AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 294: H736-H749, 2008. First published November 16, 2007; doi:10.1152/ajpheart.00316.2007
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Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes

Lee K. Landeen,1 Dorothy A. Dederko,1 Colleen S. Kondo,2 Betty S. Hu,1 Nakon Aroonsakool,1 Jason H. Haga,1 and Wayne R. Giles1,2

1Department of Bioengineering, University of California, San Diego, La Jolla, California; and 2Department of Kinesiology, University of Calgary, Calgary, Alberta, Canada

Submitted 13 March 2007 ; accepted in final form 12 November 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K+ current (IKACh) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by ~25% (vs. untreated controls) in field-stimulated myocytes. S1P1 was shown to be involved by using the S1P1-selective agonist SEW2871 on myocytes isolated from S1P3-null mice. However, in these myocytes, S1P3 can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P1 antagonist VPC23019. Since S1P1 activates Gi exclusively, whereas S1P3 activates both Gi and Gq, these results strongly implicate the involvement of mainly Gi. Additional experiments using the IKACh blocker tertiapin demonstrated that IKACh can contribute to the negative inotropy following S1P activation of S1P1 (perhaps through Giβ{gamma} subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by IKACh) can reduce L-type calcium current and thus can decrease the intracellular Ca2+ concentration ([Ca2+]i) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via Gi to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca2+]i; and 2) a second pathway that acts via Gi to activate IKACh and reduce APD. This decrease in APD is expected to decrease Ca2+ influx and reduce [Ca2+]i and myocyte contractility.

calcium; contraction; cell shortening; inhibitory G protein; acetylcholine-sensitive potassium; myocyte

SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that binds with high affinity to five distinct G-coupled protein receptor isoforms (S1P1–5). S1P1 has been detected in abundance in neonatal rat cardiomyocytes (39). In these cells, exposure to S1P (500 nM) results in an initial negative inotropic effect (reduction of systolic calcium). However, this may be followed by calcium overload (increased diastolic calcium) and then a cessation of contractility. In isolated atrial myocytes, S1P has been shown to activate a weakly inwardly rectifying potassium (K+) current. This K+ conductance is very similar to the K+ current activated by ACh (IKACh). Activation of this current can shorten action potential (AP) duration (APD), inhibit excitability by reducing Na+ current, and increase the minimum stimulus current needed to generate an AP (20, 35, 43). In mammalian in vivo experiments, S1P activation can induce a transient bradycardia (47).

S1P receptor activation, through coupling to G proteins and generation of both direct and indirect (second-messenger mediated) responses, can also activate a number of distinct intracellular signaling pathways and result in altered calcium handling. S1P1 couples exclusively via Gi, whereas S1P2 and S1P3 have been shown to couple to Gi, Gq, or G12/13 (51). Gi can inhibit cAMP-mediated increases in L-type calcium channel current (ICaL), whereas Gq can cause intracellular calcium concentration ([Ca2+]i) to increase through PLC pathways.

Excitation-contraction coupling is tightly regulated in the adult cardiac myocyte and strongly depends on [Ca2+]i during systole and diastole (4, 5). Several well-studied signaling pathways can modulate Ca2+ handling within the myocyte. Many of these act through G-protein-coupled receptors. For example, isoproterenol (Iso) is a ligand for β1-adrenergic receptor (β1-AR), a G-protein-coupled receptor that couples to Gs. Carbamoylcholine or carbachol (CCh) is a ligand for the M2 muscarinic receptor (M2R), which couples to Gi. Activation of β1-AR by Iso stimulates cAMP formation, which activates specific protein kinase A (PKA) isoforms. PKA, in turn, phosphorylates L-type Ca2+ channels, and this augments entry of Ca2+ from the extracellular space. PKA also contributes to an increased rate of relaxation lusitropy by phosphorylating phospholamban (PLB), thus accelerating the turnover rate of a Ca2+ pump, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2A (4). Conversely, activation of M2R by CCh inhibits cAMP formation, reduces cAMP-mediated calcium increase, and diminishes contraction strength (3, 26, 52).

The objectives of the present experiments were as follows: 1) to study the possibility that S1P can modulate ventricular myocyte contractility; and 2) to gain information concerning signaling pathways and ion channels that are involved following activation of S1P receptors. We have concentrated on known or putative Gi-coupled pathways, since Gq coupling appears to be less prominent in the adult mouse heart (18). Our results demonstrate that S1P1 is the most highly expressed S1P receptor subtype on adult mouse ventricular myocytes. S1P1-mediated effects of S1P were, therefore, investigated in detail in an attempt to define its role in negative inotropic actions by S1P.


   METHODS
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RESULTS
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Isolation of ventricular myocytes. Hearts from heparinized adult C57Bl/6 or S1P3-null mice (28) were isolated under isoflurane anesthesia in accordance with protocols approved by the Institutional Animal Care and Use Committee at UCSD. Guidelines for the Care and Use of Laboratory Animals (copyright 1996) were adhered to. Each isolated heart was first rinsed in Ca2+-free Tyrode solution (130 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, 5.5 mM glucose; pH 7.2–7.4) and then cannulated through the aorta for retrograde perfusion using a Langendorff apparatus. The heart was enzymatically digested for 8 min in Tyrode solution containing 50 µM CaCl2 and Liberase Blenzyme IV (125 µg/ml; Roche, Indianapolis, IN). Liberase is a mixture of purified Clostridium histolyticum collagenase isoforms I and II (~3 Wunsch units/mg protein) and Bacillus thermoproteolyticus thermolysin (~2,500 caseinase units/mg protein) and contains very low levels of endotoxin (<5 enzyme units/mg). After enzymatic digestion, the ventricles were removed, gently separated, and then shaken while being superfused with a modified Krebs-Henseleit solution (100 mM potassium-glutamate, 10 mM potassium-aspartate, 25 mM KCl, 10 mM KH2PO4, 2 mM MgSO4, 0.5 mM EGTA, 5 mM HEPES, 1% BSA, 20 mM glucose, 20 mM taurine, and 5 mM creatine; pH 7.2–7.4). This procedure consistently liberated populations of individual myocytes from the myocardium. These were separated and purified by filtering through 100-µm nylon mesh and mild centrifugation (1 min, 50 g). Pelleted myocytes were then placed in Krebs-Henseleit solution and stored at 4–8°C (for ≥1 h) until use, some 1–6 h later.

Measurements of unloaded myocyte shortening. All cell shortening measurements were conducted at room temperature (22–25°C) to maximize their stability under superfused, as opposed to perfused, conditions. Populations of isolated ventricular myocytes were placed at low density on laminin-coated (25 µg/ml) glass slides, allowed to adhere, and then superfused (0.5 ml/min) in a flow chamber with Ca2+-free Tyrode solution for ~5 min. Next, Tyrode solution containing 1 mM CaCl2 (or 1.8 mM in selected experiments) was applied for ~5 min, after which field stimulation was initiated using a custom-designed electrode (Crescent Electronics, Sandy, UT) (Fig. 1). This electrode consisted of two Ag-AgCl wires: one housed within a Tyrode solution-containing micropipette tip and the other located externally and ~1 mm from the tip opening. This terminal advance localized the stimulation to only those myocytes in its immediate vicinity. In addition, it was constructed so that it could be positioned quickly and reliably using a standard micromanipulator. The cells were stimulated with 5-ms current pulses (~10–20% above threshold) at 2 Hz.


