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Am J Physiol Heart Circ Physiol 290: H96-H106, 2006. First published July 29, 2005; doi:10.1152/ajpheart.00324.2005
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Angiotensin II-induced negative inotropy in rat ventricular myocytes: role of reactive oxygen species and p38 MAPK

Julieta Palomeque,1 Luciana Sapia,1 Roger J. Hajjar,2 Alicia Mattiazzi,1 and Martín Vila Petroff1

1Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina; and 2Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts

Submitted 1 April 2005 ; accepted in final form 20 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The octapeptide angiotensin II (ANG II) can modulate cardiac contractility and is increased in heart failure, where contractile function is impaired. In rat cardiac myocytes, 1 µM of ANG II produces a negative inotropic effect (NIE) (24.6 ± 5% reduction). However, the subcellular signaling involved in this effect remains elusive. We examined the mechanisms and signaling events involved in the reduction in contractile function induced by the peptide in indo-1-loaded rat cardiomyocytes. The results showed that the NIE of ANG II was not associated with a parallel decrease in the intracellular Ca2+ transient, indicating that a decrease in myofilament responsiveness to Ca2+ underlies the reduction in contractility. We assessed the role of PKC, tyrosine kinases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs) in the NIE of the peptide. Pretreatment of cells with the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride or with the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid did not affect the ANG II-induced NIE. Moreover, ANG II-induced ROS production, after 20 min of incubation with the peptide, could not be detected with the use of either the fluorophore 5-(6)-chloromethyl-2', 7'-dichlorodihydrofluorecein diacetate or lucigenin-enhanced chemiluminescence. In contrast, the ANG II-induced NIE was abrogated by the inhibitors of PKC (calphostin C), tyrosine kinase (genistein), and p38 MAPK (SB-202190). Furthermore, the NIE was significantly exacerbated (60 ± 10% reduction) by p38 MAPK overexpression. These results exclude the participation of ROS in the NIE of the peptide and point to PKC and tyrosine kinase as upstream mediators. Furthermore, they reveal p38 MAPK as the putative effector of the reduction in myofilament responsiveness to Ca2+ and the decrease in contractility induced by the peptide.

myocardium; calcium transients; mitogen-activated protein kinase

THE OCTAPEPTIDE ANGIOTENSIN II (ANG II) is the effector molecule of the renin-angiotensin system, and it has been shown to be implicated in many cellular physiological and pathological processes. In addition to its well-established role in vascular and electrolyte homeostasis, ANG II has been shown to modulate contractility and cardiac excitation-contraction coupling, in the near term, and to induce expression of genes responsible for cardiac hypertrophy, in the long term. Furthermore, it is now well recognized that ANG II is associated not only with hypertension but also with coronary disease and heart failure.

During the development of cardiovascular disorders, such as ischemic heart disease and heart failure, there is a continuous detriment of cardiac function, which triggers the activation of a variety of compensatory mechanisms. Among these, the most relevant phenomena are an increase in blood levels of catecholamines and endothelin and the activation of the renin-angiotensin-aldosterone system. Although, initially, these mechanisms serve to maintain an adequate cardiac output, the prolonged exposure of cardiac muscle to these hormones results in decreased contractility, contributing to an even greater decline of cardiac function. Accordingly, ANG II has been shown to exacerbate contractile dysfunction in failing hearts of rats and dogs (6, 10) and numerous studies have reported that the inhibition of the angiotensin-converting enzyme or treatment with the ANG II type 1 (AT1) receptor blocker losartan attenuates cardiac dysfunction accompanying heart failure (10, 12). Although these reports clearly establish that ANG II contributes to the contractile dysfunction of the failing heart, surprisingly, the underlying mechanisms and the signaling pathways involved have not, at present, been elucidated. Moreover, currently, not even the effects of ANG II on cardiac contractility are completely understood. Indeed, the peptide has been reported to increase (6, 13, 19, 20, 26, 28, 34, 38), not change (22) or even decrease (23, 27, 28, 29, 32, 33), contractility. This large heterogeneity in the type of inotropic response to ANG II in cardiac muscle suggests the possibility that the final effect of ANG II on cardiac excitation-contraction coupling and inotropism may result from a complex balance between several opposing mechanisms. Whereas in species that respond to ANG II with a pronounced positive inotropic effect, the subcellular mechanisms involved have been examined in detail (13, 19, 20, 26, 34, 38), those involved in the negative contractile effect of the peptide, as observed in the rat and mouse heart (27, 29, 32, 33), remain a largely uncharted territory.

