mRNA To investigate the signal pathway for the downregulation of Cav1.2 and ICa.L by OT, when cardiomyocytes were pretreated with a specific inhibitor of the Gi protein, pertussis we examinedtoxin (100 ng/mL),the possible for 24 role h. However,s of OTR inhibition focusing of on Gq-PLC-induced G proteins, because phosphoinositide OTR differentially signalhydrolysiss via Gq withand aGi compound proteins U73122 [12]. Interestingly, (2 µM) could not OT block was the unable effectsof to OT. reduce Cav1.2 mRNA when cardiomyocytGiven our results,es were it is pretreated suggested that with OT/OTR a specific activates inhibitor the Gi of protein the Gi to interfereprotein, pertussis toxinwith (100 cellular ng/mL) cAMP, for productions, 24 h. However, which is similarinhibition to the of action Gq of-PLC acetylcholine-induced binding phosphoinositide to hydrolysismuscarinic with receptors a compound to inhibit U73122 activation (2 of μ adenylateM) could cyclase. not block To confirm the effects this hypothesis, of OT. we then applied isoproterenol, forskolin, or acetylcholine to cardiomyocytes for 24 h to observeGiven theour changes results, of Cav1.2it is suggested mRNA. Importantly, that OT isoproterenol/OTR activates and forskolin the Gi significantly protein to interfere with increasedcellular cAMP the expression productions, of Cav1.2 which mRNA, is and similar acetylcholine to the action significantly of acetylcholine decreased the binding to muscarinicexpression receptor of Cav1.2s to mRNA inhibit (Figure activation3). Of note, of OTadenylate significantly cyclase. attenuated To confirm the increases this of hypothesis, we thenCav1.2 applied mRNA causedisoproterenol, by isoproterenol forskolin, or forskolin, or acetylcholine suggesting the to importance cardiomyocytes of [cAMP] Ifor 24 h to modulation by OT to regulate Cav1.2 transcription. Electrophysiological evaluation of I observe the changes of Cav1.2 mRNA. Importantly, isoproterenol and forskolinCa.L signifi- supports RT-PCR data demonstrating that OT and acetylcholine downregulated ICa.L, and cantlyisoproterenol increased upregulated the expression ICa.L in cardiomyocytes of Cav1.2 mRNA, when applied and acetylcholine for 24 h (Figure 3 significantlyC,D). decreased the expression of Cav1.2 mRNA (Figure 3). Of note, OT significantly attenuated the increases2.4. Effects of OT Cav1.2 on Protein mRNA Kinase caused Activity by isoproterenol or forskolin, suggesting the im- Multiple G protein-coupled receptors in the heart act through cAMP/PKA pathways portance of [cAMP]I modulation by OT to regulate Cav1.2 transcription. Electrophysio- to regulate a number of cellular proteins, including the L-type Ca2+ channel. To investigate logicalwhether evaluation downregulation of ICa.L supports of Cav1.2 RT expression-PCR data by OT demonstrating was dependent that on PKA OT signaling, and acetylcholine downweregulated measured I cytosolicCa.L, and PKA isoproterenol enzyme activity upregulated in OT-treated ICa.L cardiomyocytes in cardiomyocytes in conjunction when applied for 24with h (Fig severalure compounds3C,D). that regulate β-adrenergic and cholinergic receptor functions (Figure4). Long-term treatment with OT (10 −7 M) significantly decreased PKA activity (−47 ± 3%) in cardiomyocytes in comparison with vehicle treatment in experiment condition A (Figure4A ). Consistent with the result in Figure2A, OTRA completely blocked the effect of OT in downregulating PKA activity. Similarly, when cardiomyocytes were pretreated with pertussis toxin, OT was unable to reduce PKA activity. These observations tightly confirmed the action of the Gi protein as a downstream signal of OT/OTR pathway. Because assay kit enzyme activities vary upon product lot numbers, results using different

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PKA activity assay kits were evaluated independently as experiment A (panel A) and B (panel B). Likewise, long-term treatment with OT (10−7 M) significantly decreased PKA activity in cardiomyocytes (−64 ± 8%) in comparison with vehicle treatment in experiment condition B (Figure4B). In this condition, we applied isoproterenol, forskolin, and a non- selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in the culture medium to confirm their action to increase PKA activity in cardiomyocytes as positive controls. Importantly, OT significantly attenuated the PKA activity caused by isoproterenol and forskolin. In addition, we confirmed the action of acetylcholine to reduce PKA activity Membranes 2021as, 11 a, x negative/positive FOR PEER REVIEW control. In the present study, we verified that OT reduced basal PKA 5 of 13

activity likely through the OTR/Gi protein pathway.

