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Am J Physiol Heart Circ Physiol 285: H735-H744, 2003. First published May 1, 2003; doi:10.1152/ajpheart.01098.2002
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Positive inotropic effect of ceramide in adult ventricular myocytes: mechanisms dissociated from its reduction in Ca2+ influx

Shi J. Liu and Richard H. Kennedy

Department of Pharmaceutical Sciences and Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 7220

Submitted 17 December 2002 ; accepted in final form 25 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Ceramide, a sphingolipid metabolite produced by activation of sphingomyelinase, has been previously shown to reduce L-type Ca2+ channel current (ICa,L) in adult rat ventricular myocytes; however, its effect on contractile function is unknown. In this study, we investigated the effects of ceramide on excitation-contraction coupling in adult ventricular myocytes and on left ventricular (LV) function in isolated hearts. Surprisingly, in patch-clamped myocytes, ceramide increased contraction concomitant with reductions in ICa,L. In intact myocytes, ceramide increased cell shortening (CS) concurrently with enhancing maximum rates of shortening and relaxation and the duration of contraction. Ceramide also increased the amplitudes of postrest potentiated (PRP) contraction. In fura-PE3-loaded myocytes, ceramide increased systolic Ca2+ and the magnitude and maximum rates of the rising and declining phases of Ca2+ transients. Ceramide-elicited decreases in magnitudes of PRP relative to steady-state contraction and the Ca2+ transient suggest an increased fractional Ca2+ release from the sarcoplasmic reticulum (SR). However, ceramide slightly reduced the caffeine-induced Ca2+ transient and had no significant effect on the amplitude of the PRP-elicited Ca2+ transient. Additionally, the ceramide-induced upward shift in the relationship of contraction and the Ca2+ transient and increase in the Ca2+ responsiveness of CS suggest an increase in myofilament Ca2+ sensitivity. In isolated hearts, ceramide increased LV developed pressure and maximum rates of contraction and relaxation at balloon volumes of 30–50 µl. In summary, regardless of decreasing ICa,L, ceramide elicits distinct positive inotropic and lusitropic effects, resulting probably from enhanced SR Ca2+ release and uptake, and increased Ca2+ sensitivity of ventricular myocytes.

calcium; lipid metabolites; excitation-contraction coupling; contractile function; sphingolipid; heart

CERAMIDE (N-acylated sphingosines), a naturally occurring sphingolipid, is produced predominantly by the activation of neutral and/or lysosomal acidic sphingomyelinase (4, 20, 22). With the use of a natural form of ceramide and exogenous, membrane-permeable, synthetic ceramides with a short carbon chain (e.g., C2-ceramide), studies have shown that ceramides mediate some biological effects of IL-1{beta} and TNF-{alpha}, two primary proinflammatory cytokines (32). Ceramides have been shown to stimulate the production of other cytokines (e.g., IL-2 and IL-6) (28) and prostaglandin E2 (2, 3), induce transcription factors (e.g., NF-{kappa}B) (3, 11, 32, 37), suppress cell growth (19), and induce apoptosis (15, 16, 21, 23). Moreover, studies in cardiac myocytes have shown that intracellular ceramide levels are increased in response to IL-1{beta} stimulation (31), during ischemia-reperfusion (8), after treatment with doxorubicin (1), and during hypoxia/reoxygenation (18). It has also been suggested that ceramide is associated with the TNF-{alpha}-induced and ischemia-reperfusion- and doxorubicin-associated apoptosis (1, 8, 24). While ceramide has been shown to exert an antiproliferative effect, its effect on cardiac cell function is largely unknown.

Our previous study (31) has shown that IL-1{beta} increases intracellular ceramide production in 5 min and that C2-ceramide mediates the IL-1{beta}-induced decrease in L-type Ca2+ channel current (ICa,L) in adult rat ventricular myocytes. Other studies in rat ventricular myocytes have also shown that C2-ceramide reduces ICa,L (29), whereas sphingosine, an interconvertible metabolite of ceramide, completely abolishes Ca2+ transients (29) and inhibits ICa,L (29, 38). Sphingosine has also been shown to inhibit Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) (10, 35). In contrast, studies in smooth muscle cells and Swiss 3T3 fibroblasts have shown that sphingosine increases intracellular Ca2+ () by enhancing Ca2+ release from intracellular stores (33). It is unclear whether sphingosine has different effects on different stores; little is also known about how ceramide affects homeostasis and contractile function in the heart. Thus, in this study, we investigated the effects of ceramide on contractile function and excitation-contraction (E-C) coupling of adult ventricular myocytes and on left ventricular (LV) function of isolated adult rat hearts. We found that ceramide exerted positive inotropic and lusitropic effects despite reductions in ICa,L.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Myocyte isolation. Single ventricular myocytes were isolated from hearts of adult (3–6 mo old) male Sprague-Dawley rats using enzymatic dissociation as described previously (25). Isolated myocytes were harvested and plated onto culture dishes containing culture medium. Rod-shaped cells with clear striations were used for experiments, and all experiments were carried out at 35–37°C. The protocol for the use of animals in this study conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee.