At present, the general consensus among most authors is that the majority of the cardiac actions of ANG II are mediated by the activation of the {beta}-isoenzymic form of phospholipase C. The PKC limb of this transduction cascade, via the phosphorylation of several intracellular proteins, has been implicated in both the positive and negative inotropic effects of the peptide (33, 34). However, only limited information is available on the signaling events downstream from PKC. In this context, increasing evidence has accumulated suggesting that reactive oxygen species (ROS) may play a physiological role in receptor signaling, and only very recently, ROS and mitogen-activated protein kinases (MAPKs) have been implicated as downstream mediators of ANG II signaling, leading to cell growth and apoptosis (31). However, as yet, the impact of these second messengers on the contractile behavior induced by the peptide has not been evaluated. Recently, coupling of ANG II receptors to NAD(P)H oxidase, resulting in superoxide formation and p38 MAPK activation, has been recognized in ventricular myocytes (40). Interestingly, in experiments with transgenic mice, it has been demonstrated that the cardiac-specific activation of p38 MAPK markedly attenuates cardiac contractility (8, 24). These results suggest a previously unrecognized link between ANG II, NAD(P)H oxidase-ROS generation, p38 MAPK-activation, and contractile regulation of the heart. Therefore, the present study was conducted in rat cardiac myocytes to determine whether alterations in Ca2+ handling and/or in myofilament responsiveness mediate the negative inotropic effect of ANG II and to investigate the role of PKC, ROS, and p38 MAPK in this effect.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Myocyte isolation. All experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and experimental procedures were reviewed and approved by the Ethics Committee for Animal Experiments of the University of La Plata School of Medicine. Single cardiac myocytes were isolated from hearts of adult Wistar rats with the use of a collagenase-based enzymatic digestion via a previously described technique (38). Myocytes were kept in Krebs-Henseleit solution (K-H) of the following composition (in mM) and at room temperature (20°–22°C) until use: 146.2 NaCl, 4.7 KCl, 1.0 CaCl2, 10.0 HEPES, 0.35 NaH2PO4, 1.05 MgSO4, and 10.0 glucose (pH adjusted to 7.4 with NaOH). Rod-shaped myocytes with clear and distinct striations and obvious marked shortening and relaxation on stimulation were used. Experiments were performed at room temperature.

Indo-1 fluorescence and cell shortening measurements. The isolated myocytes were loaded at room temperature with the cell-permeant acetomethyl ester form of indo-1 (17 µM for 9 min), according to the previously described method (38), and left for deesterification for 45 min. Cells were then placed on the stage of an inverted microscope (Nikon Diaphot 200) adapted for epifluorescence. Myocytes were continuously superfused with K-H (pH 7.4) at a constant flow of 1 ml/min and field stimulated via two platinum electrodes on either side of the bath (square waves, 2-ms duration, and 20% above threshold) at 0.5 Hz. The excitation light was centered at 350 nm, and emission was collected at 410 and 490 nm. Background fluorescence was subtracted from each signal before obtaining the 410/490 fluorescence ratio. The ratio of the indo-1 emission at the two wavelengths was taken as an indicator of the intracellular Ca2+.

The stage of the microscope was illuminated with red light (640–750 nm) through its normal brightfield illumination optics to allow simultaneous measurements of fluorescence and shortening.

Resting cell length and cell shortening were measured by a video-based motion detector (Crescent Electronics) and stored by software (PowerLab/400 ADInstruments) for an off-line analysis.

To assess changes in myofilament responsiveness to Ca2+, the instantaneous myocyte length was plotted versus the simultaneously measured indo-1 fluorescence during single twitch contractions (phase-plane plot) (38). The position of the trajectory of the relaxation phase of these plots reflects the relative myofilament responsiveness to Ca2+.

Intracellular pH measurements. Intracellular pH (pHi) was measured with the use of the fluorescent indicator SNARF-1 AM, as previously described (38). Emissions at 590 and 640 nM, acidic and basic forms of the indicator, respectively, were obtained by exciting the fluorophore at 530 nM. Absolute pH values were determined with the use of a standard curve obtained from an in vivo calibration.

Measurement of intracellular ROS. The membrane-permeable probe 5-(6)-chloromethyl-2', 7'-dichlorodihydrofluorecein diacetate (CM-H2DCFDA) enters the cell and produces a fluorescent signal after intracellular oxidation by ROS. Cells were loaded with 1 µM CM-H2DCFDA for 30 min at 37°C and then washed with K-H solution. Intracellular oxidative stress in response to ANG II was monitored on an inverted microscope via the epifluorimetric technique (39). Excitation wavelength was set at 485 nm, and emission was collected at 530 nm. The fluorescent signals were digitized and stored by software for off-line analysis. Measurements were taken every 5 min for 30 min, and H2O2 was used as a control for ROS detection.

ROS production was also measured by lucigenin-enhanced chemiluminescence (1). After treatment with drugs and inhibitors [i.e., ANG II, diphenyleneiodonium chloride (DPI), 4, 5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), phenylephrine], myocytes were lysed in K-H solution and sonicated with three 10-s bursts on ice. Protein concentration was determined with the use of the Bradford assay, and aliquots containing 100 µg protein were brought to a volume of 100 µl with lysis buffer. A nonredox cycling dose of lucigenin (5 µM) was added to vials and allowed to equilibrate for several minutes at 37°C. Luminescence was measured in a liquid scintillation counter (Packard 1900TR) with a single active photomultiplier tube positioned in the out-of-coincidence mode. After dark adaptation, vials containing lucigenin solution (blanks) were counted once and then recounted two times after cell lysates were added to each vial. Blanks of {approx}103 counts were then subtracted from the average of the relatively constant levels of chemiluminescence produced under the different conditions to obtain the basal data presented as counts per minute. NADPH (100 µM) was added as substrate, and samples were recounted two times within 2 min.

