- Email: [email protected]
Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide
peptides 27 (2006) 1415–1419
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide Jun Ren *, David P. Relling Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota School of Medicine, Grand Forks, ND 58203, United States
article info
abstract
Article history:
Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation
Received 13 October 2005
of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and
Received in revised form
leptin participate in the regulation of cardiac function and are speculated to play roles in
18 November 2005
obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of
Accepted 18 November 2005
ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes
Published on line 18 January 2006
were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C2-dihydroceramide (25 mM). Contractile and intracellular Ca2+ properties were evaluated using an IonOptix MyoCam1 system
Keywords: Leptin
including peak shortening (PS), maximal velocity of shortening/relengthening (dL/dt),
Ceramide
time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise (D[Ca2+]) and
Cardiac
intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and
Myocyte shortening Intracellular Ca
2+
transients
intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, dL/dt, D[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, dL/dt, D[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, dL/dt, D[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Leptin, the product of the obese ob gene, is a peptide hormone mainly found in adipose tissue [28]. Leptin exerts a wide variety of biological actions including inhibition of food intake, promotion of energy expenditure and regulation of cardiovascular function [5,12,16,27]. Leptin may participate in the regulation of cardiac function through sympathetic stimulation and its myogenic inhibition of cardiomyocyte contraction [12,15,16], and has been speculated to contribute
to obesity-related cardiac contractile dysfunction [11,16,27]. The levels of leptin in circulation are reported to be significantly elevated under insulin-resistant states such as obesity, overfeeding and hypertension [4,9,10,14,16,26,27]. Furthermore, plasma leptin levels are closely correlated with insulin resistance and increased myocardial wall thickness, independent of body composition and blood pressure levels [6,13]. However, the precise mechanism of action involved in leptin-induced cardiac contractile dysfunction in hyperleptinemic conditions such as obesity is still lacking. Since
* Corresponding author. Present address: Center for Cardiovascular Research & Alternative Medicine, and Division of Pharmaceutical Science, University of Wyoming, Laramie, WY 82071, United States. Tel.: +1 307 766 6131; fax: +1 307 766 2953. E-mail address: [email protected] (J. Ren). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.11.022
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peptides 27 (2006) 1415–1419
obesity is a lipotoxic disease with dyslipidemia including overtly elevated ceramide levels, the de novo ceramide pathway has been postulated to be key to the lipoapoptosis of pancreatic b-cells and cardiomyocytes in obese individuals [24,29]. Nevertheless, the direct effect of ceramide on metabolic and other biological actions of leptin has not been elucidated. The purpose of this study was to examine the effect of short-term ceramide incubation on low (5 nM) and high (50 nM) leptin-induced cardiac contractile response and intracellular Ca2+ handling properties using a serum-free cardiomyocyte primary culture model established in our laboratory [17].
2.
Materials and methods
2.1. Isolation of adult rat ventricular myocytes and primary cell culture The experimental procedures were approved by the animal investigation committees of the University of North Dakota (Grand Forks, ND) and University of Wyoming (Laramie, WY). Single ventricular myocytes were isolated from adult male Sprague–Dawley rats (200–225 g) as described previously [17]. In brief, animals were sacrificed under ketamine/xylazine sedation (5:3, 1.32 mg/kg, i.p.). Hearts were removed and perfused (at 37 8C) with oxygenated (5% CO2–95% O2) Krebs– Henseleit bicarbonate (KHB) buffer (NaCl 118 mM, KCl 4.7 mM, CaCl2 1.25 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, N-[2-hydro-ethyl]-piperazine-N0 -[2-ethanesulfonic acid] (HEPES) 10 mM, glucose 11.1 mM, pH 7.4). Hearts were subsequently perfused with a nominally Ca2+-free KHB buffer for 2–3 min until spontaneous contractions ceased followed by a 20 min perfusion with Ca2+-free KHB containing 223 U/ml collagenase (Worthington Biochemical Corp., Freehold, NJ) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St. Louis, MO). After perfusion, the left ventricle was removed, minced and incubated with fresh enzyme solution (Ca2+-free KHB containing 223 U/ml collagenase) for 3–5 min. The cells were further digested with 0.02 mg/ml trypsin (Sigma) before being filtered through a nylon mesh (300 mm) and collected by centrifugation (60 g for 30 s). Myocytes were rinsed with Ca2+-free KHB buffer to remove remnant enzyme and extracellular Ca2+ was added incrementally back to 1.25 mM. Isolated myocytes were maintained at 37 8C in a serum-free medium consisting of Medium 199 (Sigma) with Earl’s salts containing 25 mM HEPES and NaHCO3 supplemented with BSA (2 mg/ml), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), glucose (5 mM), insulin (0.1 mM), penicillin (100 U/ml), streptomycin (100 mg/ml) and gentamicin (100 mg/ml). Myocytes were incubated at 37 8C with 100% humidity and 5% CO2 for 24 h. A cohort of myocytes was incubated with low (5 nM) or high (50 nM) levels of leptin in the absence or presence of the active ceramide analog C2dihydroceramide (C2-ceramide, 25 mM) for 24 h. C2-ceramide was dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was <0.1%, which did not affect cell mechanics [3]. Cells remained viable for 24 h and were not used if they had any obvious sarcolemmal blebs or spontaneous contractions.