Electrophysiological measurements. Ventricular myocytes were placed on the heated stage of an inverted microscope (Nikon Diaphot) and perfused with normal Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2,10 HEPES-Tris, and 5.6 glucose (pH 7.40 at 37°C, 290 mosM). Myocytes were patch clamped using conventional whole cell patch techniques (14) with a patch-clamp amplifier (Axopatch 200A, Axon Instruments) (25). The simultaneous measurement of whole cell ICa,L and cell shortening (CS) was measured in K+-rich pipette solutions containing (in mM) 120 K-aspartate, 25 KCl, 2 MgCl2, 5 Na2ATP, 0.4 Li4GTP, 10 HEPES-Tris, and 5.6 glucose (pH 7.10 at 37°C) using the conventional whole cell patch-clamp techniques as described previously (25).

Measurement of CS. Unloaded CS or contraction of myocytes was elicited in normal Tyrode solution and measured as described previously (26). Briefly, cells were then superfused with Tyrode solution containing appropriate concentrations of DMSO for 5–7 min and subsequently with Tyrode solution containing different concentrations of C2-ceramide for 3–10 min before being switched back to the DMSO control. CS was monitored with a video edge-motion detector system (Crescent Electronics; Sandy, UT). The measured parameters of contractile function in single myocytes included the peak magnitude of CS, maximum rates of contraction (+dL/dtmax) and relaxation (–dL/dtmax), and the duration of CS.

Measurement of intracellular free Ca2+ concentration. Intracellular free Ca2+ concentration in ventricular myocytes was measured as described previously (26). Briefly, ventricular myocytes seeded on 25-mm coverslips in culture medium were loaded with 2 µM fura-PE3-AM for 30 min in a culture incubator at 37°C. Myocytes were then transferred to a perfusion chamber on the stage of an inverted microscope (Nikon TE300; Irving, TX) and superfused with normal Tyrode solution. After subtraction of the background signal, fluorescent signals were recorded as the intensity ratio (R or F340/F380), i.e., the fluorescent intensity when fura-PE3 was excited at 340 nm (F340) divided by that when excited at 380 nm (F380). The measured parameters of the Ca2+ transient included peak magnitude, maximum rates of the rising phase (+dR/dtmax) and the declining phase (–dR/dtmax), and rise and decay times between 10% and 90% of the peak amplitude. In some experiments, contraction of ventricular myocytes was recorded simultaneously with Ca2+ transients.

Langendorff-perfused hearts. Hearts were isolated from adult male Sprague-Dawley rats (300–350 g) and perfused via the aorta with an oxygenated Krebs-Henseleit solution (37°C) of the following composition (in mM): 118.0 NaCl, 27.1 NaHCO3, 3.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 1.0 KH2PO4, and 11.1 glucose (pH 7.4 was maintained by saturation with 95% O2-5% CO2 gas). Hearts were perfused at a constant flow rate of 7.0 ml · g heart–1 · min–1, similar to that observed when flow is examined at a constant pressure of 80 mmHg. Coronary perfusion pressure was monitored continuously throughout the experiment with a Statham pressure transducer. Both atria were removed, and the ventricles were paced electrically at 250 beats/min by platinum contact electrodes positioned on the interventricular septum. A fluid-filled latex balloon catheter was placed in the LV to measure intraventricular pressure. Steady-state contractile function was monitored throughout experiments and recorded 10–15 min after in control, DMSO-containing solutions and subsequent perfusion with ceramide-containing buffer solutions. Measured parameters of LV function included peak systolic and diastolic pressures, maximum rates of pressure development (+dP/dtmax) and relaxation (–dP/dtmax), and developed pressure (LVDP) at various preload balloon volumes (10–90 µl, a range that elicited maximum contractility in all preparations). In addition to a polygraph recording, all data were acquired and analyzed with a software program (CODAS, DataQ Instruments; Akron, OH).

Chemicals. Most reagents were purchased from Sigma Chemical (St. Louis, MO) and directly added when needed. Ceramides were purchased from Matreya (Present Gap, PA). Stock solutions of ceramide (10–2 M) were prepared in DMSO; the final concentration in solutions was <=0.1%.

Statistics. In all experiments, data in response to ceramide were compared with the steady-state control before the treatment in each individual cells and thus expressed as a ratio or percentage of each control value before being combined for statistical analysis. Values are presented as means ± SE. Statistical significance (P < 0.05) was evaluated by the twotailed Student's paired t-test, or, when more than two conditions were compared, by one- or two-way ANOVA with Duncan's multiple-range test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of ceramide on contraction in patch-clamped adult ventricular myocytes. We (31) have previously shown that C2-ceramide decreases ICa,L concentration dependently in adult rat ventricular myocytes. This ceramide-induced decrease in ICa,L led to the hypothesis that ceramide elicits a negative inotropy. Thus we examined the inotropic effect of ceramide by monitoring contraction simultaneously with ICa,L in patch-clamped myocytes. Figure 1, A and B, shows a typical voltage dependency of ICa,L and contraction (or CS) in the same cell, respectively, in the absence and presence of a natural-form ceramide (n-ceramide). Results show that the voltage dependency of CS in control (before exposure to ceramide) was almost identical to that of simultaneously recorded ICa,L. Figure 1A also shows that exposure for 8 min to 10 µM n-ceramide reduced ICa,L, consistent with our previous findings with C2-ceramide (31). However, to our surprise, Fig. 1B shows that in the presence of ceramide, the amplitude of CS, which was associated with voltage-dependent activation of ICa,L, was increased concurrently with an increase in the duration (see Fig. 1B, inset, measured at 0 mV). The combined data showed that, whereas n-ceramide reduced the amplitude of ICa,L to 70 ± 8% (n = 10, measured at 0 mV) of control, it increased contraction by 56 ± 21% in five of six cells. These results suggest that ceramide alters handling and/or Ca2+ sensitivity of the contractile apparatus.



