Systemic Immunosuppression Fails to Suppress Cardiac Cytokine Induction in Pressure Overload Hypertrophy in Rats

Immunobiol. (2002) 205, pp. 51– 60 © 2002 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol

Klinik und Poliklinik für Innere Medizin II, and 2 Klinik und Poliklinik für Innere Medizin I, Klinikum der Universität Regensburg, Regensburg, Germany

Systemic Immunosuppression Fails to Suppress Cardiac Cytokine Induction in Pressure Overload Hypertrophy in Rats ANDREAS JERON, RAINER H. STRAUB2, TANJA KAISER, GÜNTER A. J. RIEGGER, and FRANK MUDERS Received July 31, 2001 · Accepted in revised form October 31, 2001

Abstract Background: Activation of cytokines such as interleukin-6 (IL-6) has been implicated in the pathogenesis of left ventricular dysfunction and hypertrophy since they have been shown to mediate cell proliferation, negative inotropic effects and myocardial hypertrophy. However, the effects of immunosuppressive therapy on cytokines in the treatment of heart failure and hypertrophy are unclear. Aims: To test the hypothesis that systemic immunosuppresion may influence serum and myocardial IL-6 and, thereby, may affect progression of myocardial hypertrophy. We studied the effects of chronic treatment with methotrexate (MTx) and with the ACE inhibitor ramipril on IL-6 in rats with pressure overload left ventricular hypertrophy (LVH) due to aortic banding. Methods: Animals were treated with either vehicle (n = 6) or methotrexate (MTx 1: 0.3 mg/kg BW/week; MTx 2: 0.9 mg/kg BW/week; i.p.; n = 6 each group) once a week during weeks 4–12 after aortic banding; sham-operated rats served as controls (CTRL; n = 8). During the development of LVH, serum IL-6 was determined by rat-specific ELISA and 12 weeks after aortic banding myocardial IL-6 was measured using a tissue superfusion technique or determining of protein concentration. Results: Aortic banding significantly lowered blood pressure, increased left ventricular weight and resulted in elevated serum IL-6 levels (27.6 ± 5.1 vs 19.1 ± 2.3 pg/ml, p < 0.05) compared to CTRL. MTx treatment normalised the initially elevated serum IL-6 levels after 8 weeks of treatment. The significant increase in IL-6 concentration in the superfusate of all aortic banding groups compared to CTRL (< 30%, p < 0.05) was not altered by prior MTx therapy. Accordingly, both doses of MTx failed to prevent LVH progression (1.67 ± 023g vs. 2.32 ± 0.31 g, p < 0.05). In contrast, chronic inhibition of the RAAS not only prevents LVH but also reduces myocardial IL-6 concentration (6898 ± 355 vs. 3073 ± 366 pg/mg protein, p < 0.05). Conclusion: Pressure overload LVH in rats is characterized by an increase in serum levels of IL-6 as well as myocardial IL-6. Chronic immunosuppressive therapy normalized systemic IL-6 levels, but failed to reduce cardiac IL-6 expression and the progression of LVH, while ACE inhibition is sufficient to modify LVH and thereby normalises myocardial IL-6 expression.

Introduction Cytokines are overexpressed during human cardiac-related illnesses such as congestive heart failure or dilated cardiomyopathy (1, 2, 3). Tumor necrosis factor (TNF) is a well 0171-2985/02/205/01-051 $ 15.00/0

