Short-Term Lenalidomide (Revlimid) Administration Ameliorates Cardiomyocyte Contractile Dysfunction in ob/ob Obese Mice

Authors

  • Linlin Li,

    1. Department of Pharmacology, Xinjiang Medical University, Urumqi, Xinjiang, P. R. China
    2. Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
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  • Yinan Hua,

    1. Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
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  • Maolong Dong,

    1. Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
    2. Department of Cutaneous and Burn Surgery, Xijing Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, P. R. China
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  • Quan Li,

    1. Department of Anesthesiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China
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  • Derek T. Smith,

    1. Division of Kinesiology and Health, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
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  • Ming Yuan,

    1. Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, P. R. China
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  • Kyla R. Jones,

    1. Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
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  • Jun Ren

    Corresponding author
    1. Center for Cardiovascular Research and Alternative Medicine, School of Pharmacy, University of Wyoming College of Health Sciences, Laramie, Wyoming, USA
      (jren@uwyo.edu)
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  • The first three authors contributed equally to this article.

(jren@uwyo.edu)

Abstract

Lenalidomide is a potent immunomodulatory agent capable of downregulating proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and upregulating anti-inflammatory cytokines. Lenalidomide has been shown to elicit cardiovascular effects, although its impact on cardiac function remains obscure. This study was designed to examine the effect of lenalidomide on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were given lenalidomide (50 mg/kg/day, p.o.) for 3 days. Body fat composition was assessed by dual-energy X-ray absorptiometry. Cardiomyocyte contractile and intracellular Ca2+ properties were evaluated. Expression of TNF-α, interleukin-6 (IL-6), Fas, Fas ligand (FasL), the short-chain fatty acid receptor GPR41, the NFκB regulator IκB, endoplasmic reticulum (ER) stress, the apoptotic protein markers Bax, Bcl-2, caspase-8, tBid, cytosolic cytochrome C, and caspase-12; and the stress signaling molecules p38 and extracellular signal-regulated kinase (ERK) were evaluated by western blot. ob/ob mice displayed elevated serum TNF-α and IL-6 levels, fat composition and glucose intolerance, the effects of which except glucose intolerance and fat composition were attenuated by lenalidomide. Cardiomyocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening, prolonged time-to-PS and time-to-90% relengthening as well as intracellular Ca2+ mishandling, which were ablated by lenalidomide. Western blot analysis revealed elevated levels of TNF-α, IL-6, Fas, Bip, Bax, caspase-8, tBid, cleaved caspase-3 caspase-12, cytochrome C, phosphorylation of p38, and ERK in ob/ob mouse hearts, the effects of which with the exception of Bip, Bax, and caspase-12 were alleviated by lenalidomide. Taken together, these data suggest that lenalidomide is protective against obesity-induced cardiomyopathy possibly through antagonism of cytokine/Fas-induced activation of stress signaling and apoptosis.

Introduction

Obesity, if uncorrected, contributes to the onset and development of insulin resistance, cardiac hypertrophy and myocardial dysfunction, resulting in enhanced cardiovascular morbidity and mortality (1,2). Several theories including inflammation, oxidative stress, dyslipidemia, and hyperleptinemia have been speculated for obesity-induced cardiac morphological and functional defects (1,2,3). However, the proper management of obesity and insulin resistance has not been successful to drastically reduce the cardiovascular risks in obesity (4,5). Current therapeutic regimens against type 2 diabetes including pharmacological treatment (such as biguanides and thiazolidinedione), caloric restriction and exercise have proven to be somewhat effective (4,5), although none is deemed ideal. Search for novel effective drugs is in high demand for a better management of obesity and insulin resistance. Onset and progression of obesity is linked to a proinflammatory state with overt accumulation of proinflammatory molecules such as tumor necrosis factor-α (TNF-α), high sensitive C reactive peptide, fibrinogen and interleukin-6 (IL-6), among others (6,7,8). The link between inflammation and obesity is not fully understood with cytokine release into the circulation from adipose tissue being a commonly speculated dogma (6,7,8). Pharmaceutical therapy targeted against inflammation has shown some promises in the management of obesity-related complications (4,6,8). In particular, TNF-α has been demonstrated to provoke an insulin desensitization phenomenon to propel the onset of metabolic syndrome that could in turn evolve into type 2 diabetes (9). Therefore, experimental and clinical studies to evaluate the beneficial effect of anti-TNF-α therapy have drawn some special attention recently (10,11,12).

