HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 294/6/H2540    most recent 00046.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, C. Articles by Wang, H.-C. Search for Related Content PubMed PubMed Citation Articles by Wang, C. Articles by Wang, H.-C.

Apelin decreases the SR Ca²⁺ content but enhances the amplitude of [Ca²⁺]_i transient and contractions during twitches in isolated rat cardiac myocytes

Departments of ¹Cardiology and ²Gastrointestinal Surgery, Xi Jing Hospital, the Fourth Military Medical University, Xi'an, People's Republic of China

Submitted 14 January 2008 ; accepted in final form 10 April 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Apelin has been reported to have a positive inotropic action in the isolated rat heart. However, the effect of apelin on sarcoplasmic reticulum (SR) Ca²⁺ content and its influence on intracellular Ca²⁺ transient during excitation-contraction coupling remains poorly understood. In the present study, we determined the effect of apelin on Ca²⁺ transient and contractions in isolated rat cardiomyocytes. When compared with control, treatment with apelin caused a 55.7 ± 13.9% increase in sarcomere fraction shortening and a 43.6 ± 4.56% increase in amplitude of electrical-stimulated intracellular Ca²⁺ concentration (E[Ca²⁺]_i) transients (n = 14, P < 0.05). But SR Ca²⁺ content measured by caffeine-induced [Ca²⁺]_i (C[Ca²⁺]_i) transient was decreased 8.41 ± 0.92% in response to apelin (n = 14, P < 0.05). Na⁺/Ca²⁺ exchanger (NCX) function was increased since half-decay time of C[Ca²⁺]_i was decreased 16.22 ± 1.36% in response to apelin. Sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity was also increased by apelin. These responses can be partially or completely blocked by chelerythrine chloride, a PKC inhibitor. In addition, to confirm our data, we used indo-1 as another Ca²⁺ indicator and rapid cooling as another way to measure SR Ca²⁺ content, and we observed similar results. So we conclude that apelin has a positive inotropic effect on isolated myocytes, and increased amplitude of E[Ca²⁺]_i is at least partially involved in the mechanism. NCX function and SERCA activity are increased by apelin, and the SR Ca²⁺ content is decreased by apelin during twitches. PKC played an important role in these signaling mechanisms.

excitation-contraction coupling; intracellular calcium concentration; positive inotropic action; protein kinase C; sarcoplasmic reticulum

Contraction of the cardiac myocyte is initiated by Ca²⁺ influx via the L-type Ca²⁺ current (I_Ca,L), which subsequently triggers a much larger Ca²⁺ release from the sarcoplasmic reticulum (SR) by Ca²⁺-induced Ca²⁺ release (16). Membrane depolarization during an action potential activates L-type Ca²⁺ channels (LTCCs). These channels open transiently and serve as the major pathway for Ca²⁺ entry into myocytes. The subsequent local elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i) activates Ca²⁺-release channels [ryanodine receptors (RyR)] in the SR, providing the Ca²⁺ required for contraction. This whole sequence of events determines the excitation-contraction (EC) coupling. We hypothesized that apelin had a positive inotropic effect on isolated myocytes and that the mechanism may involve the enhancement of amplitude of [Ca²⁺]_i transient during EC coupling.

The aims of our study were to test whether apelin has positive inotropic action on individual myocytes and to evaluate the effect of apelin on EC coupling by direct measurement of the cell contraction; the amplitude and the time course of the electrical-stimulated [Ca²⁺]_i (E[Ca²⁺]_i) transients, sarco(endo) plasmic reticulum Ca²⁺-ATPase (SERCA) activity, and caffeine-induced [Ca²⁺]_i (C[Ca²⁺]_i), and rapid cooling (RC) of the cell to calculate the SR Ca²⁺ content in isolated myocytes. Since PKC has been shown to alter Ca²⁺ transients, contractility, and SR Ca²⁺ handling (7, 21, 33, 43) and is involved in the apelin effect on myocardium-positive inotropic action (46), we also studied the possible role of PKC in these effects.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Myocyte isolation. Myocytes were isolated using standard procedures (55). Briefly, hearts were rapidly excised from adult male Sprague-Dawley rats (250–280 g) under pentobarbital sodium anesthesia (140 mg/kg ip), mounted on a Langendorff perfusion apparatus, and perfused with Ca²⁺-free Tyrode solution containing (in mmol/l) 143.0 NaCl, 5.4 KCl, 0.5 MgCl₂, 0.3 NaH₂PO₄, 5.0 HEPES, and 5.0 glucose (pH 7.4, equilibrated with O₂) for 5 min at 37°C. The heart was then perfused with the same solution containing 0.4 g/l collagenase II (283 U/mg; Worthington Biochemical; Lakewood, NJ) and 0.7 g/l bovine serum albumin until it became flaccid (10 to 30 min). After perfusion with Ca²⁺-free Tyrode solution for 5 min to remove enzymes, the digested tissues were separated and filtered. The resultant cell suspension was rinsed several times with progressive increases in [Ca²⁺] to 1.8 mmol/l. The experimental protocol was approved by the China Institutional Ethics Review Committee for Animal Experimentation.