2.2.
Cell shortening/relengthening measurements
Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam1 system (IonOptix, Milton, MA) as described previously [17]. In brief, coverslips with cells attached were placed in a chamber mounted on the stage of an inverted microscope (Olympus X-70) and superfused (2 ml/min at 25 8C) with a buffer containing: NaCl 131 mM, KCl 4 mM, CaCl2 1 mM, MgCl2 1 mM, glucose 10 mM, HEPES 10 mM, at pH 7.4. The cells were field stimulated at a frequency of 0.5 Hz, 3 ms in duration. A video-based edge-detector was used to capture and convert changes in cell length during shortening and relengthening into an analog voltage signal. Cell shortening and relengthening were assessed using the following indices: peak twitch amplitude (PS), time-to-90% PS (TPS) and time-to-90% relengthening (TR90), maximal velocities of shortening (+dL/dt) and relengthening (dL/dt), respectively.
2.3.
Intracellular Ca2+ transient measurement
For these experiments myocytes were loaded with fura-2/AM (0.5 mM) for 10 min at 25 8C. Fluorescence measurements were recorded with a dual-excitation single-emission fluorescence photomultiplier system (IonOptix). Myocytes were placed on an inverted microscope and imaged through an Olympus Fluor 40 oil objective. Myocytes were exposed to light emitted by a 75 W halogen lamp through either a 360 or 380 nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol. The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration (D[Ca2+]) were inferred from the ratio of fluorescence intensity obtained at both wavelengths. Intracellular Ca2+ decay time was calculated from the fluorescence decay curve as an indicator for intracellular Ca2+ clearing.
2.4.
Data analysis
Data were expressed as mean S.E.M. Statistical comparisons were performed by analysis of variance (ANOVA) followed by Newman–Keuls post hoc test. p < 0.05 was considered statistically significant.
3.
Results
As shown in Fig. 1, 24 h incubation of leptin (5 nM) with cardiac myocytes did not exhibit any obvious effect on cell shortening. However, higher concentration of leptin (50 nM) significantly depressed peak shortening (PS) amplitude, maximal velocities of shortening/relengthening (dL/dt) and prolonged duration of relengthening (TR90) without affecting duration of shortening (TPS). Twenty-four hours incubation of C2-ceramide (25 mM) itself did not exhibit any effect on cell shortening mechanics, consistent with our previous finding [3]. Interestingly, C2ceramide significantly potentiated leptin-induced cardiac contractile response. In the presence of C2-ceramide, low concen-
peptides 27 (2006) 1415–1419
1417
Fig. 1 – Effect of 24 h incubation of leptin (5 and 50 nM) on myocyte shortening in the absence or presence of C2-ceramide (25 mM). (A) Peak shortening amplitude (% of resting cell length), (B) maximal velocity of shortening/relengthening (WdL/dt), (C) time-to-peak shortening (TPS) and (D) time-to-90% relengthening (TR90). Mean W S.E.M., n = 75 cells per group. *p < 0.05 vs. control group; #p < 0.05 vs. leptin 50 nM group.