Lenalidomide (Revlimid) is an analogue of thalidomide as a member of a new drug group commonly known as IMiDs. IMiDs family displays a potent cytokine modulatory activity via inhibition of cytokines such as TNF-α and IL-6 (13,14,15). Recent finding have depicted that lenalidomide and IMiDs may suppress the action of cytokines TNF-α, IL 1β, 6, 12, and granulocyte macrophage-colony stimulating factor (13,14). Other main biological actions of lenalidomide and other IMiDs include antiangiogenesis, antiproliferation, proapoptosis, anticarcinogenesis, inhibition of microenvironment support for tumor cells, and immunomodulation (16,17). These agents have been used as inhibitors of α glucosidase and could be potential drugs for treatment of obesity and diabetes mellitus (14). Nonetheless, the effect of lenalidomide and its analogues on cardiac contractile function has not been examined in proinflammatory states such as obesity. Therefore the present study was designed to evaluate the effect of short-term lenalidomide treatment on cardiomyocyte contractile function and potential mechanisms involved with a focus on apoptosis, endoplasmic reticulum (ER) stress, as well as activation of stress signaling including extracellular signal-regulated kinase (ERK) and p38 MAP kinases.

Methods and Procedures

Experimental mice and lenalidomide treatment

The experimental procedures described in this study were approved by the University of Wyoming (Laramie, WY) Animal Use and Care Committee. In brief, homozygous B6.V-lep/J male mice were obtained from the Jackson Laboratory (Bar Harbor, ME) at 5 weeks of age and were housed in the School of Pharmacy Animal Facility at the University of Wyoming with free access to food and water. Seven-week-old male ob/ob obese (leptin deficient) and age-matched wild-type C57BL/6J mice were randomly divided into two groups to receive lenalidomide (50 mg/kg/day, Selleck Chemicals LLC, Houston, TX) via oral gavage for 3 days (18). The dosage and treatment duration chosen for lenalidomide were essentially based on the reported effective dosage range and the relatively quick onset of drug efficacy (13,18,19). Control mice received similar volume of saline.

Measurement of body fat composition, bone mineral density and determination of bone mineral density z-scores

Body composition was measured using dual-energy X-ray absorptiometry, which is a clinical measure of lean tissue mass, adipose tissue mass, and bone mineral mass and density. A low level pencil-beam X-ray moved transversely from the head to the tail across the sedated mouse. Difference in absorbance of the X-ray was detected according to tissue density. Percent fat was calculated using fat and body mass (20). In 13 mice (N = 3 in each of the following groups: lean, ob/ob, and ob/ob + lenalidomide; and N = 4 in lean + lenalidomide), bone mineral density (g/cm2) was detectable within the sensitivity range of dual-energy X-ray absorptiometry. Bone mineral density z-scores were determined using computed mean and standard deviations from a wild-type lean mouse reference dataset (N = 43).

Serum analysis

To determine the effect of obesity and lenalidomide on systemic factors in the blood, we measured TNF-α and IL-6 in the serum of each mouse using the Millipore's multiplex MAP mouse serum adipokine panel kit (Millipore, Billerica, MA) per the instruction by the manufacturer (21).

Oral glucose tolerance test

Two days before sacrifice, mice fasted for 12 h were given an intraperitoneal challenge of glucose (2 g/kg body weight). Blood samples were drawn from the tail veins immediately before the glucose challenge, as well as 15, 60, 90, and 120 min thereafter. Blood glucose levels were determined using a glucometer. Area underneath the intraperitoneal glucose tolerance test curve was determined using the GraphPad Prism Software (GraphPad Software, La Jolla, CA) (22).

Isolation of mouse cardiomyocytes

Single cardiomyocytes were enzymatically isolated as described (22). Briefly, hearts were removed and perfused (at 37 ºC) with oxygenated (5% CO2–95% O2) Krebs-Henseleit bicarbonate buffer containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, and 11.1 glucose. Hearts were subsequently perfused with a Ca2+-free Krebs-Henseleit bicarbonate buffer containing 223 U/ml collagenase D (Worthington Biochemical, Freehold, NJ) for 20 min. After perfusion, left ventricles were removed and minced to disperse individual cardiomyocytes in Ca2+-free Krebs-Henseleit bicarbonate buffer. Extracellular Ca2+ was added incrementally back to a final concentration of 1.25 mmol/l. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used. Only rod-shaped myocytes with clear edges were selected for mechanical measurement. Neither obesity nor lenalidomide treatment significantly affected the yield of murine cardiomyocytes.