Measurement of intracellular Ca²⁺. We used two different Ca²⁺ indicators and protocols to measure intracellular Ca²⁺. First, freshly isolated myocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 min before they were loaded with fura-2 AM (0.5 µmol/l; Alexis Biochemicals, San Diego, CA) for 30 min. All fura-2 experiments were conducted at room temperature. The free [Ca²⁺]_i of loaded cardiac myocytes was measured as the fluorescence ratio (360 to 380 nm) (55). The myocytes were superfused with Ca²⁺-containing Tyrode solution and then with 1 nM (17, 46) apelin-16 (Phoenix) or apelin + 5 µM chelerythrine chloride (CHE, PKC inhibitor). The E[Ca²⁺]_i transients were elicited by field stimulation (0.5 Hz). The myocytes were allowed to equilibrate for about 10 min after added apelin or apelin + CHE. The application of CHE alone without apelin had no effect on the myocytes (Table 1). Second, the cells were loaded during 30 min with 5 µmol/l indo-1 AM and 0.01% pluronic before each experiment (1, 2). Myocytes were washed twice with fresh HEPES solution (without albumin) and kept for 15 min to complete deesterification of indo-1 AM. The loaded myocytes were attached to a poly-D-lysine (0.1 g/l)-treated coverslip placed on a microscope stage of an inverted fluorescence microscope (Olympus, Tokyo, Japan). A temperature-controlled perfusion chamber with two needles at opposite sides for perfusion purposes (height, 0.4 mm; diameter, 10 mm; and volume, 30 µl) was tightly positioned over the coverslip. The contents of the chamber could be replaced within 100 ms. The field stimulation (0.5 Hz) was elicited, and indo-1 fluorescence was measured in a dual emission mode, excited at 340 nm with xenon lamp flashes (100 W). Dual wavelength emission was measured at 410 and 516 nm, respectively. Fluorescence signals were recorded at a sample rate of 1 kHz. We calculated free cellular [Ca²⁺]_i according to the ratio equation (11, 18, 19): [Ca²⁺]_i = K_d·β(R – R_min)/(R_max – R) nmol/l, where R is the ratio of fluorescence signals at 410 and 516 nm, R_max is the ratio at saturating [Ca²⁺], R_min is the ratio at zero [Ca²⁺], K_d was 250 nmol/l, and β is 2.1. In indo-1 AM-loaded myocytes, the final free indo-1 is compartmentalized mainly in the cytosol and mitochondria (45). To calculate "true" cytosolic free [Ca²⁺]_i from measured overall values, both the fraction of mitochondrially localized indo-1 and mitochondrial free [Ca²⁺]_i were measured (32). "True" free cytosolic [Ca²⁺] was calculated from measured cellular [Ca²⁺]_i, correcting measured values for compartmentalization and mitochondrial [Ca²⁺]. Total cytosolic Ca²⁺ content (cytosolically bound plus free cytosolic Ca²⁺) was calculated using "true" free [Ca²⁺]_i and data on cytosolic Ca²⁺ buffer capacity in the literature (24). In the second protocol, the same cell was superfused in turn with Ca²⁺-containing Tyrode solution (control solution) and then with 1 nM apelin-16 and then washout with control solution and lastly with apelin + 5 µM CHE. We tested three cells to find out that, repeatedly, the application of electric stimulation and RC would not affect the baseline of the Ca²⁺ activity (data not shown).

Measurement of caffeine contracture. C[Ca²⁺]_i transient amplitude was used as a measurement of SR Ca²⁺ content (22, 51, 54). In the presence of caffeine, the elimination of Ca²⁺ is mainly attributed to NCX. The time course of the decay of C[Ca²⁺]_i transient is used as an index of NCX function (38, 39, 51). A rapid application of caffeine to the myocyte was performed just 2 s after the stimulation stopped to measure SR Ca²⁺ content during electrical stimulation twitches.

RC used to estimate SR Ca²⁺ content. SR Ca²⁺ content was measured by another method since this was the most important observation in the current study. RC causes complete depletion of Ca²⁺ from SR- and Ca²⁺-released remains confined to the cytoplasm (5, 6). RC was carried out by a rapid superfusion with ice-cold Tyrode solution of the same composition; a low temperature (0–1°C) was reached within 200 ms. SR Ca²⁺ content was calculated from the increase of total cytosolic Ca²⁺ following RC and a fractional SR volume of 10%. RC was applied 2 s after the cessation of stimulation.