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  		Fig. 1. Effects of ceramide on L-type Ca2+ channel current (ICa,L) and contraction. The current-voltage (I-V) relationships of peak ICa,L (A) and the voltage dependency of cell shortening (CS; B) were simultaneously recorded in response to 250-ms voltage pulses to potentials between –60 and +60 mV from a holding potential of –40 mV (right inset) before and during exposure to 10 µM of the natural form of ceramide (n-ceramide). Left inset: raw traces of peak ICa,L (A) and CS (B) in response to a voltage pulse to 0 mV. Cell membrane capacitance = 163 pF.

 

Effects of ceramide on contraction of intact adult ventricular myocytes. Because ventricular myocytes became very arrhythmogenic during exposure to ceramide under patch-clamped conditions, we examined the inotropic effect of ceramide in intact ventricular myocytes under more physiological conditions. Figure 2A shows that exposure to 3 µMC2-ceramide caused an initial, small, transient decrease in contraction, followed by an increase in systolic and diastolic levels, and reached a steady state in 10 min. Figure 2B shows that C2-ceramide elicited a positive inotropic effect in a concentration-dependent manner. Data also show that n-ceramide had a similar positive inotropic effect (increasing by 47%) to C2-ceramide. In contrast, C8-ceramide and dihydro-C2-ceramide, a biologically inactive analog of C2-ceramide, had little effect on CS (1.04 ± 0.03%, n = 3, and 1.05 ± 0.08%, n = 4, vs. control, respectively).



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  		Fig. 2. Inotropic effects of ceramide in intact adult ventricular myocytes. A: CS recordings in one myocyte in response to C2-ceramide (C2) stimulation. Inset, single CS traces where indicated were an average from 5 single traces in the steady state. B: concentration dependency of the C2-ceramide-induced increase in the amplitude of CS. n-Ceramide had a comparable inotropic effect. Data are means ± SE; numbers in parentheses are numbers of cells. *P < 0.05 compared with the level before exposure to ceramide.

 

The kinetic parameters of contraction were then analyzed in the presence of C2-ceramide. For example, in the results shown in Fig. 2A, after the diastolic level was offset to 0, steady-state CS traces (averaged from 5 to 6 traces) before and during exposure to C2-ceramide were superimposed and are shown in Fig. 3A. The first derivatives of each CS trace are shown in Fig. 3A, inset, to obtain +dL/dtmax and –dL/dtmax. C2-ceramide increased +dL/dtmax and –dL/dtmax by 34% and 20%, respectively, and lengthened the time to peak ~17% and the duration of CS. The data summarized in Fig. 3B show that C2-ceramide enhanced +dL/dtmax, –dL/dtmax, and the duration of CS (or the area under the CS trace) concomitant with a reduction in the decay time in a concentration-dependent manner. These results suggest that C2-ceramide elicited positive inotropic and lusitropic effects in adult rat ventricular myocytes.



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  		Fig. 3. Effects of ceramide on the kinetics of contraction. A: super-imposed CS traces before and during exposure to ceramide (as shown in Fig. 2A) after the diastolic level was offset to 0. Inset, superimposed first derivatives of each CS trace. B: relative C2-ceramideinduced changes in the maximum rates of shortening (+dL/dtmax) and relaxation (–dL/dtmax) and area under the CS trace. Data are means ± SE from 9, 26, and 17 experiments for 1, 3, and 10 µM, respectively. *P < 0.05 compared with control (C).

 

The inotropic effect of ceramide could involve the enhancement of SR function and of the kinetics and/or Ca2+ sensitivity of contractile proteins. Postrest potentiation (PRP) protocols have been used as an indirect indicator for handling by the SR (7) and the Ca2+ response of contractile proteins. After a short period of cessation of electrical stimulation (rest interval), rat ventricular myocytes display a PRP of contraction followed by a recovery (7). Thus we used this PRP protocol to examine whether ceramide alters handling. Figure 4A shows that 10 µM C2-ceramide significantly enhanced the potentiated contraction after a 30-s rest (PRP30); however, the degree of potentiation relative to prerest CS level (i.e., the ratio of the amplitude of PRP30 to that of prerest steady-state CS) was decreased. The combined data show that compared with the control PRP30, C2-ceramide increased the amplitude and duration of PRP30 by ~22% and 36%, respectively (Fig. 4B), and reduced the relative potentiation of PRP30 by 18 ± 4% (n = 9, P < 0.005, paired t-test; Fig. 4C). These data suggest increases in fractional Ca2+ release from the SR and the Ca2+ response of contractile proteins in the presence of ceramide. Moreover, the time course of the recovery of potentiated systolic shortening was best fit by a double-exponential function (Fig. 4B, inset). The fast time constant (but not the slow time constant) of recovery from PRP30 was significantly increased in the presence of C2-ceramide.