Cell shortening/relengthening

Mechanical properties of cardiomyocytes were assessed using an IonOptix MyoCam system (IonOptix, Milton, MA ref. 22). In brief, myocytes were placed in a chamber mounted on the stage of an inverted microscope and superfused with a buffer containing (in mmol/l) 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES at pH 7.4. The cells were field stimulated with suprathreshold voltage and at a frequency of 0.5 Hz (3-min duration) with the use of a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE). The polarity of stimulatory electrodes was reversed frequently to avoid possible buildup of the electrolyte byproducts. The myocyte being studied was displayed on the computer monitor with the use of an IonOptix MyoCam camera (IonOptix), which rapidly scans the image area at every 8.3 min, such that the amplitude and velocity of shortening/relengthening is recorded with good fidelity. A SoftEdge software (IonOptix) was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indexes: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), maximal velocities of shortening (+dL/dt) and relengthening (−dL/dt).

Intracellular Ca2+ transients

A cohort of myocytes was loaded with fura-2/AM (0.5 µmol/l) for 10 min, and florescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocytes were placed onto an Olympus IX-70 (Olympus, Tokyo, Japan) inverted microscope and imaged through a Fluor 40 oil objective. Cells were exposed to light emitted by a 75W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm, and qualitative change in fura-2 florescence intensity (FFI) was inferred from the FFI ratio at the two wavelengths (360/380). Fluorescence decay time was measured as an indication of the intracellular Ca2+ clearing rate. Single exponential decay rate and area underneath the curve (AUC) using the intracellular Ca2+ transient curve were used to evaluate the intracellular Ca2+ decay (22).

Analysis of cytosolic cytochrome c content

The ventricles were minced and homogenized by Polytron in the ice-cold MSE buffer (220 mmol/l mannitol, 70 mmol/l sucrose, 2 mM EGTA, 5 mmol/l 3-(4-morpholino) propane sulfonic acid, pH 7.4, 0.2% bovine serum albumin and a protease inhibitor cocktail containing 4-(2-aminoethyl) benzenesulfonyl fluoride, E-64, bestatin, leupeptin, aprotinin, and EDTA obtained from Sigma Chemicals (St. Louis, MO)). The homogenates were centrifuged for 10 min at 600g to remove unbroken tissue and nuclei, and the supernatants were centrifuged for 30 min at 100,000g to obtain cytosolic fraction. Fifty micrograms of cytosolic protein was separated by 15% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was analyzed by western blot using the anticytochrome c antibody (1:1,000; Upstate, Waltham, MA ref. 23).

Western blot analysis

Left ventricles were rapidly removed and homogenized in a lysis buffer containing 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1 mM EDTA, 1 mmol/l EGTA, 1% Triton, 0.1% SDS, and 1% protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000g for 20 min at 4 °C. The protein concentration of the supernatant was evaluated using the Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts (50 µg protein/lane) of protein and prestained molecular weight marker (Gibco-BRL, Gaithersburg, MD) were loaded onto 7–10% SDS polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II; Bio-Rad) before being separated and transferred to nitrocellulose membranes (0.2 µm pore size; Bio-Rad). Membranes were incubated for 1 h in a blocking solution containing 5% nonfat milk in Tris-buffered saline, washed in Tris-buffered saline and incubated overnight at 4 °C with anti-TNF-α (1:1,000), anti-IL-6 (1:1,000), anti-Fas (1:1,000), anti-Fas ligand (FasL, 1:1,000), anti-GPR41 (1:1,000), anti-IKB (1:1,000), anti-pIKB (1:1,000), anti-Bip (1:1,000), anti-Bax (1;1,000), anti-Bcl-2 (1:1,000), anti-caspase-8 (1:1,000), anti-tBid (1:1,000), anti-cleaved caspase-3 (1:1,000), anti-caspase-12 (1:1,000), anti-p38 (1:1,000), anti-pp38 (1:1,000), anti-ERK (1:1,000), anti-phosphorylated ERK (1:1,000), and α-tubulin (1:5,000, loading control) antibodies. All antibodies were obtained from Cell Signaling (Beverly, MA) or Santa Cruz (Santa Cruz, CA). After incubation with the primary antibody, blots were incubated with an antimouse IgG horseradish peroxidase linked antibody at a dilution of 1:5,000 for 1 h at room temperature. Immunoreactive bands were detected using the Super Signal West Dura Extended Duration Substrate. The intensity of bands was measured with a scanning densitometer (Model GS-800; Bio-Rad) coupled with Bio-Rad PC analysis software (Bio-Rad)(22).

