Atorvastatin blocks increased l-type Ca²⁺ current and cell injury elicited by angiotensin II via inhibiting oxide stress

Abstract

The l-type Ca²⁺ current (I_Ca,l) plays a crucial role in shaping action potential and is involved in cardiac arrhythmia. Statins have been demonstrated to contribute to anti-apoptotic and anti-arrhythmic effects in the heart. Here, we examined whether atorvastatin regulates the I_Ca,l and cell injury induced by angiotensin II (AngII) as well as the putative intracellular cascade responsible for the effects. Cultured neonatal rat ventricular myocytes were incubated with AngII for 24 h, and then cell injury and expression levels of Nox2/gp91^phox, p47^phox_, and Cav1.2 were analyzed. In addition, I_Ca,l was recorded using the whole-cell patch-clamp technique, and mechanisms of atorvastatin actions were also investigated. It was found that the number of apoptotic cardiomyocytes was increased and cell viability was significantly decreased after AngII administration. AngII also augmented the expressions of Nox2/gp91^phox and p47^phox compared with control cardiomyocytes. Exposure to AngII evoked I_Ca,l in a voltage-dependent manner without affecting the I–V relationship. In addition, AngII enhanced membrane Cav1.2 expression. These effects were abolished in the presence of the reactive oxygen species (ROS) scavenger, manganese (III)-tetrakis 4-benzoic acid porphyrin [Mn(III)TBAP], or the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, atorvastatin. These results suggested that atorvastatin mediates cardioprotection against arrhythmias and cell injury by controlling the AngII–ROS cascade.

Introduction

Statins, inhibitors of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-CoA reductase in the liver, are used in the treatment of hypercholesterolemia and have been well demonstrated to have pleiotropic effects. Treatment with statins has been reported to markedly reduce the relative risk of coronary events and attenuate angiotensin II (AngII)-induced atherosclerosis through upregulation of the anti-inflammatory status in lesions. Rugale et al. [1] reported that statins had antioxidant properties and attenuated target organ damage by reducing oxidative stress. In addition, statin treatment was found to be beneficial in preventing arrhythmia after ischemia-reperfusion, and the mechanisms had been elucidated. However, the effects of statins on cardiac electrical activity are complex. Ozturk et al. [2] reported that rosuvastatin prevented electrical remodeling in the diabetic heart. Seto et al. [3,4] demonstrated that acute simvastatin administration inhibited KATP channels of porcine coronary artery myocytes and modulated the vascular iberiotoxin-sensitive Ca²⁺-activated K(+) [BK(Ca)] channels of porcine coronary artery smooth muscle cells. Su et al. [5] found that the anti-arrhythmic effect of simvastatin was carried out by its effect on Kv4.3. Rosuvastatin could block the potassium current I (Kr) and prolong cardiac repolarization [6]. Ischemia-reperfusion had been shown to induce significant upregulation of the l-type calcium current (I_Ca,l), and simvastatin pretreatment could attenuate I_Ca,l without lowering the serum cholesterol level [7]. Laszlo et al. [8] found that atorvastatin treatment decreased I_Ca,l, similar to rapid atrial pacing in atrial myocytes. These studies indicated that the effect of statin treatment on arrhythmia is mediated through different ion channels independent of a decrease in cholesterol [9].

In the heart, I_Ca,l is the main calcium channel that contributes to numerous cellular processes, and its dysregulation can produce aberrant electrophysiological processes, resulting in arrhythmia in cardiomyocytes. Treatment with statins has been demonstrated to inhibit I_Ca,l and reduce intracellular Ca²⁺ concentration [10], but the mechanism of the effect of statins on I_Ca,l still needs to be clarified. AngII has been reported to induce cell injury and Ca²⁺ release from intracellular stores and elevate the concentration of intracellular Ca²⁺ in the heart, and patch-clamp studies have shown that AngII also enhances I_Ca,l.

In the present study, we aimed to analyze the putative acute effects of atorvastatin on cell injury and I_Ca,l induced by AngII in neonatal rat ventricular myocytes (NRVMs) and to clarify the intracellular signaling pathway involved in its effects.

Materials and Methods

Animals

Neonatal Sprague-Dawley (SD) rats were obtained from the Department of Experimental Animals of Hebei Medical University. This study was approved by the Animal Care and Use Committees of Bethune International Peace Hospital in accordance with the Helsinki Declaration of 1975 and the guidelines of Harris and Atkinson.