Figure 3. Changes of Cav1.2 mRNA and ICa.L by OT. (A) Regulation of Cav1.2 expression (ratio) by OT (100 nM) was Figure 3. Changes of Cav1.2 mRNA and ICa.L by OT. (A) Regulation of Cav1.2 expression (ratio) by assessed with/without an inhibitor of the Gi subunit of heterotrimeric G proteins pertussis toxin (PTX) of 100 ng/mL or OT (100 nM) was assessed with/without an inhibitor of the Gi subunit of heterotrimeric G proteins an inhibitor of the phospholipase C pathway (U73122) of 2 μM. (B) Regulation of Cav1.2 expression (ratio) by OT was pertussis toxin (PTX) of 100 ng/mL or an inhibitor of the phospholipase C pathway (U73122) of 2 µM. assessed with/without 100 nM isoproterenol (ISO), 10 μM forskolin (FSK) or 1 μM acetylcholine (ACh). (C) Typical ICa.L traces recorded(B) Regulation at a test potential of Cav1.2 of expression 0 mV from (ratio)a holding by OTpotential was assessedof −50 mV with/without in cardiomyocytes 100 nM cultured isoproterenol with the vehicle, OT, isoproterenol(ISO), 10 µM (ISO) forskolin, or acetylcholine (FSK) or 1 µ (MACh) acetylcholine at the same (ACh).concentrations (C) Typical in panel ICa.L (Btraces) for 24 recorded h. (D) Group at a testdata for ICa.L recordedpotential at 0 mV of 0in mV cardiomyocytes from a holding treated potential with OT, of − isoproterenol50 mV in cardiomyocytes (ISO), or acetylcholine cultured (A withCh). Data the vehicle, are expressed as mean  SE. * p < 0.05 vs. vehicle, # p < 0.05 vs. ISO, † p < 0.05 vs. FSK. The numbers of cells are indicated in OT, isoproterenol (ISO), or acetylcholine (ACh) at the same concentrations in panel (B) for 24 h. parentheses. (D) Group data for ICa.L recorded at 0 mV in cardiomyocytes treated with OT, isoproterenol (ISO),

or acetylcholine (ACh). Data are expressed as mean ± SE. * p < 0.05 vs. vehicle, # p < 0.05 vs. ISO, † p < 0.05 vs. FSK.2.4. The Effects numbers of OT of on cells Protein are indicated Kinase Activity in parentheses. 2.5. Effects of OT on CREBMultiple Phosphorylation G protein-coupled receptors in the heart act through cAMP/PKA pathways 2+ Because theto cAMP/PKA regulate a number pathway of cellular is now proteins, clariﬁed including to regulate the L expression-type Ca channel. of Cav1.2 To investigate whether downregulation of Cav1.2 expression by OT was dependent on PKA signaling, gene, and because PKA phosphorylates and consequently activates a transcription fac- we measured cytosolic PKA enzyme activity in OT-treated cardiomyocytes in conjunction tor cyclic AMP response element binding protein (CREB), we then examined whether with several compounds that regulate β-adrenergic and cholinergic receptor functions downregulation of Ca 1.2 mRNA expression by OT was associated with lower CREB (Figure v4). Long-term treatment with OT (10−7 M) significantly decreased PKA activity phosphorylation level in the nucleus. Western blot analysis revealed that phosphorylated (−47  3%) in cardiomyocytes in comparison with vehicle treatment in experiment condi- CREB in the nucleustion Awas (Figure signiﬁcantly 4A). Consistent decreased with the by result OT when in Figure assessed 2A, OTRA at 24 completely h after OT blocked the treatment, and theeffect reduction of OT in was down evenregulating larger whenPKA activity. the treatment Similarly, was when prolonged cardiomyocytes for 48 h were pre- (Figure5A ). Similarly,treated thewith result pertussis of PKA toxin, downregulation OT was unable by to OTreduce through PKA OTRactivity. in FigureThese 4observations, a decrease of phosphorylatedtightly confirm CREBed the action by OT of was the completelyGi protein as blocked a downs bytream OTRA signal (Figure of OT/OTR5B ), pathway. Because assay kit enzyme activities vary upon product lot numbers, results using different PKA activity assay kits were evaluated independently as experiment A (panel A) and B (panel B). Likewise, long-term treatment with OT (10−7 M) significantly decreased PKA activity in cardiomyocytes (−64  8%) in comparison with vehicle treatment in experiment condition B (Figure 4B). In this condition, we applied isoproterenol, forskolin, and a non- selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in the culture medium to confirm their action to increase PKA activity in cardiomyocytes as positive