tration leptin (5 nM) significantly suppressed peak shortening amplitude, dL/dt and prolonged TR90 without affecting TPS. The high concentration leptin (50 nM)-induced depression of peak shortening, dL/dt, D[Ca2+] and prolongation of TR90 was further potentiated by C2-ceramide co-incubation. In addition, 50 nM leptin significantly shortened TPS in the presence of C2ceramide, the effect of which was not seen in the absence of C2ceramide. To determine whether C2-ceramide-induced potentiation of leptin-induced cardiac contractile response was due to reduced availability of intracellular-free Ca2+ concentration ([Ca2+]), we used fura-2 to evaluate changes in intracellular Ca2+ levels in response to electrical stimuli. The time course of fluorescence signal decay was calculated to assess intracellular Ca2+ clearing rate. Similar to our observation in cell shortening, 5 nM leptin did not affect baseline [Ca2+], peak [Ca2+], changes of intracellular Ca2+ (D[Ca2+]) and intracellular Ca2+ decay rate, whereas 50 nM leptin significantly reduced baseline [Ca2+], peak [Ca2+], D[Ca2+] without affecting intracellular Ca2+ decay rate (Fig. 2). Interestingly, although ceramide itself failed to affect intracellular Ca2+ handling properties, it significantly sensitized leptin-induced response on intracellular Ca2+ transients. In the presence of C2-ceramide, low concentration leptin (5 nM) significantly suppressed D[Ca2+] without affecting the baseline or peak [Ca2+] as well as intracellular Ca2+ decay rate. Although the high concentration leptin (50 nM)-induced depression of
baseline and peak [Ca2+] was not affected by C2-ceramide, depression of D[Ca2+] elicited by 50 nM leptin was significantly augmented by C2-ceramide. While 50 nM leptin itself failed to affect the intracellular Ca2+ decay rate, it significantly slowed intracellular Ca2+ clearing rate in the presence of C2-ceramide (Fig. 2).
4.
Discussion
This study demonstrated that 24 h co-incubation of the ob gene product leptin with cardiac myocytes led to depressed cell shortening and intracellular Ca2+ transients at high (50 nM) but not low (5 nM) leptin concentration, consistent with our previous finding [12]. Although ceramide itself failed to elicit any overt effect on cell shortening and intracellular Ca2+ transients following 24 h incubation, it significantly potentiated the cardiac contractile response of the adipokine leptin. As circulating levels of leptin are increased in almost all types of obesity except in ob/ob mice [4,6,7,10] accompanied with elevated cellular ceramide level and de novo synthesis [23–25,29], the depressed cardiac contractility seen in hyperleptinemic obesity [1,2,18,19] may be due, in part, to the synergistic action of the two lipid mediators on cardiomyocyte contractile function.
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peptides 27 (2006) 1415–1419
Fig. 2 – Effect of 24 h incubation of leptin (5 and 50 nM) on intracellular Ca2+ transients in the absence or presence of C2ceramide (25 mM). (A) Baseline intracellular Ca2+ level, (B) peak intracellular Ca2+ level, (C) change of intracellular Ca2+ level in response to electrical stimuli (D[Ca2+] = peak S baseline) and (D) intracellular Ca2+ decay rate. Mean W S.E.M., n = 39–40 cells per group. *p < 0.05 vs. control group; #p < 0.05 vs. leptin 50 nM group.