Statistical analysis

Data were presented as mean ± s.e.m. Statistical significance (P < 0.05) for each variable was evaluated by analysis of variance followed by a Dunnett's post hoc analysis. Dunnett's test compares group means and is designed for situations where all groups with somewhat similar sample sizes are to be pitted against one “reference” control group. It is widely used after analysis of variance has rejected the hypothesis of equality of the means of the distributions. Therefore Dunnett's test is capable of identifying groups whose means are significantly different from the mean of the reference group.

Results

General features of lean and ob/ob mice treated with lenalidomide

As expected, ob/ob mice displayed a significantly greater body weight and body fat composition compared with the age-matched lean controls. Short-term lenalidomide treatment failed to affect body weight and body fat composition. Likewise, there was no effect of lenalidomide treatment or adiposity on bone mineral density (in g/cm2; lean: 0.068 ± 0.004, lean + lenalidomide: 0.074 ± 0.005, ob/ob: 0.073 ± 0.002, and ob/ob + lenalidomide: 0.074 ± 0.003; P > 0.05 among any groups) or z-scores. z-scores were similar across all four groups (F = 0.39, P = 0.55). Serum levels of proinflammatory cytokines including TNF-α and IL-6 were significantly higher in ob/ob mice compared with the lean controls, the effect of which was significantly attenuated by lenalidomide treatment. Basal blood glucose levels were overtly higher in ob/ob mice compared with the lean controls, the effect of which was unaffected by lenalidomide. Following acute oral glucose challenge, the blood glucose levels in lean mice started to drop after peaking at around 30 min, and returned to nearly baseline after 120 min. However, the postchallenge glucose levels maintained at much high levels from 30 to 120 min in ob/ob mice, indicative of glucose intolerance and insulin resistance. Lenalidomide treatment did not affect the glucose clearance rate in lean or ob/ob mice, nor did the TNF-α inhibitor affect obesity-induced increase in AUC, indicating little effect of the drug on glucose intolerance (Figure 1).

Figure 1.

Body weight, body fat composition, serum levels of proinflammatory cytokines, oral glucose tolerance test (OGTT, 2 g/kg body weight) in lean and ob/ob mice treated with or without lenalidomide (50 mg/kg/day, p.o.) for 3 days. (a) Body weight data displaying a higher body weight in ob/ob mice regardless of the lenalidomide treatment; (b) Body fat composition data displaying a higher body fat composition in ob/ob mice regardless of the lenalidomide treatment; (c) Serum tumor necrosis factor-α (TNF-α) levels depicting elevated TNF-α levels in ob/ob mice, which are partially attenuated by lenalidomide; (d) Serum interleukin-6 (IL-6) levels depicting elevated IL-6 levels in ob/ob mice, which are partially reversed by lenalidomide; (e) OGTT curve depicting glucose intolerance in ob/ob mice, which is not affected by lenalidomide; and (f) Area underneath the curve (AUC) plotted in e depicting glucose intolerance in ob/ob mice, which is unaffected by lenalidomide. Mean ± s.e.m., n = 5–7 mice per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Mechanical and intracellular Ca2+ properties of cardio myocytes from lean and ob/ob mice