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suggesting again that OT exerted its effects through OT/OTR pathways. Moreover, a decrease of phosphorylated CREB by OT was completely blocked by a specific inhibitor of Gi protein pertussis toxin (Figure5C ), suggesting that inactivation of PKA activity by OT is sufficient to reduce phosphorylation levels of CREB in the nucleus in this study. The Membranes 2021, 11, x FOR PEER REVIEW 6 of 13 role of the G-Protein-coupled receptor signaling pathway on the CREB phosphorylation was finally confirmed by three ligands, OT, isoproterenol, and acetylcholine (Figure5D). Given all together, these results indicate that OT can modulate myocardial Ca2+ homeostasis via regulatingcontrols. sarcoplasmic Importantly, membrane OT significantly expression attenuated levels the of thePKA voltage-gated activity caused Ca by2+ isoproterenol and forskolin. In addition, we confirmed the action of acetylcholine to reduce PKA channel, which isactivity qualitatively as a negative/positive comparable withcontrol. parasympathetic/acetylcholine In the present study, we verified that signals OT reduced in cardiomyocytes.basal PKA activity likely through the OTR/Gi protein pathway.

FigureFigure 4. Changes 4. Changes of PKA ofactivity PKA by activity OT assess byed OT with/without assessed with/withoutcellular OTR/Gi/cAMP cellular regulators. OTR/Gi/cAMP (A,B) PKA regula-activity in neonatal cardiomyocytes were assessed after treatment with/without OT (100 nM), OTRA (1 μM), pertussis toxin (PTX, tors. (A,B) PKA activity in neonatal cardiomyocytes were assessed after treatment with/without 100 ng/mL), isoproterenol (ISO, 100 nM), forskolin (10 μM), a non-selective inhibitor of phosphodiesterase isobutylmethylxanthine,OT (100 1- nM),methyl OTRA-3-isobutylxanthine (1 µM), pertussis (IBMX, 100 toxin μM) or (PTX, Acetylcholine 100 ng/mL), (ACh, 1isoproterenol μM) for 24 h. Data (ISO, are expressed 100 nM), as meanforskolin  SE (n = (106). * µpM), < 0.05 a non-selectivevs. vehicle, # p < inhibitor 0.05 vs. ISO, of phosphodiesterase† p < 0.05 vs. FSK. isobutylmethylxanthine, 1-methyl- Membranes 2021, 11, x FOR PEER REVIEW 7 of 13 3-isobutylxanthine (IBMX, 100 µM) or Acetylcholine (ACh, 1 µM) for 24 h. Data are expressed 2.5. Effects of OT on CREB Phosphorylation as mean ± SE (n = 6). * p < 0.05 vs. vehicle, # p < 0.05 vs. ISO, † p < 0.05 vs. FSK. Because the cAMP/PKA pathway is now clarified to regulate expression of Cav1.2 gene, and because PKA phosphorylates and consequently activates a transcription factor cyclic AMP response element binding protein (CREB), we then examined whether downregulation of Cav1.2 mRNA expression by OT was associated with lower CREB phosphorylation level in the nucleus. Western blot analysis revealed that phosphorylated CREB in the nucleus was significantly decreased by OT when assessed at 24 h after OT treatment, and the reduction was even larger when the treatment was prolonged for 48 h (Figure 5A). Similarly, the result of PKA downregulation by OT through OTR in Figure 4, a decrease of phosphorylated CREB by OT was completely blocked by OTRA (Figure 5B), suggesting again that OT exerted its effects through OT/OTR pathways. Moreover, a decrease of phosphorylated CREB by OT was completely blocked by a specific inhibitor of Gi protein pertussis toxin (Figure 5C), suggesting that inactivation of PKA activity by OT is sufficient to reduce phosphorylation levels of CREB in the nucleus in this study. The role of the G-Protein-coupled receptor signaling pathway on the CREB phosphorylation was finally confirmed by three ligands, OT, isoproterenol, and acetylcholine (Figure 5D). Given all together, these results indicate that OT can modulate myocardial Ca2+ homeostasis via regulating sarcoplasmic membrane expression levels of the voltage-gated Ca2+ channel, which is qualitatively comparable with parasympathetic/acetylcholine signals in cardiomyocytes.

Figure 5. Cont.