Leptin possesses a wide variety of biological responses ranging from cell proliferation, differentiation, to functional activation of hemopoietic and embryonic cells [5,20,22]. Recent evidence also depicted a role of leptin in cardiac contractile regulation either directly through cardiomyocyte contraction or indirectly via enhanced sympathetic nerve activity [12,15,16]. Low nanomolar levels of leptin are considered between physiological and pathophysiological range [9,13]. Our current study did not reveal any overt cardiac contractile response for leptin at concentrations below 10 nM, consistent with our earlier observations [12,27]. Therefore, there is a seemingly disconnection between in vivo and in vitro leptin levels and window of regulation for cardiac contractile function. Since obesity is often associated with elevated synthesis and cellular levels of the lipotoxic mediator ceramide [23,25,29], our observation that ceramide may potentiate or augment cardiac depressive effect of leptin may provide an explanation for hyperleptinemiaassociated cardiac dysfunction. It is believed that elevated triglyceride and fatty acid levels in overfeeding and obesity stimulate serine palmitoyl transferase activity resulting in ceramide synthesis de novo and elevated ceramide levels [23,25]. Compromised ventricular function is commonly seen in obesity with insulin resistance and decreased adrenergic responsiveness being the most predominant contributing
factors [8,21]. Recent evidence has implicated a role of the satiety factor leptin in cardiac contractile dysfunction in obesity characterized by decreased contractility, reduced diastolic compliance and prolonged relaxation [1,2,18,19]. Results from our current study indicated that short-term incubation of leptin with cardiac myocytes reduced peak shortening amplitude, maximal velocity of shortening/ relengthening and prolonged duration of relaxation. Perhaps the most important finding is that a low level of leptin, which displays no effect on cardiac contraction by itself, significantly depresses cardiac contractile response in the presence of ceramide. This may put individuals with normal leptin levels but raised ceramide levels at risk of developing heart dysfunction. The higher level (50 nM) leptin-induced cardiac contractile responses were further exacerbated by ceramide. Ceramide also unmasked additional effects of leptin on duration of shortening and intracellular Ca2+ clearing rate. These observations are supported by shortened contraction duration [27] and reduced rate of intracellular Ca2+ clearing [18] reported in cardiomyocytes from hyperleptinemic individuals. It is worthy mentioning that the mechanical effects are supported by the fact of reduced intracellular Ca2+ rise in response to electrical stimuli, suggesting an alteration of intracellular Ca2+ handling is likely responsible for the synergistic effect of ceramide and leptin on the cardiac contractile response in single cardiac myocytes.
peptides 27 (2006) 1415–1419
In conclusion, our present study demonstrated, for the first time, a synergistic effect between leptin and ceramide on cardiac contractile function and intracellular Ca2+. These data should shed some light on the elucidation of the role of leptin in hyperleptinemia-induced cardiac dysfunction. Further study is warranted to examine the cellular machineries such as sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger in this culprit synergy between leptin and ceramide.
Acknowledgements This work was supported by grants from American Heart Association Northland and Pacific Mountain Affiliates. DPR held a predoctoral fellowship from American Heart Association Northland Affiliate during the course of this work. The authors wish to sincerely thank Ms. Bonnie Zhao for her assistance in data analysis.
[12]
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references [19] [1] Carroll JF, Jones AE, Hester RL, Reinhart GA, Cockrell K, Mizelle HL. Reduced cardiac contractile responsiveness to isoproterenol in obese rabbits. Hypertension 1997;30:1376– 81. [2] Carroll JF, Summers RL, Dzielak DJ, Cockrell K, Montani JP, Mizelle HL. Diastolic compliance is reduced in obese rabbits. Hypertension 1999;33:811–5. [3] Colligan PB, Relling DP, Ren J. Ceramide attenuates high glucose-induced cardiac contractile abnormalities in cultured adult rat ventricular myocytes. Cell Mol Biol (Noisy-le-grand) 2002;48 [online Pub: OL251–7]. [4] Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1995;1:1311–4. [5] Friedman JM. The function of leptin in nutrition, weight, and physiology. Nutr Rev 2002;60:S1–4. [6] Girard J. Is leptin the link between obesity and insulin resistance? Diabetes Metab 1997;23(Suppl. 3):16–24. [7] Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269:543–6. [8] Haynes WG, Morgan DA, Walsh SA, Sivitz WI, Mark AL. Cardiovascular consequences of obesity: role of leptin. Clin Exp Pharmacol Physiol 1998;25:65–9. [9] Hintz KK, Aberle NS, Ren J. Insulin resistance induces hyperleptinemia, cardiac contractile dysfunction but not cardiac leptin resistance in ventricular myocytes. Int J Obes Relat Metab Disord 2003;27:1196–203. [10] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–61. [11] Mark AL, Correia ML, Rahmouni K, Haynes WG. Selective leptin resistance: a new concept in leptin physiology