52 · A. JERON et al. characterized cytokine with negative inotropic effects on myocytes, most likely by inducing iNOS (4), that is capable of stimulating fibroblasts and mycocytes, resulting in cardiac remodeling and hypertrophy (5, 6). Transgenic mice overexpressing TNF in the heart show increased left ventricular mass and a progressive dilatation of the heart, resulting in congestive heart failure (7). Elevated serum levels of TNF in patients with heart failure and hypertensive heart disease are linked to poor outcome of these patients (8). The association between TNF and heart failure was confirmed in a recent study which demonstrated that the soluble TNF-receptor I seems to be an independent prognostic marker for survival of heart failure patients (9). Many drugs used in the treatment of heart failure like ACE inhibitors and amiodarone have various modulatory effects on the production of cytokines (10). Nevertheless, it is unknown whether some of the beneficial effects of these drugs are mediated by cytokines. The effects of increased cytokine levels on cardiovascular function in vivo and the progression of heart disease is still a matter of debate, because the origin of elevated serum cytokines (11) is unknown. Another multifunctional cytokine that modulates inflammatory responses is Interleukin-6 (IL-6); in cardiac hypertrophy, congestive heart failure as well as acute myocardial infarction elevated serum levels were found (12, 13). IL-6 activates the Janus-kinase-signal transducer after binding to its receptor and subsequent formation of the gp130 receptor complex, which is abundant in the heart. The importance of the gp130 signaling pathway in myocyte hypertrophy and cardiac development is indicated by results from transgenic animals. Mice, overexpressing IL-6 and the IL-6 receptor in the heart, are characterized by a continuously activated gp130, resulting in cardiac hypertrophy (14). Furthermore, disruption of the gp130 gene is lethal in mice due to the development of a hypoplastic ventricular myocardium (15). Therefore, inhibition of cardiac IL-6 by immunomodulating therapy might be a promising tool in the therapy of left ventricular hypertrophy. Our hypothesis is that lowdose immunosuppressive therapy with methotrexate (MTx) will inhibit production of cardiac and serum IL-6 and thereby may reduce left ventricular hypertrophy in rat model of pressure overload left ventricular hypertrophy. MTx was chosen as immunosuppressive drug since it is effective in modulating cytokine expression in various rat models of chronic arthritis (16) at low doses without side effects. Furthermore, there is no hypertensive blood pressure during MTx therapy compared to Cyclosporin A (CyA) therapy, which may contribute to LVH progression. The renin-angiotensin-aldosteron-system (RAAS) is well known to contribute to left ventricular hypertrophy and heart failure in humans. Consequently, inhibition of the RAAS by ACE inhibition or aldosteron antagonists is the standard therapy for hypertension as well as heart failure (17, 18). Therefore, we measured myocardial IL-6 concentration in aortic banding rats treated with the ACE inhibitor ramipril for 8 weeks to compare these results with MTx treated rats. ACE inhibition is known to reduce wall stress and the progression of hypertrophy in rodents as well as in men, but the effects on cytokines are poorly understood (19, 20). For evaluation of cardiac IL-6 production, a superfusion system, enabling the measurement of IL-6 production in living tissues from spleen (21, 22), kidney (23) and heart (24), was used to compare the effects of different doses of MTx on cytokine production. The heart slice model allows assessment of intact tissue architecture, normal transport of substances within the tissue and the possibility of using primary cells without the need of artificial digestion procedures.

Immunosuppression and cardiac cytokines · 53

Methods Study protocol

Left ventricular hypertrophy was induced by supravalvular aortic banding in young male Wistar rats (8 weeks, 80-90 g, n = 27; Charles River, Sulzfeld, Germany) as previously described (25). Six sham operated, healthy rats served as controls (CTRL). Four weeks after surgery rats were either treated with Methotrexate (0.3 mg/kg BW (MTx 0.3) and 0.9mg/kg BW (MTx 0.9) i.p., n = 9 per group) or NaCl (n = 9, (Vehicle)) once a week for 8 weeks in total. These dosages were sufficient in a rat model of chronic arthritis (16) and are comparable with the human dose of 10–15mg/week for treatment of rheumatic arthritis. Furthermore, 8 more rats were treated with the ACE inhibitor ramipril (10 mg/kgBW/d) p.o. for 8 weeks, starting 4 weeks after operation. Blood samples for determination of serum IL-6 (ELISA, Endogen, Boston, USA) from the tail vein were obtained at week 4, week 8 and week 12 after surgery. Heart rate and systolic blood pressure were measured three times with a tail cuff at week 12. The rats were sacrificed at the end of week 12, the hearts removed and left ventricular myocardial slices (0.35 mm) were prepared for the superfusion study. Cardiac IL-6 secretion