Figure 5. Changes of phosphorylated CREB protein level in the nucleus of cardiomyocytes. Densitometric analysis of phosphorylated-CREB (p-CREB) and CREB protein expression normalized to β-actin. The amount of p-CREB per total CREB proteins (phosphorylated CREB/CREB) was assessed and compared among the groups. (A) Time-dependent suppression of p-CREB by OT (100 nM). Upper panel, an immunoblot demonstrating the effect of OT (100 nM, 24 h or 48 h) on total-CREB, p-CREB, and β-actin expression in neonatal rat cardiomyocytes. (B,C) Inhibitory actions of OTR antagonist (OTRA, 1 μM) and/or pertussis toxin (PTX, 100 ng/mL) on p-CREB. (D) Changes of p-CREB by OT (100 nM), isoproterenol (ISO, 100 nM), and acetylcholine (ACh, 10 μM). Data are expressed as mean  S.E (n = 6). * p < 0.05, vs. non-treated myocytes (vehicle).

3. Discussion Our study demonstrates that OT downregulate the Cav1.2 channel leading to a reduction of ICa.L in cardiomyocytes as a long-term effect, which is accompanied by a decrease of p-CREB in the nucleus. The reduction of Cav1.2 by OT was prevented by pertussis toxin, but not by a Gq/11 inhibitor U73112, suggesting that the signal is mediated by Gi in suppressing PKA-dependent CREB phosphorylation. Moreover, isoproterenol- or forskolin-treatment demonstrated an opposite result, whereas acetylcholine-treatment demonstrated a similar effect to that of OT on Cav1.2 expression, which indicates that OT/OTR signal pathway on the myocardial Ca2+ modulation is similar to those of parasympathetic stimulation. This study provides new information shedding light on OT as a cardiovascular-regulating hormone by modulating transcription of the Cav1.2 channel.

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Figure 5. Changes of phosphorylated CREB protein level in the nucleus of cardiomyocytes. Densit- ometric analysis of phosphorylated-CREB (p-CREB) and CREB protein expression normalized to Figure 5. Changes of phosphorylated CREB protein level in the nucleus of cardiomyocytes. Densitometric analysis of β-actin. The amount of p-CREB per total CREB proteins (phosphorylated CREB/CREB) was assessed phosphorylated-CREB (p-CREB) and CREB protein expression normalized to β-actin. The amount of p-CREB per total CREB proteinsand (phosphorylated compared among CREB/CREB) the groups. was (A )assessed Time-dependent and compared suppression among the of p-CREBgroups. ( byA) OTTime (100-dependent nM). suppression of Upperp-CREB panel, by OT an (100 immunoblot nM). Upper demonstrating panel, an immunoblot the effect demonstrating of OT (100 nM, the 24 effect h or 48 of h)OT on (100 total-CREB, nM, 24 h or 48 h) on total-CREB,p-CREB, p-CREB and, andβ-actin β-actin expression expression in in neonatal neonatal rat rat cardiomyocytes.cardiomyocytes. ( (BB,,CC)) Inhibitory Inhibitory actions actions of ofOTR OTR antagonist (OTRA, 1 μM)antagonist and/or pertussis (OTRA, toxin 1 µM) (PTX, and/or 100 ng pertussis/mL) on toxinp-CREB. (PTX, (D) 100 Changes ng/mL) of p on-CREB p-CREB. by OT (D (100) Changes nM), isoproterenol of (ISO, 100 nM)p-CREB, and acetylcholine by OT (100 ( nM),ACh, isoproterenol10 μM). Data are (ISO, expressed 100 nM), as mean and acetylcholine  S.E (n = 6). * (ACh,p < 0.05, 10 vs.µ M).non- Datatreated are myocytes (vehicle). expressed as mean ± S.E (n = 6). * p < 0.05, vs. non-treated myocytes (vehicle).

3. Discussion3. Discussion Our study demonstratesOur study thatdemonstrate OT downregulates that OT thedown Cav1.2regulate channel the Cav1.2 leading channel to a reduc- leading to a re- tion of ICa.L induction cardiomyocytes of ICa.L in as cardiomyocytes a long-term effect, as a whichlong-term is accompanied effect, which by is aaccompanied decrease by a de- of p-CREB increase the nucleus. of p-CREB The in reduction the nucleus. of Cav1.2The reduction by OT of was Cav1.2 prevented by OT was by pertussis prevented by pertus- toxin, but notsis by toxin a Gq/11, but not inhibitor by a Gq/11 U73112, inhibitor suggesting U73112, that suggest the signaling that is mediatedthe signal byis mediated by Gi in suppressingGi in PKA-dependent suppressing PKA CREB-dependent phosphorylation. CREB phosphorylation. Moreover, isoproterenol- Moreover, isoproterenol or - or forskolin-treatmentforskolin demonstrated-treatment demonstrated an opposite result,an opposite whereas result, acetylcholine-treatment whereas acetylcholine-treatment demonstrateddemonstrated a similar effect a similar to that effect of OT to on that Cav1.2 of OT expression, on Cav1.2 which expression indicates, which that indicates that OT/OTR signalOT/OTR pathway signa on thel pathway myocardial on the Ca 2+myocardialmodulation Ca is2+ similarmodulation to those is similar of parasym- to those of para- pathetic stimulation.sympathetic This stimulation. study provides This study new information provides new shedding information light shedding on OT as light a on OT as a cardiovascular-regulatingcardiovascular hormone-regulating by modulating hormone by transcription modulating transcription of the Cav1.2 of channel. the Cav1.2 channel.