Isolation of myocytes and cell culture

Single ventricular myocytes were obtained by enzymatic dissociation using collagenase (Type II) and pancreatin as described previously [11] with some modifications. Ventricular myocardial tissues from neonatal SD rats (aged 1–2 days) were homogenized and dissociated with collagenase II and pancreatin six times for 20 min each time. The first cell suspension was discarded, whereas the rest of the cell suspensions were dispensed with fetal bovine serum (FBS) and then mixed together. These cell suspensions were placed in 10-cm plates for 1.5 h to allow enrichment for cardiomyocytes by differential adhesion. The supernatant was then plated onto a new dish with DMEM containing 10% FBS at 37°C. NRVMs were divided into four groups: control cells; control cells treated with AngII only; cells pretreated with a superoxide scavenger, manganese (III)-tetrakis 4-benzoic acid porphyrin [Mn(III)TBAP] before being treated with AngII; and cells pretreated with atorvastatin before being treated with AngII. Cells were treated with AngII at a final concentration of 0.1 µM for 24 h. Atorvastatin was obtained from Jialin Pharmaceutical Company (Beijing, China) and used at a concentration of 10 µM. Mn(III)TBAP was purchased from Cayman Chemical (Ann Arbor, USA) and used at a concentration of 2.5 µM.

Recording techniques

Whole-cell patch-clamp techniques were used to record the I_Ca,l in the voltage-clamp modes. An EPC-8 patch-clamp amplifier (HEKA, Lambrecht, Germany) was used for these recordings. Fire-polished pipettes were pulled from borosilicate glass capillaries (Narishige Scientific Instrument Lab., Tokyo, Japan). The micropipettes had a resistance of 2.0–3.5 mΩ when they are filled with the pipette solution, and the patch-clamp experiments were performed at room temperature. For Ca²⁺ current recordings, the external solution contained 120 mM tetraethylammonium chloride (TEA-Cl), 10 mM HEPES, 1 mM MgCl₂, 0.001 mM TTX, 10 mM CsCl, 2 mM CaCl₂, and 10 mM glucose, pH 7.4. The pipette solution contained 120 mM CsCl, 10 mM EGTA, 10 mM HEPES, and 5 mM MgATP, pH 7.4.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Cell viability was estimated using the MTT assay (Sigma, St Louis, USA). Cells (2 × 10⁴ cells/ml) were cultured in a complete medium for 24 h in 96-well plates. The medium in the cell cultures was replaced by a fresh medium containing 0.5% FBS, and then the cells were subject to different treatments. Then, 20 µl of MTT at a concentration of 5 mg/ml was added to each well and incubated for 4 h. Thereafter, the medium was discarded, and 150 µl of DMSO was added and incubated for 10 min. The absorbance of each well was recorded at 570 nm using a VERSAmax ELISA microplate reader (Molecular Devices, Sunnyvale, USA). All assays were performed in triplicate and repeated three times.

Caspase-3/7 activity assay

The Apo-ONE^® Homogeneous Caspase-3/7 Assay (Promega, Madison, USA) was used to measure apoptosis of NRVMs. Briefly, cells seeded in 96-well plates at a density of 2 × 10⁴ cells/well were pretreated with Mn(III)TBAP or atorvastatin for 1 h, and then NRVMs were treated for 24 h with AngII. Thereafter, 100 µl of Apo-ONE^® Homogeneous Caspase-3/7 reagent was added to each well. The plate was incubated for 1 h at room temperature using a plate shaker (350 rpm). Finally, fluorescence was measured in a fluorometer at excitation wavelength of 499 nm and emission wavelength of 521 nm. All assays were performed in triplicate and repeated three times.

Immunoblotting analysis

After being rinsed with cold phosphate buffer saline (PBS) three times, cells were homogenized in RIPA buffer when required, and the membrane fractions were prepared. The supernatant was then centrifuged at 120,000 g for 15 min at 4°C. Samples (10–20 mg) were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred to polyvinylidene fluoride (PVDF) membranes and analyzed by western blotting with monoclonal antibodies against p47^phox (Biorbyt, Cambridge, UK) and Nox2/gp91phox (Biorbyt, Cambridge, UK). PVDF membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. The membrane was developed with an ECL-Plus chemiluminescence reagent kit and visualized with a UVP Bio-Imaging System (Upland, USA). Immunoblot densities were analyzed using Image J software.

Statistical analysis

Data are expressed as the mean ± SD or the mean ± SE. All data were subject to ANOVA followed by Dunnett's t test, whereas data obtained from the same myocyte were evaluated by the two-tailed paired Student's t-test. Western blot densities were analyzed with Image J software. P< 0.05 was considered as statistically significant.