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  		Fig. 4. Effects of ceramide on postrest potentiation (PRP) of contraction. A: representative recording of PRP after a 30-s rest (PRP30) before and during exposure to C2-ceramide. B: ceramide-induced changes in the magnitude, area, and recovery of PRP30-elicited CS. Inset, recovery of systolic shortening from PRP30 in the absence and presence of C2-ceramide (as shown in A) was best fit by a double exponential function. {tau}f and {tau}s, fast and slow time constants, respectively. C: magnitude of PRP (relative to prerest steady-state level) in the absence and presence of C2-ceramide. Data are means ± SE from 9 experiments. *P < 0.05.

 

Effects of ceramide on the Ca2+ transient. The effects of ceramide on handling were examined using fura-PE3-loaded ventricular myocytes. Figure 5A shows that exposure to 1 µM C2-ceramide significantly increased systolic and diastolic shortening and the magnitude of contraction, consistent with those in nonfura-PE3-loaded cells (Fig. 2A) before contracture. C2-ceramide slightly increased systolic and diastolic free and the magnitude of the Ca2+ transient (i.e., the difference between systolic and diastolic level) (Fig. 5B). In contrast to a 80% increase in CS, Fig. 5C shows that ceramide increased the magnitude of the Ca2+ transient only by 9% with an increase in +dR/dtmax of the Ca2+ transient but with a smaller effect on –dR/dtmax at 1 µM (Fig. 5C, inset). Under these conditions, the increased –dL/dtmax with little change in –dR/dtmax could be due to an underestimate of the maximum rate of Ca2+ return (–dCa/dtmax) by –dR/dtmax. Figure 5, E and F, shows that ceramide-induced changes in the magnitude and kinetics of the Ca2+ transient were concentration dependent. Ceramide (at concentrations >=3 µM) significantly increased +dR/dtmax and –dR/dtmax. The results also showed that n-ceramide had a similar but smaller effect (12.2 ± 2.2%, n = 4) than C2-ceramide at 10 µM, whereas dihydro-C2-ceramide displayed little effect (103.2 ± 1.3% of control, n = 3). The differential effect of ceramide on CS and the Ca2+ transient suggests an increase in the Ca2+ sensitivity of contractile proteins.



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  		Fig. 5. Effects of ceramide on the Ca2+ transient. Simultaneous recordings of contraction (A) and intracellular free Ca2+ (B) were obtained from a fura-PE3-loaded myocyte. C: superimposed traces of Ca2+ transients before and during exposure to C2-ceramide (* in A). Inset, first derivatives of each trace obtained from C. D: phase-plane plot of CS as a function of the simultaneously recorded fluorescence ratio (R) in the absence and presence of C2-ceramide. Inset, replot of CS versus the fluorescence ratio after both being scaled to 1. E and F: concentration dependency of ceramide-induced increases in the Ca2+ transient and its kinetic parameters, respectively. F340 and F380, fluorescence at 340 and 380 nm, respectively. Data are means ± SE. Only the dihydro-C2 group in A and –dR/dt in B were not significantly different from control. *P < 0.05 compared with control.

 

After a phase-plane plot of CS and simultaneously measured free (or the fluorescence ratio), Fig. 5D shows that a hysteresis relationship between CS and fluorescence ratio was shifted upward in the presence of C2-ceramide. Figure 5D, inset, shows that when this relationship was replotted using relative changes in the CS-vs.-fluorescence ratio (normalized to each peak magnitude), a leftward shift in the contraction- trajectory was revealed during the early phase of relaxation, suggesting an increase in the myofilament sensitivity, as described previously by others (34). Furthermore, the effect of ceramide on the response of CS to extracellular Ca2+ () was examined and is shown in Fig. 6A. In a representative experiment, both the systolic and diastolic states of CS were increased in response to increasing the concentration ([Ca2+]o) to 2 mM. Figure 6B shows that the -induced increase in CS was enhanced in the presence of 3 µM C2-ceramide. Figure 6C shows that the time courses of the response of CS before, during, and after exposure to C2-ceramide were curve fit by a single exponential function. The time constant of the on rate ({tau}on) in response to 2 mM [Ca2+]o was 12.0 and 13.8 s in the absence and presence of C2-ceramide, respectively. The time constant of the off rate ({tau}off) was also increased in the presence of C2-ceramide (from 14.2 to 18.9 s). The amplitude of CS and time constants of the Ca2+ response only partially recovered after removal of C2-ceramide. The combined data show that C2-ceramide (3 µM) increased {tau}on and {tau}off of the response by 57 ± 18% and 75 ± 23% (n = 10), respectively. These results support the suggestion that ceramide increases the Ca2+ sensitivity of contractile proteins.