3.1. Myocardial Ca2+ Homeostasis Modulated by OT Oxytocin, a female reproductive hormone, has well-established functions in parturi-

tion, lactation, and mental health. The present study explored the actions of OT beyond its traditionally recognize central (e.g., social behaviors) and peripheral (e.g., uterine contraction) actions. We have previously reported that OT is a cardiovascular modulator that acts primarily to downregulate Cav1.2-L-type Ca2+ channel, but not Cav1.3, Cav3.1, or Cav3.2 channels in cardiomyocytes [11]. In addition, OT decreased ICa.L by the long-term effect (24–72 h), but not by the short-term effect (5 min) [11] Although these long-term effects of OT on the Cav1.2 channel was obvious, the underlying mechanism was not fully understood. Here we extend our previous observations and show that OT/OTR signals stimulate the Gi protein to interfere with PKA activity to reduce CERB phosphorylation, causing a disruption of its transcription activity for Cav1.2 expression. This action of OT is well comparable to parasympathetic (vagal) neuronal agonist acetylcholine, and adver- sarial to sympathetic β-adrenergic agonist isoproterenol (Figure6). According to recent studies, OT and OTR are widely recognized in the heart and vessels [13] in consistent with our study. The OTR can be a couple to distinct G proteins leading to different functional Membranes 2021, 11, 234 8 of 13

effects [14]. OTR coupling to the Gq/11 protein activates the PLC pathway, which leads to diacylglycerol (DAG)-protein kinase C (PKC) activation and inositol 1,4,5-trisphosphate 2+ (IP3)-dependent cellular [Ca ]I elevation [14]. These signal pathways have been implicated in processes thought to underlie smooth muscle contraction and NO production. These signal pathways leading to functions that result in a reduction of heart rate and contraction, and vasodilation are reportedly regulated by actions of ANP-guanosine-30,50-cyclic monophosphate (cGMP), and NO-cGMP systems [15]. Therefore, molecular signaling of OT in the heart is estimated to be largely dependent on the Gq/11 protein and its downstream targets. The other signaling of OT in the heart which may account for the cardiovascular effect is the pertussis toxin-sensitive GTP-binding protein, Gi [14]. Our results in this study ﬁrmly support the action of the Gi protein to modify the expression of the Cav1.2 channel mediated by a transcription factor CREB. The effect of OT on the phosphorylation of CREB in the brain has widely been recognized [14]. OT regulates stress-induced corticotropin releasing factor (CRF) gene transcription via a cAMP/PKA-dependent mechanism through CREB-regulated transcription coactivator 3 [16]. In the heart, ICa.L reduction was observed in CREB knockout cardiomyocytes along with a reduction of the Cav1.2 encoding mRNA CACNA1C [17]. The obvious question of importance asks where and how CREB inter- acts with the CACNA1C gene. Target genes of CREB reportedly include consensus sites for CREB-binding on their promotor region, whose molecular structure contains eight- base-pair palindrome (TGACGTCA) or single (CGTCA) motif CREs [18]; there are four CREB-binding sites in the putative promotor region of CACNA1C within −2000 bp from transcription starting site, which is required for activation of transcriptional activity [19,20]. CREB is activated by phosphorylation on Ser−133. Consistent with these observations, CREB could be a candidate factor for CACNA1C transcription downregulation by OT. Membranes 2021, 11, x FOR PEER REVIEW 9 of 13 Taken together, it is postulated that OT plays a crucial role in regulating CREB expression in the heart, which is important for the physiological function of the myocardium, including intracellular Ca2+ homeostasis.