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with cardiovascular implications. J Hypertens 2002;20:1245–50. Nickola MW, Wold LE, Colligan PB, Wang GJ, Samson WK, Ren J. Leptin attenuates cardiac contraction in rat ventricular myocytes. Role of NO. Hypertension 2000;36:501–5. Paolisso G, Tagliamonte MR, Galderisi M, Zito GA, Petrocelli A, Carella C, et al. Plasma leptin level is associated with myocardial wall thickness in hypertensive insulin-resistant men. Hypertension 1999;34:1047–52. Piatti P, Monti LD. Insulin resistance, hyperleptinemia and endothelial dysfunction in coronary restenosis. Curr Opin Pharmacol 2005;5:160–4. Rahmouni K, Haynes G. Leptin and the central neural mechanisms of obesity hypertension. Drugs Today (Barc) 2002;38:807–17. Ren J. Leptin and hyperleptinemia—from friend to foe for cardiovascular function. J Endocrinol 2004;181:1–0. Ren J, Duan J, Hintz KK, Ren BH. High glucose induces cardiac insulin-like growth factor I resistance in ventricular myocytes: role of Akt and ERK activation. Cardiovasc Res 2003;57:738–48. Ren J, Sowers JR, Walsh MF, Brown RA. Reduced contractile response to insulin and IGF-I in ventricular myocytes from genetically obese Zucker rats. Am J Physiol Heart Circ Physiol 2000;279:H1708–14. Ren J, Walsh MF, Jefferson L, Natavio M, Ilg KJ, Sowers JR, et al. Basal and ethanol-induced cardiac contractile response in lean and obese Zucker rat hearts. J Biomed Sci 2000;7:390–400. Saad MF, Khan A, Sharma A, Michael R, Riad-Gabriel MG, Boyadjian R, et al. Physiological insulinemia acutely modulates plasma leptin. Diabetes 1998;47:544–9. Strassheim D, Houslay MD, Milligan G. Regulation of cardiac adenylate cyclase activity in rodent models of obesity. Biochem J 1992;283(Pt 1):203–8. Umemoto Y, Tsuji K, Yang FC, Ebihara Y, Kaneko A, Furukawa S, et al. Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells. Blood 1997;90:3438–43. Unger RH, Orci L. Diseases of liporegulation: new perspective on obesity and related disorders. FASEB J 2001;15:312–21. Unger RH, Orci L. Lipoapoptosis: its mechanism and its diseases. Biochim Biophys Acta 2002;1585:202–12. Unger RH, Zhou YT. Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes 2001;50(Suppl. 1):S118–21. Wang J, Obici S, Morgan K, Barzilai N, Feng Z, Rossetti L. Overfeeding rapidly induces leptin and insulin resistance. Diabetes 2001;50:2786–91. Wold LE, Relling DP, Duan J, Norby FL, Ren J. Abrogated leptin-induced cardiac contractile response in ventricular myocytes under spontaneous hypertension: role of Jak/ STAT pathway. Hypertension 2002;39:69–74. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–32. Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, et al. Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci USA 2000;97:1784–9.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide Jun Ren *, David P. Relling Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota School of Medicine, Grand Forks, ND 58203, United States
article info
abstract
Article history:
Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation
Received 13 October 2005
of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and
Received in revised form
leptin participate in the regulation of cardiac function and are speculated to play roles in
18 November 2005
obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of
Accepted 18 November 2005
ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes
Published on line 18 January 2006
were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C2-dihydroceramide (25 mM). Contractile and intracellular Ca2+ properties were evaluated using an IonOptix MyoCam1 system
Keywords: Leptin
including peak shortening (PS), maximal velocity of shortening/relengthening (dL/dt),
Ceramide
time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise (D[Ca2+]) and
Cardiac
intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and
Myocyte shortening Intracellular Ca
2+
transients
intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, dL/dt, D[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, dL/dt, D[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, dL/dt, D[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Leptin, the product of the obese ob gene, is a peptide hormone mainly found in adipose tissue [28]. Leptin exerts a wide variety of biological actions including inhibition of food intake, promotion of energy expenditure and regulation of cardiovascular function [5,12,16,27]. Leptin may participate in the regulation of cardiac function through sympathetic stimulation and its myogenic inhibition of cardiomyocyte contraction [12,15,16], and has been speculated to contribute
to obesity-related cardiac contractile dysfunction [11,16,27]. The levels of leptin in circulation are reported to be significantly elevated under insulin-resistant states such as obesity, overfeeding and hypertension [4,9,10,14,16,26,27]. Furthermore, plasma leptin levels are closely correlated with insulin resistance and increased myocardial wall thickness, independent of body composition and blood pressure levels [6,13]. However, the precise mechanism of action involved in leptin-induced cardiac contractile dysfunction in hyperleptinemic conditions such as obesity is still lacking. Since
* Corresponding author. Present address: Center for Cardiovascular Research & Alternative Medicine, and Division of Pharmaceutical Science, University of Wyoming, Laramie, WY 82071, United States. Tel.: +1 307 766 6131; fax: +1 307 766 2953. E-mail address: [email protected] (J. Ren). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.11.022
1416
peptides 27 (2006) 1415–1419
obesity is a lipotoxic disease with dyslipidemia including overtly elevated ceramide levels, the de novo ceramide pathway has been postulated to be key to the lipoapoptosis of pancreatic b-cells and cardiomyocytes in obese individuals [24,29]. Nevertheless, the direct effect of ceramide on metabolic and other biological actions of leptin has not been elucidated. The purpose of this study was to examine the effect of short-term ceramide incubation on low (5 nM) and high (50 nM) leptin-induced cardiac contractile response and intracellular Ca2+ handling properties using a serum-free cardiomyocyte primary culture model established in our laboratory [17].