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  		Fig. 6. Effect of ceramide on the Ca2+ response of CS. In a representative experiment, CS of a myocyte was recorded in response to increasing the external Ca2+ concentration (Cao) from 1 to 2 mM (A). After recovering from 2 mM Ca2+, the myocyte was exposed for 5 min to 3 µMC2-ceramide, followed by another exposure to 2 mM Ca2+ (B). C: time course of changes in CS amplitude (difference between systole and diastole) in response to 2 mM Ca2+ before ({circ}), during ({bullet}), and after ({triangleup}) exposure to C2-ceramide. The on and off responses to 2 mM Ca2+ were curve fit by a single exponential function to obtain the time constants of the on and off rate (solid lines).

 

We then examined whether ceramide alters the SR Ca2+ load by applying a brief pulse (~5s)of10–15mM caffeine, which has been used to estimate SR Ca2+ content (6, 12), after a 15-s termination of electrical stimulation. Figure 7A shows an experiment in which a caffeine pulse was applied to the cell before, during, and after exposure to C2-ceramide. In addition, a PRP30 protocol was repetitively performed under the same conditions. Figure 7B shows that in addition to increasing the amplitude and +dR/dtmax of the Ca2+ transient, 10 µM C2-ceramide increased –dR/dtmax (Fig. 7B, inset). Figure 7A shows that the magnitude of the caffeine-induced Ca2+ transient was slightly reduced in the presence of C2-ceramide (also see Fig. 7C). Upon the removal of C2-ceramide, the caffeine-induced Ca2+ transient was transiently increased before returning to the control level. The combined data of the caffeine-induced Ca2+ transient shown in Fig. 7D show that 10 µM C2-ceramide reduced the magnitude of the caffeine-induced Ca2+ transient by 11%. Consequently, the steady-state fractional SR Ca2+ release (a ratio of steady-state systolic amplitude to caffeine-induced amplitude) was greater in the presence of C2-ceramide than in control (67% vs. 56% in control), supporting the results obtained from CS. Figure 7D also shows that the time constant of the decline phase of the caffeineinduced Ca2+ transient was increased by 18% in the presence of C2-ceramide, suggesting a reduction in Ca2+ efflux via sarcolemmal Na+/Ca2+ exchange. In addition, upon the removal of caffeine (postcaffeine), the magnitude of the first electrically elicited Ca2+ transient in the presence of C2-ceramide was 17 ± 4% (n = 6) smaller than that in control, suggesting a reduced Ca2+ influx, which is consistent with a ceramide-induced reduction in ICa,L.



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  		Fig. 7. Effects of ceramide on PRP30- and caffeineinduced Ca2+ transients. A: a caffeine pulse was applied before, during, and after exposure to C2-ceramide. PRP15, PRP after a 15-s rest. B: superimposed steady-state Ca2+ transient traces before and during C2-ceramide stimulation (* in A). Inset, first derivatives of Ca2+ transient traces. C: superimposed caffeine-induced Ca2+ transients obtained where indicated in A (1–3), with trace 4 being obtained 8 min after the removal of ceramide. D, left: magnitude of caffeineinduced Ca2+ transients before and during exposure to ceramide. Right, ratios of the magnitude and decay time constant ({tau}) of the caffeine-induced Ca2+ transient in the presence of ceramide to those in control. E: ratio of the magnitude of the PRP30-elicited Ca2+ transient in the presence of ceramide to that of the prerest steady-state level. *P < 0.05.

 

handling by the SR was also examined in fura-PE3-loaded myocytes using the PRP30 protocol. C2-ceramide slightly increased the magnitude of the PRP30-elicited Ca2+ transient by 7 ± 3% (n = 6, P = 0.045, one-tailed paired t-test). Similar to the PRP30 of CS (in Fig. 4C), Fig. 7E shows that the relative amplitude (to the prerest level) of the PRP30-associated Ca2+ transient was significantly smaller in the presence of C2-ceramide than in control. These results reinforced that C2-ceramide increases the fractional SR Ca2+ release in regular twitches.

Effects of C2-ceramide on LV contractile function in isolated hearts. We next examined whether C2-ceramide affects LV function in isolated whole heart preparations. In control experiments, isolated hearts were perfused with normal Tyrode solution for 10 min before the addition of 0.1% DMSO for 30–45 min, and the results showed that DMSO produced no significant changes in LV contractile function. Figure 8A shows a representative experiment in which perfusion for 10 min with 10 µM C2-ceramide enhanced systolic LV pressure accompanied by increases in +dP/dtmax and –dP/dtmax at a balloon volume of 50 µl. Figure 8B shows combined data from three hearts of C2-ceramide-induced increases in LV systolic pressure at balloon volumes of 30–80 µl and diastolic pressure only at balloon volumes of 70–80 µl. C2-ceramide also increased LVDP (i.e., the difference between systolic pressure and diastolic pressure) by ~15% (filled circles in Fig. 8D). The kinetic parameters of LV contractile function were analyzed under these conditions. C2-ceramide significantly enhanced +dP/dt with a smaller effect on –dP/dt (Fig. 8C). The summarized data shown in Fig. 8D show that C2-ceramide increased +dP/dtmax by 20–25% throughout the tested volumes (filled squares) but increased –dP/dtmax only at balloon volumes of 30–50 µl (filled triangles). Similarly, in three other hearts, 3 µM C2-ceramide increased LVDP, +dP/dtmax, and –dP/dtmax by 22.8 ± 3.7%, 20.9 ± 1.3%, and 14.2 ± 4.6%, respectively, at a balloon volume of 40 µl. These results suggest that C2-ceramide exerts a positive inotropic effect on LV myocardium with a maximum effect at 3 µM. Moreover, it is worth mentioning that 3 and 10 µM C2-ceramide caused an initial, transient decrease in LVDP by 6.6 ± 1.3 and 7.0 ± 0.9 mmHg, respectively, in the first 2 min during perfusion concomitantly with a small drop in coronary pressure.