Measurement of Ca²⁺-ATPase activity. SR was prepared according to the methods of Jones (25) as modified by Kodavanti et al. (27) and Pande et al. (36). The myocytes were put in homogenizing medium containing (in mmol/l) 50.0 Na₂HPO₄, 10.0 Na₂EDTA, and 25.0 NaF (pH 7.4). The minced ventricle tissue was placed in 10 ml of ice-cold homogenizing medium and homogenated three times. An additional 5 ml of homogenizing medium was added, and the homogenate was sedimented twice for 20 min at 14,000 g at 4°C. The supernatant was recentrifuged at 45,000 g for 30 min. The pellet obtained after this centrifugation, consisting of crude membrane vesicles (SR), was suspended in storage buffer containing 30.0 mmol/l histidine, 0.25 mol/l sucrose, 10.0 mmol/l EDTA, and 10.0 mmol/l NaF (pH 7.4) to a final concentration of 30–40 mg/ml protein and stored at –80°C until used. The activity of Ca²⁺-ATPase was determined with a kit (Jiancheng, Nanjing, China) by measuring the inorganic phosphate (P_i) liberated from ATP hydrolysis (27). Ca²⁺-ATPase activity was assayed in a medium containing (in mmol/l) 50.0 histidine, 3.0 MgCl₂, 100.0 KCl, 5.0 sodium azide, and 3.0 ATP and 50.0 µmol/l CaCl₂ (pH 7.0) (36). Cardiac SR membranes were added to the reaction mixture at a final concentration of 20–25 µg of protein per milliliter, preincubated for 10 min at 37°C, and the reaction was initiated by the addition of ATP. The ATP hydrolysis that occurred in the absence of Ca²⁺ (1 mmol/l EGTA) was subtracted to determine the activity of Ca²⁺-stimulated ATPase. Ouabain was added fresh to a final concentration of 1 mmol/l in the media, which remained unchanged throughout the incubation. Mitochondrial contamination was assessed by determining the activity of azide-sensitive ATPase, that is, that activity inhibited by 5 mmol/l sodium azide (29).

Statistical analysis. All values are presented as means ± SE. Differences were compared by one-way ANOVA followed by Student-Newman-Keuls test as appropriate. P < 0.05 was considered to be statistically significant; n represents the number of cardiomyocytes or hearts. All of the statistical tests were carried out with SPSS (v. 11.0).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Single-myocyte sarcomere fractional shortening (FS, normalized by resting cell sarcomere length) and E[Ca²⁺]_i transients were measured simultaneously when myocyte twitches were under steady state at 0.5 Hz stimulation. Cell [Ca²⁺]_i transient and shortening data (Table 1) showed in Figs. 2 and 3 are normalized by data under the control condition (myocytes in Tyrode solution). Apelin condition means the addition of 1 nM apelin-16 in Tyrode solution, and apelin + CHE condition means the addition of 1 nM apelin-16 and 5 µM CHE in Tyrode solution. In the indo-1 protocol, the diastolic "true" free [Ca²⁺]_i was not different in four solutions (Fig. 4B).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Effect of apelin on electrical-induced [Ca2+]i (E[Ca2+]i) transients and sarcomere shortening of myocytes gotten from fura-2 protocol. A: amplitude of E[Ca2+]i transients in apelin group is larger than in control but less than in apelin + CHE group. B: sarcomere fractional shortening (FS) during twitches. The enhancement of apelin effect on FS is partially abolished by CHE. C: effect of apelin and CHE on time to peak (TTP) of E[Ca2+]i. D: effect of apelin on half-decay time (T50) of E[Ca2+]i transient. Apelin (n = 14), apelin + CHE condition (n = 14), control condition group (n = 28). Data are normalized by the same cell in control condition and shown as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. apelin + CHE.

View larger version (27K): [in this window] [in a new window]   Fig. 3. A: original traces of C[Ca2+]i transient. The mark line means the presence of 10 mM caffeine. B: sarcoplasmic reticulum (SR) Ca2+ content assessed by amplitude of C[Ca2+]i transient after stimulation stopped (apelin, n = 14; apelin + CHE, n = 14; and control, n = 28). C: T50 of C[Ca2+]i transient was reduced significantly in apelin group. Data are normalized by control and shown as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. apelin + CHE.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Intracellular Ca2+ measured by indo-1 and rapid cooling (RC) protocol. A: total cytosolic Ca2+ change traces calculated from measured data (see MATERIALS AND METHODS). The traces were from the same cell that was perfused in turn with control, apelin, washout (control), and apelin + CHE solution. 1°C, RC solution presented 2 s after stimulation stopped. B: diastolic cytosolic "true" free Ca2+ is not different in the 4 solutions. C: data from the total cytosolic Ca2+ change amplitude induced by field stimulation (white bars) and RC (black bars) in the 4 solutions. The change amplitude of the total cytosolic Ca2+ induced by RC was used to calculate the SR Ca2+ content (in mmol/l). Values are shown as means ± SE; n = 5. *P < 0.05 vs. control; #P < 0.05 vs. apelin + CHE.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Original traces gotten from fura-2 protocol show the effect of apelin on isolated myocytes. A: original traces of Ca2+ transient induced by electrical stimulation. B: original traces of apelin effect on sarcomere contraction of single cardiac myocytes. [Ca2+]i, intracellular Ca2+ concentration; CHE, chelerythrine chloride; F360/F380, fluorescence ratio of 360 to 380 nm.