The resting cell length was significantly greater in cardiomyocytes from the ob/ob mice. Lenalidomide treatment did not affect resting cell length in lean or ob/ob mice. Cardiomyocytes from ob/ob mice displayed significantly depressed PS amplitude and maximal velocity of shortening/relengthening (±dL/dt) as well as prolonged TPS and TR90 compared with those from lean controls. Interestingly, lenalidomide treatment effectively corrected the abnormal PS, ±dL/dt, TPS, and TR90 in cardiomyocyte from ob/ob mice. Lenalidomide treatment failed to affect any of the tested mechanical indices in lean cardiomyocytes (Figure 2). To explore the possible mechanism of action behind lenalidomide and obesity-induced cardiac mechanical responses, intracellular Ca2+ homeostasis was evaluated in cardiomyocytes using the intracellular Ca2+ fluorescent dye fura-2. Data presented in Figure 3 depicted that ob/ob obesity significantly elevated baseline FFI and suppressed electrically stimulated rise in FFI (ΔFFI) as well as slowed intracellular Ca2+ decay (manifested as both elevated intracellular Ca2+ decay rate and AUC). Although lenalidomide itself did not affect these intracellular Ca2+ parameters, it abolished ob/ob obesity-induced changes in intracellular Ca2+ handling anomalies.

Figure 2.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on cell shortening in cardiomyocytes from lean and ob/ob mice. (a) Resting cell length exhibiting a greater cell length in ob/ob mice, which is unaffected by lenalidomide; (b) Peak shortening (% of resting cell length) depicting a depressed peak shortening capacity in ob/ob mice, which is ablated by lenalidomide; (c) Maximal velocity of cell shortening (+dL/dt) depicting depressed + dL/dt in ob/ob mice, which is mitigated by lenalidomide; (d) Maximal velocity of relengthening (−dL/dt) depicting depressed −dL/dt in ob/ob mice, which is obliterated by lenalidomide; (e) Time-to-peak shortening (TPS) depicting prolonged TPS in ob/ob mice, which is reversed by lenalidomide; and (f) Time-to-90% relengthening (TR90) depicting prolonged TR90 in ob/ob mice, which is rescued by lenalidomide. Mean ± s.e.m., n = 109 cells per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Figure 3.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on intracellular Ca2+ handling in cardiomyocytes from lean and ob/ob mice. (a) Resting fura-2 fluorescence intensity (FFI) depicting elevated resting FFI levels in ob/ob mice, which are reversed by lenalidomide; (b) Electrically stimulated rise of FFI (ΔFFI) depicting suppressed ΔFFI in ob/ob mice, which is ablated by lenalidomide; (c) Intracellular Ca2+ decay rate depicting delayed intracellular Ca2+ clearance in ob/ob mice, which is reversed by lenalidomide; and (d) Area underneath the Ca2+ fluorescence curve (AUC) supporting data shown in c with regards to intracellular Ca2+ clearance. Mean ± s.e.m., n = 52 cells per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Effect of lenalidomide treatment on TNF-α, IL-6, Fas, FasL, and caspase-3

To determine if the TNF-α signaling cascade plays a role in lenalidomide-induced cardioprotection, expression of the proinflammatory cytokines TNF-α and IL-6, the extracellular death pathway molecules Fas and FasL as well as the ultimate apoptotic marker cleaved caspase-3 was determined. Our data revealed upregulated expression of TNF-α, IL-6, Fas, and caspase-3 with unchanged FasL expression in myocardium from ob/ob mice. Although lenalidomide itself did not exert any effect on the expression of these proteins, it significantly attenuated or ablated obesity-induced upregulation of TNF-α, IL-6, Fas, and cleaved caspase-3 without any effect on FasL (Figure 4).

Figure 4.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), Fas, Fas ligand (FasL), and cleaved caspase-3 in myocardium from lean and ob/ob mice. (a) Representative gel blots of TNF-α, IL-6, Fas, FasL, cleaved caspase-3 and α-Tubulin (as loading control) using specific antibodies; (b) TNF-α; (c) IL-6; (d) Fas; (e) FasL; and (f) cleaved caspase-3. These data depict upregulated levels of TNF-a, IL-6, Fas and Cleaved caspase-3 (but not FasL) in ob/ob mice which are reversed by lenalidomide treatment. Mean ± s.e.m., n = 5 to 6 per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Effect of lenalidomide treatment on ER stress, apoptosis, GRP41, and IκB phosphorylation