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  		Fig. 8. Effects of ceramide on left ventricular (LV) function in the isolated adult rat heart. A: results of an experiment examining representative LV systolic and diastolic pressure at LV balloon volumes of 50 µl before and after 10-min perfusion with C2-ceramide. B: ceramide-induced increases in LV systolic and diastolic pressure. C: maximum rates of LV pressure (LVP) development and relaxation (±dP/dtmax) at various balloon volumes before and during perfusion with ceramide. D: ceramide-induced changes in LV developed pressure and ±dP/dtmax. Data are means ± SE from 3 experiments. *P < 0.05 compared with the value before exposure to ceramide.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
We (31) have previously reported that ceramide production is increased in response to IL-1{beta} stimulation in adult rat ventricular myocytes. C2-ceramide has also been shown to reduce ICa,L in these preparations (29, 31). The present study demonstrated that despite its reduction in ICa,L, ceramide induced a positive inotropic effect and an increase in systolic free , a positive lusitropic effect, and an increase in the Ca2+ sensitivity of contractile proteins. Ceramide also enhanced LV systolic and diastolic function, but with less effect on diastolic function.

In patch-clamped myocytes, n-ceramide, a naturally occurring ceramide, produced an inhibitory effect on ICa,L, similar to C2-ceramide, as described previously (31). This effect, however, was accompanied by increases in systolic amplitude and the duration of contraction. n-Ceramide also produces a positive inotropic effect in intact myocytes, again similar to that induced by C2-ceramide. These results support the notion that C2-ceramide mimics the naturally occurring ceramide in altering cardiac electric and mechanical function. The present study also suggests that the ceramideinduced inotropic effect is specific and probably independent of the length of the N-acyl hydrocarbon chain because dihydro-C2-ceramide and C8-ceramide have little inotropic effect.

The ceramide-induced reduction in ICa,L suggests that the accompanying positive inotropic effect is mediated by alterations in handling and/or in the Ca2+ sensitivity of contractile proteins. During cardiac action potentials, Ca2+ influx via sarcolemmal Ca2+ channels triggers CICR, thereby initiating a contraction that is followed by relaxation resulting from Ca2+ removal by the SR Ca2+ pump and sarcolemmal Na+/Ca2+ exchange (for a review, see Ref. 6). The ceramideinduced decreases in ICa,L and the first electrically stimulated postcaffeine Ca2+ transient indicate that its increases in contraction and systolic result from an increase in SR Ca2+ cycling and/or SR Ca2+ content rather than an increased Ca2+ influx. Our data showed that ceramide augments 1) +dL/dtmax and +dR/dtmax, an index of SR Ca2+ release (36), and –dL/dtmax and –dR/dtmax, an index of SR Ca2+ uptake (5), in intact ventricular myocytes; and 2) LVDP, +dP/dtmax, and –dP/dtmax in Langendorff-perfused heart preparations. These results support the suggestion that ceramide enhances handling in myocytes by increasing both SR Ca2+ release and uptake. However, ceramide did not increase the caffeine-induced Ca2+ transient (or SR Ca2+ content), possibly because ceramide has a more profound effect on SR Ca2+ release than Ca2+ uptake or interferes with the action of caffeine. Other possibilities, which require further investigation, include the fact that ceramide enhances the kinetics of interaction between Ca2+ and the contractile machinery and Ca2+ release from intracellular caffeine-insensitive Ca2+ pool. The magnitude of the steady-state CS or Ca2+ transient relative to that of the respective PRP-elicited CS or PRP30- and caffeineinduced Ca2+ transient is smaller in the presence of ceramide reinforces the suggestion that ceramide enhances fractional Ca2+ release from the SR in regular twitches.

Rest-dependent potentiation of contraction has been suggested to result from a recovery of the E-C coupling from a refractory state with an increased fraction of SR Ca2+ release, relatively independent of SR Ca2+ content (7). In addition to increased fractional Ca2+ release, ceramide increases the duration and magnitude of PRP30-elicited CS without a significant change in the respective Ca2+ transient. While ceramide might facilitate the recovery of refractoriness of E-C coupling, our data strongly suggest that it increases the Ca2+ sensitivity or active state of contractile machinery for the following reasons: 1) the degree of the ceramide-induced increase in CS (e.g., the systole and the duration) is greater than in the Ca2+ transient in regular twitches; 2) the ceramide-induced increase in the diastolic level of CS without a significant change in diastolic free is comparable to that observed in response to 2 mM (Fig. 6); 3) ceramide enhances the response of CS; 4) recoveries of CS from PRP30-associated potentiation and from 2 mM Ca2+ exposure in the presence of ceramide are slower than in its absence (Figs. 4B and 6C); and 5) ceramide induces an upward and leftward shift in the CS-tofluorescence ratio phase-plane trajectory, consistent with previous findings by others (34), who showed that such shifts in the -cell length trajectory during the relaxation phase of contraction reflects an increase in myofilament Ca2+ sensitivity in adult rat ventricular myocytes.