2.
Materials and methods
2.1. Isolation of adult rat ventricular myocytes and primary cell culture The experimental procedures were approved by the animal investigation committees of the University of North Dakota (Grand Forks, ND) and University of Wyoming (Laramie, WY). Single ventricular myocytes were isolated from adult male Sprague–Dawley rats (200–225 g) as described previously [17]. In brief, animals were sacrificed under ketamine/xylazine sedation (5:3, 1.32 mg/kg, i.p.). Hearts were removed and perfused (at 37 8C) with oxygenated (5% CO2–95% O2) Krebs– Henseleit bicarbonate (KHB) buffer (NaCl 118 mM, KCl 4.7 mM, CaCl2 1.25 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, N-[2-hydro-ethyl]-piperazine-N0 -[2-ethanesulfonic acid] (HEPES) 10 mM, glucose 11.1 mM, pH 7.4). Hearts were subsequently perfused with a nominally Ca2+-free KHB buffer for 2–3 min until spontaneous contractions ceased followed by a 20 min perfusion with Ca2+-free KHB containing 223 U/ml collagenase (Worthington Biochemical Corp., Freehold, NJ) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St. Louis, MO). After perfusion, the left ventricle was removed, minced and incubated with fresh enzyme solution (Ca2+-free KHB containing 223 U/ml collagenase) for 3–5 min. The cells were further digested with 0.02 mg/ml trypsin (Sigma) before being filtered through a nylon mesh (300 mm) and collected by centrifugation (60 g for 30 s). Myocytes were rinsed with Ca2+-free KHB buffer to remove remnant enzyme and extracellular Ca2+ was added incrementally back to 1.25 mM. Isolated myocytes were maintained at 37 8C in a serum-free medium consisting of Medium 199 (Sigma) with Earl’s salts containing 25 mM HEPES and NaHCO3 supplemented with BSA (2 mg/ml), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), glucose (5 mM), insulin (0.1 mM), penicillin (100 U/ml), streptomycin (100 mg/ml) and gentamicin (100 mg/ml). Myocytes were incubated at 37 8C with 100% humidity and 5% CO2 for 24 h. A cohort of myocytes was incubated with low (5 nM) or high (50 nM) levels of leptin in the absence or presence of the active ceramide analog C2dihydroceramide (C2-ceramide, 25 mM) for 24 h. C2-ceramide was dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was <0.1%, which did not affect cell mechanics [3]. Cells remained viable for 24 h and were not used if they had any obvious sarcolemmal blebs or spontaneous contractions.
2.2.
Cell shortening/relengthening measurements
Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam1 system (IonOptix, Milton, MA) as described previously [17]. In brief, coverslips with cells attached were placed in a chamber mounted on the stage of an inverted microscope (Olympus X-70) and superfused (2 ml/min at 25 8C) with a buffer containing: NaCl 131 mM, KCl 4 mM, CaCl2 1 mM, MgCl2 1 mM, glucose 10 mM, HEPES 10 mM, at pH 7.4. The cells were field stimulated at a frequency of 0.5 Hz, 3 ms in duration. A video-based edge-detector was used to capture and convert changes in cell length during shortening and relengthening into an analog voltage signal. Cell shortening and relengthening were assessed using the following indices: peak twitch amplitude (PS), time-to-90% PS (TPS) and time-to-90% relengthening (TR90), maximal velocities of shortening (+dL/dt) and relengthening (dL/dt), respectively.
2.3.