Results

Atorvastatin prevents cell injury induced by AngII in NRVMs

Previous studies have shown that AngII causes cell injury. In this study, NRVMs were pre-incubated with atorvastatin for 1 h and then incubated with AngII for 24 h. As shown in Fig. 1A, at 24 h after addition of AngII, cell viability decreased significantly as evidenced by the MTT assay. Atorvastatin at the concentration of 10 µM significantly increased the cell viability during the exposure to AngII. The number of apoptotic cardiomyocytes increased after AngII administration, and atorvastatin markedly reduced the percentage of apoptotic cardiomyocytes (Fig. 1B). Similar results were observed in the expression of cleaved caspase-3, which was enhanced by AngII and attenuated by atorvastatin (Fig. 1C).

Figure 1.

Effects of atorvastatin on viability and apoptosis of NRVMs (A) NRVMs were pre-incubated with atorvastatin for 1 h and then incubated with AngII for 24 h. Cell viability was determined by the MTT assay (n = 6/group). (B) Cardiomyocyte apoptosis was determined by caspase-3/7 activity assay (n = 6/group). (C) Cleaved caspase-3 expression was stimulated by AngII and inhibited by atorvastatin (n = 4/group). **P < 0.01 versus PBS group, ^#P< 0.05 versus AngII group, and ^##P < 0.01 versus AngII group. Data are expressed as the mean ± SD.

Atorvastatin inhibits I_Ca,l rise and Cav1.2 expression during exposure to AngII

The I_Ca,l is mediated by the l-type Ca²⁺ channel, and the α1C (Cav1.2) subunit is considered to be the most important polypeptide of the Ca²⁺ channel-forming proteins, since it forms the channel pore for ion flow. I_Ca,l was measured by whole-cell patch clamping, and the current was stimulated at a holding potential of −50 mV with a series of 300 ms depolarizing steps from −50 to +50 mV in 10 mV steps, then back to −50 mV. As shown in Fig. 2A, representative tracings of the inward Ca²⁺ currents were recorded at 0 mV with or without AngII or atorvastatin from NRVMs. Comparison of the top and middle panels shows that AngII markedly elicited the peak amplitudes of I_Ca,l, which were markedly reduced by the subsequent application of atorvastatin (bottom panel). The current densities were obtained by normalizing the current amplitude to cell capacitance in picoamps (pA)/picofarads (pF). Current density–voltage (I–V) relationships were constructed using mean values of I_Ca,l at different potentials (−50 to 50 mV). The I–V relationship showed a significant increase in I_Ca,l density after addition of AngII compared with control cells at most of the voltages tested, whereas atorvastatin suppressed the increase caused by AngII (Fig. 2B). Next, the membrane Cav1.2 expression was analyzed, and results showed that its expression was enhanced by AngII, but was reversed by atorvastatin (Fig. 2C).

Figure 2.

Enhancement of l-type Ca²⁺ current (I_Ca,l) by AngII in NRVMs and its suppression by atorvastatin (A) Superimposed current traces of I_Ca,l during 300-ms voltage-clamp steps to potentials of −50 through +50 mV in 10 mV steps, recorded before (control) and during exposure to AngII, and after further addition of atorvastatin (AngII + atorvastatin). The voltage-clamp protocol is given above these traces. (B) Current–voltage (I–V) relationships for peak amplitude of I_Ca,l, measured before (control, circle) and during exposure to AngII (square), and after further addition of atorvastatin (AngII + atorvastatin, triangle). (C) The expression level of Cav1.2 was decreased with AngII incubation overnight, but atorvastatin reversed it during exposure to AngII (^#P< 0.05 versus AngII, and **P < 0.01 versus PBS group, n = 4). Whole-cell patch-clamp data are expressed as the mean ± SE, and western blot data are expressed as the mean ± SD.

Atorvastatin attenuates AngII-induced reactive oxygen species (ROS) production

ROS production is stimulated by AngII. To assess whether atorvastatin acts by reducing ROS production induced by AngII, the expressions of NOX2/gp91^phox and p47^phox were analyzed in cardiomyocytes. As shown in Fig. 3A, AngII markedly enhanced NOX2/gp91^phox expression at the protein level by western blot analysis, whereas atorvastatin suppressed its expression. The expression of p47^phox was significantly increased after exposure to AngII without atorvastatin, and atorvastatin significantly decreased its expression in the presence of AngII (Fig. 3B). NRVMs were pretreated with a highly selective ROS scavenger, Mn(III)TBAP, for 1 h before AngII administration. As shown in Fig. 3C, the AngII-induced cell apoptosis was abolished by addition of Mn(III)TBAP. These results suggested that the oxide stress pathway is involved in the atorvastatin-mediated inhibition of AngII-induced cell injury in cardiomyocytes.