A study (9) using transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger showed that 15 µM C2-ceramide inhibited Na+/Ca2+ exchange activity. Our data showed that ceramide prolonged the rate of the decline phase of the caffeine-induced Ca2+ transient, an indirect measure of sarcolemmal Na2+/Ca2+ exchange due to the absence of SR uptake function (5), suggesting a decrease in Ca2+ efflux via sarcolemmal Na+/Ca2+ exchange. However, the return of to the baseline level during diastole depends primarily on the SR Ca2+ pump (contributing 92%) and the normal mode of Na+/Ca2+ exchange (7%) in rat ventricular myocytes (5). Thus the reduction in the small fraction (i.e., Na+/Ca2+ exchange) in return during diastole could account for the observed little change in the diastolic level in the presence of ceramide.

The mechanism involved in ceramide-induced alterations in Ca2+ sensitivity and SR Ca2+ cycling and in the biophysical properties of the cell membrane remains unknown. The dissociation of its inotropic effects from its electrophysiological effect could result from its diversity of cell signaling mechanisms and alterations in the lipid microenvironment of ion channels (17). For example, ceramide has been reported to activate protein kinases (including PKC and MAPK) as well as protein phosphatases (13, 16, 27, 30). Activation of protein phosphatases could reduce ICa,L, whereas activation of protein kinases could enhance contractile protein activities. Similarly, the target proteins involved in the ceramide-induced positive inotropic effect in ventricular myocytes also remain largely unknown and require further investigation.

In summary, the increase in ceramide resulting from stimulation of proinflammatory cytokines causes an increase in intracellular free Ca2+ and contractility, probably due to increases in SR Ca2+ release and uptake, and in the Ca2+ sensitivity of contractile proteins, despite a reduction of ICa,L. Ceramide-elicited effects can be beneficial (as a Ca2+-sensitizing and positive inotropic agent) or deleterious (as a potential arrhythmogenic agent). The cardiac effects of ceramide could play an important role in cytokine-related pathophysiological conditions such as ischemia-reperfusion, hypoxia, and sudden cardiac death.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-62226.


   ACKNOWLEDGMENTS
 
We thank Meei-Yueh Liu and Kerrey A. Roberto for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Liu, Dept. of Pharmaceutical Sciences, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St. MS 522-3, Little Rock, AR 72205 (E-mail: sliu{at}uams.edu).