Figure 1
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  		Fig. 1. Myocyte stimulating electrode. The electrode used for field-stimulating adult mouse ventricular myocytes is shown. This custom-designed electrode consisted of two wire leads threaded into a hollow metallic shaft, affixed to a standard three-axis micromanipulator. The positive lead was connected to the Ag-AgCl wire of an Axopatch electrode (arrow) and centered through an adjoining plastic micropipette tip filled with Tyrode solution. The ground lead (arrowhead; shown in inset) consisted of an insulated Ag-AgCl wire, coiled around the outside of the electrode/micropipette, with the insulation removed near the tip. Bar = 35 mm. Inset bar = 3.5 mm.

 
Unloaded shortening of individual myocytes was recorded using an edge-detection device (model VED-205, Crescent Electronics) coupled to a charge-coupled device camera (model FTM800NH/HGI, Phillips, Atlanta, GA). The output of this edge detector was linear over a range of 0–110 µm (R2 = 1.0). Signals were acquired through a Digidata 1322A 16-bit (Axon Instruments, Sunnyvale, CA) or MP150 (BioPac, Goleta, CA) data-acquisition system using either Clampex software (Axon; 10-kHz sampling rate) or Acknowledge software (BioPac; 2-kHz sampling rate), respectively. After ~5-min equilibration under field stimulation, a control data set of measurements was collected. Only those myocytes that had rod-shaped morphology, clearly defined sarcomere striations, and stable and steady-state cell shortening (≥5% of resting length, for ≥5 min) were included in this data analysis. Responses to Iso (100 nM) and to CCh (100 nM) (52) were obtained from selected myocytes, followed by washout and then repeated application of the experimental compound. Data collected using the BioPac system was first analyzed by the Acknowledge software to signal average and determine maximal shortening values. Contraction/relaxation rates were then obtained using Kaleidograph graphing software (Synergy Software, Reading, PA). Data collected by the Digidata system were analyzed by Clampfit software (Axon) and then saved as text files. Text files were imported into MatLab 7 (MathWorks, Natick, MA) and analyzed using a program specifically written to calculate contractility parameters as a function of time (contraction/relaxation rates and maximum cell shortening).

Pharmacological agents. S1P (Avanti Polar Lipids, Alabaster, AL) was dissolved in dimethyl sulfoxide (DMSO) to make a 1–10 mM stock solution and was applied at 1–1,000 nM. Before each experiment, Iso (Sigma-Aldrich, St. Louis, MO) and CCh (Sigma) were each dissolved in Ca2+-Tyrode solution at 500 µM and then diluted to a final 50–200 nM concentration. SEW2871 (Calbiochem, La Jolla, CA) was dissolved in DMSO to make a 1 mM stock solution and applied at 10–1,000 nM. VPC23019 (Avanti Polar Lipids) was dissolved in DMSO/1 N HCl (95:5 vol/vol) to make a 1 mM stock solution and applied at 100 nM. Tertiapin (Peptides International, Louisville, KY) was dissolved in water to make a 100 µM stock solution and was applied at 100 nM. Verapamil (Calbiochem) was dissolved in Ca2+-Tyrode solution to make a 2 mM stock solution and applied at 5 µM. All stock reagents (except verapamil) were kept as frozen (–20°C) aliquots before use. These agents were then diluted to the selected final concentrations in Ca2+-containing Tyrode solution.

PCR. Populations of myocytes were lysed, and total RNA was isolated on silica gel columns using RNeasy kits (QIAGEN, Valencia, CA) and eluted in water. cDNA was reverse transcribed from mRNA by oligo(dT) priming and Omniscript reverse transcriptase (QIAGEN). Expression levels of S1P receptors were performed using Applied Biosystems (Foster City, CA) TaqMan primer/probe sets as follows: S1P1 (Mm00514644_m1), S1P2 (Mm01177794_m1), S1P3 (Mm00515669_m1), and GAPDH (Mm99999915_g1). Primer sets for genotyping S1P3-null mice were as previously reported (28).

Mathematical modeling. A published mathematical model of the mouse ventricular myocyte AP (7) provided the starting point for the computations included in this paper. This model was validated against experimental data by our laboratory (D. A. Dederko and W. R. Giles, unpublished observations). Sets of computations using this model were useful for revealing and beginning to understand the relationship between APD, the size and time course of the ICaL, and the [Ca2+]i transient. The output from these simulations was utilized to provide plausible explanation for experimental data describing the effects of S1P on cell shortening in these ventricular myocytes.

Statistical analyses. Data from sets of experiments involving myocytes from multiple animals were averaged. These results are presented as arithmetic means and their SDs. Results were analyzed using ANOVA analyses (with Bonferroni t-test post-hoc testing), a Student's t-test, or paired t-test as appropriate, using statistical software packages (GraphPad Prism, San Diego, CA). Significant differences between data sets were accepted when the means of the results were P < 0.05.


   RESULTS
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S1P receptor isoforms expressed in ventricular myocytes. Isolated ventricular myocytes from adult mouse myocardium were analyzed by quantitative PCR (qPCR) to determine the relative expression levels of S1P receptor isoforms 1–3 (Fig. 2A). These results demonstrated that the S1P1 receptor isoform is expressed at much higher levels than S1P2 or S1P3 in both the left and the right ventricles. Additional measurements showed that S1P3 expression levels were approximately one-half of those observed for S1P1, in both the right and left ventricles. In contrast, S1P2 expression levels were very much lower and, in some cases, not detectable. Myocytes from both ventricles showed a similar pattern of S1P receptor expression. As a result, in subsequent experiments, tissues from both the left and right ventricles were pooled.


Figure 2
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  		Fig. 2. Sphingosine-1-phosphate (S1P) receptor subtype mRNA expression levels in adult mouse ventricular myocytes based on quantitative PCR analyses. A: expression levels of S1P1 in myocytes isolated from the left ventricle (LV) and right ventricle (RV) of adult wild-type (WT) mice were both approximately twofold larger than expression levels of S1P3. In contrast, the expression levels of S1P2 were much smaller (near detection limits) in these myocytes. Values are means ± SD (n = 3). B: genotyping of S1P3-null mice demonstrated a single band (predicted 380 kDa) of the deleted S1P3– allele after amplification by PCR, separation by agarose electrophoresis, and visualization by ethidium bromide. Amplified gene products from WT mice demonstrated a single band (predicted 130 kDa) for the intact S1P3+ allele. No template controls (NTC), in which cDNA was omitted from the amplification procedure, are absent of either gene product. This demonstrates that the primer sets utilized do not generate nonspecific amplification products. MW Std, molecular weight standard. C: analysis of S1P3-null myocytes demonstrate a total absence of S1P3 mRNA expression and extremely low levels of expression for S1P2. Values are means ± SD (n = 3).