Intracellular Ca2+ transient measurement
For these experiments myocytes were loaded with fura-2/AM (0.5 mM) for 10 min at 25 8C. Fluorescence measurements were recorded with a dual-excitation single-emission fluorescence photomultiplier system (IonOptix). Myocytes were placed on an inverted microscope and imaged through an Olympus Fluor 40 oil objective. Myocytes were exposed to light emitted by a 75 W halogen lamp through either a 360 or 380 nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol. The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration (D[Ca2+]) were inferred from the ratio of fluorescence intensity obtained at both wavelengths. Intracellular Ca2+ decay time was calculated from the fluorescence decay curve as an indicator for intracellular Ca2+ clearing.
2.4.
Data analysis
Data were expressed as mean S.E.M. Statistical comparisons were performed by analysis of variance (ANOVA) followed by Newman–Keuls post hoc test. p < 0.05 was considered statistically significant.
3.
Results
As shown in Fig. 1, 24 h incubation of leptin (5 nM) with cardiac myocytes did not exhibit any obvious effect on cell shortening. However, higher concentration of leptin (50 nM) significantly depressed peak shortening (PS) amplitude, maximal velocities of shortening/relengthening (dL/dt) and prolonged duration of relengthening (TR90) without affecting duration of shortening (TPS). Twenty-four hours incubation of C2-ceramide (25 mM) itself did not exhibit any effect on cell shortening mechanics, consistent with our previous finding [3]. Interestingly, C2ceramide significantly potentiated leptin-induced cardiac contractile response. In the presence of C2-ceramide, low concen-
peptides 27 (2006) 1415–1419
1417
Fig. 1 – Effect of 24 h incubation of leptin (5 and 50 nM) on myocyte shortening in the absence or presence of C2-ceramide (25 mM). (A) Peak shortening amplitude (% of resting cell length), (B) maximal velocity of shortening/relengthening (WdL/dt), (C) time-to-peak shortening (TPS) and (D) time-to-90% relengthening (TR90). Mean W S.E.M., n = 75 cells per group. *p < 0.05 vs. control group; #p < 0.05 vs. leptin 50 nM group.
tration leptin (5 nM) significantly suppressed peak shortening amplitude, dL/dt and prolonged TR90 without affecting TPS. The high concentration leptin (50 nM)-induced depression of peak shortening, dL/dt, D[Ca2+] and prolongation of TR90 was further potentiated by C2-ceramide co-incubation. In addition, 50 nM leptin significantly shortened TPS in the presence of C2ceramide, the effect of which was not seen in the absence of C2ceramide. To determine whether C2-ceramide-induced potentiation of leptin-induced cardiac contractile response was due to reduced availability of intracellular-free Ca2+ concentration ([Ca2+]), we used fura-2 to evaluate changes in intracellular Ca2+ levels in response to electrical stimuli. The time course of fluorescence signal decay was calculated to assess intracellular Ca2+ clearing rate. Similar to our observation in cell shortening, 5 nM leptin did not affect baseline [Ca2+], peak [Ca2+], changes of intracellular Ca2+ (D[Ca2+]) and intracellular Ca2+ decay rate, whereas 50 nM leptin significantly reduced baseline [Ca2+], peak [Ca2+], D[Ca2+] without affecting intracellular Ca2+ decay rate (Fig. 2). Interestingly, although ceramide itself failed to affect intracellular Ca2+ handling properties, it significantly sensitized leptin-induced response on intracellular Ca2+ transients. In the presence of C2-ceramide, low concentration leptin (5 nM) significantly suppressed D[Ca2+] without affecting the baseline or peak [Ca2+] as well as intracellular Ca2+ decay rate. Although the high concentration leptin (50 nM)-induced depression of
baseline and peak [Ca2+] was not affected by C2-ceramide, depression of D[Ca2+] elicited by 50 nM leptin was significantly augmented by C2-ceramide. While 50 nM leptin itself failed to affect the intracellular Ca2+ decay rate, it significantly slowed intracellular Ca2+ clearing rate in the presence of C2-ceramide (Fig. 2).
4.
