Effect of Clofibric Acid on Desmin and Vimentin Contents in Rat Myocardiocytes

Abstract

The aim of this experimental study was to analyze in vitro effects of clofibric acid on vimentin and desmin contents in rat myocardiocytes, which was carried out in primary myocardiocyte cells that were treated only with clofibric acid at 0.1 mM. The measurement of vimentin and desmin were done by Western blotting and densitometry. This study showed that myocardiocytes exposed to clofibric acid exhibit a 26.3% decrease in vimentin and a 42.1% decrease in desmin. Considering the role that these intermediate filaments play in the anchorage and cellular organization of myocardiocytes, the decrease of desmin and vimentin observed in cells treated with clofibric acid may be partially responsible for the adverse effects observed in patients. In conclusion, the alteration of cytoskeletal proteins may be a cause of cardiopathy in patients treated with these compounds.

Clofibrate has been used as an antilipidemic drug since 1962 (Thorp and Waring 1962). Subsequently, gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate were developed. The active metabolite of clofibrate is clofibric acid also known as 2-p-clorophenoxyisobutyric acid, CPIB (Thorp and Waring 1962).

Fibrates are the drugs of choice in the management of hypertriglyceridemia but the efficacy of fibric acid derivatives in both the primary and secondary prevention of atherosclerosis has remained widely in doubt (Luc 2003). Early primary prevention studies of atherosclerosis using the fibric acid derivative clofibrate showed only modest effects on atherosclerosis and an alarming increase in mortality in the intervention group (Krakoff, Vela, and Brinton 2000; Eghdamian and Ghose 1998). Moreover, the use of fibrates has been associated with fatal myocardial vascular accidents. Additionally, fibrates have been reported to induce arrhythmia, which resolves by the 4th week after treatment is stopped (González et al. 1991).

Gould et al. (1995) found that fibrates decreased coronary heart disease (CHD) mortality by 13% but increased non-CHD mortality by 30%. This study concludes that the decrease in cholesterol is beneficial, but that fibric acid derivatives increase the risk of mortality in non-CHD; cholesterol lowering confers an overall benefit by the reductions in CHD and totally mortality. Therefore patients on certain fibrates treatment, specifically clofibrate, appear to have an increased risk of non-CHD and total mortality. Therefore patients on certain fibrates treatment, specifically clofibrate, appear to have an increased risk of non-CHD and total mortality. This suggest that fibric acid derivatives make profound changes in myocardiocytes structure and function.

On the other hand, the ability of eukaryotic cells to adopt a great variety of forms and carry out coordinate movements depends on a complex mesh of filamentous proteins present in the cytoplasm. This mesh is known as the cytoskeleton and it has a dynamic structure that is responsible for cell movement, cellular shape, cell division, and the cell’s response to external stimuli. The different functions of the cytoskeleton depend on the three types of filamentous proteins such as actin, microtubules, and intermediate filaments such as vimentin and desmin. The organization of desmin provides evidence for its supportive function for the maintenance of structural integrity and function of cardiac muscle cells (Nag and Huffaker 1998). Therefore it has been reported that desmin-related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy. Study conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. They identified a novel heterozygous Q389P desmin mutation. This study showed that Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant-negative effect on filament formation. Q389P mutation is the molecular event leading to the development of desmin-related myopathy (Goudeau et al. 2001).

Cytoplasmic intermediate filaments containing vimentin are the most widely distributed. Vimentin is a 57-kDa protein that interconnects the nuclear and plasma membranes, maintaining the position of the nucleus within the cell. Desmin (53 kDa) and vimentin filaments are able to copolymerize in order to form intermediate filaments that contain both types of subunits. Intermediate filaments provide mechanical strength and maintain the cellular structure (Karp 1987).

The loss of desmin filament function in cardiac and skeletal muscle myocytes would be expected to affect not only mitochondrial function (Capetanaki 2002) but also active force generation (Balogh et al. 2002) in the cardiomyocytes. Several myopathies and cardiomyopathies have been characterized by the presence of altered intermediate filaments (D’Amati et al. 1992).