Recombinant adenoviruses. Two first-generation type 5 recombinant adenoviruses were used in this study. The method used to construct the recombinant adenovirus has been described previously (16). Briefly, the backbone vector, which contains most of the adenoviral (Adv) genome (pAdv.EASY1), was used and the recombination performed in Escherichia coli. p38{alpha} and {beta}-galactosidase ({beta}-Gal) cDNA were subcloned into the adenoviral shuttle vector (pAdv.TRACK), which uses the cytomegalovirus long-terminal repeat as a promoter. pAdv.TRACK also has a concomitant green fluorescent protein (GFP) under the control of a separate cytomegalovirus promoter. The adenoviruses were propagated in human embryonic kidney 293 cells. The titers of stocks used for these studies measured by plaque assays were 4 x 109 plaque-forming units (pfu)/ml for the {beta}-galactosidase gene (Adv.{beta}-Gal) and 2 x 109 pfu/ml for Adv.p38 with particle-to-pfu ratios of 20:1. These recombinant adenoviruses were tested for the absence of wild-type virus by polymerase chain reaction of the early transcriptional unit E1.

Culture and adenovirus infection of adult ventricular cardiomyocytes. After isolation, the cells were resuspended in DMEM medium containing (in g/l) 0.017 ascorbic acid, 2 BSA, 0.4 L-carnitine, 0.66 creatine, 0.62 taurine, 50 U/ml penicillin, and 50 U/ml streptomycin, and counted. The adenoviral infection was performed at a multiplicity of infection (MOI) of 100. Myocytes were plated at a density of 5 x 105 rod-shaped cells/ml onto culture dishes and cultured for 48 h. The infection efficiency of both viruses was monitored by fluorescence microscopy at an excitation wavelength of 480 nm, to detect GFP fluorescence.

Electrophoresis and Western blot analysis. Isolated adult rat cardiomyocytes were incubated in the HEPES buffer, 1 mM Ca2+, with 1 µM ANG II, 1 µM ANG II plus 1 µM genistein, 1 µM ANG II plus 1 µM calphostin C, or in the absence of drug for the control condition, at room temperature for 20 min. Myocytes were then lysed with ice-cold lysis buffer. Lysates (70 µg of total protein) were separated by 12% Tris-glycine/SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were first probed with the antibody reacting with phospho-p38 MAPK (New England BioLabs). The same membrane was then stripped and reprobed with a second primary antibody reacting with total p38 MAPK (Santa Cruz Biotechnology) to determine the total protein abundance. Immunoreactivity was visualized by peroxidase-conjugated antibodies with the use of a peroxidase-based chemiluminescence detection kit (PerkinElmer Life Sciences). The signal intensity of the bands on the film was quantified with the use of National Institutes of Health Image 1.60 software. The activity of p38 MAPK was normalized against its corresponding total protein.

Materials. Collagenase type B was purchased from Worthington Biochemical Lakewood, NJ; Pronase from Boerhinger Mannheim, Mannheim, Germany; bovine serum albumin (BSA) essentially fatty acid free, Tiron, and ANG II from Sigma Chemical, St. Louis, MO; indo-1 AM, SNARF-1 AM, and CM-H2DCFDA from Molecular Probes, Eugene, OR; calphostin C from Research Biochemical International, Natick, MA; genistein, DPI, SB-202190, and PD-98059 from Calbiochem, La Jolla, CA. All other chemicals were of the purest reagent grade available.

Statistics. All data are presented as means ± SE. Comparisons within groups were assessed by either paired or unpaired Student's t-test, as appropriate. A value of P < 0.05 was taken to indicate statistical significance.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Effect of ANG II on myocyte contraction and calcium transient. Using electrically stimulated indo-1-loaded cardiac myocytes, we investigated the effect of 1 µM ANG II on the unloaded contraction and the intracellular Ca2+ transient (CaiT). This concentration of ANG II was chosen on the basis of the results of pilot experiments, which indicated that the maximal negative inotropic effect of ANG II occurred in the concentration range of 0.1 to 1 µM, in agreement with results reported by Sakurai et al. (33) in mouse myocytes.

Figure 1A shows a representative example of the effect of ANG II on the unloaded myocyte contraction and the associated CaiT. In the rat isolated myocyte, ANG II produced a slow developing, negative inotropic effect that reached steady state after 20 min of incubation with the drug. In contrast, CaiT amplitude was unaffected by the presence of ANG II, suggesting that the negative inotropic effect of the peptide is mediated by a reduction in myofilament responsiveness to Ca2+. Similar results were obtained in five other cells by using a lower concentration of ANG II (0.5 µM; data not shown). ANG II (1 µM) produced a similar negative inotropic effect when cells were stimulated at a higher, more physiological frequency of 1 Hz (30 ± 6% reduction in the amount of shortening; n = 13 cells from 5 hearts). To reinforce the conclusion that a decrease in myofilament responsiveness to Ca2+ was the mechanism responsible for the negative inotropic effect of the peptide and that this was not concluded on the basis of a failure to detect small decreases in the CaiT in our experimental conditions, two different approaches were used. 1) Phase-plane diagrams (loops) of the instantaneous cell length versus the simultaneous indo-1 fluorescence (see METHODS) at control and after 20 min of ANG II application were compared as illustrated in Fig. 1A, right. The relaxation phase of the loop obtained after 20 min of ANG II application is shifted to the right compared with the control loop, indicating that the response of the myofilaments to Ca2+ has diminished after ANG II administration. Similar phase-plane diagrams were observed in 5 indo-1-loaded cells from five different hearts exposed to ANG II (1 µM). 2) The second approach consisted in verifying whether a decrease in the CaiT amplitude could be detected when a similar negative inotropic effect to that produced by ANG II was induced by lowering extracellular Ca2+ concentration ([Ca2+]o). Figure 1B depicts a representative example showing a continuous recording of cell length and the associated twitch contractions and individual Ca2+ transients. For a similar decrease in contraction amplitude to that induced by ANG II, lowering [Ca2+]o produced a negative inotropic effect that was associated with a parallel decrease in the amplitude of the CaiT. The bar graph (Fig. 1C) represents the overall data of these experiments showing that at steady state, the negative inotropic effect of the peptide occurs without changes in the CaiT, whereas lowering [Ca2+]o is associated with a significant decrease in peak fluorescence. The average effects of either reducing [Ca2+]o (from 1 mM to 0.75 or 0.5 mM) or ANG II (1 µM) administration on contraction and CaiT amplitude and kinetics are provided in Table 1. Taken together, these results are consistent with a failure of ANG II to affect the CaiT and point to a reduction in myofilament responsiveness to Ca2+ as the mechanism responsible for the negative inotropic effect of the peptide.