 
In an important subset of experiments that examined the effects of S1P receptor subtype contributions to myocyte contractility, S1P3-null mice were utilized (28). Myocytes isolated from S1P3-null mice were also characterized for S1P receptor expression levels. As shown in Fig. 2B (and as expected), S1P3-positive alleles were absent in S1P3-null mice, and expression of S1P3 mRNA was undetectable by qPCR (Fig. 2C). S1P2 expression levels did not increase in the S1P3-null mice and were negligible, as observed in measurements from wild-type mice. Thus S1P3-null mice essentially have only S1P1 expression. This provides a very useful tool for characterizing S1P signaling pathways via S1P1. In summary, based on qPCR, S1P1 and S1P3 are the dominant isoforms in adult mouse ventricular myocytes. The contributions of these receptor subtypes and associated signaling pathways in modulating myocyte contractility formed the focus of the remaining experimental and theoretical work in this paper.

ICaL and G-protein-coupled regulation of cell shortening. In murine ventricular myocytes, contractility is strongly regulated by Ca2+ release from the sarcoplasmic reticulum (SR) (32). The ICaL, however, is an essential trigger signal for SR release. The magnitude of ICaL is dependent on the number of L-type calcium channels that open, their open time, and their conductance. Since conductance is a function of the concentration of permeant ions, experiments were conducted to validate the relationship between extracellular calcium concentration ([Ca2+]o) and cell shortening in adult mouse ventricular myocytes. Parameters describing peak cell shortening and rates of shortening and relaxation in field-stimulated (2 Hz, 22–25°C) myocytes were recorded during exposure to 1.8 mM [Ca2+]o in the superfusate. Next, [Ca2+]o in the superfusate was lowered to 1.0 mM, and cell shortening was recorded again. After subsequent exposure to 0.5 mM [Ca2+]o, the cells were returned to 1.8 mM [Ca2+]o in the superfusate. The results of this regimen are presented in Fig. 3A. It is evident that changes in [Ca2+]o cause significant changes in the maximum cell shortening. This relationship is convincingly demonstrated by the strong correlation coefficients that were calculated between cell shortening and [Ca2+]o or Ca2+ electrochemical equilibrium potential (using the Nernst equation) (R2 > 0.99 and R2 > 0.93, respectively). A similar pattern of results was obtained using myocytes isolated from S1P3-null mice (data not shown).


Figure 3
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  		Fig. 3. Extracellular Ca2+ levels and L-type Ca2+ channel fluxes affect cell shortening in adult mouse ventricular myocytes. A: measurements of peak (maximum) cell shortening made on individual, field-stimulated (2 Hz, 22–25°C) WT ventricular myocytes that were superfused with selected concentrations of extracellular calcium ([Ca2+]o). As expected, the maximum unloaded myocyte shortening was strongly correlated with [Ca2+]o (R2 > 0.99). B: measurements of peak (maximum) cell shortening made on individual, field-stimulated (2 Hz, 22–25°C) WT myocytes superfused with 1.8 mM [Ca2+]o in the presence or absence of verapamil (5 µM), a blocker of L-type Ca2+ current (ICaL). Representative records of myocyte shortening are shown.

 
To negatively regulate Ca2+ flux through L-type channels, experiments were conducted using the L-type calcium channel blocker verapamil (24). In these experiments, cell shortening measurements were made before and after application of verapamil (5 µM) and also after a 10-min washout period. Verapamil effectively blocked ICaL and resulted in a significant decrease in myocyte shortening (Fig. 3B). Washout recovered ~70–80% of peak contraction values. Similar results were obtained using myocytes isolated from S1P3-null mice (data not shown).

The pharmacological analogs to the autonomic transmitters norepinephrine and ACh, Iso and CCh (respectively), are well-known inotropic agents (52). Both can modulate cAMP concentrations by changing adenylyl cyclase (AC) turnover rates. Stimulation of the β1-AR by Iso activates Gs to induce cAMP formation and thus positively regulate ICaL. Agonists that activate Gi-coupled receptors, in contrast, inhibit cAMP formation. Accordingly, these compounds were used as positive and negative controls, respectively, to alter cell shortening in isolated myocytes. Compared with control values (100%), Iso (100 nM) increased maximal cell shortening by 100 ± 24.5%, whereas CCh (100 nM) decreased cell shortening by 20.7 ± 4.6% when applied to naive wild-type myocytes (Fig. 4A). Each of these differences was statistically significant (P < 0.001). Increases in cell shortening by Iso and decreases in cell shortening by CCh in S1P3-null mice (Fig. 4B) were similar to those results observed with wild-type mice, indicating that AC-cAMP signaling was intact and functioning normally in both genotypes, which formed the basis for the experimental design. Furthermore, significant differences between Iso and S1P (P < 0.001) and significant changes in rates of relaxation were noted between myocytes treated with Iso vs. either CCh or S1P (P < 0.001). This latter effect, a decrease in the rate of relaxation in the presence of either CCh or S1P, was observed in both myocytes from wild-type and S1P3-null mice (Fig. 4, C and D, respectively). The increased rate of relaxation upon β-adrenergic stimulation agrees with experimental and modeling data regarding the involvement of PLB/SERCA (27). The antagonistic action of S1P or CCh has not been reported previously.


Figure 4
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  		Fig. 4. Comparison of positive and negative inotropic agents in adult mouse ventricular myocytes. Measurements of peak (maximum) cell shortening (A and B) and relaxation rates (C and D) were made on individual, field-stimulated (2 Hz, 22–25°C) WT (A and C) or S1P3-null (B and D) myocytes, before and after application of isoproterenol (Iso; 100 nM), carbachol (CCh; 100 nM), or S1P (100 nM). A: note that the negative inotropic effect of S1P (n = 21) was not significantly different than CCh (n = 5), but that both of these data sets were significantly different than those for Iso (n = 9) (*P < 0.001). Additionally, each treatment was significantly different than its paired control values (P < 0.01 for Iso, P < 0.03 for CCh, and P < 0.0001 for S1P). B: in S1P3-null preparations, S1P (n = 10) results were not significantly different than those for CCh (n = 7), but results for both S1P and CCh were significantly different than the Iso (n = 3) results (*P < 0.001). Additionally, each treatment was significantly different than its paired control values (P < 0.003 for Iso, P < 0.009 for CCh, and P < 0.006 for S1P). C: relaxation rates in the presence of Iso were significantly different than either S1P or CCh (*P < 0.001). Specifically, Iso increased the rate of relaxation, whereas this was decreased for both S1P and CCh. D: relaxation rates in S1P3-null preparations showed similar differences between Iso and either S1P or CCh (*P < 0.001). Values are means ± SD.

 
Taken together, these results demonstrate that the rate and extent of shortening in adult mouse ventricular myocytes is regulated by both [Ca2+]o and L-type calcium channel activity. Furthermore, these findings establish that G-protein activation (e.g., via Iso-Gs) and inhibition (e.g., via CCh-Gi) of L-type calcium channels are fully functional in these ventricular myocytes from the adult mouse heart.

S1P attenuates cell shortening in ventricular myocytes. When S1P was added to field-stimulated (2 Hz, 22–25°C), isolated adult mouse ventricular myocytes, significant decreases in cell shortening were observed. This pattern of results was similar to the negative inotropy due to CCh application (Fig. 4). The average change in myocyte length in cells during control recordings was 8.3 ± 2.8 µm (Fig. 5, A and B). After application of S1P (100 nM), the average change in myocyte length during a field-stimulated contraction decreased to 6.0 ± 2.2 µm. Thus S1P-treated myocytes contract at 72.0 ± 15.6% of control values. This change was consistent and highly statistically significant (P < 0.0001). Very similar differences were also noted between control and S1P-treated myocytes when measured in terms of peak shortening (P < 0.001) or relaxation rates (P < 0.001) (Fig. 5C).