Figure 6. Schematic diagram of hypothetical OT signaling pathways targeting the cytoplasmic membrane and the nucleus in cardiomyocytes. OT-mediated downregulation of the L-type Ca2+ channel Cav1.2 expression through a reduction of Figure 6.CREB Schematic phosphorylation. diagram of hypothetical OT signaling pathways targeting the cytoplasmic membrane and the nucleus in cardiomyocytes. OT-mediated downregulation of the L-type Ca2+ channel Cav1.2 expression through a reduction of CREB phosphorylation.

3.2. Action of OT on the Cardiovascular System though Gi-Dependent Pathway OT is a potent uterine stimulant that is usually used for the induction of labor and control of postpartum hemorrhage. Acute application OT (intravenous injection) acts on the smooth muscle of the uterus to stimulate contractions through DAG-PKC activation and IP3-dependent cellular [Ca2+]i elevation [14]. However, OT does not usually possess cardiovascular effects, such as elevation of blood pressure. Meanwhile, plasma OT concentration in nonpregnant women was <1 μU/mL. The mean plasma OT concentration between weeks 4 and 40 of pregnancy was 66–165 μU/mL [21]. Nevertheless, maternal blood pressure during pregnancy does not correspond to the action of OT through Gq/11- IP3-dependent elevation of intracellular Ca2+ concentration. Although serum circulating OT is believed to prevent hypertension, and to be cardioprotective by reducing the inflammatory response and improving cardiovascular function [15], signaling pathway of OT through Gq/11-IP3-dependent activation of PKC and IP3 may not be appropriate enough to illustrate the beneficial effect of OT in the cardiovascular system. In this context, downregulation of the Cav1.2 channel in cardiomyocytes, as shown in this study and possibly in vascular smooth muscle cells, may account for beneficial action of OT in cardioprotection and control of blood pressure during pregnancy. In accordance with these mechanisms, our results open to the understanding of the novel action of OT as a cardiovascular hormone.

Membranes 2021, 11, 234 9 of 13

3.2. Action of OT on the Cardiovascular System though Gi-Dependent Pathway OT is a potent uterine stimulant that is usually used for the induction of labor and control of postpartum hemorrhage. Acute application OT (intravenous injection) acts on the smooth muscle of the uterus to stimulate contractions through DAG-PKC activation 2+ and IP3-dependent cellular [Ca ]i elevation [14]. However, OT does not usually possess cardiovascular effects, such as elevation of blood pressure. Meanwhile, plasma OT concentration in nonpregnant women was <1 µU/mL. The mean plasma OT concentration between weeks 4 and 40 of pregnancy was 66–165 µU/mL [21]. Nevertheless, maternal blood pressure during pregnancy does not correspond to the action of OT through Gq/11- 2+ IP3-dependent elevation of intracellular Ca concentration. Although serum circulating OT is believed to prevent hypertension, and to be cardioprotective by reducing the inflammatory response and improving cardiovascular function [15], signaling pathway of OT through Gq/11-IP3-dependent activation of PKC and IP3 may not be appropriate enough to illustrate the beneficial effect of OT in the cardiovascular system. In this context, downregulation of the Cav1.2 channel in cardiomyocytes, as shown in this study and possibly in vascular smooth muscle cells, may account for beneficial action of OT in cardioprotection and control of blood pressure during pregnancy. In accordance with these mechanisms, our results open to the understanding of the novel action of OT as a cardiovascular hormone.

3.3. Study Limitation Various limitations of our study should be addressed: (1) This was an experimental study by use of rodent, and the results may not be extrapolated to human subjects, (2) down- regulations of phosphorylated-CREB and Cav1.2 by OT were assessed only in cardiomyocytes, but not in vascular smooth muscle cells, (3) actions of OT on other CREB-associated transcription regulators, such as CREB-binding protein (CBP) and CREB-regulated transcriptional coactivators (CRTCs), were not assessed in this study, and (4) possible changes of Ca2+ channel β and α2/δ subunits were not examined. These questions should be addressed thoroughly in future studies. In addition, it is important to keep in mind that the concentration of OT in this study (100 nM) was much higher than the serum OT concentration in humans in pregnancy and postpartum (0.5–2.5 nM) [22]. Therefore, it is important to exercise caution when interpreting data from this animal model.

3.4. Conclusions In the present study, we have successfully demonstrated a novel signal pathway of OT to regulate cardiac L-type Ca2+ expression, which is largely associated with cardiac excitability and intracellular Ca2+ homeostasis. This pathway is expected to attenuate Ca2+ overload in cardiomyocytes and smooth muscle cells when the Gq/IP3 pathway is overly activated by excessive OT. With regard to the pharmaceutical aspect, these mechanisms of OT on cardiomyocytes provide the precise pharmacological rationale for the prevention and treatment of cardiovascular diseases based on the Ca2+-overload pathophysiological condition of the heart.

4. Materials and Methods All experimental protocols were approved in advance by the Ethics Review Com- mittee for Animal Experimentation of Oita University School of Medicine (No. C004003, No. G004006), and were carried out according to the guidelines for animal research of the Physiological Society of Japan to minimize the number of animals used, as well as their suffering.

4.1. Animals and Surgery Adult male Wistar rats (300–400 g) used for this study were obtained from Kyudo Co., Ltd. (Fukuoka, Japan). All rats were housed three per cage in a temperature-controlled room (23 ◦C) with a constant light/dark cycle (lights on 8:00–20:00 h). Tissues from adult rat atrium or ventricle were dissected in liquid nitrogen and stored at −80 ◦C until real-time Membranes 2021, 11, 234 10 of 13

PCR analysis was performed. Neonatal rat ventricular myocytes were prepared from 1- to 2-day-old Wistar rats as described previously [23]. The cardiomyocytes were maintained at ◦ 37 C under 5% CO2 in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum for 24 h, then the medium was changed to serum-free condition containing oxytocin (10−8 M to 10−6 M) or oxytocin with other signal inhibitors (see the section on Inhibitor experiments). The spontaneously beating myocytes were subjected to electrophysiological experiments 24 h after treatment with OT.

4.2. Chemicals 2 4 9 OT and OTRA [d(CH2)5Tyr(Me) ,Thr ,Tyr-NH2 ]-vasotocin were purchased from Bachem (Bachem Peninsula Laboratories, Inc., Bubendorf, Switzerland). U73122 was obtained from Sigma (St. Louis, MO, USA). Isoproterenol, pertussis toxin, acetylcholine, forskolin, and other chemicals were from Wako Pure Chemical Industries (Osaka, Japan). OT and OTRA were dissolved in 5% acetic acid, while U73122 and forskolin were dissolved in dimethyl sulfoxide (DMSO) as stock solutions (5–20 mM), and then diluted to final concentrations in cell culture solutions. The final concentration of DMSO or acetic acid in the bathing solution was 0.01% or less. Prior to electrophysiological and real-time PCR analyses, these concentrations of DMSO or acetic acid were confirmed not to significantly influence ICa.L or Cav1.2 mRNA expression.

4.3. Inhibitor Experiments Several inhibitors were used to identify the role of intracellular signaling pathways in the action of OT. The concentration of these inhibitors used in this study was optimized based on the results of previous studies [13,19,24]: A highly speciﬁc oxytocin receptor 2 4 9 antagonist, [d(CH2)5Tyr(Me) ,Thr ,Tyr-NH2 ]-vasotocin 1 µM, a speciﬁc inhibitor of the Gi class pertussis toxin (PTX) 100 ng/mL, a phospholipase C (PLC) inhibitor (U73122) 2 µM, and an adenylate-cyclase activator forskolin (FSK) 10 µM. Each inhibitor was added alone or simultaneously with OT for 24 h to determine L-type Ca2+ channel expression and to assess the level of CREB phosphorylation.

4.4. Electrophysiological Measurements Whole-cell voltage clamp experiments were performed as described previously [25]. 2+ L-type Ca channel current (ICa.L) was recorded from a holding potential (VH) of −50 mV followed by various test potentials. ICa.L density was obtained by normalizing ICa.L to ◦ the cell capacitance. All experiments were conducted at 37 C. For measuring ICa.L, the bath solution was composed by Na+- and K+-free solution contained (mM): Tetraethy- lammonium chloride (TEA-Cl) 120, CsCl 6, 4-Aminopyridine (4-AP) 5, MgCl2 0.5, 4,4P- diisothiocyanostilbene-2,2P-disulfonic acid (DIDS) 0.1, HEPES 10, CaCl2 1.8, and glucose 10 (pH of 7.4 adjusted with TEA-OH). The pipette solution contained (mM): CsCl 130, Mg-ATP 2, EGTA 5, and HEPES 10 (pH of 7.2 adjusted with 1 M CsOH).