Figure 3.

Atorvastatin attenuation of ROS production induced by AngII (A) Western blot analysis of Nox2/gp91^phox expression in NRVMs (n = 4/group). (B) Western blot analysis of p47^phox expression in NRVMs (n = 4/group). (C) Mn(III)TBAP attenuates AngII-induced myocyte apoptosis. ^## P< 0.01 versus AngII, and **P < 0.01 versus PBS group. Data are expressed as the mean ± SD.

Role of ROS in atorvastatin modulation of I_Ca,l induced by AngII

Atorvastatin was shown to suppress ROS production in NRVMs. Therefore, the role of ROS in the anti-arrhythmic action of atorvastatin on NRVMs was investigated. As shown in Fig. 4A, AngII markedly increased I_Ca,l, and during exposure to Mn(III)TBAP the current decreased rapidly and markedly. The I–V relationship showed a significant elevation in I_Ca,l density after addition of AngII when compared with control cells at most of the voltages tested, whereas the AngII-induced current was inhibited by Mn(III)TBAP (Fig. 4B). ROS, including H₂O₂, causes intracellular calcium overload. In this study, exogenous H₂O₂ also rapidly activated I_Ca,l, similar to the effect caused by AngII (Fig. 4C,D). Figure 4E shows that expression of Cav1.2 was increased after AngII or H₂O₂ administration, but was decreased by addition of Mn(III)TBAP.

Figure 4.

Requirement of ROS for I_Ca,l activation in NRVMs (A) Currents and (B) I–V relationships before (circle) and after eliciting I_Ca,l with AngII (square) and after addition of Mn(III)TBAP (triangle). (C, D) H₂O₂ activated peak current of I_Ca,l (control, circle; H₂O₂, square). (E) AngII that enhanced Cav1.2 expression was suppressed by Mn(III)TBAP (n = 4/group). **P < 0.01 versus PBS group, and ^##P< 0.01 versus AngII group. Whole-cell patch-clamp data are expressed as the mean ± SE, and western blot data are expressed as the mean ± SD.

Discussion

The present study demonstrated that atorvastatin could inhibit I_Ca,l and cell injury induced by AngII via suppressing ROS production in NRVMs. During AngII administration, NRVMs showed increased levels of cell injury and a significant rise of I_Ca,l. Additionally, Cav1.2 expression was increased, and ROS production was enhanced under this condition, whereas atorvastatin could block these effects elicited by AngII.

Experimental evidence indicated that the level of AngII was increased under pathophysiological conditions, such as congestive heart failure, cardiac hypertrophy, and myocardial ischemia. It also has been reported to exert apoptotic effects on cardiomyocytes by coupling to several important intracellular signaling pathways, such as ROS, or by increasing intracellular Ca²⁺. AngII itself may contribute to the mechanism of atrial fibrillation (AF) through increasing Na∼+/Ca∼(2+) exchanger (NCX) expression and augmenting calcium transient, which is PKC- and CREB-dependent. AngII was found to modulate Kv4.3 mRNA via NADPH oxidase (NOX)-dependent ROS production in neonatal rat myocytes [12], as well as to increase expression of l-type calcium channel and augment calcium transient depending on the activation of PKC and CREB [13]. In this study, our results showed that AngII-induced NRVM injury as evidenced by increased caspase-3/7 activity, cleaved caspase-3 expression, and decreased cell viability. Moreover, AngII elicited a rise of I_Ca,l in NRVMs. NOX2/gp91^phox and P47^phox expressions were also enhanced by AngII, which implicates that ROS is downstream effector of AngII.

Many studies have revealed that ROS generation controls biological processes, including cell proliferation, differentiation, and migration. ROS evidently participates in signal transduction processes that control gene expression, cell growth, and apoptosis. Therefore, a change of the ROS level would play an important role in many cellular functions, including ionic homeostasis and signal transduction. A moderate increase of ROS levels is not detrimental, whereas an excessive increase of ROS can cause oxidative damage. It has been demonstrated that ROS contributes to adverse outcomes after myocardial infarction and heart failure, which can be inhibited by statin therapy [14]. The level of AngII is increased in cardiac disease, and it contributes to ROS production in cardiac myocytes [15]. Meanwhile, AngII antagonist drugs have been shown to reduce mortality in patients with structural heart disease [16,17]. In the present study, exposure to AngII significantly augmented NOX2/gp91^phox and P47^phox expressions, and inhibition of ROS production by Mn(III)TBAP substantially suppressed cellular injury. Moreover, redox modulation of ion channel and pump activity has been suggested to be directly responsible for these pathological processes in the heart [18,19], and ROS is known to play a crucial role in Ca²⁺ homeostasis. Accumulation of ROS leads to cardiac Ca²⁺ overload. In addition to the well-recognized role of ROS in acute ischemia/reperfusion-related Ca²⁺ overload, the AngII-induced I_Ca,l elevation studied here must also depend on ROS production, since the block of ROS by Mn(III)TBAP suppresses the AngII-induced I_Ca,l in ventricular myocytes.