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Andrieu-Abadie N, Jaffrezou JP, Hatem S, Laurent G, Levade T, and Mercadier JJ. L-Carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation. FASEB J 13: 1501–1510, 1999.[Abstract/Free Full Text]
  2. Ballou LR, Chao CP, Holness MA, Barker SC, and Raghow R. Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide. J Biol Chem 267: 20044–20050, 1992.[Abstract/Free Full Text]
  3. Ballou LR, Laulederkind SJF, Rosloniec EF, and Raghow R. Ceramide signalling and the immune response. Biochim Biophys Acta 1301: 273–287, 1996.[Medline]
  4. Bankers-Fulbright JL, Kalli KR, and McKean DJ. Interleukin-1 signal transduction. Life Sci 59: 61–83, 1996.[ISI][Medline]
  5. Bassani JWM, Bassani RA, and Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol 476: 279–293, 1994.[Abstract/Free Full Text]
  6. Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res 87: 275–281, 2000.[Free Full Text]
  7. Bers DM, Bassani RA, Bassani JWM, Baudet S, and Hryshko LV. Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium. J Mol Cell Cardiol 25: 1047–1057, 1993.[ISI][Medline]
  8. Bielawska AE, Shapiro JP, Li JA, Melkonyan HS, Piot C, Wolfe CL, Tomei LD, Hannun YA, and Umansky SR. Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion. Am J Pathol 151: 1257–1263, 1997.[Abstract]
  9. Condrescu M and Reeves JP. Inhibition of sodium-calcium exchange by ceramide and sphingosine. J Biol Chem 276: 4046–4054, 2001.[Abstract/Free Full Text]
  10. Dettbarn CA, Betto R, Salviati G, Palade P, Jenkins GM, and Sabbadini RA. Modulation of cardiac sarcoplasmic reticulum ryanodine receptor by sphingosine. J Mol Cell Cardiol 26: 229–242, 1994.[ISI][Medline]
  11. Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J 8: 1314–1325, 1994.[Abstract]
  12. Frampton JE, Orchard CH, and Boyett MR. Diastolic, systolic and sarcoplasmic reticulum [Ca2+] during inotropic interventions in isolated rat myocytes. J Physiol 437: 351–375, 1991.[Abstract/Free Full Text]
  13. Grunicke HH and Uberall F. Protein kinase C modulation. Semin Cancer Biol 3: 351–360, 1992.[Medline]
  14. Hamill OP, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[ISI][Medline]
  15. Hannun YA and Obeid LM. Ceramide: an intracellular signal for apoptosis. Trends Biochem Sci 20: 73–77, 1995.[ISI][Medline]
  16. Hannun YA, Obeid LM, and Wolff RA. The novel second messenger ceramide: identification, mechanism of action, and cellular activity. Adv Lipid Res 35: 43–64, 1993.
  17. Hazen SL and Gross RW. Principles of membrane biochemistry and their application to the pathophysiology of cardiovascular disease. In: The Heart and Cardiovascular System, edited by Fozzard HA, Haber E, Jennings RB, Katz AM, and Morgan HE. New York: Raven, 1992, p. 839–860.
  18. Hernandez OM, Discher DJ, Bishopric NH, and Webster KA. Rapid activation of neutral sphingomyelinase by hypoxiareoxygenation of cardiac myocytes. Circ Res 86: 198–204, 2000.[Abstract/Free Full Text]
  19. Jayadev S and Hannun YA. Ceramide: role in growth inhibitory cascades. J Lipid Mediat Cell Signal 14: 295–301, 1996.[ISI][Medline]
  20. Jayadev S, Linardic CM, and Hannun YA. Identification of arachidonic acid as a mediator of sphingomyelin hydrolysis in response to tumor necrosis factor alpha. J Biol Chem 269: 5757–5763, 1994.[Abstract/Free Full Text]
  21. Ji L, Zhang G, Uematsu S, Akahori Y, and Hirabayashi Y. Induction of apoptotic DNA fragmentation and cell death by natural ceramide. FEBS Lett 358: 211–214, 1995.[ISI][Medline]
  22. Kolesnick R and Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77: 325–328, 1994.[ISI][Medline]
  23. Krönke M, Schütze S, Wiegmann K, and Machleidt T. Sphingomyelinases and TNF-induced apoptosis. Cell Physiol Biochem 6: 337–344, 1996.
  24. Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJE, and Sabbadini RA. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes–involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest 98: 2854–2865, 1996.[ISI][Medline]
  25. Liu S and Schreur KD. G protein-mediated suppression of L-type Ca2+ current by interleukin-1{beta} in cultured rat ventricular myocytes. Am J Physiol Cell Physiol 268: C339–C349, 1995.[Abstract/Free Full Text]
  26. Liu SJ, Kennedy RH, Creer MH, and McHowat J. Alterations in Ca2+ cycling by lysoplasmenylcholine in adult rabbit ventricular myocytes. Am J Physiol Cell Physiol 284: C826–C838, 2003.[Abstract/Free Full Text]
  27. Mathias S and Kolesnick R. Ceramide: a novel second messenger. Adv Lipid Res 25: 65–90, 1993.[ISI][Medline]
  28. Mathias S, Younes A, Kan CC, Orlow I, Joseph C, and Kolesnick RN. Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1{beta}. Science 259: 519–522, 1993.[Abstract/Free Full Text]
  29. McDonough PM, Yasui K, Betto R, Salviati G, Glembotski CC, Palade PT, and Sabbadini RA. Control of cardiac Ca2+ levels: inhibitory actions of sphingosine on Ca2+ transients and L-type Ca2+ channel conductance. Circ Res 75: 981–989, 1994.[Abstract/Free Full Text]
  30. Ogretmen B and Hannun YA. Updates on functions of ceramide in chemotherapy-induced cell death and in multidrug resistance. Drugs 4: 368–377, 2001.
  31. Schreur KD and Liu S. Involvement of ceramide in inhibitory effect of IL-1{beta} on L-type Ca2+ current in adult rat ventricular myocytes. Am J Physiol Heart Circ Physiol 272: H2591–H2598, 1997.[Abstract/Free Full Text]
  32. Schutze S, Machleidt T, and Kronke M. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J Leukoc Biol 56: 533–541, 1994.[Abstract]
  33. Spiegel S, Olivera A, and Carlson RO. The role of sphingosine in cell growth regulation and transmembrane signaling. Adv Lipid Res 25: 105–129, 1993.[ISI][Medline]
  34. Spurgeon HA, DuBell WH, Stern MD, Sollott SJ, Ziman BD, Silverman HS, Capogrossi MC, Talo A, and 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]
  35. Webster RJ, Sabbadini RA, Dettbarn CA, and Paolini PJ. Sphingosine effects on the contractile behavior of skinned cardiac myocytes. J Mol Cell Cardiol 26: 1273–1290, 1994.[ISI][Medline]
  36. Wier WG, Egan TM, Lopez-Lopez JR, and Balke CW. Local control of excitation-contraction coupling in rat heart cells. J Physiol 474: 463–471, 1994.[Abstract/Free Full Text]
  37. Yang Z, Costanzo M, Golde DW, and Kolesnick RN. Tumor necrosis factor activation of the sphingomyelin pathway signals nuclear factor kappa B translocation in intact HL-60 cells. J Biol Chem 268: 20520–20523, 1993.[Abstract/Free Full Text]
  38. Yasui KJ and Palade P. Sphingolipid actions on sodium and calcium currents of rat ventricular myocytes. Am J Physiol Cell Physiol 270: C645–C649, 1996.[Abstract/Free Full Text]



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