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  		Fig. 1. Effect of ANG II and low extracellular Ca2+ concentration ([Ca2+]o) on indo-1 transient and contraction amplitudes. A: representative continuous recording of cell length and associated Ca2+ transient showing that, typically, ANG II produces a negative inotropic effect without a parallel decrease in indo-1 fluorescence. Right: diagram of indo-1 fluorescence vs. cell length (phase-plane plots) for contractions a and e used to assess myofilament responsiveness to Ca2+. B: typical continuous recording of cell length showing that lowering [Ca2+]o produces a negative inotropic effect associated with the decrease in a Ca2+ transient and without changes in trajectory of phase-plane plots (right). C: overall data of effect of ANG II (n = 8 cells from 7 hearts) and low [Ca2+]o (n = 6 cells from 6 hearts) on contraction amplitude and peak indo-1 fluorescence.

 

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  		Table 1. Effect of ANG II and low extracellular Ca2+ on contraction and Ca2+ transient parameters of single rat myocytes

 
Additional experiments evaluating the effect of ANG II on myocyte contraction were performed in the physiological buffer bicarbonate. In these conditions, ANG II elicited a negative inotropic effect similar in magnitude to that produced by the peptide in HEPES buffer (25 ± 6% n = 5 cells from 4 hearts).

Role of PKC and tyrosine kinases in the negative inotropic effect of ANG II. To examine the possible role of PKC on the negative inotropic effect of ANG II, the effect of this peptide was studied in myocytes, where cell shortening was monitored in the continued presence of 1 µM calphostin C. The bar graph depicted in Fig. 2A shows that calphostin C significantly attenuates the decrease in cell shortening induced by ANG II. Calphostin C at the concentration employed did not affect basal cell shortening.



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  		Fig. 2. Negative inotropic effect of ANG II is PKC and tyrosine kinase dependent. A: effect of ANG II in presence of 1 µM calphostin C (CC). CC significantly inhibited the negative inotropic effect induced by peptide (n = 5 cells from 5 hearts). B: effect of ANG II in presence of genistein (Gen). Genistein (1 µM) prevented the ANG II-induced negative intropic effect (n = 5 cells from 5 hearts).

 
To further investigate the intracellular signals involved in the ANG II-induced negative inotropic effect, experiments were performed with the use of the tyrosine kinase inhibitor genistein. Figure 2B shows the overall results of these experiments. Application of 1 µM genistein produced a small, although significant, increase in contractility (24.3 ± 9.1% above control). When ANG II was administered after the positive inotropic effect of genistein had reached steady state, it failed to reduce myocyte contractility, indicating that tyrosine kinases are also involved in the signaling leading to the negative inotropic effect of the peptide.

Role of ROS in the ANG II-induced negative inotropic effect. Figure 3 shows representative continuous recordings of cell length in the absence and presence of the NAD(P)H oxidase inhibitor DPI or the superoxide scavenger Tiron. Treatment of cells with these inhibitors neither affected basal contraction parameters nor the negative inotropic effect induced by ANG II. The bar graph in Fig. 3 shows the overall results of these experiments. In additional experiments, the effect of 1 µM ANG II on intracellular ROS production was assessed with the use of the fluorophore CM-H2DCFDA. Figure 4, A and B, shows that H2O2 dose dependently enhanced CM-H2DCFDA fluorescent intensity, whereas ANG II did not significantly affect basal fluorescence, at least during the time frame in which the negative inotropic effect of the peptide becomes manifest. The lack of effect of ANG II on ROS production during the time period in which the negative inotropic effect of the peptide becomes stable (20 min) was confirmed by using lucigenin chemiluminescence. Figure 4C shows that myocytes exposed for 20 min to 1 µM ANG II did not increase NADPH-dependent O2– production compared with nontreated controls. However, both 60 min of exposure to the peptide or to 10 µM of the {alpha}-adrenoceptor agonist phenylephrine significantly enhanced ROS production.