Effect of apelin on amplitude and time course of C[Ca²⁺]_i transients. During a normal twitch, the amplitude of the E[Ca²⁺]_i transient highly depends on the SR Ca²⁺ content. To assess the SR Ca²⁺ content, we used rapid caffeine application. Caffeine keeps the RyR open, and all Ca²⁺ stored in the SR is released. Application of caffeine caused a rapid increase of [Ca²⁺]_i (C[Ca²⁺]_i transient) as a result of SR Ca²⁺-release (Fig. 3A). The amplitude of C[Ca²⁺]_i transient is an index of Ca²⁺ content in SR (22). It was surprising to find that SR Ca²⁺ content estimated by amplitude of C[Ca²⁺]_i transient, showed in Fig. 3, A and B, was slightly but significantly (P < 0.05) reduced (8.41 ± 0.92%) by apelin compared with control (n = 14). Thus we subsequently analyzed the relationship between the amplitude of E[Ca²⁺]_i and the SR Ca²⁺ content (C[Ca²⁺]_i). The ratio of E[Ca²⁺]_i to C[Ca²⁺]_i (E[Ca²⁺]_i/C[Ca²⁺]_i) in the presence of apelin was largely enhanced versus control (0.325 ± 0.003 vs. 0.204 ± 0.002, n = 14, P < 0.05). In our study, apelin reduced the SR Ca²⁺ content but increased the amplitude of E[Ca²⁺]_i transient during twitches. During a relaxation course after a twitch, close to 7% of [Ca²⁺]_i is pumped out of the cell via NCX (4, 44), but in the presence of caffeine, the RyR of SR remains open continuously and the extrusion of [Ca²⁺]_i from the cytoplasm across the sarcolemma is mainly through the NCX. To assess NCX function, we measured the T₅₀ of C[Ca²⁺]_i transient. Figure 3C showed that apelin reduced T₅₀ of C[Ca²⁺]_i transients by 16.22 ± 1.36% versus control condition (n = 14, P < 0.05). The decrease of C[Ca²⁺]_i T₅₀ reflects the increase of NCX function.

Effect of apelin on RC-induced total cytosolic Ca²⁺ change. The same cell in different solutions got different total cytosolic Ca²⁺ change induced by RC 2 s after the stimulation stopped (shown in Fig. 4A). The total cytosolic Ca²⁺ was calculated from measured free [Ca²⁺]_i, correcting the mitochondrially localized indo-1 and mitochondrial free [Ca²⁺]_i and then, in addition, the reported cytosolic Ca²⁺ buffer capacity. The SR volume was about 10% of the cell, so 10 times of the RC-induced total cytosolic Ca²⁺ content was the SR Ca²⁺ content (Fig. 4C). Apelin decreased the SR Ca²⁺ content versus control (n = 5, P < 0.05, Fig. 4C), similarly to the result gotten from the caffeine experiment.

Effect of apelin on SERCA. T₅₀ of E[Ca²⁺]_i transient reduced by apelin means that apelin probably increased the SERCA activity. To study whether SERCA is involved in the apelin-induced improvement of contractile function, the present study examined Ca²⁺-ATPase activity by an optical assay in crude SR extracted from myocytes. As shown in Fig. 5, apelin increased the SR Ca²⁺-ATPase activity in myocytes versus control (n = 7, P < 0.05), but the effect was abolished by CHE completely (n = 7, P < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity was increased by apelin, and CHE blocked the effect. Values are shown as means ± SE; n = 7. *P < 0.05 vs. control; #P < 0.05 vs. apelin + CHE.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Consistent with previous study in whole isolated rat heart (46), our results showed that apelin had positive inotropic action on individual rat myocytes. With the positive inotropic action, apelin increased the amplitude of Ca²⁺ transients and decreased the SR Ca²⁺ content during electrical stimulations. Furthermore, we found that PKC played an important role in the mechanism of the action of apelin on isolated rat myocytes.

Contractions and amplitude of Ca²⁺ transients. In perfused rat hearts, apelin has a positive inotropic effect (46). In our study, we demonstrated that apelin also has a positive inotropic effect in isolated single rat cardiomyocytes at concentrations of 1 nM (Fig. 1B). The positive inotropic effect of apelin could in principle be due to Ca²⁺ availability and/or Ca²⁺ responsiveness of the myofilaments. The amplitude of [Ca²⁺]_i transient has been shown to be increased in heart muscles with apelin, and it was concluded that the increase in force development was due to increased Ca²⁺ availability rather than changes in myofilament Ca²⁺ responsiveness (12). In our study, we used two different methods to find that the increased shortening induced by apelin was accompanied by the increased amplitude of Ca²⁺ transients. Furthermore, we found that the effect of apelin on the amplitude of E[Ca²⁺]_i transients was not decreased but that the sarcomere FS was reduced in the presence of CHE, which provided evidence that apelin increases the Ca²⁺ transient through a PKC-independent way and also gave the feasibility that there are other PKC-dependent mechanisms, reported as NHE activity and intracellular PH value (17), involved in the positive inotropic effect of apelin. These findings could explain the result that the suppression of PKC with staurosporine and GF-109203X markedly attenuates the apelin inotropic effect (46). Therefore, we suggest that apelin acts on myocyte contraction through at least two different ways: 1) a PKC-independent way with a Ca²⁺ transient increase and 2) a PKC-dependent way without a Ca²⁺ transient increase.