Our further study revealed overt ER stress and apoptosis as evidenced by significantly enhanced expression of Bip, Bax, caspase-8, and caspase-12 with unchanged Bcl-2. Although lenalidomide failed to alter the expression of these ER stress (Bip and Caspase-12) and apoptotic proteins, it mitigated obesity-induced increase in caspase-8 expression without affecting that of Bip, Bax, and caspase-12. Neither obesity nor lenalidomide affected the expression of Bcl-2. However, combination of the two significantly decreased the expression of Bcl-2 (Figure 5). To determine the possible interplay between TNF-α-mediated extrinsic death pathway (through Fas and FasL) and mitochondrial (intrinsic) pathway for apoptosis, the truncated form of the BH3-only protein Bid interconnecting the two apoptotic pathways (24) was scrutinized. In addition, the short-chain fatty acid receptor GPR41, the mitochondrial apoptotic marker cytosolic cytochrome C and phosphorylation of the NFKB regulator IKB were determined in ob/ob mouse hearts with or without lenalidomide treatment. Our data revealed upregulated expression of tBid, cytosolic cytochrome C, and IKB phosphorylation (either absolute or normalized to pan IKB) with unchanged GPR41 in myocardium from ob/ob mice. Although lenalidomide itself did not exert any effect on the expression of these proteins, it significantly attenuated or ablated obesity-induced upregulation of tBid, cytosolic cytochrome C, and IKB phosphorylation. Expression of pan IKB was unaffected by either ob/ob obesity or lenalidomide. Interestingly, lenalidomide elicited a significant increase in the expression of GPR41 in ob/ob obese mice (Fig. 6).

Figure 5.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on expression of cell stress and apoptotic proteins including Bip, Bax, Bcl-2, caspase-8, and caspase-12 in myocardium from lean and ob/ob mice. (a) Representative gel blots of Bip, Bax, Bcl-2, caspase-8, caspase-12, and α-Tubulin (as loading control) using specific antibodies. (b) Bip; (c) Bax; (d) Bcl-2; (e) caspase-8; and (f) Caspase-12. The elevated levels of Bip, Bax, and caspase-12 in ob/ob mice are unaffected by lenalidomide treatment whereas lenalidomide reverses upregulated caspase-8 levels in ob/ob mice. Lenalidomide and obesity display a synergistic reduction in Bcl-2 levels. Mean ± s.e.m., n = 5 to 6 per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Figure 6.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on expression of truncated Bid (tBid), GRP41, cytosolic cytochrome C, pan IKB, phosphorylated IKB (pIKB), and pIKB-to-IKB ratio in myocardium from lean and ob/ob mice. (a) tBid; (b) GRP41; (c) cytosolic cytochrome C; (d)pan IKB; (e): pIKB; and (f) pIKB-to-IKB ratio. Insets: Representative gel blots depicting levels of tBid, GRP41, cytochrome C, IKB, pIKB, and α-Tubulin (as loading control) using specific antibodies. The elevated levels of cytochrome C (cytosolic) and IKB phosphorylation in ob/ob mice are ablated or attenuated by lenalidomide treatment. Lenalidomide and obesity display a synergistic rise in GRP41 levels. Mean ± s.e.m., n = 5–7 per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Effect of lenalidomide treatment on stress signaling

Given that stress signaling such as MAP kinases plays an essential role in obesity-associated heart dysfunction (25), stress signaling cascades including p38 and ERK were examined in lean and ob/ob hearts with or without lenalidomide treatment. Data shown in Figure 7 indicated that ob/ob obesity significantly enhanced the expression of both pan and phosphorylated p38 (although the pp38-to-p38 ratio remained unchanged) as well as ERK phosphorylation without affecting the pan ERK expression. Although lenalidomide treatment did not exert any notable effect on pan and phosphorylated proteins in lean mice, it significantly attenuated the ob/ob obesity-induced phosphorylation in ERK, but not p38.

Figure 7.

Effect of lenalidomide treatment (50 mg/kg/day, p.o. for 3 days) on expression of pan and phosphorylated p38 and extracellular signal-regulated kinase (ERK) in myocardium from lean and ob/ob mice. (a) and (b): Pan p38 and pan ERK; (c) and (d): Phosphorylated p38 (pp38) and phosphorylated ERK (pERK); (e) and (f): p38-to-pp38 and pERK-to-ERK ratios. Insets: Representative gel blots of pan and phosphorylated p38 and ERK as well as the loading control α-Tubulin using specific antibodies. Levels of pan and phosphorylated p38 exhibit a parallel increase in ob/ob mice, which are unaffected by lenalidomide treatment. Phosphorylation of ERK is higher in ob/ob mice the effect of which is partially reversed by lenalidomide treatment. Mean ± s.e.m., n = 5 to 6 per group, *P < 0.05 vs. lean group, #P < 0.05 vs. ob/ob group.