For determination of myocardial IL-6 synthesis, left ventricular heart slices from the free left wall (6 slices per heart) were transferred immediately after harvesting the heart to minisuperfusion chambers with a volume of 80 ml. In detail, superfusion was performed for six hours at a temperature of 37° Celsius and a flow rate of 66 ml/min (one slice per chamber, 48 chambers in parallel) with culture medium containing RPMI 1640, 25mM HEPES, 5% FCS, 30 mM mercaptoethanol, 0.57 mM ascorbic acid, 1.3mM calcium and 100 IU/ml penicillin and 100 mg/ml streptomycin. Superfusate aliquots were collected hourly for 6 hours and immediately frozen at –80°C until measurements by rat-specific ELISA (Endogen, Boston, USA) were performed according to the manufacture’s protocol with a sensitivity of > 8pg/ml. In order to ensure constant superfusion conditions as well as tissue viability, following parameters were measured hourly in the superfusate for up to 8 hours: Sodium, potassium, pH, pO2, glucose, lactate dehydrogenase (LDH) and creatine kinase (CK). Furthermore, the active production of myocardial IL-6 was evaluated in an additional experiment with 12 left ventricular myocardial slices from a healthy rat by adding the protein synthesis blocker cycloheximide (10 mg/ml) to the superfusion solution. In addition, transstenotic pressure gradients were determined in untreated anesthetized and mechanically ventilated rats 12 weeks after surgery. Arterial pressure was measured after preparation of the right carotid artery and LV pressure was recorded after thoracotomy and intraventricular placement of a 20-gauge tube through the apex of the heart. Pressures were recorded simultaneously on a multichannel recorder (Recomed, PPG Hellige). Transstenotic gradients were calculated by substraction of the aortic systolic pressure from peak LV pressure. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by an independent ethical committee as well as the domestic regulatory authorities for use of laboratory animals. Statistical analysis

All data are given as mean ± SD. The data of six rats in each intervention group as well as the control group was used. Since in the superfusion study six heart slices were obtained from each heart, the results were calculated from 36 measurements for every group for every hour. The results are expressed as percentage of the 1 hour value to account for myocardial slice variation. Using 48 superfusion chambers, we were able to investigate 8 rats per day in a random fashion. Student’s t-test or ANOVA for repeated measurements were used when appropriate. A value of p < 0.05 was considered statistically significant.

54 · A. JERON et al. Results Hemodynamic data

Twelve weeks after aortic banding, systolic blood pressure, body weight and relative left ventricular weight were significantly different between the controls and the three aortic banding groups (Table 1). Treatment with MTx in both doses did not alter these parameters significantly compared to vehicle-treated LVH rats. This points to a failure of systemic immunosuppression to modify the progression of left ventricular hypertrophy in the aortic banding model. Furthermore, similar numbers of rats died during the progression of hypertrophy in all three banding groups (2–3 out of 9). The LV peak pressure and the aortic blood pressure was measured invasively in a small number of control rats (n = 5) 12 week after surgery to calculate transstenotic gradients and to quantify the hemodynamic stress caused by aortic banding. The mean systolic LV pressure was 227 ± 7 mmHg, the aortic pressure 131±6 mmHg, resulting in a substantial mean gradient of 96 ± 9 mmHg. Table 1. Haemodynamic parameters and body weight after 12 weeks Parameter

CTRL

Vehicle

MTx 0.3

MTx 0.9

p

Heart rate (bpm) Blood pressure (mmHg) Body weight (g) Heart weight (g) Relative HW (g/kg BW)

386 ± 33 136 ± 12 532 ± 57 1.67 ± 0.23 3.15 ± 0.34

396 ± 39 114 ± 10 474 ± 47 2.35 ± 0.74 4.91 ± 1.23

387 ± 33 120 ± 10 469 ± 18 2.32 ± 0.31 4.97 ± 0.78

397 ± 29 120 ± 12 478 ± 40 2.24 ± 0.26 4.70 ± 0.49

n.s 0.05 0.05 0.05 0.05

CTRL: Sham-operated rats; Vehicle: Aortic banding rats, placebo treated; MTx 0.3: Aortic banding plus 0.3 mg/kg methotrexate per week i.p.; MTx 0.9: Aortic banding plus 0.9 mg/kg methotrexate per week i.p.