Analysis of the time course of E[Ca²⁺]_i transients. The Ca²⁺ release by the SR is graded (8): the rate and the amount of Ca²⁺ released from the SR is variable and depends on the Ca²⁺ current flowing through the I_Ca,L LTCC, the amount of Ca²⁺ stored in the SR, and the availability of RyR for activation. The previous result has demonstrated that I_Ca,L via LTCC is not changed by apelin (46). In the present study, we showed that SR Ca²⁺ content was decreased but that the TTP of E[Ca²⁺]_i transient was reduced. Thus the reasonable explanation is that apelin increased the availability of RyR for activation. The speed of Ca²⁺ release via RyR of SR was significantly faster in the apelin group, which presented the possibility of increasing the amplitude of E[Ca²⁺]_i transients even when the SR Ca²⁺ content was reduced. The mechanism of this effect is not clear, but our data show that this effect is PKC independent (Fig. 2C). T₅₀ of E[Ca²⁺]_i transient was decreased by apelin, indicating that SERCA activity was most likely enhanced. We know that the function of SERCA is regulated by phospholamban (PLB) and that phosphorylation of PLB causes a dissociation of PLB from SERCA, allowing for faster rates of SR Ca²⁺ uptake and relaxation and enhanced contractility (53). In our study, we do not know whether the PKC phosphorylation of the PLB mechanism was involved, but we tested SERCA activity by an optical assay, and we found that apelin does increase the SERCA activity through a PKC-dependent pathway.

Apelin on the SR Ca²⁺ content. A rapid application of caffeine induces a [Ca²⁺]_i transient in myocytes, the amplitude of C[Ca²⁺]_i represents the SR Ca²⁺ content, and the T₅₀ of C[Ca²⁺]_i is an index of NCX function. We showed that apelin caused a decrease in SR Ca²⁺ content (Fig. 3, A and B) PKC dependently, and we confirmed this with the RC of myocytes (Fig. 4). The decrease of SR Ca²⁺ content seems incompatible with the increase of SERCA, but we find that apelin increases the NCX activity, too (Fig. 3C). A plausible explanation is that the increase of NCX activity is greater than that of SERCA activity, so the enhanced activity of NCX extrudes more Ca²⁺ and a smaller fraction of the Ca²⁺ will be uptaken by the SERCA during the relaxation result in the decrease of SR Ca²⁺ content in presence of apelin. In our study, apelin caused a 30% and 16% reduction in E[Ca²⁺]_i T₅₀ and C[Ca²⁺]_i T₅₀, respectively. The T₅₀ of E[Ca²⁺]_i mainly reflects the SERCA activity because the efficiency of SERCA is much higher than that of NCX (4, 44), and in the rat cardiomyocytes, immediately after contraction, most (>90%) of the Ca²⁺ is taken back to the SR via SERCA. However, when caffeine is present, SR cannot hold Ca²⁺ anymore; the extrusion of cytosolic Ca²⁺ is mainly trough NCX, so the speed of decline in C[Ca²⁺]_i is slower than in E[Ca²⁺]_i. The 30% reduction in E[Ca²⁺]_i T₅₀ suggested that the SERCA activity was enhanced, which was confirmed by the measurement of SERCA activity directly, but the 16% reduction in C[Ca²⁺]_i suggested that NCX function increased. In the present study, the measurement of the NCX activity is not direct. For this reason, future studies are needed to test the NCX activity more directly and try to give solid evidence to confirm the relationship between apelin and NCX.

Role of PKC in the effect of apelin. The activation of PKC has many cardiomyocyte effects (10, 35, 48, 57), but the mechanisms by which PKC mediates its effects are not fully understood. In our study, apelin decreased the SR Ca²⁺ content in a PKC-dependent fashion, but the positive effect of apelin on the amplitude of E[Ca²⁺]_i is likely PKC independent. In addition, the increasing effect of CHE on E[Ca²⁺]_i transient showed in Fig. 2A may be secondary to the larger SR Ca²⁺ content showed in Fig. 3B, since the amplitude of E[Ca²⁺]_i/C[Ca²⁺]_i was not different between apelin group and apelin + CHE group. The possible explanation, shown in Fig. 6, is that apelin not only has an increase effect on E[Ca²⁺]_i but also has a decrease effect on SR Ca²⁺ content, and the decrease effect on SR Ca²⁺ content could decrease the E[Ca²⁺]_i indirectly. When we used apelin + CHE solution, the PKC-independent increase effect on E[Ca²⁺]_i was still working, but the decrease effect on SR Ca²⁺ was stopped, so the SR Ca²⁺ content was increased. And consequently, CHE further enhances the E[Ca²⁺]_i. The PKC-involved enhancement effect on Ca²⁺ extrusion via NCX, shown in Fig. 3C, could explain the SR Ca²⁺ content decrease, but the decreased T₅₀ of E[Ca²⁺]_i, shown in Fig. 2D, by apelin is mainly due to the enhancement of SERCA activity. PKC inhibitor CHE partially blocked the positive inotropic action of apelin (shown in Fig. 2B) without decreasing the amplitude of E[Ca²⁺]_i, which indicates that a PKC-dependent mechanism was partially involved in the positive intropic action of apelin. The possible pathways of these effects are shown in Fig. 6.