Figure 5
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  		Fig. 5. S1P decreases cell shortening in adult mouse ventricular myocytes. Measurements of peak (maximum) myocyte shortening were made on individual, field-stimulated (2 Hz, 22–25°C) myocytes before and after application of S1P (100 nM). A: representative myocyte shortening record for each experimental condition. B: myocyte shortening data. *P < 0.0001. C: contraction and relaxation rates. #P < 0.001. Values are means ± SD (n = 21).

 
S1P1 has a reported Kd for S1P of 8 nM (31). In experiments in which S1P was applied at lower concentrations (e.g., 10 nM), a similar pattern of findings was observed. Cell length changes averaged 77.0 ± 13.1% of control values (n = 4, data not shown). Thus S1P at physiological concentrations can result in significantly reduced cell contractility.

We next examined whether S1P might act similarly to the well-known accentuated antagonistic phenomenon for β-adrenergic vs. muscarinic effects in the ventricular myocardium (52). In these studies (Fig. 6A), myocytes were first exposed to Iso. After an approximate twofold increase in myocyte shortening developed, the cells were superfused with Iso and S1P (100 nM). After addition of S1P, cell shortening was significantly reduced (P < 0.003) (Fig. 6B), indicating that S1P opposes the positive inotropic effects of Iso. A similar pattern of results was observed when CCh was coadministered after prechallenging the cells with Iso (P < 0.05) (Fig. 6C). These results suggest that a signaling pattern via Gi is involved in the negative inotropy observed after administration of S1P (18).


Figure 6
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  		Fig. 6. S1P and CCh can significantly reduce Iso effects on cell shortening in adult mouse ventricular myocytes. Measurements of peak (maximum) myocyte shortening were made on individual, field-stimulated (2 Hz, 22–25°C) myocytes. Iso (100 nM) was applied before superfusion with S1P (100 nM) in the continued presence of Iso. A: representative time course of cell shortening measurements. B: summarized S1P data. *P < 0.003. C: comparative measurements were also made using CCh (100 nM) instead of S1P. *P < 0.04. Values are means ± SD; n = 10 (B) and 4 (C).

 
S1P can attenuate cell shortening by activating S1P3 receptors. As noted, S1P3 receptor expression is ~50% of that of S1P1 in wild-type adult mouse ventricular myocytes (see Fig. 2A). It was, therefore, of interest to examine the effects of S1P on myocyte contractility, which are mediated by S1P3 receptors. Unfortunately, the use of genetically manipulated mice is not an option for these types of studies, as knockout of the S1P1 receptor subtype is embryonically lethal (34). As an alternative approach, the population of S1P1 receptors on the ventricular myocyte was blocked by pretreatment with the antagonist VPC23019 (12). Subsequent application of S1P in these myocytes from wild-type mice was used to study the contributions of S1P3 to cell shortening.

We first evaluated the effectiveness of VPC23019 as an S1P1 antagonist. S1P3-null mice effectively have receptor isoforms limited only to S1P1. This is due to knockout of S1P3 and negligible expression of S1P2 (see Fig. 2C). If pretreatment with VPC23019 effectively prevents S1P from activating S1P1 receptors, then cell shortening in myocytes isolated from S1P3-null mice, which have been pretreated with VPC23019, should be very similar, before and after application of S1P. This expectation was borne out: S1P (100 nM) application to S1P3-null myocytes pretreated with VPC23019 (100 nM, 5 min) resulted in average cell shortening waveforms that were 95.5 ± 5.0% of that of control values (Fig. 7A), and this small apparent decrease was not significant (P = 0.243, n = 4). Therefore, VPC23019 appears to be an effective antagonist against S1P1. On this basis, it was used as a pharmacological tool with which to study signaling effects in myocytes from wild-type mice due to S1P3.


Figure 7
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  		Fig. 7. S1P can decrease cell shortening via activation of S1P3 receptors. A: myocytes from the ventricles of adult S1P3-null mice were pretreated with the S1P1-selective antagonist VPC23019 (VPC; 100 nM), and cell shortening measurements were made. S1P (100 nM) was then administered in the continued presence of VPC (S1P+VPC). Note that the values obtained after application of S1P were not significantly different than the values obtained before S1P application (95.5 ± 5.0% of VPC-only values) in these S1P3-null myocytes. B: next, myocytes from WT mice were pretreated with VPC as described above. In contrast to results from S1P3-null myocytes, peak (maximum) cell shortening values in WT myocytes after application of S1P were (nearly) significantly different than before S1P application (*P = 0.0547). Values are means ± SD; n = 4 (A) and 3 (B).

 
Pretreatment of wild-type myocytes with VPC23019 (100 nM) did not affect control contractions. Maximal cell shortening was 5.8 ± 0.3 µm, which fell within the normal range of control values observed during these experiments. Application of S1P (100 nM) in the continued presence of VPC23019 reduced cell shortening to 3.9 ± 0.4 µm, or 32.5% below control values (P = 0.0547, n = 3) (Fig. 7B). These results suggest that activation of S1P3 may reduce myocyte contractility.

S1P1 receptor and Gi involvement in S1P-activated negative inotropy. It is known that both S1P1 and S1P3 can signal through Gi. However, S1P3 can also activate other G-proteins. To begin to evaluate the relative contributions of S1P1 exclusively (and Gi partially) to decreased myocyte contractility, initial experiments were conducted using myocytes isolated from S1P3-null mice. As mentioned, S1P3-null mice have receptor isoforms limited to S1P1 almost exclusively, due to knockout of S1P3 and negligible expression of S1P2. When myocytes isolated from these genetically altered mice were superfused with S1P (100 nM), a decrease in cell shortening of 23.53% compared with untreated controls was observed (Fig. 4B).

Further confirmation of S1P1 involvement in S1P-activated negative inotropy was performed using the S1P1-selective agonist SEW2871. SEW2871 has an EC50 that is ~10-fold higher than S1P (47). SEW2871 was, therefore, evaluated at concentrations similar to, and also 10-fold higher to, those observed with S1P to have a negative inotropic effect. When SEW2871 (0.1 µM) was applied to myocytes, cell shortening was reduced to 72.0 ± 19.4% with respect to control values (Fig. 8). SEW2871 administered at 1 µM was also effective at reducing cell shortening and produced a similar steady-state negative inotropy (77.0 ± 15.7% of control values). The results of SEW2871 at 0.1 or 1 µM were not significantly different from each other or from the values obtained after S1P treatment. Moreover, additional experiments were conducted to demonstrate the ability of Iso to overcome the reduced contractility caused by SEW2871. In these experiments, myocytes were pretreated with SEW2871 (10.11 ± 4.54 µm maximum cell shortening), then superfused with both SEW2871 and Iso (16.55 ± 4.89 µm maximum cell shortening) (Fig. 8B). There was a significant increase in maximum cell shortening when Iso was coadministered in this way (P < 0.007).


