Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-ß-estradiol (E2, 10 nM) or G-1 (1 µM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 µM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

The functional association between the sodium/bicarbonate cotransporter (NBC) and the soluble adenylyl cyclase (sAC) modulates cardiac contractility.

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.

Cardiac up-regulation of NBCe1 emerges as a beneficial consequence of voluntary wheel running in mice.

Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, µm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice. These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.

The inhibition of voltage-gated H+ channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H+ extrusion to maintain cell viability. In this sense, many H+ transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H+ outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn2+ and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase.

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30.

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3(-)/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3(-); control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

Characterization of the Na/HCO3(-) cotransport in human neutrophils.

BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

Elevated carbon dioxide upregulates NBCn1 Na+/HCO3(-) cotransporter in human embryonic kidney cells.

The NBCn1 Na(+)/HCO3(-) cotransporter catalyzes the electroneutral movement of 1 Na(+):1 HCO3(-) into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10-15%). After acid loading, in the presence of HCO3(-), ∼50% of the pHi recovery phase was blocked by the Na(+)/H(+) exchanger inhibitors EIPA (10-50 µM) and amiloride (1 mM) and was fully cancelled by 30 µM EIPA under nominally HCO3(-)-free conditions. In addition, in the presence of HCO3(-), pHi recovery after acid loading was completely blocked when Na(+) was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner (Ki = 30 µM, full inhibition at 60 µM), which confirmed NBC Na(+)/HCO3(-) cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3(-)-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na(+)/HCO3(-) cotransporter as the only HCO3(-)-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3(-) transport.

Is N-methylacetazolamide a possible new therapy against ischemia-reperfusion injury?

The increase of intracellular Ca2+ concentration, produced principally by its influx through the L-type Ca2+ channels, is one of the major contributors to the ischemia-reperfusion injury. The inhibition of those channels in different experimental models was effective to ameliorate the post-ischemic damage. However, at a clinical level, the results were contradictory. Recent results of our group obtained in an ¨ex vivo¨ heart model demonstrated that a chemical derived from acetazolamide, the N-methylacetazolamide (NMA) protected the heart against ischemia-reperfusion injury, diminishing the infarct size and improving the post-ischemic recovery of myocardial function and mitochondrial dynamic. A significant inhibitory action on L-type Ca2+ channels was also detected after NMA treatment, suggesting this action as responsible for the beneficial effects on myocardium exerted by this compound. Although these results were promising, the effectiveness of NMA in the treatment of ischemic heart disease in humans as well as the advantages or disadvantages in comparison to the classic calcium antagonists needs to be investigated.

Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3- cotransporter enhances NBCe1-mediated HCO3- influx in the rat heart.

Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 µM) or 6-ethoxyzolamide (ETZ; 100 µM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current.

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 µM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aß were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aß decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.

In vivo Overexpression of Electrogenic Sodium/Bicarbonate Cotransporter (NBCe1) by AAV9 Modifies the Cardiac Action Potential and the QT Interval in Mice.

Cardiac cells depend on specific sarcolemmal ion transporters to assure the correct intracellular pH regulation. The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms. In the heart two different NBC isoforms have been described: the electroneutral NBCn1 (1Na+:1 HCO 3 - ) and the electrogenic NBCe1 (1Na+:2 HCO 3 - ). NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). In addition to regulating the pH, the NBC is a source of sodium influx. It has been postulated that NBC could play a role in the development of hypertrophy. The aim of this research was to study the contribution of NBCe1 in heart electrophysiology and in the development of heart hypertrophy in an in vivo mouse model with overexpression of NBCe1. Heart NBCe1 overexpression was achieved by a recombinant cardiotropic adeno-associated virus (AAV9) and was evidenced by western-blot and qPCR. AAV9-mCherry was used as a transduction control. NBCe1 overexpression fails to increase heart growth. Patch clamp and electrocardiogram were performed. We observed a reduction on both, ventricular myocytes APD and electrocardiogram QT interval corrected by cardiac rate, emphasizing for the first time NBCe1 relevance for the electrical activity of the heart.