View larger version (6K): [in this window] [in a new window]   Fig. 6. The possible pathway of the action of apelin on cell contractions. "1", PKC dependent; "2", PKC independent; "3", reported (Ref. 17) but not confirmed directly in our study whether through the pH, Na+/H+ exchanger (NHE), and myofilament response. During "2", there is no direct evidence to show whether and how apelin acts on ryanodine receptor (RyR). If CHE stopped the "1" pathway, the effect of apelin of decreasing SR Ca2+ content will disappear and the apelin increase of Ca2+ transient will be greater than without CHE, but "3" will be stopped at the same time, so the cell contraction is downregulated lastly. The "1" pathway that decreases SR Ca2+ content will indirectly decrease the E[Ca2+]i and counteract the increased E[Ca2+]i effect of the "2" pathway. The counteraction effect will disappear when blockade "1" with CHE, so CHE could further increase the apelin effect on E[Ca2+]i. NCX, Na+/Ca2+ exchanger.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H.-C. Wang, Dept. of Cardiology, Xi-Jing Hospital, 17 W. Chang-Le Rd., Xian, PR China, 710032 (e-mail: wanghc{at}fmmu.edu.cn)

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.

^* C. Wang and J.-F. Du contributed equally to this work.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Baartscheer A, Schumacher CA, Belterman CN, Coronel R, Fiolet JW. SR calcium handling and calcium after-transients in a rabbit model of heart failure. Cardiovasc Res 58: 99–108, 2003.[Abstract/Free Full Text]
2. Baartscheer A, Schumacher CA, Fiolet JW. SR calcium depletion following reversal of the Na⁺/Ca²⁺-exchanger in rat ventricular myocytes. J Mol Cell Cardiol 32: 1025–1037, 2000.[CrossRef][Web of Science][Medline]
3. Baker DL, Hashimoto K, Grupp IL, Ji Y, Reed T, Loukianov E, Grupp G, Bhagwhat A, Hoit B, Walsh R, Marban E, Periasamy M. Targeted overexpression of the sarcoplasmic reticulum Ca²⁺-ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res 83: 1205–1214, 1998.[Abstract/Free Full Text]
4. Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res 87: 275–281, 2000.[Free Full Text]
5. Bers DM. Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures. Am J Physiol Cell Physiol 253: C408–C415, 1987.[Abstract/Free Full Text]
6. Bers DM, Bridge JH, Spitzer KW. Intracellular Ca²⁺ transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol 417: 537–553, 1989.[Abstract/Free Full Text]
7. Capogrossi MC, Kaku T, Filburn CR, Pelto DJ, Hansford RG, Spurgeon HA, Lakatta EG. Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca²⁺ in adult rat cardiac myocytes. Circ Res 66: 1143–1155, 1990.[Abstract/Free Full Text]
8. Carmeliet E. Intracellular Ca²⁺ concentration and rate adaptation of the cardiac action potential. Cell Calcium 35: 557–573, 2004.[CrossRef][Web of Science][Medline]
9. Cayabyab M, Hinuma S, Farzan M, Choe H, Fukusumi S, Kitada C, Nishizawa N, Hosoya M, Nishimura O, Messele T, Pollakis G, Goudsmit J, Fujino M, Sodroski J. Apelin, the natural ligand of the orphan seven-transmembrane receptor APJ, inhibits human immunodeficiency virus type 1 entry. J Virol 74: 11972–11976, 2000.[Abstract/Free Full Text]
10. Chen W, London R, Murphy E, Steenbergen C. Regulation of the Ca²⁺ gradient across the sarcoplasmic reticulum in perfused rabbit heart. A 19F nuclear magnetic resonance study. Circ Res 83: 898–907, 1998.[Abstract/Free Full Text]
11. Cobbold PH, Rink TJ. Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem J 248: 313–328, 1987.[Web of Science][Medline]
12. Dai T, Ramirez-Correa G, Gao WD. Apelin increases contractility in failing cardiac muscle. Eur J Pharmacol 553: 222–228, 2006.[CrossRef][Web of Science][Medline]
13. De Falco M, De Luca L, Onori N, Cavallotti I, Artigiano F, Esposito V, De Luca B, Laforgia V, Groeger AM, De Luca A. Apelin expression in normal human tissues. In Vivo 16: 333–336, 2002.[Web of Science][Medline]
14. De Mota N, Lenkei Z, Llorens-Cortes C. Cloning, pharmacological characterization and brain distribution of the rat apelin receptor. Neuroendocrinology 72: 400–407, 2000.[CrossRef][Web of Science][Medline]
15. De Mota N, Reaux-Le Goazigo A., El Messari S, Chartrel N, Roesch D, Dujardin C, Kordon C, Vaudry H, Moos F, Llorens-Cortes C. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release. Proc Natl Acad Sci USA 101: 10464–10469, 2004.[Abstract/Free Full Text]
16. Fabiato A, Fabiato F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat, and frog hearts and from fetal and new-born rat ventricles. Ann NY Acad Sci 307: 491–522, 1978.[Medline]