Circulating IL-6

The IL-6 serum concentration was already elevated in aortic-banded rats compared to controls at the beginning of MTx therapy (week 4 after operation: 27.4 ± 3.0 pg/ml vs 19.1 ± 2.2 pg/ml, p < 0.05), indicating an increased inflammatory process after a few weeks of left ventricular pressure overload. In contrast, IL-6 serum levels were normalised in the two MTx groups as well as the ramipril group after 8 weeks of treatment (week 12 of the study), while the untreated aortic banding group was characterized by sustained, significantly elevated IL-6 concentration (26.3 ± 1.8 pg/ml vs. 19.7 ± 2.2 pg/ml of controls, p < 0.05, Figure 1). Protein concentration

The myocardial IL-6 protein concentrations were determined by the method of Loewry. Measurements of LV myocardial IL-6 (ELISA) revealed a significant increase in aortic-

Immunosuppression and cardiac cytokines · 55

Figure 1. Serum IL-6 concentration. Serum IL-6 concentration in pg/ml ± SD, obtained from the tail vein before methotrexate (MTx) therapy starts (week 4 after operation), during MTx therapy (week 8) and at the end of therapy (week 12). Black bars: Vehicle treated group; Horizontal lined bars: MTx 0.3 mg/kg treated group; Vertical lined bars: MTx 0.9 mg/kg treated group; White bars: Controls * = p < 0.05 (Anova).

banding rats as compared to vehicle treated controls (6898 ± 355 vs. 3436 ± 432 pg/mg protein, p < 0.05). ACE inhibition by ramipril strongly suppressed myocardial IL-6 concentration (3073 ± 366 pg/mg protein, p < 0.05 vs. aortic-banding rats). In contrast, MTx-treated rats showed similar IL-6 concentration as the vehicle-treated LVH rats. Superfusion studies

The superfusion conditions (Sodium: 121 ± 1 mmol; Potassium: 5.5 ± 0.2 mmol; Glucose: 175 ± 2 mg/dl; pO2: 90 ± 7 mmHg; pH: 7.41 ± 0.04; lactate: 7.4 ± 0.5 mg/dl) remained stable for the entire 6 hours of tissue superfusion. Preliminary results demonstrated vital tissue for up to 24 hours in case the slices were thinner than 0.5 mm (data not shown). The concentration of LDH and CK were elevated in the first hour (LDH: 73 ± 13 U/l; p < 0.001 vs. hour 2–8; CK: 36 ± 3 U/l, p < 0.05 vs. hour 2–8) and remained stable during the following hours (LDH hour 2: 12 ± 4 U/l vs. hour 3–8: 8 ± 2 U/l, n.s.; CK hour 2: 8 ± 3 U/l vs. hour 3–8: 7 ± 3 U/l, n.s.), most likely caused by cell damage during preparation. IL-6 concentration in the superfusate was significantly elevated beginning from hour 2, peaking at hour 4 and continuously elevated until hour 6. This increase can be completely blocked by administration of the protein synthesis blocker cycloheximide, indicating de novo synthesis of IL-6 during the superfusion study (Fig. 2). Cytokine concentration in the superfusate was at least 30% higher in all aortic banding groups compared to controls at the various time points normalised to the 1 hour value (Fig. 3). These differences reached statistical significance starting from the second

56 · A. JERON et al.

Figure 2. Reduction of IL-6 concentration in the superfusate during superfusion with the protein synthesis inhibitor cycloheximide. Average IL-6 concentration in pg/ml in the superfusate from eight slices of control rats (black line) compared to eight slices of the same rats superfused with cycloheximide beginning at 2 hour (dotted line). * = P < 0.05.