The cardiac voltage-dependent, dihydropyridine-sensitive l-type calcium channel (l-VDCC) is the main calcium channel in the heart, where it contributes to the plateau of the action potential. Since I_Ca,l is a major determinant of the plateau phase of the action potential and plays a central role in excitation–contraction coupling [20]. Modification of its properties has proved to be detrimental to cardiomyocyte function [21]. Cardiac l-type Ca²⁺ channels are composed of four polypeptide subunits (α1, β, α2, and δ), and the α1 subunit is the most important one, as it plays a critical role in forming the channel pore for ion flow, opening of voltage-dependent Ca²⁺ channel, and Ca²⁺ ion channel selectivity [22,23]. At least 10 different α1 subunit genes are known to exist, of which the α1C (Cav1.2) isoform is highly expressed in cardiomyocytes [24]. The whole-cell patch-clamp experiment in this study clearly demonstrated that AngII elicited I_Ca,l in NRVMs, and this finding was further strengthened by the finding that membrane Cav1.2 expression increased after AngII administration. These results are consistent with a previous study showing that elevated Cav1.2 protein expression may be the main cause of the increase in I_Ca,l [25]. The enhanced expression of Cav1.2 was fully suppressed by addition of Mn(III)TBAP in the presence of AngII, indicating the involvement of enhanced ROS production in modulating I_Ca,l in NRVMs.

Clinical trials have demonstrated that statins could reduce the risk of major coronary events in patients regardless of whether they had coronary heart disease and high cholesterol or not, and those with higher risk received a greater absolute benefit. The mechanisms have been well-studied, including plasma lipoprotein reduction, endothelial function improvement, plaque stability, and anti-inflammation. Apparently, the clinical benefits of statins independent of lowering low-density lipoproteins play an important role. Furthermore, statins may also be beneficial in reducing oxidative stress in addition to lowering cholesterol level [26,27]. Therefore, we tried to evaluate whether atorvastatin provides protection via inhibiting ROS production in this study. Our results showed that the presence of atorvastatin during AngII exposure suppressed the expressions of both NOX2/gp91^phox and P47^phox augmented by AngII. Furthermore, atorvastatin and Mn(III)TBAP both fully blocked cell injury induced by AngII, indicating that atorvastatin exerts cardioprotection through normalizing ROS levels induced by AngII in NRVMs.

Statins have been proved to be anti-arrhythmic agents in recent years. Previous studies have recognized that treatment with statins could affect atrial ion currents, causing significant reductions in the Na(+)-K(+) pump current [I(p)] in the myocardium and skeletal muscle. However, this effect was not related to cardiac cholesterol content [28]. Our results showed that atorvastatin administered under superfusion conditions attenuated the AngII-induced I_Ca,l increase in a fully or partially reversible manner without affecting the I–V relationship.

In conclusion, our results demonstrated that atorvastatin produced an inhibition of the rise in I_Ca,l and cell injury stimulated by AngII in NRVMs, exerting cardioprotection on these cells through the oxide stress pathway. Further analysis of the pathways utilized by atorvastatin in carrying out its cardioprotection in cardiomyocytes would be of interest. Upregulation of AngII has been shown to occur in cardiac disease, such as congestive heart failure, cardiac hypertrophy, myocardial ischemia, and AF, which leads to upregulation of ROS [10,16]. Some of these patients are treated with atorvastatin, and we suspect that the same signaling cascade described here may modulate the I_Ca,l and cell injury in the human heart, which may influence the duration of action potentials, as well as cell growth and apoptosis. Moreover, ROS production triggered by AngII may influence other ion channels and cell apoptosis in the heart. Thus, more studies are needed to clarify the possible pro-arrhythmic and/or anti-arrhythmic effect of the inhibition produced by atorvastatin on the adverse effects induced by AngII via the oxide stress pathway demonstrated here.