Discussion

The salient findings from our study revealed that treatment with the TNF-α inhibitor lenalidomide offers beneficial effects against ob/ob obesity-associated cardiomyocyte contractile function independent of changes in body weight and body fat composition. The lenalidomide-offered cardioprotective effects against obesity-associated cardiac contractile dysfunction may be associated with attenuation of obesity-induced increase in serum levels of the proinflammatory cytokines TNF-α and IL-6, upregulation of TNF-α and IL-6, the Fas-dependent death receptor apoptosis, mitochondrial death receptor activation (increase in cytosolic cytochrome C content), and stress signaling activation, but not ER stress. These data revealed potential benefits of lenalidomide and its analogues against proinflammatory obesity-induced cardiac complications.

Obesity impairs cardiac contractile function in a manner reminiscent of prediabetic insulin resistance and full-blown diabetes (26,27). This is supported by our observation of depressed PS amplitude, reduced ± dL/dt as well as prolonged TPS and TR90 in ob/ob obese hearts. These findings are consistent with our previous findings using ob/ob or the diet-induced obesity models (27,28). Our data revealed that lenalidomide rescued obesity-associated cardiomyocyte contractile dysfunction, suggesting the beneficial role of TNF-α inhibition against cardiac dysfunction in obesity. We also observed that impaired intracellular Ca2+ homeostasis (elevated baseline FFI, reduced ΔFFI, and prolonged intracellular Ca2+ decay) in cardiomyocytes from ob/ob obese mice, consistent with our previous findings using the same model (27). Intriguingly, lenalidomide treatment effectively reconciled ob/ob obesity-induced intracellular Ca2+ mishandling, in a manner similar to cardiomyocyte contractile function. These findings have implicated a possible role of intracellular Ca2+ homeostasis in lenalidomide-offered cardioprotection against the leptin-deficient obesity.

The unchanged body weight, body fat composition, and bone mineral density (z-scores) in ob/ob obesity in response to lenalidomide treatment suggest that the beneficial effect of lenalidomide may be independent of the body mass, body fat regulation, or bone loss. Albeit preliminary and underpowered, lenalidomide treatment does not appear to influence bone mineral density. However, this finding should be interpreted with caution, because adiposity and lean mass are positively associated with bone mineral density (29) and leptin has been shown to stimulate bone formation and metabolism (30). Data from our study also suggested that the leptin deficiency-induced obesity is accompanied with elevated serum and myocardial levels of the proinflammatory cytokines namely TNF-α and IL-6 associated with elevated Fas—FasL extrinsic death pathway apoptotic signaling in TNF-α family (31). Fas is widely expressed in various tissues including thymus, heart, lung, liver, kidney, and ovaries (32). Binding of Fas to FasL plays a pivotal role in activation-induced cell death. Antagonists for Fas, the neutral antibodies for FasL and the Fas-induced apoptosis pathway inhibitors may all possess therapeutic potential in pathological conditions with the extrinsic death pathway-dependent apoptosis (33,34). Leptin deficiency results in cardiac apoptosis linked to an increase in cardiac Fas activation. In our hand, the TNF-α inhibitor lenalidomide ablated obesity-induced upregulation of Fas and apoptosis (as evidenced by the caspase-8 and cleaved capase-3 levels). Our finding that lenalidomide rescues against ob/ob obesity-induced increase in cytosolic cytochrome c content supports a possible role of mitochondrial death pathway in lenalidomide- and obesity-induced cardiac responses. Upon proapoptotic stress, cytochrome c, an essential component of the electron transport chain in mitochondria, is released into cytosol representing the activation of mitochondrial death pathway (35). This is in line with the finding that lenalidomide ablated upregulated tBid levels in ob/ob hearts. The BH3-only protein Bid, whose truncated form (tBid) is generated by caspase-8, can target mitochondria to trigger loss of mitochondrial membrane potential. As a result, tBid serves as an essential molecule interconnecting extrinsic (death receptor via caspase-8) and intrinsic (mitochondrial) apoptotic pathways (24,35). Taken together, our data suggests that leptin deficiency-induced obesity is linked to the increased cardiac apoptosis through upregulated Fas activity, which was reversed by the TNF-α inhibitor lenalidomide. The inconsistent findings in the change of Bax and Bcl-2 in obese mice following lenalidomide treatment suggest possible contribution of certain Bax/Bcl-2-independent apoptotic signaling molecules to the TNF-α inhibition-induced beneficial effect in the heart. TNF-α is known to promote apoptosis through direct activation of NFKB, leading to the nuclear entry of the nuclear factor and cell death (36).