4.5. Quantitative Real-Time PCR Total RNA was extracted from rat neonatal ventricular cardiomyocytes and adult hearts using TRIzol (Invitrogen, Carlsbad, CA, USA) 24 h after the treatment with agents described above. The single-stranded cDNA was synthesized from 1 µg of total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Molecular System Inc, Alameda, CA, USA). Real-time PCR was performed on Light Cycler (Roche) using the FastStart DNA Master SYBR Green I (Roche) as a detection reagent. The oligonucleotide primers used for real-time quantitative PCR are described in our previous reports [11]. Rat glyceraldehydes- 3-phosphate dehydrogenase (GAPDH; GeneBank accession no. M17701) mRNA was used as an internal control. Data were calculated by 2−∆∆CT and presented as fold change of transcripts for Cav1.2 genes in myocytes and normalized to GAPDH (deﬁned as 1.0 fold). The size of the PCR products was conﬁrmed by 2% agarose gel electrophoresis. Membranes 2021, 11, 234 11 of 13

4.6. Western Blot Analysis Cultured neonatal rat cardiomyocytes were treated with OT (10−7 M) and OT with other signal inhibitors or agonists for 24–48 h in DMEM. All the subsequent steps were performed at 4 ◦C. Cardiomyocytes were washed twice with ice-cold PBS, scrapped, and transferred to a centrifuge tube. Cytoplasmic or nuclear proteins from cardiomyocytes were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer’s instruction (Pierce, Rockford, IL, USA). The extracted protein concentration was determined by the BCA protein assay kit (Pierce). Samples containing 40 µg were denatured at 95 ◦C for 5 min in loading buffer [Tris-HCl (pH 6.8) 250 mM, 4% SDS, 1% β-mercaptoethanol, 1% bromophenol blue, and 20% glycerol], separated by SDS- polyacrylamide gel electrophoresis using 8% polyacrylamide gel, and then transferred from the gel to a PVDF membrane (Hybond-P; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). To prevent nonspecific binding, the blotted membranes were blocked with 5% skim milk in tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) for 1 h at room temperature and then probed overnight at 4 ◦C with an anti-CREB antibody (1:1000, Cell Signaling, Beverly, MA, USA) and phosphospecific antibody against anti-pCREB (Ser 133) (1:1000, Cell Signaling). The blot was visualized with anti-rabbit IgG horseradish peroxidase- conjugated secondary antibodies (1:2000, American Qualex, CA, USA) and an ECL plus Western Blotting Detection System (GE Healthcare Bio-Sciences), and the results were exposed on Biomax Light film (Eastman Kodak; Rochester, NY, USA). Band density was measured with Scion image software and background subtraction algorithms. Protein loading was controlled by probing Western blots with anti-β-actin (1:1000, Cell Signaling) and normalizing ion channel protein band intensities to β-actin values.

4.7. Protein Kinase A (PKA) Assay Cultured neonatal rat cardiomyocytes were treated with OT (10−7 M) and OT with other signal inhibitors for 24 h in DMEM, and cells then underwent lysis in a commercial lysis buffer containing protease inhibitors and phosphatase inhibitors. A 15-µg quantity of cytoplasmic protein was used for PKA reaction with a PepTag assay kit (ProMega, Madison, WI, USA), following the manufacturer’s protocol. After phosphorylation, the PKA-speciﬁc ﬂuorescent peptide substrate kemptide alters the net charge from +1 to −1, and the phosphorylated and non-phosphorylated forms can be separated on agarose gels. Enzyme activity was expressed as substrate catalyzed unit/mg protein.

4.8. Statistical Analysis Data are expressed as mean ± S.E. Between groups and among groups comparisons were conducted with one-way ANOVA followed by a Scheffe test. A p value < 0.05 was considered signiﬁcant.

Author Contributions: M.M. and K.O. designed the study. M.M., S.T. and Y.W. performed experiments. M.M. and Y.W. analyzed the data. M.M. and K.O. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by JSPS KAKEN 21590934 (K.O.) from the Japan Society for the Promotion of Science, Tokyo. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Review Committee for Animal Experimentation of Oita University School of Medicine (No. C004003, No. G004006), and were carried out according to the guidelines for animal research of the Physiological Society of Japan to minimize the number of animals used, as well as their suffering. Informed Consent Statement: Not applicable. Data Availability Statement: The data in this study are available from the corresponding author on reasonable request. Acknowledgments: The authors thank Yoko Miyanari for her technical supports. Membranes 2021, 11, 234 12 of 13

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

OT oxytocin OTR oxytocin receptor OTRA oxytocin receptor antagonist cAMP adenosine-30,50-cyclic monophosphate CREB cAMP response element binding protein PKA protein kinase A PKC protein kinase C Gi inhibitory GTP-binding regulators of adenylate cyclase PLC phospholipase C DAG diacylglycerol IP3 inositol 1,4,5-trisphosphate 2+ ICa.L L-type Ca channel current NO nitric oxide ANP atrial natriuretic peptide

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