Role of autocrine/paracrine mechanisms in response to myocardial strain.

Myocardial strain triggers an autocrine/paracrine mechanism known to participate in myocardial hypertrophy development. After the onset of stretch, there is a rapid augmentation in developed tension due to an increase in myofilament calcium sensitivity (the Frank Starling mechanism) followed by a gradual increase in tension over the next 10-15 min. This second phase is called the slow force response (SFR) to stretch and is known to be the result of an increase in calcium transient amplitude. In the present review, we will discuss what is known thus far about the SFR, which is the in vitro equivalent of the Anrep effect and the mechanical counterpart of the autocrine/paracrine mechanism elicited by myocardial stretch. The chain of events triggered by myocardial stretch comprises: (1) release of angiotensin II, (2) release/formation of endothelin, (3) NADPH oxidase activation and transactivation of the EGFR, (4) mitochondrial reactive oxygen species production, (5) activation of redox-sensitive kinases, (6) NHE-1 hyperactivity, (7) increase in intracellular Na(+) concentration, and (8) increase in Ca(2+) transient amplitude through the Na(+)/Ca(2+) exchanger. The evidence for each step of the intracellular signaling pathway leading to the development of SFR and their relationship with the mechanisms proposed for cardiac hypertrophy development will be analyzed.

Angiotensin II inhibits the electrogenic Na+/HCO3- cotransport of cat cardiac myocytes.

The Na(+)/HCO(3)(-) cotransporter (NBC) plays an important role in intracellular pH (pH(i)) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K(+) [K(+)](o) from 4.5 to 45 mM (K(+) pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19 ± 0.008, n=6; data expressed as an increase of pH(i) units after 14 min of applying the K(+) pulse). This alkalization was completely abrogated by the NBC blocker S0859 (-0.004 ± 0.016*, n=5; * indicates p<0.05 vs control) but not by the Na(+)/H(+) exchanger blocker HOE642 (0.185 ± 0.04, n=4), indicating that we are exclusively measuring eNBC. The K(+) pulse induced alkalization was canceled by 100 nM Ang II (-0.008 ± 0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT(1) receptor blocker, 0.18 ± 0.02; n=4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n=5). Neither chelerythrine (PKC inhibitor, -0.06 ± 0.04*; n=4), nor U0126 (ERK inhibitor, -0.07 ± 0.04*; n=4) nor MPG (ROS scavenger, -0.02 ± 0.05*; n=8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT(1) receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.

The activation of the G protein-coupled estrogen receptor (GPER) prevents and regresses cardiac hypertrophy.

Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling.

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3- cotransport.

The sarcolemmal Na+/HCO3- cotransporter (NBC) plays an important role in intracellular pH (pH(i)) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH(i) was measured in single cells in a medium with HCO3- using the fluorescent pH indicator BCECF. The NH4+ pulse method was used to induce an intracellular acid load and the acid efflux (JH) in the presence of the Na+/H+ exchanger blocker HOE642 (10 microM) was calculated as indicator of NBC activity. The following JH data are presented at pH(i) of 6.8 (* and # indicate p<0.05 after ANOVA vs. control and Ang II, respectively). The basal JH (1.03+/-0.12 mM/min, n=11) was significantly increased in the presence of 100 nM Ang II (1.70+/-0.15 mM/min, n=8*). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80+/-0.08 mM/min, n=11#), 300 microM apocynin (NADPH oxidase blocker; 0.80+/-0.13 mM/min, n=6#), 500 microM 5-hydroxidecanoate (mitochondrial ATP dependent K+ channel, mK(ATP), blocker; 0.97+/-0.21 mM/min, n=9#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 microM; 0.56+/-0.18 mM/min, n=6#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK(ATP) opening and mitochondrial ROS production ("ROS-induced ROS-release mechanism"). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.

Calcineurin/P38MAPK/HSP27-dependent pathways are involved in the attenuation of postischemic mitochondrial injury afforded by sodium bicarbonate co-transporter (NBCe1) inhibition.

The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110 min of perfusion; 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10 min of R; 4) SB + a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca2+ response (CaR), Ca2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations.