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Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels have not yet been explored. In this work we study the role of CaVï¢ï€ subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveal changes in the CaVï¢ï€ subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual ï¢ï€ subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the CaVβ subunit isoform, with CaVï¢1b containing channels being more strongly regulated. CaVï¢2a containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a CaVï¢ï€ subunit-dependent manner. These data demonstrate that CaVï¢ï€ subunits alter the magnitude of inhibition of L-type current by Angiotensin II.
Key Words: ï¢ subunits; CaV1.2; Angiotensin II; Neonatal cardiomyocytes; L-type calcium current; arachidonic acid.
Angiotensin II (AngII) is a well known hormone that plays a crucial role in physiology and physiopathology by regulating the function of many cells types1. In particular, the acute effect of AngII on L-type calcium currents has been shown to be dependent on the cell type, while in smooth muscle is accepted that AngII induce activation of L-type calcium current2-4; in neurons and kidney, the opposing effect (inhibition of L-type calcium current) is observed5-7. On the other hand, the effect of this hormone in cardiac muscle is still controversial as AngII was reported to induce an increase8, 9 or a decrease10, 11 of L-type calcium current in heart cells. Although the reason for this disagreement is not known, it is proposed that the experimental approach is relevant12, 13.
The Angiotensin type I receptor (AT1) is responsible for most of the classical actions associated with AngII. This receptor belongs to the super-family of seven membrane domain Gq-protein coupled receptor (GqPCR) and its activation is linked to phospholipase C (PLC) activation and to the production of inositol triphosphate (IP3) and diacylglycerol (DAG), which in turn activates the classic and novel isoforms of protein kinase C (PKC). In parallel to PKC activation, PLC activation induces a decrease in phosphoinositol 4,5-bisphosphate (PIP2) membrane levels and DAG production induces an increase in arachidonic acid (AA) levels trough the action of DAG lipase (DAGL). Interestingly, while the role of PKC in the AngII-dependent modulation of L-type calcium channel is controversial10, 13-15, recent reports show that either PIP2 depletion16 or DAGL-dependent AA production17 are able to modulate L-type calcium channels.
In the heart, L-type Ca2+-currents are carried by a multi-subunit membrane complex that includes CaV1.2 as the pore-forming subunit that co-assembles with the auxiliary CaVï¡2ï¤1 and CaVï¢ subunits 18, 19. To date, four genes that encode the CaVï¢1-4 isoforms have been identified and shown to differentially alter channel behavior, including open probability, gating kinetics activation, and inactivation over L-type Ca2+-current, either in heterologous expression systems 20 or in cardiomyocytes 21. While these CaVβ subunit effects have been extensively studied, less is known about their ability to fine tune the modulation of L-type calcium channels by neurotransmitters and hormones.
Here we show that the magnitude of AngII inhibition of L-type calcium current in neonatal rat cardiomyocytes correlate with the rate of inactivation of the currents and is dependent on DAGL activity, moreover, we demonstrate that this correlation is due to a CaVβ subunit dependence of AngII action. Finally, we show that calcium transients in spontaneous beating cells are modulated by AngII in a CaVï¢ subunit manner. In conclusion, we postulate that the effect of AngII over L-type calcium current is significantly dependent on CaVï¢ subunits and arachidonic acid production.
Constructs cDNA encoding the angiotensin receptor 1, AT1, was obtained from the University of Missouri-Rolla cDNA resource center. cDNAs for calcium channel subunits were kindly provided by Dr. Snutch. The palmitoylation deficient CaVβ2a was described previously22.
Cardiomyocytes Isolation Rats were bred in the Animal Breeding Facility from the Facultad Ciencias Químicas y Farmacéuticas, Universidad de Chile (Santiago, Chile). All studies were done with the approval of our Institutional Bioethical Committee. Cardiac myocytes were prepared from hearts of 1-3 day-old Sprague Dawley rats as described previously23. Briefly, cardiomyocytes were dissociated in a solution of collagenase (0.2 mg/ml) and pancreatin (1.2 mg/ml). The collected cells were plated in nitrocellulose-coated glass cover slips and cultured in DMEM containing 10% Horse Serum, 5% FBS, 2 mmolar/L L-glutamine and 50 U/ml penicillin-streptomycin (Sigma, USA)
Transfection Tissue culture and transfection of tsA-201 cells was described previously in detail 24. Briefly, cells were grown at 37 °C (5% CO2) in DMEM (+5% FBS, 50 U/ml penicillin-streptomycin) and plated on glass cover slips. Transfection solutions for individual culture dishes contained a mixture of cDNA expression vectors (2 ïg for each L-type calcium channel subunit and 0.2 μg of a pEGFP marker vector (Clontech)) and were transfected into cells by the calcium phosphate method. Cells were transferred to 30°C 24 h after transfection, and recordings were conducted 2-3 days later. Cardiomyocytes were transfected the same day of isolation with the same protocol described above with the exception that the cells were kept at 37° C and experiments were performed 48 hr after transfection.
Electrophysiology Prior to recordings, cells were transferred into an external bath solution of 100 mM sodium (mM): 100 NaCl, 2 CaCl2, 1 MgCl2, 5 glucose, 95 sorbitol and 10 Hepes, pH 7.4, adjusted with Tris. Borosilicate glass pipettes were pulled and polished to 2-4-MΩ resistance and filled with internal solution contained (mM): 108 CsCl, 4 MgCl2, 2 CaCl2 10 EGTA, and 10 HEPES (pH 7.2 adjusted with CsOH). Data were acquired at room temperature using an Axopatch 200B amplifier and pClamp 8 software (Axon Instruments), low pass-filtered at 1 kHz, and digitized at 10 kHz. Series resistance was compensated to 85%, leak currents were negligible. In all experiments the perforated-patch configuration was obtained by supplementing the pipette internal solution with Nystatin25 to a final concentration of 800 μg/ml. Ramp protocols (-120 mV to +40 mV, 0.8 mV/ms every 15 sec) were used to monitor the gradual increase of the sodium current. After a stable current was achieved (≈10 min, access resistance (Ra) of 15 ± 8 MΩ) bath solution was replaced with a solution contained (mM): 20 BaCl2, 1 MgCl2, 10 HEPES, 40 TEA-Cl, 10 glucose, and 65 CsCl (pH 7.2 adjusted with TEA-OH) changes in liquid junction potential were calculated26, and voltages corrected for.
Semi-quantitative PCR. Real-time semi-quantitative PCR was performed using a Strategene Mx300P thermal cycler (Stratagene, La Jolla, CA). Briefly, cDNAs amplified out of total RNA from cardiomyocytes in culture at different days. PCR amplification of the GADPH RNA was used as internal control. PCR reaction was done with Brilliant SYBR Green according with manufacturer's directions, primers used for qPCR were:
CaVï¢1b S: ATGGTCCAGAAGAGCGGCATGTCC; AS: TTGATGTGCAGGAAGTCCTTGGG
CaVï¢2a S: ATGCAGTGCTGCGGGCTGGTAC; AS: TCCGAACTGCAAATGCAACAGG
CaVï¢3 S: ATGTATGACGACTCCTACGTGCC; AS: TTGACTCCAGAGCCCTGGACTGG
To confirm amplification specificity, PCR products were subjected to a melting curve program. Relative RNA amount was calculated with the ï„ï„Ct method and normalized with the amount of GADPH for each sample, measured in triplicates.
Calcium Imaging Plated cardiomyocytes were mounted in a perfusion chamber on the stage of an inverted microscope (Olympus IX-81, UPLFLN 40XO 40 x/1.3 oil-immersion objective). Cells were incubated with Fura-2 AM (Molecular Probes; 1 ïM) and then superfused for 10-20 min with a solution contained (mM): 100 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 90 sorbitol, 5 glucose and 10 Hepes, pH 7.4, adjusted with Tris. AngII was applied by local perfusion system. Fura-2 was alternately excited at 340 and 400 nm, and the fluorescence filtered at 510 nm was collected and recorded at 5 Hz using a CCD-based imaging system (Olympus DSU) running CellR software (Olympus). For every experiment, signals were recorded and the background intensity was subtracted, using a same-size region of interest outside the cells. Results are expressed as the ratio between the 340 nm and 400 nm (R340/400) signals27.
Reagents All reagents were of analytical grade and were purchased from Sigma (USA) and Merck (Germany).
Statistical Analysis All results are presented as means ± SEM. Statistical analysis of the data was performed using Statgraphics Plus 5.0 (Statistical Graphics Corp., USA). Statistically significant differences between means were assessed with Student's t tests or a one-way ANOVA (Dunnett's method) and considered significant at P<0.05
Macroscopic barium currents were recorded from dispersed beating cardiomyocytes using the nystatin perforated patch-clamp method to ensure minimal disruption of the cytosolic environment. Only cells that were beating at the onset of experiments were chosen, and to verify proper voltage-control, voltage-dependent Na+ currents were measured before each experiment (see methods and Figure 1).
We found that L-type currents with fast inactivation kinetics are more sensitive to AngII, while barium currents with slow inactivation are barely inhibited by AngII (Figure 2A, B). This observation was confirmed after plotting the residual current after a 250 ms pulse to 10 mV (R250, a measure of current inactivation) against the percentage of current inhibition after AngII (100 nM) exposure. Figure 2C shows that these two variables correlate (R>0.8) suggesting that the percentage of current inhibition by AngII is linked to the inactivation rate of the L-type current.
It has been reported that calcium currents in neonatal cardiomyocytes change throughout the time of culture28, therefore we examined whether changes in R250, and consequently AngII sensitivity could be attributable to different days in culture. We found that both parameters are correlated with the time in culture of the cardiomyocytes after isolation (Figure 3A, B). As the speed of inactivation (and therefore R250) is correlated with the CaVï¢ subunit expressed, semi-quantitative PCR was performed to detect changes in CaVï¢ subunit expression. As seen in Figure 3C-E there is a decrease in CaVï¢1b expression after cells isolation while CaVï¢2a increase it expression and CaVï¢3 remain constant.
The later results could implicate that changes in the magnitude of AngII inhibition is in fact due to changes in CaVï¢ subunits. To address this point directly, the cardiac form of CaV1.2 (+CaVï¡2ï¤1) was over-expressed in a stable cell line that over-expresses the AT1 receptor (HEK-AT1) with different CaVï¢ subunits. As seen in Figure 4, when cells over-expressing CaVï¢1b were exposed to AngII (100 nM) a fast current inhibition (~70%) was observed (Figure 4A, B, and Figure 5C). Cells expressing CaVï¢3 showed a lower degree of current inhibition (~50%) (Figure 4E, F, and Figure 5C); however, in CaVï¢2a expressing cells, almost no inhibition was observed (Figure 4C, D, and Figure 5C), demonstrating that CaVï¢ subunits are potent regulators of AngII mediated effects on L-type currents.
CaVβ2a differs from other CaVï¢ subunits by the existence of two cysteine residues near the N-terminus which form palmitoylation sites29, and many of the unique properties of this subunit are dependent on these residues29. Elimination of the putative palmitoylation sites via site-directed mutagenesis22 (ï¢2aC(3,4)S) resulted in a significant enhancement of the AngII mediated modulation, albeit not to the levels observed with other CaVβ subtypes (Figure 5A-C). This indicates that the palmitoyl groups of CaVï¢2a contribute to the insensitivity of CaVβ2a containing L-type channels to AngII.
Next, we decided to explore the intracellular signaling pathways responsible for the effect of AngII on L-type currents. In order to focus on cardiomyocytes exhibiting a fast rate of inactivation and therefore, with higher L-type current inhibition by AngII, only cardiomyocytes with less of 48 hours (0-1 days) in culture were used (Figure 3B). As shown in Figure 6, PLC inhibition with the generic inhibitor U73122 prevented the effect of AngII on L-type currents. However, in cardiomyocytes treated with the PKC inhibitor chelrytrine, the AngII-dependent L-type current inhibition was unaffected. Interestingly, inhibition of DAGL was enough to prevent the effect of AngII, suggesting a role for AA in L-type calcium channel inhibition (Figure 6).