Figure 8
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  		Fig. 8. S1P decreases myocyte shortening via activation of S1P1 receptors. A: myocyte shortening measurements were made after application of S1P (n = 21) or the S1P1-selective agonist SEW2871 (SEW) at concentrations of either 0.1 µM (n = 5) or 1 µM (n = 6) on separate populations of field-stimulated (2 Hz, 22–25°C) WT myocytes. Note that the mean of the data set for each treatment was significantly different than its paired control values (P < 0.0001 for S1P, P < 0.02 for SEW 0.1 µM, and P < 0.003 for SEW 1 µM). However, no significant differences between data sets could be demonstrated. These results strongly suggest that these effects were mediated through S1P1 and imply an involvement of a Gi-mediated mechanism. B: myocyte shortening measurements made after application of SEW (0.1 µM) and subsequent coapplication of Iso (100 nM) demonstrate that Iso is able to counter the negative inotropic effects of SEW in field-stimulated (2 Hz, 22–25°C) WT myocytes. n = 4, *P < 0.007. Values are means ± SD.

 
Collectively, the results obtained using SEW2871 and S1P3-null mice demonstrate that S1P1 is an important and significant signaling pathway for S1P-induced negative inotropy. Furthermore, since S1P1 signals exclusively through Gi, these results strongly suggest that Gi is involved in S1P-mediated reductions in cell shortening.

Does activation of IKACh via Gi contribute to S1P-mediated cell shortening? S1P is known to activate IKACh. This outward K+ current would be expected to reduce APD in mammalian myocytes (43). In an effort to reveal the contributions of IKACh to the observed decrease in cell shortening following application of S1P, a series of experiments were conducted using the IKACh-blocking peptide tertiapin (13, 25). To further restrict the potential effects to S1P1 and Gi, myocytes isolated from S1P3-null mice were used in these experiments. After pretreatment with tertiapin (100 nM) for 5 min, control data were recorded. Next, S1P (100 nM) was applied (in the continued presence of tertiapin), and the pattern of field-stimulus-induced shortening was recorded. As shown in Fig. 9A, following blockage of IKACh with tertiapin, S1P activation of S1P1 caused a 12.5% reduction in maximal shortening compared with control values. This reduction in the extent of cell shortening was significant (P < 0.007), as was the decrease in contraction rate (P < 0.008) (Fig. 9B). In additional studies, tertiapin reduced the changes from control values observed in S1P3-null myocytes in the presence of S1P. Specifically, these changes were less (12.5%) than those changes observed without blockade (i.e., S1P alone; 23.5%; Fig. 9C). These results suggest that, at the concentrations of agents used, S1P induces activation of a K+ current (IKACh) that contributes significantly to the negative inotropy following S1P activation of S1P1.


Figure 9
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  		Fig. 9. S1P activation of S1P1 receptors activates a K+ current activated by ACh (IKACh) and modulates myocyte shortening. After pretreatment with the IKACh-selective blocking peptide tertiapin (Tert; 100 nM), measurements were made on individual, field-stimulated (2 Hz, 22–25°C) myocytes from S1P3-null mice in the continued presence of Tert (100 nM), either with or without concurrent application of 100 nM S1P (Tert+S1P). A: peak (maximum) myocyte shortening. n = 4, *P < 0.007. B: corresponding contraction and relaxation rates. n = 4, #P < 0.008. C: comparison of Tert and non-Tert components of S1P1-mediated negative inotropic effects of S1P. Note that Tert-treatment (block of IKACh) only partially restores the S1P1-mediated negative inotropic effects of S1P. D: effect of coadministration of Tert (100 nM) on myocytes from WT mice prechallenged with Iso (100 nM) and CCh (100 nM). n = 4, *P < 0.05. Values are means ± SD.

 
Further experiments were conducted to determine the response of tertiapin during β-AR challenge with Iso. We had previously demonstrated using myocytes isolated from wild-type mice that CCh decreases cell shortening when coadministered with Iso (see Fig. 6C). When tertiapin was coadministered with Iso and CCh, however, myocyte shortening increased (Fig. 9D), and this response was found to be reversible upon washout of tertiapin. These additional results suggest that the ability of tertiapin to alleviate muscarinic-mediated decreases in myocyte shortening (by blocking IKACh) is independent of β-AR agonist.

Exploring the relationship between APD and alterations in [Ca2+]i with mathematical modeling. It is well established that S1P can shorten APD in mammalian atrial myocytes (43). Furthermore, it is also known that shortening APD in rat ventricular myocytes can reduce [Ca2+]i and decrease cell shortening (8). In an attempt to relate these experimental findings to our experimental results, we have utilized a mathematical model of the mouse ventricular myocyte AP and [Ca2+]i homeostatis (7). Our working hypothesis was that activation of an outward K+ current by S1P-mediated IKACh would enhance repolarization, leading to decreased APD. The reduction in APD would produce smaller [Ca2+]i transients and lowered [Ca2+]i. An important second tenant was that these changes, in addition to inhibition of ICaL, would contribute to the functional changes observed in myocyte cell shortening.

We proceeded as follows. Upon stimulation, the "myocyte" in this model will initiate an AP that quite closely resembles experimentally derived AP (using whole cell clamp techniques). To begin to evaluate the utility of this approach, we first assessed the robustness of this model by evaluating an in silico myocyte that had been exposed to a simulated Iso challenge. Iso acts via Gs to significantly increase ICaL (41). Accordingly, we increased ICaL in the model parameter set to twice that of control values (since we observed nearly a twofold increase in cell shortening with Iso; see Figs. 4 and 6) and then stimulated the cell to initiate an AP. As shown in Fig. 10, A–C, increasing ICaL resulted in an enhancement of the "plateau" phase and altered the rate of repolarization while also increasing APD. Conversely, when ICaL was decreased to 75% of that of control values (to simulate reductions in cell shortening upon treatment with CCh or S1P) (see Fig. 4), APD was decreased (Fig. 10, A–C).


Figure 10
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  		Fig. 10. Integration of S1P actions of ventricular myocytes using mathematical modeling. Mathematical simulation of the transmembrane ionic currents, intracellular Ca2+ concentration ([Ca2+]i) transient, and action potential waveform [action potential duration (APD)] under control conditions and then following parameter changes, which were made to mimic the following: 1) Iso application (a twofold increase in ICaL), or 2) S1P or CCh (a 25% decrease in ICaL). A: control or treatment simulations. B and C: changes in the action potential waveform measured at 50% repolarization (APD50) or at 90% repolarization (APD90), respectively. D and E: changes in ICaL and [Ca2+]i, respectively, as a consequence of maneuvers 1 or 2 described above.

 
The next set of computations attempted to evaluate changes in ICaL and [Ca2+]i in the in silico myocyte. As expected, Iso yielded an approximately twofold increase in ICaL, and CCh/S1P was ~25% less than control (Fig. 10D). This pattern of computational results provided an initial positive indication of the applicability and validity of our model. In addition, it illustrated the relationship between APD and ICaL under these conditions. Peak [Ca2+]i values also were increased and decreased, respectively, with Iso and CCh/S1P in this model (Fig. 10E).