Figure 3. IL-6 concentration in the superfusate. Average IL-6 concentration in the superfusate solution in percentage of the 1-hour value to account for slice variation. Quadrangle: Sham-operated controls; Triangular: Aortic banding + MTx 0.3 mg/kg; Rhomb: Aortic banding + MTx 0.9 mg/kg; Point: Aortic banding with vehicle; N = 36 for each group (6 rats per group, 6 slices per heart); * = p < 0.05 (C vs. aortic banding groups).

Immunosuppression and cardiac cytokines · 57

hour of the superfusion studies. Interestingly, the myocardial IL-6 production was unaffected by prior MTx therapy since no significant differences were obtained between the three aortic banding groups. Discussion The contribution of elevated serum levels of pro-inflammatory cytokines, found in patients with hypertensive heart disease or congestive heart failure, to the progression of these cardiac disorders is still a matter of debate (19). Previous studies have shown deleterious effects of cytokines on cardiomyocyte contractility in vitro, most likely by induction of iNOS, and on cardiac function in vivo (4, 26). Furthermore, TNF and IL-6 are capable to induce hypertrophy in cardiomyocytes as well as in fibroblasts (27, 28). These studies indicate that therapeutic inhibition of proinflammatory cytokines may be a promising therapy of hypertensive heart disease or congestive heart failure (29). The model of pressure overload left ventricular hypertrophy is characterised by a transition of LVH to heart failure, as well as alteration of serum neurohumoral factors such as an increase in natriuretic peptides and vasopressin (30, 31, 32). Furthermore, elevated serum concentration of IL-6 is an additional characteristic feature of this model, as indicated by the current study. It has been demonstrated that immunosuppression with low dose MTx treatment is sufficient to suppress serum cytokines in a rat model of chronic arthritis (16). In accordance with these results, the present study clearly demonstrates that the normalisation of elevated IL-6 levels by chronic immunosuppression with MTx is achievable in the two MTx – treated aortic banding groups compared to the LVH group. Interestingly, not only MTx treatment but also ACE inhibition is sufficient to normalise elevated IL-6 serum concentration to control values. This observation points to the heart as potential source of cytokines in left ventricular hypertrophy. In contrast to this suppressive systemic effect, chronically MTx administration failed to normalize cardiac IL-6 secretion and to modify progression of left ventricular hypertrophy. Different activated pathways may contribute to this failure in the pressure overload hypertrophy model. First of all, it was shown that cardiac Angiotensin II, which is elevated in the aortic banding model (32), can enhance IL-6-like cytokine-induced hypertrophy of neonatal rat cardiac myocytes (33) and increase mRNA IL-6 expression in fibroblasts (34). Additionally, mechanical stretch augments mRNA expression of cardiac IL-6 (35). Progressive left ventricular hypertrophy results in increased sympathetic activity and finally in myocardial hypoxy. Recently, it was shown that chronic beta-adrenergic stimulation induces myocardial, but not systemic, IL-6 expression in rat hearts (36). Furthermore, IL-6 is induced by hypoxic stimulation in cardiac myocytes (37). In contrast to chronic immunosuppressive therapy, ACE inhibition was sufficient to reduce elevated myocardial IL-6 expression. Since the ACE inhibitor ramipril has no direct immunosuppressive properties, reduction of wall stress and myocardial angiotensin II may cause this beneficial effect. Taken together, the current data support the hypothesis that cardiac IL-6 might be regulated differently from immune cell-derived IL-6. As demonstrated, the superfusion system enables cytokine secretion studies within living tissue of normal and diseased myocardium. The advantages of this approach compared to studies of cultured cells are numerous. The architecture of the tissue of interest

58 · A. JERON et al. remains unaltered, enabling the physiological interaction of the different cell lines. No artificial procedures like digestion and passages of cultured cells are necessary, which may modify the physiological response of these cells to different stimuli. Furthermore, feedback mechanisms can take place at similar interstitial flow rates of about 33–66 ml/min as occur in vivo. In summary, systemic immunosuppressive therapy with drugs like MTx seems to be insufficient to modify myocardial cytokine expression, as demonstrated by superfusion experiments of intact heart tissue. Furthermore, cardiac function and progression of hypertrophic heart disease are unaltered by chronic administration of MTx.

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