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  		Fig. 3. Failure of diphenyleneiodonium chloride (DPI) and 4, 5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) to inhibit the negative inotropic effect of ANG II. Typical continuous recordings of cell length showing the effect of ANG II alone (top) and in continued presence of either DPI (middle) or Tiron (bottom). Bar graph depicts overall data showing that the negative inotropic effect of the peptide is not affected by DPI or Tiron. Data are means ± SE for n = 6 cells from 6 hearts per group. *P < 0.05 vs. control.

 


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  		Fig. 4. Effect of ANG II on intracellular reactive oxygen species (ROS) generation. A: typical experiment showing the effect of ANG II and H2O2 on 5-(6)-chloromethyl-2', 7'-dichlorodihydrofluorecein diacetate (CM) fluorescence. H2O2 dose dependently increased CM fluorescence, whereas ANG II had no effect. B: overall results indicating that ANG II failed to significantly increase ROS production, whereas H2O2, at concentrations as low as 10 µM, was able to significantly enhance CM fluorescence. C: effect of DPI, Tiron, ANG II, phenylephrine (Phe), and ANG II + Tiron on basal and NADPH-stimulated ROS production measured by lucigenin-enhanced chemiluminescence. Data are means ± SE for n = 5 cells from 4 hearts (B). *P < 0.05 vs. control. CPM, counts per minute.

 
NADPH-dependent chemiluminescence was virtually abolished by the flavoprotein inhibitor DPI and the O2– scavenger Tiron. Furthermore, Tiron abrogated the increase in ROS production induced by 60 min of exposure to 1 µM ANG II. Taken together, these results indicate that neither NAD(P)H oxidase-mediated ROS production nor ROS coming from other sources is involved in the negative intropic response induced by the peptide.

Specific inhibition of p38 MAPK abolishes the ANG II-induced negative inotropic effect. To study the possible role of p38 MAPK activation on the reduction of contraction induced by ANG II, cells were treated with 10 µM of the specific p38 MAPK inhibitor SB-202190 (SB). Figure 5 depicts representative recordings of the effect of ANG II on cell length in the absence and presence of SB. Treatment of cells with SB significantly increased the cell contraction amplitude (Fig. 5, right). This effect, which within ~10 min reaches steady state, has been attributed to the inhibition of endogenous p38 MAPK activity (24). When ANG II was applied after the effect of SB had reached steady state, the peptide failed to induce a negative inotropic effect. Moreover, in another series of experiments in which ANG II was applied until the typical negative inotropic effect had settled in and SB was then given in the continued presence of ANG II, the inhibitor was able to revert the negative inotropic effect induced by ANG II, returning contractility to a level slightly above baseline, similar to the one observed by SB alone. Moreover, incubation of myocytes with ANG II for 20 min induced an increase in p38 MAPK activity, as revealed by the increase in the ratio of phosphorylated p38 MAPK to nonphosphorylated p38 MAPK. ANG II produced a 68 ± 24% increase in p38 MAPK activity with respect to control. Interestingly, the ANG II-induced increase in p38 MAPK activity was significantly suppressed by calphostin C and genistein (12 ± 9% and –8 ± 8% with respect to control, respectively). These results suggest that p38 MAPK activation mediates the negative inotropic response induced by the peptide and that PKC and tyrosine kinases are involved as intermediate messengers.



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  		Fig. 5. Inhibition of p38 MAPK activity prevents the ANG II-induced negative inotropic effect. Typical continuous recordings of cell length showing the effect of ANG II alone (A) or in presence of SB-202190 (SB; B and C). Bar graphs (right) depict overall data of each experimental series showing that ANG II alone produces a significant negative inotropic effect (6 cells from 6 hearts). This effect is either prevented or reversed when ANG II is administered in continuous presence of SB (n = 6 cells from 5 hearts) or when the inhibitor is applied after the negative inotropic effect of peptide has reached steady state (n = 6 cells from 4 hearts), respectively. Data are means ± SE. *P < 0.05 vs. control.

 
In additional experiments, we examined the role of the ERK MAPK with the use of the inhibitor PD-98059. In this series, 1 µM ANG II reduced contractility by 26 ± 3% and 28 ± 5% in the absence and presence of 10 µM PD-98059, respectively (n = 4 cells from 4 hearts), indicating that ERK is not involved in the negative inotropic effect induced by the peptide.

p38 MAPK overexpression exacerbates the ANG II-induced negative inotropic effect. In search of additional confirmation for the involvement of p38 MAPK in the ANG II-induced negative intropic effect, adult rat ventricular myocytes were infected with an adenovirus vector carrying the p38 MAPK gene (Adv.p38) at a multiplicity of infection of 100. Forty-eight hours after infection, myocytes (nearly 100%) presented a robust expression of the reporter gene GFP, detected by the green fluorescence on an inverted fluorescence microscope (Fig. 6A), and retained their rod-shaped morphology and functional integrity. Functional experiments were then carried out to examine the effect of ANG II on contraction and CaiT amplitude in Adv.p38-infected cells and in cells infected in similar conditions but with the adenovirus carrying Adv.{beta}-Gal to serve as controls. Figure 6B shows representative tracings of the effect of ANG II on the contraction and the associated CaiT of {beta}-Gal- and p38 MAPK-overexpressing cells. Basal contraction of Adv.p38 MAPK-infected cells was slightly but significantly reduced compared with {beta}-Gal-overexpressing controls (7.3 ± 0.4 and 5.7 ± 0.6% of resting cell length for {beta}-Gal- and p38-overexpressing cells, respectively). ANG II produced a typical negative inotropic effect in myocytes infected by the Adv.{beta}-Gal, whereas in cells infected with the Adv.p38, the peptide produced a much more pronounced reduction in contraction amplitude. In neither {beta}-Gal- nor p38-overexpressing cells was CaiT amplitude affected by ANG II. The phase-plane diagrams show that ANG II shifts the loop to the right in {beta}-Gal cells and that there is a further shift in p38-overexpressing cells. The overall data of these experiments, shown in the bar graph of Fig. 6, depict that the significantly larger negative inotropic effect induced by ANG II in the Adv.p38-infected cells (60 ± 10%; n = 6 cells from 5 hearts) with respect to the control Adv.{beta}-Gal-infected cells (26 ± 6%; n = 6 cells from 5 hearts) is produced without significant differences in the CaiT. These data serve to confirm that p38 MAPK is functionally linked to the ANG II-induced reduction in myofilament responsiveness and contractility and that the enhanced negative inotropic effect in p38-overexpressing cells is due to a greater reduction in myofilament Ca2+ sensitivity.



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  		Fig. 6. Overexpression of p38 MAPK exacerbates the ANG II-induced negative inotropic effect. A: forty-eight hours after infection, adult rat ventricular myocytes were visualized to detect green fluorescent protein (GFP) fluorescence. Coexpression of GFP demonstrates visually that {beta}-galactosidase gene (Adv.{beta}-Gal; left) and Adv.p38 MAPK (right) are being expressed in the cell. B: recordings of twitch contractions and associated intracellular Ca2+ transient (CaiT) show that in {beta}-Gal-expressing cells, ANG II produces a typical negative inotropic effect, whereas in p38 MAPK-overexpressing cells, the peptide provokes a more pronounced reduction in twitch amplitude. In both cell types, no effect of ANG II was observed in CaiT amplitude. C: bar graph shows overall data indicating that the negative inotropic effect induced by ANG II in Adv.p38-infected cells is significantly larger than the effect observed in Adv.{beta}-Gal-infected cells and is due to a greater decrease in myofilament responsiveness to Ca2+. Data are means ± SE for n = 6 cells from 5 hearts per group.

 
In additional experiments, with the use of the fluorescent pHi indicator SNARF-1, the effect of ANG II on pHi was measured in p38- and {beta}-Gal-overexpressing cells. Basal pH values for {beta}-Gal and p38 cells were 7.19 ± 0.04 and 7.20 ± 0.03, respectively, and were not significantly different after 20 min of incubation with 1 µM ANG II [7.12 ± 0.04 (n = 4 cells from 4 hearts) and 7.10 ± 0.03 (n = 4 cells from 4 hearts), respectively].


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The importance of the renin-angiotensin system in the pathophysiology of heart failure has been highlighted by the vast number of clinical and experimental investigations. However, the mechanisms underlying its detrimental effect on cardiac function remain, at present, poorly understood. Moreover, although it has been reported that in rat isolated myocytes (27, 29), as well as in mouse cells (33), ANG II induces a negative inotropic effect, the subcellular signaling events have not been examined in detail. Considering that this ANG II-induced negative inotropic effect could, in principle, be one of the mechanisms underlying the decline in cardiac function of the failing heart, understanding the subcellular signaling involved becomes both timely and necessary. Therefore, in the present study, we focused on characterizing the negative inotropic effect of the peptide and the associated Ca2+ dynamics, in addition to establishing the second messengers and signaling events involved. Our results indicate that in rat isolated cardiac myocytes, ANG II induces a negative inotropic effect determined by a reduction in myofilament responsiveness to Ca2+. Furthermore, we provide evidence indicating that p38 MAPK is the effector molecule of this event and demonstrate that both PKC and tyrosine kinases, but not ROS, are involved as intermediate messengers.

ANG II-induced negative inotropic effect: diminished intracellular Ca2+ or myofilament responsiveness? ANG II is a G protein-coupled receptor agonist and, as such, is linked to phospholipase C and to the hydrolysis of phosphoinositide. The activation of these receptors is characterized by a wide range of agonist- and species-dependent variations in their contractile response. In this scenario, ANG II has been shown to elicit a positive inotropic effect in rabbit (13, 19, 20) and cat myocytes (26, 34, 38), whereas negative contractile effects have been observed in rat (27, 29) and mouse cells (33). The positive inotropic effect of ANG II has been attributed to an increase in the CaiT (38), although in some cases, an increase in myofilament responsiveness to Ca2+ has also been shown to participate (13, 19). The results presented herein show that in the rat myocyte, ANG II elicits a decrease in cell shortening without a parallel decrease in the CaiT (Fig. 1), suggesting that the mechanism behind the negative inotropic effect of the peptide is a decrease in myofilament responsiveness to Ca2+. This contention is further supported by the phase-plane analysis of cell shortening versus intracellular Ca2+. Our results using low [Ca2+]i also argue in favor of the negative inotropic effect of the peptide being mediated by a decrease in myofilament responsiveness to Ca2+ and invalidate the argument that this conclusion could result from a failure to detect small decreases in the CaiT.

ANG II has been shown to produce an increase in pH (19, 24). Our previous observations indicate that intracellular alkalinization does not contribute to ANG II-induced inotropy (38). However, others have postulated that ANG II-induced intracellular alkalinization could, in some species, account for at least part of the positive inotropic effect of the peptide as a result of an increase in myofilament responsiveness to Ca2+ (13, 19). This mechanism, obviously, cannot explain the negative inotropic effect observed in the rat myocyte. Moreover, in rat cells, the ANG II-induced negative inotropic effect occurs even when the more physiological bicarbonate buffer is used (where ANG II fails to produce significant changes in pHi as has been shown in different species, including the rat) (5, 14, 26). Furthermore, as will be discussed below, the peptide fails to modify pHi in control {beta}-Gal-overexpressing cells. Our results suggest, therefore, that pH-dependent mechanisms are not involved in the decrease in myofilament responsiveness to Ca2+ that mediates the ANG II-induced negative inotropic effect.

PKC mediates the ANG II-induced negative inotropic effect. PKC is known to mediate some of the contractile effects of ANG II in cardiac muscle. Indeed, in cat and rabbit isolated myocytes, the positive inotropic effect of ANG II was abolished by inhibitors of PKC (20, 34). In contrast, in chick and rat isolated myocytes, as well as in rat papillary muscles and whole hearts, the activation of PKC, with the use of phorbol esters, has been shown to produce a negative inotropic effect (7). Furthermore, in isolated mouse cardiac myocytes, the PKC inhibitor chelerythrine was shown to significantly suppress the ANG II-induced negative inotropic effect (33). Similarly, our results with the PKC inhibitor calphostin C demonstrate that in the rat myocyte, PKC is also the upstream mediator of the ANG II-induced contractile response. Interestingly, the fact that both positive and negative inotropic effects of the peptide can be abrogated by inhibitors of PKC could suggest that distinct isoforms of PKC may be responsible for the multiple contractile actions of ANG II.

Recently, it was shown that PKC{alpha} modulates the development of lethal cardiomyopathy (3). Further results showed that PKC{alpha} acts as a negative regulator of ventricular systolic and diastolic function by modulating calcium handling. Interestingly, in the present study, there were no changes in Ca2+ handling, which would lead to the conclusion that PKC{alpha} may not be responsible for the negative inotropic effect of ANG II. In contrast, our observations are consistent with findings in PKC{beta}-overexpressing mice, where the activation of PKC{beta} has been shown to produce a decrease in contractility via a reduction in myofilament sensitivity to Ca2+ (36). Indeed, activation of PKC in vitro leads to phosphorylation of several myofibrilar proteins [i.e., troponin I (TnI), troponin T, and actomyosin ATPase] (9, 21, 30, 36), any of which could potentially lead to a reduction in myofilament responsiveness to Ca2+. Although ANG II has been shown to be able to induce the phosphorylation of these proteins, the intermediate signaling events involved are presently unknown.

Role of MAPK in the ANG II-induced negative intropic effect. In this study, we found that neither DPI nor Tiron was able to prevent the negative inotropic effect induced by ANG II (Fig. 3). Moreover, although in cells loaded with the fluorescent probe CM-H2DCFDA we were able to detect intracellular oxidative stress when probing the cells with H2O2, we failed to detect ANG II-induced ROS formation in the same cells (see Fig. 4, A and B). Similar results were obtained when O2– production was measured by lucigenin-enhanced chemiluminescence after incubating cells for 20 min with ANG II (Fig. 4C). These results are in agreement with those of a recent report by Hool et al. (18) showing that short-term exposure to ANG II (30 min) does not alter cellular superoxide production in adult ventricular myocytes. Taken together, these results indicate that ANG II-induced ROS formation is not involved in the negative inotropic effect induced by the peptide. However, they do not rule out the participation of p38 MAPK, which can also be activated directly by PKC (33), in the contractile response of the peptide. Therefore, we next assessed the role of p38 MAPK on the decrease in contraction amplitude evoked by ANG II. We found that 1) ANG II increases p38 MAPK activity; 2) SB, a pharmacological inhibitor of p38 MAPK, was able to either prevent or even revert the ANG II-induced negative inotropic effect; and 3) overexpression of p38 MAPK via adenovirus-directed gene transfer exacerbates the negative inotropic response elicited by ANG II. These findings, combining pharmacological inhibition and genetic manipulation, provide substantial evidence indicating that p38 MAPK is mechanistically involved in the negative inotropic effect produced by the peptide. Although we did not observe an ANG II-induced increase in ROS production during the development of the negative inotropic effect of the peptide (see Fig. 4C), we were able to detect a significant increase in ROS formation after 60 min of incubation with the peptide. It is plausible, therefore, that under pathological conditions such as hypertrophy and heart failure, where NADPH oxidase expression has been shown to be increased (17), ANG II could evoke enhanced and sustained ROS production, which could contribute to p38 MAPK activation and exacerbate the negative inotropic effect of the peptide.