Having established this relationship between APD, ICaL, and [Ca2+]i with this model of the mouse ventricular myocyte and using simulated positive and negative inotropic agents, we next explored how changes in APD could affect Ca2+ homeostasis and ultimately cell contraction, as judged by the simulated [Ca2+]i transient. Triangular voltage-clamp commands (which simulate the shape of the AP in actual adult mouse ventricular myocytes) were used to "voltage clamp" the in silico myocyte. The rate of repolarization was adjusted to generate APDs between 40 and 5 ms (Fig. 11A). These ranges of APD were used because they approximated experimental data, which has been shown to affect calcium (8) and to be involved with S1P application (43), respectively. We first established that a single voltage command or a train of 10 voltage commands showed no time-dependent changes in either APD, ICaL, or [Ca2+]i (data not shown). Thereafter, a single voltage clamp command waveform was "applied". Decreasing the APD from 40 to 5 ms resulted in marked changes in ICaL (Fig. 11B) and [Ca2+]i (data not shown).


Figure 11
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  		Fig. 11. Illustration of the association between APD and ICaL based on a mathematical model of the mouse ventricular myocyte action potential and Ca2+ homeostasis. In these simulations, the action potential waveform (APD) was approximated as a right triangle, and its duration was varied systematically to 1) mimic the known effect of S1P on APD, and 2) explore the resulting changes in ICaL and [Ca2+]i. A: APD were varied between 40 and 5 ms using a triangular-shaped action potential (holding potential of –80 mV, depolarization step to +40 mV, then ramped to –80 mV over APD). B: normalized ICaL time course for each APD. C: relationship between the integral of ICaL and APD.

 
In ventricular myocytes, contraction strength is modulated by changes in ICaL or other maneuvers, which alter amplitude or duration of the Ca2+ transient (3, 8). We, therefore, explored the relationship between APD and ICaL. In these simulations, the relationships between S1P, APD, ICaL, [Ca2+]i, and cell shortening in the adult mouse ventricular myocyte were explored. As shown in Fig. 11C, there was a strong correlation (R2 > 0.92) between APD and total Ca2+ flux through L-type Ca2+ channels. Thus any alteration in transmembrane Ca2+ flux or change in APD can have significant effects on myocyte Ca2+ homeostasis in the adult mouse ventricle.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Summary of major findings. These experiments were done to characterize the major changes in, and then explore, key aspects of the signaling pathways involved in S1P-mediated negative inotropic effects in adult mouse ventricular myocytes. qPCR demonstrated that adult mouse ventricular myocytes express primarily the S1P1 and S1P3 receptor isoforms. Both of these receptor isoforms are coupled to Gi. Gi-coupled receptors, for example M2R, which are responsive to CCh, are known to inhibit cAMP-mediated ICaL, to reduce cell shortening, and to shorten APD (11, 18, 20, 54). An M2-Gi effect in the sinoatrial node and the atrium also involves activation of an inwardly rectifying K+ current: IKACh. We, therefore, investigated responses in cell shortening in field-stimulated adult mouse ventricular myocytes after application of S1P.

S1P (10–100 nM) caused significant and maintained decrease in peak cell shortening within several minutes after application. Furthermore, the well-known positive inotropy due to Iso application was reduced markedly by S1P, similar to the pattern of results observed by others with CCh (11). By using the S1P1-selective agonist SEW2871, as well as the S1P1-selective antagonist VPC23019, and/or myocytes isolated from S1P3-null mice, we have demonstrated that the S1P-mediated negative inotropic effect is mediated by activation of both S1P1 and S1P3 receptor isoforms. In addition, the effects of the IKACh-selective inhibitor tertiapin (25) demonstrated that activation of IKACh can also contribute to the negative inotropy due to S1P.

In an attempt to integrate these findings and relate them to contemporary knowledge and understanding of the ionic mechanism(s) of the mouse ventricular AP, we have adapted and utilized a mathematical model of the adult mouse ventricular myocyte. Results of the positive and negative inotropic responses to Iso and CCh/S1P, respectively, in myocytes were used to adjust the initial ICaL model parameters for ICaL, Ca2+ release, and Ca2+ binding to replicate the observed changes in cell shortening. These simulations suggest that Iso can lengthen APD at a fixed heart rate. Similar findings have been reported in models of the rat ventricular myocyte (48). In contrast, CCh/S1P results in a decrease in APD. These changes in APD can result in either an increased or decreased calcium influx through ICaL. When APD waveforms were approximated in "voltage clamp" so that APD at 70% repolarization was between 40 and 5 ms, a strong correlation between APD and Ca2+ influx due to ICaL was observed. In the ventricular myocardium, ICaL is mainly responsible for initiating calcium-induced calcium release (CICR) and thus altering [Ca2+]i. Furthermore, [Ca2+]i and contraction are tightly coupled in mammalian myocytes (3, 4). In summary, therefore, our experimental results and related mathematical modeling strongly suggest that shortening of the APD through direct activation of IKACh or through Gi-coupled inhibition of ICaL are plausible and additive mechanisms that regulate negative inotropy by S1P in adult mouse ventricular myocytes.

Modulation of [Ca2+]i and inotropy in the mouse myocyte. In the adult mouse myocyte, [Ca2+]i is tightly regulated. Peak [Ca2+]i levels are coupled to contraction and can regulate CICR, which is initiated through activation of L-type Ca2+ channels, a corresponding Ca2+ influx, and activation of the SR release mechanism (3, 4). It takes very few active Ca2+ channels to open to activate a ryanodine receptor cluster and initiate CICR. Hence, this positive feedback system is highly efficient. We observed a strong correlation between [Ca2+]o and contraction strength (see Fig. 3A).

During diastole, Ca2+ is removed from the myoplasm. Much of this is mediated by SERCA, sarcolemmal Ca2+ pumps, mitrochondrial Ca2+ pumps, or Na+/Ca2+ exchange (NCX) mechanisms in the sarcolemma. In the mouse ventricle, it has been suggested that ~90% of the increase in [Ca2+]i is resequestrated into the SR via SERCA, with 10% removed by NCX and the remainder via Ca2+ pumps in the mitochondria (32). Studies on ventricular myocytes isolated from adult NCX-null mice have demonstrated unchanged Ca2+ dynamics (46), suggesting that NCX may be less important during the adult mouse ventricular myocyte AP than in other mammalian species. In the mouse myocyte, a small Ca2+ influx may occur due to reverse NCX activity if ICaL is inhibited (3). This may account for the small contractions still present after administration of verapamil (see Fig. 3B).

Ca2+ influx through L-type Ca2+ channels can be enhanced by their phosphorylation by protein kinases such as PKA. PKA-mediated phosphorylation increases ICaL by increasing the open-channel probability, as well as augmenting the fraction of channels that are available to open during a given transient depolarization (23). PKA also modulates other comports in the Ca2+ homeostasis in the contracting myocyte, including phosphorylating ryanodine receptors to induce CICR and phosphorylating PLB, and thus augmenting SERCA turnover to increase SR calcium uptake. PKA can also phosphorylate myosin binding protein C and troponin I, thus altering myofilament interaction and contractile parameters. Collectively, these effects can account for the positive inotropy and lusitropy observed after Gs-mediated activation, for example after application of Iso (3).

In studies on rodent ventricular myocytes, Iso has been shown to increase significantly cell shortening, ICaL, and PLB phosphorylation (11, 48, 52). Our experimental findings agree with these reports: we have observed an approximate twofold increase in the maximal cell shortening in myocytes treated with Iso (100 nM) (see Figs. 4 and 6). In addition, an approximately twofold increase in rates of relaxation with Iso was observed consistently (see Fig. 4).

In the adult heart and other tissues, agonists for Gi-coupled receptors act in opposition to those for Gs-coupled receptors, and this can decrease AC turnover rates and cAMP levels (18). CCh is widely used as an agonist of ACh receptors (mainly muscarinic) in the heart. CCh can decrease Iso-stimulated cell shortening in rat myocytes. This occurs through a pertussis toxin (PTX)-sensitive mechanism, strongly implicating involvement of Gi (11). Our results (see Fig. 4) reveal a significant negative inotropic effect when 100 nM CCh is applied to mouse ventricular myocytes. These results are comparable to those observed by others (52). Binding of ACh or CCh to M2R activates a weakly inwardly rectifying IKACh following dissociation of the holo G protein's Giβ{gamma} subunits (29, 30). ACh-sensitive K+ (KACh) gives rise to an outward K+ current at membrane potentials corresponding to the AP plateau (43). In summary, there are at least three signaling pathways that can, therefore, account for the CCh-induced negative inotropy: 1) activation of the inwardly rectifying current IKACh and APD shortening (20, 54); 2) reduction in AC turnover and cAMP levels (18); and 3) activation of PI3K activity to reduce ICaL (10).

Role of S1P receptor activation in APD changes and related inotropic effects. In 1993, Banach et al. (2) first reported that serum contains a PTX-sensitive lipid factor that could antagonize β-AR-stimulated AC activity on L-type Ca2+ channels. Later studies by this group (and others) demonstrated that S1P and other related molecules (e.g., sphingosylphosphorylcholine and lysophosphatidic acid) could depress contractility by inhibiting ICaL and/or by activating an IKACh-like K+ current in myocytes (1, 11, 16, 19, 33, 43). These important findings on S1P-induced negative inotropic effects and/or bradycardia have been replicated in a number of animal species, including rabbit (16), cats (44), rats (39), mice (14), and guinea pigs (43).

S1P receptor isoform expression in the mammalian heart is primarily limited to S1P1–3. Our studies showed that S1P1 was expressed at relatively high levels in adult mouse ventricular myocytes (see Fig. 2A). S1P1 expression levels were almost twofold greater than those of S1P3. Other investigators have also reported high levels of S1P1 in mouse heart (55) and isolated human (20, 36) and rat ventricular myocytes (33, 39). S1P3 has also been routinely identified in myocardium of various species (14, 33). In contrast, a recent study reports that S1P3 is expressed at higher levels than S1P1 in isolated adult mouse ventricular myocytes (37). Obviously, this differs from our findings and those of others. We note that our myocytes were studied shortly after isolation, whereas the myocytes in the other study (37) had been maintained in culture. These procedural differences may account for the apparent discrepancy between S1P1 and S1P3 expression levels. It is universally agreed that S1P2 is expressed at much smaller levels or may be lacking entirely (14, 20), and we concur: we found that S1P2 expression levels were negligible (see Fig. 2, A and C).

To activate S1P1 receptors selectively and then to study the resulting effects on myocyte shortening, we have used two approaches. The first was to use the S1P1-selective agonist SEW2871. Thus SEW2871 (with a reported EC50 of 20.7 nM in mouse) has minimal activity for other S1P receptor subtypes, even at concentrations up to 10 µM. Furthermore, SEW2871 causes receptor internalization and recycling similar to that induced by S1P (22, 47). Nevertheless, SEW2871 is ~10 times less potent than S1P with regard to S1P1 activation (47). We have used SEW2871 at concentrations equal to, or 10 times greater than, those of S1P. Our results show that SEW2871 is approximately as effective as S1P in reducing myocyte shortening in normal mouse ventricular myocytes (see Fig. 8A). These findings provide important evidence that S1P1 is involved in the negative inotropic effect of S1P. Our second approach was to study shortening of myocytes isolated from S1P3-null mice. In these conditions, the S1P1 receptor can be activated selectively. As show in Fig. 4B, S1P in these myocytes resulted in similar decreases in contractility.

In an attempt to identify, and then investigate, S1P3-specific responses in terms of myocyte shortening, we have used the S1P1-selective antagonist VPC23019. Although VPC23019 is a competitive antagonist for both S1P1 and S1P3, it is more potent at the S1P1 receptor (7.9 pKi for S1P1 vs. 5.9 pKi for S1P3) (12). Our results from myocytes isolated from S1P3-null mice verified that VPC23019 treatment very effectively blocked S1P1 receptor-mediated responses (see Fig. 7A). Thus pretreating wild-type myocytes with VPC23019 and then challenging with S1P (in the continued presence of VPC23019) is an effective paradigm for identifying S1P3-mediated changes in myocyte contractility. The results with VPC23019 (see Fig. 7B), when combined with those in the presence of SEW2871, suggest that the S1P3 isoform, in addition to S1P1, is involved with negative inotropy in ventricular myocytes.

Activation of S1P3 (shown through experiments with S1P3-null mice) may result in decreases in heart rate (14, 47). This is of interest because S1P3 can couple to Gq in addition to Gi. In myocytes, PLC activation mediated by Gq results in conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol can activate the PKC holoenzyme family, which, in turn, can both positively and negatively modulate ICaL (23). Inositol 1,4,5-trisphosphate receptors often localize to the SR of ventricular myocytes and result in an increase in [Ca2+]i when activated. Moreover, PIP2 has been shown to control the gating of KACh (38). However, since signaling via Gq is less prominent in the adult mouse than in the adult rat (18), the potential for S1P3 signaling through Gq may be prurient in myocytes isolated from the mouse. S1P3-mediated signaling may be through Gi. In fact, Gi is likely to be involved in the observed decrease in contractility and shortening of APD, since activation of IKACh in atrial myocytes is PTX sensitive (20).

Activation of IKACh by the Giβ{gamma} dimer requires PIP2 (as do all inwardly rectifying K+ channels) (49). Thus enzyme-mediated reactions that deplete PIP2 (e.g., Gq-PLC) may reduce the activity of these K+ channels. We have examined the role of S1P1 and IKACh in the observed decrease in myocyte shortening by taking advantage of the availability of a specific blocker for the inwardly rectifying K+ channel that is linked to the M2 and the S1P receptors in adult heart. One such compound is tertiapin, a small peptide (21 amino acids) purified from the venom of honey bees (15). Tertiapin selectively blocks IKACh in cardiac myocytes by acting on the extracellular surface of this K+ channel (13, 25, 54). Tertiapin has little activity on other inward rectifying K+ currents, such as IK1 or IKATP, and does not affect either repolarizing K+ currents or ICaL (13, 25). Tertiapin (10–100 nM) reduces both the inward and outward components of the IKACh (25) and also reduces the negative chronotropic responses induced by ACh in Langendorff-perfused hearts (13). The Kd of tertiapin is ~8 nM, and its IC50 is ~30 nM (13, 21, 25). Accordingly, we pretreated myocytes with 100 nM tertiapin before its application with S1P (100 nM). Importantly, a S1P-mediated decrease in cell shortening was still observed. However, this effect was smaller than those obtained when IKACh had not been blocked (see Fig. 9C). This data suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via Gi{alpha} to reduce ICaL, blunt CICR, and decrease [Ca2+]i; and 2) a second pathway that acts via Giβ{gamma} to activate IKACh and reduce APD. This decrease in APD is expected to decrease Ca2+ influx and reduce [Ca2+]i and myocyte contractility.