Our results showed that obesity upregulated p38 and promoted phosphorylation of p38 and ERK, consistent with the earlier finding of oxidative stress and activation of stress signaling cascades in obesity (20,22). In our hands, lenalidomide attenuated the ob/ob obesity-facilitated phosphorylation of ERK, but not p38, favoring a role for ERK, but not p38 in TNF-α inhibition-offered protection against obesity-induced cardiac contractile dysfunction. ERK and NFKB have been shown to be turned on by proinflammatory cytokines and free fatty acids present in obesity, resulting in interrupted insulin signaling and development of cardiac dysfunction (37,38). Our observation of enhanced phosphorylation of ERK and IKB (which activates NFKB due to removal of NFKB inhibition by IKB) are consistent with enhanced apoptosis in ob/ob obesity. Our data did not favor a major role of the short-chain fatty acid receptor GPR41 in lenalidomide-induced protection against obesity-associated cardiac contractile dysfunction, although further study is warranted to elucidate the upregulated GPR41 expression in lenalidomide-treated ob/ob hearts. Last but not least, although overt ER stress is apparent in ob/ob obese heart as reported previously (22), our data did not favor a role of ER stress regulation in lenalidomide-offered beneficial effect in cardiac contractile function. TNF-α inhibition by lenalidomide failed to reverse obesity-induced increase in the ER stress maker Bip or ER stress-related apoptotic marker Caspase-12 in ob/ob obese hearts. Bip has been widely used as a marker for ER stress and caspase-12 mediated apoptosis was dependent to a specific apoptotic pathway of ER, both of which are directly associated with the maintenance and regulation of cardiac contractile function (39,40).

Experimental limitations

Although our data suggest lenalidomide may rescue against cardiac dysfunction in obesity through alleviating TNF-α-Fas-mediated apoptosis, a number of experimental limitations should be considered. First, only a small numbers of TNF-α responsive apoptotic and anti-apoptotic proteins were examined. Other apoptotic proteins may also mediate the apoptotic changes seen in our experimental setting. Second, we only evaluated certain inflammatory and apoptotic events within a few days. It is quite possible that more dramatic changes may occur with a longer period of lenalidomide treatment. Third, the use of ob/ob leptin-deficient obese model may not best represent human obesity (often with hyperleptinemia ref. 3). It is possible that potential interaction between the TNF-α inhibitor and leptin deficiency may contribute to some puzzling findings (such as in Bax and Bcl-2 levels) in lenalidomide-treated ob/ob mice. The species difference between rodent and human also resulted in an inability of obtain T-score, which is the number of s.d. above or below the mean for a healthy 30-year-old adult of the same sex and ethnicity as the patient. Due to the apparent difference in lifespan between lean and ob/ob mice, it is extremely difficult to estimate a mouse age in both lean and obese mice equivalent to 30 years of age in human, letting alone the ethnicity issue. Last, C57BL/6J mice rather than the lean (+/?) mice purchased from Jackson Laboratory were used as the lean control in this study. Although we have not identified any difference in cardiomyocyte function and morphology between the two murine lines, caution must be taken when extrapolating data obtained from C57BL/6J mice for the authentic lean control of ob/ob mice as subtle differences may present in cardiomyocyte function, geometry, and inflammatory markers between these two mouse models.

In summary, this study suggests that lenalidomide rescues against cardiomyocyte contractile dysfunction in obesity possibly through a TNF-α-Fas-associated apoptotic mechanism. Given that effective management of obesity-associated health complications including heart dysfunction is still lacking at this time, the potential benefit of lenalidomide against obesity-associated cardiac anomalies suggests therapeutic promises of TNF-α inhibition and suppression of cytokines in obesity and insulin resistance-associated complications.

ACKNOWLEDGMENTS

This work was supported by American Diabetes Association (7–08-RA-130) to JR and National Natural Science Foundation of China (30760288) to L.L.

DISCLOSURE

The authors declared no conflict of interest.

See the online ICMJE Conflict of Interest Forms for this article.

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