Several mechanisms could be involved in the p38-mediated decrease in myofilament responsiveness to Ca2+. Among these, a modulation of pHi or a change in the phosphorylation status of contractile elements seem the most likely candidates. Both MAPK and ANG II have been shown to modulate the different pH-regulating proteins (as was referred to earlier) (5, 14). However, in this study, we failed to observe ANG II-mediated changes in pHi, either in {beta}-Gal- or p38-overexpressing cells. These results are in agreement with those of Liao et al. (24) showing that the p38 MAPK-mediated negative inotropic effect is unlikely to be mediated by intracellular acidification. A phosphorylation, targeted to the contractile apparatus, seems, therefore, a more likely scenario. Although phosphorylation of TnI is known to reduce myofilament responsiveness, p38 MAPK, however, is unable to directly phophorylate this protein, indicating that TnI is not a direct downstream target of p38 MAPK (24). Previous studies have indicated that heat shock proteins (HSP) such as HSP27 are activated by p38 MAPK, causing their translocation to the Z line of the sarcomeres. Further studies have shown that p38-mediated activation of HSP27 leads to a decrease in actomyosin ATPase activity and contractile depression, possibly through modifications in sarcomeric scaffolding proteins such as {alpha}-actin (8). Yet another possibility could arise from the proapoptotic activity of p38. Caspase-3 has been shown to directly target the contractile proteins and to decrease myofibrillar responsiveness to Ca2+ (11). Clearly, identifying the molecular mechanisms that are responsible for the ANG II/p38 MAPK-mediated decrease in myofilament responsiveness requires further investigation.

Our data using the inhibitor PD-98059 indicate that ERK MAPK activation is not required for the negative inotropic response induced by ANG II. These results are consistent with previous findings documenting that ERK MAPK activation by G protein receptor agonists (i.e., endothelin 1 and {alpha}-adrenoceptor stimulation) is associated with a positive rather than with a negative inotropic effect.

In the present study, we also investigated the potential role of tyrosine kinases as upstream mediators of the effect of ANG II on contraction in adult rat myocytes. Our observation that the nonselective tyrosine kinase inhibitor genistein can both suppress the activation of p38 MAPK and the negative inotropic effect of the peptide demonstrates that such a response requires the activation of these kinases. Our results are consistent with recent reports (2) revealing that during ischemic preconditioning, ANG II activates p38 MAPK in a PKC-dependent manner and that this activation also involves tyrosine kinases. The use of the nonselective tyrosine kinase inhibitor genistein does not allow the delineation of whether receptor- or nonreceptor tyrosine kinases, both of which have been shown to play critical roles in ANG II-induced signaling, are involved (41). Dissecting the subtype of tyrosine kinase involved in the negative inotropic effect induced by the peptide clearly deserves further study.

Until the present, studies that have delved into recognizing the signaling events involved in the negative inotropic effect of ANG II have failed to find messenger molecules beyond that of PKC. Our results confirm the participation of PKC and further recognize the involvement of tyrosine kinases and identify p38 MAPK activation as an important downstream target of the signaling cascade leading to a reduction in myofilament responsiveness to Ca2+ and decreased contractility.

Pathophysiological relevance of ANG II-induced negative inotropic effect. Specifically, with regard to the human heart, it is interesting to speculate that even though a negative inotropic action of ANG II has not been observed in this species, this does not necessarily imply that the negative component of the pathway does not exist. In the normal myocardium, where small positive contractile actions of ANG II have been reported (28), the positive contractile effects could prevail and be of sufficient magnitude to mask the negative events. In contrast, this negative limb of the balance could be more important in certain disease states where ANG II levels are increased and thus could either diminish or even reverse the inotropic response to ANG II of the normal heart. In this scenario, a positive inotropic response has been observed in human atrial and ventricular muscle, whereas a diminished inotropic response to ANG II has been reported in the failing human heart (28).

The importance of understanding the mechanisms involved in the negative inotropic response induced by ANG II is further highlighted by reports showing that treatment with the AT1 receptor blocker losartan attenuates the contractile dysfunction accompanying heart failure. Therefore, because circulating levels of the peptide are increased in heart failure as well as p38 MAPK activity (4, 35), the negative inotropic effect of ANG II may contribute, at least in part, to the diminished cardiac contractility under such pathological conditions. Indeed, the inhibition of p38 MAPK has been shown to improve contractile function and to provide significant cardioprotection in ischemia-reperfusion and in heart failure (4, 42).

In summary, we have presented evidence indicating that ANG II can promote a series of signaling events involving PKC, tyrosine kinases, and activation of p38 MAPK, culminating in a decrease in myofilament responsiveness to Ca2+ and decreased contractility. These findings provide new insight for the implication of ANG II and enhanced p38 MAPK signaling in cardiac dysfunction in pathological conditions such as heart failure.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants PICT 05–12368 and PICT 08592 from Agencia Nacional de Promoción Científica y Tecnológica, Argentina (to M. Vila Petroff and A. Mattiazzi, respectively), and Subsidio Laboratorios Klonal from Laboratorios Klonal SRL (to M. Vila Petroff). This study was also supported by the Sociedad Argentina de Hipertensión Arterial (SAHA). The study was also supported by National Heart, Lung, and Blood Institute Grants HL-57236, HL-71763, and HL-078691 (to R. J. Hajjar).


   ACKNOWLEDGMENTS
 
The technical assistance of Mónica Rando and the expertise of Dr. Claudia Caldiz are gratefully acknowledged.

A. Mattiazzi and M. Vila Petroff are Established Investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. G. Vila Petroff, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, La Plata 1900, Argentina (e-mail: mvila{at}atlas.med.unlp.edu.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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