17. Farkasfalvi K, Stagg MA, Coppen SR, Siedlecka U, Lee J, Soppa GK, Marczin N, Szokodi I, Yacoub MH, Terracciano CM. Direct effects of apelin on cardiomyocyte contractility and electrophysiology. Biochem Biophys Res Commun 357: 889–895, 2007.[CrossRef][Web of Science][Medline]
18. Fiolet JW, Baartscheer A, Schumacher CA. Intracellular [Ca²⁺] and O₂ after manipulation of the free-energy of the Na⁺/Ca²⁺-exchanger in isolated rat ventricular myocytes. J Mol Cell Cardiol 27: 1513–1525, 1995.[CrossRef][Web of Science][Medline]
19. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
20. Habata Y, Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Hinuma S, Kitada C, Nishizawa N, Murosaki S, Kurokawa T, Onda H, Tatemoto K, Fujino M. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta 1452: 25–35, 1999.[Medline]
21. Han R, Bakker AJ. The effect of chelerythrine on depolarization-induced force responses in skinned fast skeletal muscle fibres of the rat. Br J Pharmacol 138: 417–426, 2003.[CrossRef][Web of Science][Medline]
22. Ho JC, Wu S, Kam KW, Sham JS, Wong TM. Effects of pharmacological preconditioning with U50488H on calcium homeostasis in rat ventricular myocytes subjected to metabolic inhibition and anoxia. Br J Pharmacol 137: 739–748, 2002.[CrossRef][Web of Science][Medline]
23. Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma S, Kitada C, Honda S, Kurokawa T, Onda H, Nishimura O, Fujino M. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem 275: 21061–21067, 2000.[Abstract/Free Full Text]
24. Hove-Madsen L, Bers DM. Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes. Am J Physiol Cell Physiol 264: C677–C686, 1993.[Abstract/Free Full Text]
25. Jones LR. Mg²⁺ and ATP effects on K⁺ activation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum. Biochim Biophys Acta 557: 230–242, 1979.[Medline]
26. Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, Nishizawa N, Kitada C, Onda H, Nishimura O, Fujino M. Molecular properties of apelin: tissue distribution and receptor binding. Biochim Biophys Acta 1538: 162–171, 2001.[Medline]
27. Kodavanti PR, Cameron JA, Yallapragada PR, Desaiah D. Effect of chlordecone (Kepone) on calcium transport mechanisms in rat heart sarcoplasmic reticulum. Pharmacol Toxicol 67: 227–234, 1990.[Web of Science][Medline]
28. Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond DH, George SR, O'Dowd BF. Characterization of apelin, the ligand for the APJ receptor. J Neurochem 74: 34–41, 2000.[CrossRef][Web of Science][Medline]
29. Lindemann JP, Jones LR, Hathaway DR, Henry BG, Watanabe AM. β-Adrenergic stimulation of phospholamban phosphorylation and Ca²⁺-ATPase activity in guinea pig ventricles. J Biol Chem 258: 464–471, 1983.[Free Full Text]
30. Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, Periasamy M. Enhanced myocardial contractility and increased Ca²⁺ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase. Circ Res 83: 889–897, 1998.[Abstract/Free Full Text]
31. Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn KY, Lawrie KW, Hervieu G, Riley G, Bolaky JE, Herrity NC, Murdock P, Darker JG. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 84: 1162–1172, 2003.[CrossRef][Web of Science][Medline]
32. Miyata H, Silverman HS, Sollott SJ, Lakatta EG, Stern MD, Hansford RG. Measurement of mitochondrial free Ca²⁺ concentration in living single rat cardiac myocytes. Am J Physiol Heart Circ Physiol 261: H1123–H1134, 1991.[Abstract/Free Full Text]
33. Nicolas JM, Renard-Rooney DC, Thomas AP. Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes. J Mol Cell Cardiol 30: 2591–2604, 1998.[CrossRef][Web of Science][Medline]
34. O'Carroll AM, Selby TL, Palkovits M, Lolait SJ. Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues. Biochim Biophys Acta 1492: 72–80, 2000.[Medline]
35. Osada M, Netticadan T, Tamura K, Dhalla NS. Modification of ischemia-reperfusion-induced changes in cardiac sarcoplasmic reticulum by preconditioning. Am J Physiol Heart Circ Physiol 274: H2025–H2034, 1998.[Abstract/Free Full Text]
36. Pande M, Cameron JA, Vig PJ, Desaiah D. Phencyclidine block of Ca²⁺ ATPase in rat heart sarcoplasmic reticulum. Toxicology 129: 95–102, 1998.[CrossRef][Web of Science][Medline]
37. Pei JM, Yu XC, Fung ML, Zhou JJ, Cheung CS, Wong NS, Leung MP, Wong TM. Impaired G_s and adenylyl cyclase cause β-adrenoceptor desensitization in chronically hypoxic rat hearts. Am J Physiol Cell Physiol 279: C1455–C1463, 2000.[Abstract/Free Full Text]
38. Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca²⁺ handling and sarcoplasmic reticulum Ca²⁺ content in isolated failing and nonfailing human myocardium. Circ Res 85: 38–46, 1999.[Abstract/Free Full Text]