As calcium influx via L-type Ca2+-current is critical for Ca2+-induced Ca2+-release30, an inhibition of L-type Ca2+-currents by AngII should be reflected in a decrease in the magnitude of Ca2+-transients. Consequently, we explored whether CaVï¢ subunits could modulate AngII responses on Ca2+-transients in spontaneous beating cardiomyocytes. All these experiments were done in cardiomyocytes with less of 48 hours in culture. In control cardiomyocytes, AngII produced a strong inhibition of the magnitude of Ca2+-transients as measured with Fura-2 (Figure 7A, B, G). A similar response was observed in cardiomyocytes over-expressing CaVï¢1b (Figure 7C, D, G) whereas CaVï¢2a over-expressing cardiomyocytes showed only little reduction in the magnitude of calcium transient after exposure to AngII (Figure 7E, F, G).
In this report, we show that L-type calcium channels CaVï¢ subunits are critical modulators of L-type current response to AngII. Using primary cultures of neonatal rat cardiomyocytes we have demonstrated that AngII inhibition of L-type currents correlate with the inactivation speed of the current. At a first glance, this observation could imply that inhibition may be dependent on the inactivation state of CaV1.2 channels, however, the experimental protocol was designed to avoid this possibility with short (50 ms) depolarizing pulses and long inter-pulses intervals (15 seconds), channels are expected to be in a closed state during AngII application. Therefore, it is more likely that the correlation with the rate of inactivation may be an epiphenomenon of a different underlying molecular event, such as channel subunit composition.
Indeed, cardiomyocytes express more than one isoform of CaVï¢ subunits, the main component of the L-type calcium channel multi-protein complex that determines the time course of inactivation31. As we show here, the expression pattern of CaVï¢ subunits changes throughout the time that the cardiomyocytes remain in culture, therefore over-expression of individual CaVï¢ subunits in cells that over-express AngII receptor, AT1, and the cardiac form of CaV1.2 (+CaVï¡2ï¤1) was chosen to demonstrate the impact of these subunits in AngII modulation. As suggested from cardiomyocytes data, the magnitude of inhibition is correlated with the type of CaVï¢ subunit that is expressed. Hence, a more likely explanation for the observed effects is that cells showing a lower macroscopic rate of inactivation simply express more CaVβ2a to give rise to a greater proportion of AngII insensitive channels in a given cell.
This CaVβ subunit dependency of calcium channel modulation is reminiscent of results showing that CaVï¢ subunit subtype regulates the modulation of N-type Ca2+-current by Gq-coupled receptors32 and of the inhibition of CaV1.3 channels by AA33. In line with these results, we show that DAGL activity is necessary for AngII-mediated L-type calcium channel inhibition, suggesting that AA production is necessary for this effect and indicating that ancillary calcium channel subunits can serve to fine-tune second messenger modulation of several VDCCs.
Increases in CaVï¢2a subunit expression34 as well as increases in AngII levels35 and changes in ion channels expression36 are associated with cardiac hypertrophy. In view of our results demonstrating that AngII is unable to modulate L-type currents and calcium transients when CaVï¢2a is expressed, this could reflect a novel mechanism that may contribute to the understanding of the deleterious effects of AngII.
Sources of Funding This work was supported by Fondecyt 11080019 to DV and FONDAP-Fondecyt 15010006 to AS, Chile, and by a CIHR grant to GWZ. GWZ is a Scientist of the AHFMR and a Canada Research Chair.
Figure 1 Na+ and L-type currents in cardiomyocytes. A) Representative nystatin-perforated whole-cell voltage-dependent Na+ current traces activated by the voltage protocol shown in the bottom B) Current-Voltage relation of whole-cell currents with 100 mM Na+ external solution (black trace) and 20 mM Ba2+ (gray trace), using a 180 ms duration voltage ramp from -120 to 40 mV. C) Nystatin-perforated whole-cell L-type barium current traces activated by the voltage protocol shown in the bottom. All data was obtained from the same cardiomyocyte.
Figure 2 The magnitude of inhibition of L-type Ba2+-currents depend on the inactivation speed of the current. A) Representative L-type currents, normalized to Imax, from 3 different cardiomyocytes, recorded using the voltage protocol shown in the upper part B) Time course of L-type Ba2+-current inhibition induced by AngII (100 nM) obtained from the cardiomyocytes shown in A C) Plot of the remaining current after a pulse of 250 ms to 10 mV (R250) versus AngII inhibition obtained at 60 seconds for individual cardiomyocytes. The regression line corresponds to a linear fit of the data (R=0.84, n=16)
Figure 3 Changes on CaVï¢ subunits expression pattern in cardiomyocytes in culture A) R250 of L-type barium current (mean ± sem) in cardiomyocytes from different days in culture (n>4). B) Percent inhibition by AngII (mean ± sem) in cardiomyocytes from different days in culture (n>4). CaVï¢ subunits mRNA levels relative to GADPH mRNA in cardiomyocytes cultures at different days for CaVï¢1b (C), CaVï¢2a (D) or CaVï¢3 (E). n=4. *p<0.01, compared with day 1.
Figure 4 L-type Ba2+-current inhibition by AngII is ï¢ subunit dependent. Representative barium currents before (black line) and 60 seconds after (gray line) exposure to AngII (100 nM). The currents were recorded from a HEK-AT1 cell line over expressing the cardiac form of CaV1.2 (+CaVï¡2ï¤1) and CaVï¢1b (A) or CaVï¢2b (C) or CaVï¢3 (E). Time course of the percentage of remaining current (mean ± sem) of L-type Ba2+-current in a HEK-AT1 cell line over-expressing the cardiac form of CaV1.2 (+CaVï¡2ï¤1) and CaVï¢1b (B) or CaVï¢2a (D) or CaVï¢3 (F).
Figure 5 Palmitoyl groups of CaVï¢2a are partially responsible for the lack of response after AngII exposure. Representative currents before (black line) and 60 seconds after (gray line) exposure to AngII (100 nM) of HEK-AT1 cell line over expressing the cardiac form of CaV1.2 (+CaVï¡2ï¤1) and CaVï¢2a(C3,4S) (A). Time course of the percentage of current (mean ± sem) of L-type barium current in HEK-AT1 cell line over expressing the cardiac form of CaV1.2 (+CaVï¡2ï¤1) and CaVï¢2a(C3,4S) (B). C) Summary bar graph for percent inhibition by AngII for different CaVï¢ subunits. n>6. *p<0.01 compared with parental HEK cell lines. **p<0.01 compared with CaVï¢2a.
Figure 6 DAGL inhibition prevents AngII effect over L-type calcium channel. A) Representative cardiomyocyte barium currents before (black line) and 60 seconds after (gray line) exposure to AngII (100 nM) and treated with different drugs. B) Pharmacological strategy used to establish the enzymes involved in the effect of AngII over L-type calcium channel. C) Summary bar graph for percent inhibition by AngII for cardiomyocytes treated with different drugs. n>6. *p<0.01 compared with cardiomyocytes control.
Figure 7 Calcium transients in rat cardiomyocytes decrease upon AngII treatment. Changes of cytosolic [Ca2+] in spontaneous beating control (A); CaVï¢1b-transfected (C) or CaVï¢2a-transfected (E) cardiomyocytes loaded with Fura-2 (Ratio 340/380). To determine the magnitude of calcium transient, the fitting to two Gaussians of an all-points histogram (5 minutes duration) was used (B,D,F). The black line represents the best fitting before AngII, the gray line after AngII G) Summary bar graph showing the percentage of inhibition of calcium transient amplitude after AngII (100 nM) exposure. n>6, *p<0.01.