A relationship between APD and [Ca2+]i: mathematical simulations. Contraction strength in mammalian ventricular myocytes is strongly modulated by the frequency as well as the amplitude and duration of Ca2+ transients and APD (3, 8). It is now known, in general terms, that APD can modulate both the [Ca2+]i transient and the associated phasic contraction. We have adapted a mathematical model of the adult mouse ventricular myocyte AP and excitation-contraction coupling to integrate our experimental results; we illustrate how alterations in ADP can alter ICaL and [Ca2+]i (see Fig. 10, D and E).

In the guinea pig atrial myocyte, for example, application of S1P causes a concentration-dependent decrease in APD (43). Additionally, a model of the human atrial myocyte (42) has also been utilized by our laboratory to demonstrate that incorporation of an S1P-activated IKACh shortens APD (W. R. Giles, unpublished observations).

We have explored the relationship between APD and [Ca2+]i using this model of the adult mouse ventricular myocyte. These simulations suggested that quite small changes in APD can significantly alter Ca2+ influx due to ICaL. These simulations agree with experimental data on isolated myocytes from our laboratory showing a positive correlation between APD and Ca2+ flux through ICaL (W. R. Giles, unpublished observations). Thus a working hypothesis that can account for our experimental/modeling findings is described as follows. Activation of S1P1 (or S1P3) receptor isoforms by S1P can directly decrease ICaL by a Gi-mediated mechanism. This will shorten APD and attenuate CICR. In addition, activation of S1P1 receptors can activate IKACh. The resulting outward K+ current then increases the rate of repolarization and shortens APD. As judged from our simulations, APD decreased over the approximate range of –20 to –80 mV, in response to a simulated negative inotropic challenge (i.e., CCh or S1P). This decrease in APD resulted in a concurrent decrease in Ca2+ current as a result of its intrinsic voltage dependence (53). A previous model of L-type Ca2+ channel function in the rat ventricular myocyte has demonstrated that decreases in (less depolarized) membrane potential can decrease the open probabilities and increase the transitions to the inactivated states of these channels (50). This results in less Ca2+ influx during this part of the AP. It is now known that KACh channels have multiple binding sites for the Giβ{gamma} dimer, which could increase their mean open time and enhance repolarization (40).

Thus S1P acting in a manner similar to CCh would also be expected to cause decreased ICaL due to decreased APD and a less prominent plateau phase of the AP. The loss of [Ca2+]i by these two mechanisms lessens Ca2+-mediated myofilament contraction strength and results in reduced cell shortening. A schematic of S1P-mediated changes in APD and contraction is presented in Fig. 12.


Figure 12
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  		Fig. 12. Diagram of our working hypothesis and plausible signaling pathways for S1P receptor-mediated negative inotropic effects of S1P in adult mouse ventricular myocytes. Activation of S1P1 or S1P3 by S1P, or activation of S1P1 by SEW2871, results in a Gi{alpha}-mediated inhibition of adenylyl cyclase (AC) and, hence, reduced production of cAMP. Reduction in cAMP levels decrease activation of PKA, which, in turn, decreases ICaL (CaL). Since the Ca2+ flux is the trigger for sarcoplasmic reticulum (SR) Ca2+ release, the net result is less Ca2+ release from the SR and a reduced contractile response. This is simulated in terms of the myocyte shortening response, represented by the myocyte shortening curves on the left (control, blue). VPC23019 selectively blocks the S1P1 receptor population, thus allowing S1P to activate S1P3 only. Activation of S1P1 or S1P3 by their agonists also simultaneously activates Gi and releases the Giβ{gamma} dimer of this G-protein, which directly activates IKACh. The increased outward K+ current shortens APD (control, blue). The decreased APD reduces the voltage-dependent Ca2+ influx through L-type Ca2+ channels. The net effect is less Ca2+-mediated myofilament activation, as depicted in the shortening curves on the right (control, blue). The combined effects of reduced ICaL and IKACh activation summate, and each contributes to the observed negative inotropy due to S1P (as depicted in the contraction curves in the center). Tert can selectively block IKACh. RyR, ryanodine receptor; PLB, phospholamban.

 
Limitations of our work. In the present experiments, the orientation of the myocytes with regard to field stimulus direction was not monitored systematically. In general, the stimulating electrode was near, but not touching the myocyte. A recent study using pig myocytes has reported that the degree of alignment of the myocytes with the stimulating electrodes appeared to be an important experimental variable (17). In this paper, the field-stimulated contractions stopped if the myocyte was aligned in parallel with the electrodes, but phasic mechanical contractions resumed if the myocyte was aligned greater or equal to a 25° offset. However, we observed no qualitative differences with orientation of our field stimulation device. This may be due to the custom design of our probe (see Fig. 1), whereas the other studies may have used more traditional parallel electrodes, or mistakenly used myocyte pairs as the experimental preparation.

An additional report has suggested that field stimulation, compared with intracellular current-clamp stimulation, may result in slower times to peak contraction and that myocyte shortening is extracellular Na+ concentration independent and instead requires L-type Ca2+ channels and/or NCX (6). The reported stimulation methods in this study were similar to those used in our studies. However, this study utilized rat myocytes, and the isolation and maintenance protocol for the cells differed very significantly.

We recognize that there are limitations with the mathematical model that we have utilized. This model includes an acknowledged (7) inability to quantitatively account for inactivation kinetics of L-type Ca2+ channels. However, the original authors of this model [Bondarenko et al. (7)] have shown that it can accurately reproduce experimental data describing the Ca2+ fluxes in adult mouse ventricular myocytes in addition to accurately reproducing findings from voltage-clamp experiments. In addition, this model accurately simulates a number of other major ionic currents, including fast sodium and nearly all of the K+ currents. Thus we have confidence in the ability of this model to serve as an adjunct for investigating selected electrophysiological parameters of the adult mouse ventricular myocyte. We utilized the model to explore the relationship between APD and [Ca2+]i. When applied in this way as an "integrative tool," it can provide insight into experimental design.

Last, while we attempted to distinguish S1P signaling pathways to Gi responses through the use of selective S1P agonists and myocytes isolated from S1P-receptor subtype null mice, we recognize the complicated signaling pathways of the S1P receptors and their ability to link to multiple and distinct G proteins (9, 45).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a grant from the American Heart Association Western States Affiliate Program to W. R. Giles. L. K. Landeen was supported by a Graduate Research Fellowship from the National Science Foundation.


   ACKNOWLEDGMENTS
 
The authors sincerely thank Dr. Richard L. Proia of the National Institute of Diabetes and Digestive and Kidney Diseases for making S1P transgenic mice available to us. We also thank Dr. Masahiko Hoshijima of the University of California, San Diego (UCSD) for allowing us to use his qPCR machine, and Kim Weldy (UCSD) for maintenance of the S1P3-null breeding colonies. We are grateful to Dr. Ken Spitzer (University of Utah) for guidance and training on techniques for field-stimulation of ventricular myocytes.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. R. Giles, Dept. of Kinesiology, Univ. of Calgary, 2500 Univ. Dr. NW, Calgary, Alberta, Canada T2N 1N4 (e-mail: wgiles{at}ucalgary.ca)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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