39. Quinn FR, Currie S, Duncan AM, Miller S, Sayeed R, Cobbe SM, Smith GL. Myocardial infarction causes increased expression but decreased activity of the myocardial Na⁺-Ca²⁺ exchanger in the rabbit. J Physiol 553: 229–242, 2003.[Abstract/Free Full Text]
40. Reaux A, De Mota N, Skultetyova I, Lenkei Z, El Messari S, Gallatz K, Corvol P, Palkovits M, Llorens-Cortes C. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain. J Neurochem 77: 1085–1096, 2001.[CrossRef][Web of Science][Medline]
41. Reaux A, Gallatz K, Palkovits M, Llorens-Cortes C. Distribution of apelin-synthesizing neurons in the adult rat brain. Neuroscience 113: 653–662, 2002.[CrossRef][Web of Science][Medline]
42. Reaux-Le Goazigo A, Morinville A, Burlet A, Llorens-Cortes C, Beaudet A. Dehydration-induced cross-regulation of apelin and vasopressin immunoreactivity levels in magnocellular hypothalamic neurons. Endocrinology 145: 4392–4400, 2004.[Abstract/Free Full Text]
43. Rogers TB, Gaa ST, Massey C, Dosemeci A. Protein kinase C inhibits Ca²⁺ accumulation in cardiac sarcoplasmic reticulum. J Biol Chem 265: 4302–4308, 1990.[Abstract/Free Full Text]
44. Shigekawa M, Iwamoto T. Cardiac Na⁺-Ca²⁺ exchange: molecular and pharmacological aspects. Circ Res 88: 864–876, 2001.[Abstract/Free Full Text]
45. Spurgeon HA, duBell WH, Stern MD, Sollott SJ, Ziman BD, Silverman HS, Capogrossi MC, Talo A, Lakatta EG. Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol 447: 83–102, 1992.[Abstract/Free Full Text]
46. Szokodi I, Tavi P, Foldes G, Voutilainen-Myllyla S, Ilves M, Tokola H, Pikkarainen S, Piuhola J, Rysa J, Toth M, Ruskoaho H. Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility. Circ Res 91: 434–440, 2002.[Abstract/Free Full Text]
47. Taheri S, Murphy K, Cohen M, Sujkovic E, Kennedy A, Dhillo W, Dakin C, Sajedi A, Ghatei M, Bloom S. The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats. Biochem Biophys Res Commun 291: 1208–1212, 2002.[CrossRef][Web of Science][Medline]
48. Tani M, Asakura Y, Hasegawa H, Shinmura K, Ebihara Y, Nakamura Y. Effect of preconditioning on ryanodine-sensitive Ca²⁺ release from sarcoplasmic reticulum of rat heart. Am J Physiol Heart Circ Physiol 271: H876–H881, 1996.[Abstract/Free Full Text]
49. Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, Kawamata Y, Fukusumi S, Hinuma S, Kitada C, Kurokawa T, Onda H, Fujino M. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun 251: 471–476, 1998.[CrossRef][Web of Science][Medline]
50. Tatemoto K, Takayama K, Zou MX, Kumaki I, Zhang W, Kumano K, Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism. Regul Pept 99: 87–92, 2001.[CrossRef][Web of Science][Medline]
51. Terracciano CM, MacLeod KT. Effects of acidosis on Na⁺/Ca²⁺ exchange and consequences for relaxation in guinea pig cardiac myocytes. Am J Physiol Heart Circ Physiol 267: H477–H487, 1994.[Abstract/Free Full Text]
52. Xiao RP, Zhang SJ, Chakir K, Avdonin P, Zhu W, Bond RA, Balke CW, Lakatta EG, Cheng H. Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)—but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts. Circulation 108: 1633–1639, 2003.[Abstract/Free Full Text]
53. Yamamura K, Steenbergen C, Murphy E. Protein kinase C and preconditioning: role of the sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol 289: H2484–H2490, 2005.[Abstract/Free Full Text]
54. Yeung HM, Kravtsov GM, Ng KM, Wong TM, Fung ML. Chronic intermittent hypoxia alters Ca²⁺ handling in rat cardiomyocytes by augmented Na⁺/Ca²⁺ exchange and ryanodine receptor activities in ischemia-reperfusion. Am J Physiol Cell Physiol 292: C2046–C2056, 2007.[Abstract/Free Full Text]
55. Yu J, Zhang HF, Wu F, Li QX, Ma H, Guo WY, Wang HC, Gao F. Insulin improves cardiomyocyte contractile function through enhancement of SERCA2a activity in simulated ischemia/reperfusion. Acta Pharmacol Sin 27: 919–926, 2006.[CrossRef][Web of Science][Medline]
56. Zou MX, Liu HY, Haraguchi Y, Soda Y, Tatemoto K, Hoshino H. Apelin peptides block the entry of human immunodeficiency virus (HIV). FEBS Lett 473: 15–18, 2000.[CrossRef][Web of Science][Medline]
57. Zucchi R, Ronca-Testoni S, Yu G, Galbani P, Ronca G, Mariani M. Postischemic changes in cardiac sarcoplasmic reticulum Ca²⁺ channels. A possible mechanism of ischemic preconditioning. Circ Res 76: 1049–1056, 1995.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: 294/6/H2540    most recent 00046.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, C. Articles by Wang, H.-C. Search for Related Content PubMed PubMed Citation Articles by Wang, C. Articles by Wang, H.-C.