Discussion
This study demonstrated that 24 h co-incubation of the ob gene product leptin with cardiac myocytes led to depressed cell shortening and intracellular Ca2+ transients at high (50 nM) but not low (5 nM) leptin concentration, consistent with our previous finding [12]. Although ceramide itself failed to elicit any overt effect on cell shortening and intracellular Ca2+ transients following 24 h incubation, it significantly potentiated the cardiac contractile response of the adipokine leptin. As circulating levels of leptin are increased in almost all types of obesity except in ob/ob mice [4,6,7,10] accompanied with elevated cellular ceramide level and de novo synthesis [23–25,29], the depressed cardiac contractility seen in hyperleptinemic obesity [1,2,18,19] may be due, in part, to the synergistic action of the two lipid mediators on cardiomyocyte contractile function.
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Fig. 2 – Effect of 24 h incubation of leptin (5 and 50 nM) on intracellular Ca2+ transients in the absence or presence of C2ceramide (25 mM). (A) Baseline intracellular Ca2+ level, (B) peak intracellular Ca2+ level, (C) change of intracellular Ca2+ level in response to electrical stimuli (D[Ca2+] = peak S baseline) and (D) intracellular Ca2+ decay rate. Mean W S.E.M., n = 39–40 cells per group. *p < 0.05 vs. control group; #p < 0.05 vs. leptin 50 nM group.
Leptin possesses a wide variety of biological responses ranging from cell proliferation, differentiation, to functional activation of hemopoietic and embryonic cells [5,20,22]. Recent evidence also depicted a role of leptin in cardiac contractile regulation either directly through cardiomyocyte contraction or indirectly via enhanced sympathetic nerve activity [12,15,16]. Low nanomolar levels of leptin are considered between physiological and pathophysiological range [9,13]. Our current study did not reveal any overt cardiac contractile response for leptin at concentrations below 10 nM, consistent with our earlier observations [12,27]. Therefore, there is a seemingly disconnection between in vivo and in vitro leptin levels and window of regulation for cardiac contractile function. Since obesity is often associated with elevated synthesis and cellular levels of the lipotoxic mediator ceramide [23,25,29], our observation that ceramide may potentiate or augment cardiac depressive effect of leptin may provide an explanation for hyperleptinemiaassociated cardiac dysfunction. It is believed that elevated triglyceride and fatty acid levels in overfeeding and obesity stimulate serine palmitoyl transferase activity resulting in ceramide synthesis de novo and elevated ceramide levels [23,25]. Compromised ventricular function is commonly seen in obesity with insulin resistance and decreased adrenergic responsiveness being the most predominant contributing
factors [8,21]. Recent evidence has implicated a role of the satiety factor leptin in cardiac contractile dysfunction in obesity characterized by decreased contractility, reduced diastolic compliance and prolonged relaxation [1,2,18,19]. Results from our current study indicated that short-term incubation of leptin with cardiac myocytes reduced peak shortening amplitude, maximal velocity of shortening/ relengthening and prolonged duration of relaxation. Perhaps the most important finding is that a low level of leptin, which displays no effect on cardiac contraction by itself, significantly depresses cardiac contractile response in the presence of ceramide. This may put individuals with normal leptin levels but raised ceramide levels at risk of developing heart dysfunction. The higher level (50 nM) leptin-induced cardiac contractile responses were further exacerbated by ceramide. Ceramide also unmasked additional effects of leptin on duration of shortening and intracellular Ca2+ clearing rate. These observations are supported by shortened contraction duration [27] and reduced rate of intracellular Ca2+ clearing [18] reported in cardiomyocytes from hyperleptinemic individuals. It is worthy mentioning that the mechanical effects are supported by the fact of reduced intracellular Ca2+ rise in response to electrical stimuli, suggesting an alteration of intracellular Ca2+ handling is likely responsible for the synergistic effect of ceramide and leptin on the cardiac contractile response in single cardiac myocytes.
peptides 27 (2006) 1415–1419
In conclusion, our present study demonstrated, for the first time, a synergistic effect between leptin and ceramide on cardiac contractile function and intracellular Ca2+. These data should shed some light on the elucidation of the role of leptin in hyperleptinemia-induced cardiac dysfunction. Further study is warranted to examine the cellular machineries such as sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger in this culprit synergy between leptin and ceramide.
Acknowledgements This work was supported by grants from American Heart Association Northland and Pacific Mountain Affiliates. DPR held a predoctoral fellowship from American Heart Association Northland Affiliate during the course of this work. The authors wish to sincerely thank Ms. Bonnie Zhao for her assistance in data analysis.
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