In previous studies carried out in erythrocytes from Sprague-Dawley male rats treated with 300 mg/kg/day clofibrate for 40 days, we observed a change in the protein profile of erythrocyte cell membranes: an increase in ß-spectrin, a decrease in ankyrin, and a decrease of the protein bands 4.1 and 6. Additionally, alterations in erythrocyte morphology and a decrease in osmotic resistance were observed (Morales-Aguilera and Sampayo-Reyes 1993; Sampayo-Reyes et al. 1997). We suggest that these proteins may have an important role in the structural and functional properties of erythrocytes. Hence, it is hypothesized that clofibrate may also modify the cytoskeletal proteins in other types of cells, particularly in myocardiocytes.

It is important to point out that the effects of the fibric acids at the cellular level have not been well characterized. Based on these antecedents, the aim of this experimental study was to analyze in vitro effects of clofibric acid on desmin and vimentin contents in rat myocardiocytes.

MATERIALS AND METHODS

Reagents

Propylene glycol, clofibric acid, NaCl, Tris-Cl, CaCl₂, ATP, 2-mercaptoethanol, Triton X-100, bovine serum albumin, Coomassie brilliant blue R-250, anti-desmin or anti-vimentin monoclonal antibodies, Tween 20, alkaline phosphatase (AP)-conjugated anti-mouse immunoglobulin G (IgG), 5-bromo-4-chloro-3-indolyl phosphate (BCIP; AP substrate), nitro-blue tetrazolium (NBT), tetrazolium salts, Connaugh Medical Research Laboratories (CMRL) 1066 medium, fetal bovine serum, horse serum, collagen, cytosine B–arabinofuranoside, Trypan blue, EDTA, MnCl₂, formaldehyde, fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and propidium iodine, Cell Growth Determination Kit/MTT based were all obtained from Sigma Chemical (St Louis, MO, USA). Bradford Protein Assay was purchased from Bio-Rad (Hercules, CA, USA), sodium pentobarbital (Anestesal) from Pfizer (Mexico), and trypsin from DIFCO (Detroit, MI, USA). Methanol and acetic acid were obtained from J.T. Baker (México). All other reagents were analytical reagent grade or of the highest quality available.

Cell Cultures

Hearts were harvested under sterile conditions from 3- to 5-day-old male Sprague-Dawley rats were minced and dissociated with 0.15% trypsin. Digestion was allowed to proceed for 18 h at 4°C, after which the supernatant obtained by gravity separation was aspirated and discarded. Immediately after, 2 ml CMRL 1066 medium supplemented with 10% fetal bovine serum and 10% horse serum were added per 100 mg tissue. The cells were seeded at a concentration of 10⁶ cells/ml in 25-cm² culture flasks previously coated with 6 μg/cm² collagen (Sigma). Contamination by muscle was avoided by adding 10 μM cytosine B–arabinofuranoside. The primary culture was passed and maintained routinely in supplemented CMRL 1066 culture medium at 37°C in a humid atmosphere of 95% O₂ and 5% CO₂.

Cells grown in serum-supplemented medium were adapted to serum-free medium by initially inoculating cells into a 1:1 mixture of serum-supplemented and serum-free media, then passaged into media containing increasing proportions of serum-free medium (1:3 ratio) before being transferred to 100% serum-free medium.

Verification of Cell Viability

The viability of control and treated primary cell cultures was analyzed by Trypan blue dye exclusion. The cells were then stained with 0.4% Trypan blue and the number of Trypan blue–positive and –negative cells were counted on a hemocytometer by light microscopy. The cell viability of treated cultures was more than 90% compared to untreated controls.

Treatment of Rat Myocardial Cell with Clofibric Acid

Clofibric acid was added at least 24 h after the cells had stopped beating. At approximately 100% confluency, clofibric acid was added to each flask at a concentration of 0.1 mM in serum-free CMRL 1066 medium. Serum was excluded to prevent macromolecules present in the serum from binding clofibric acid. Free serum-supplemented control was used. The cells were then incubated in the presence of clofibric acid for 24 h. This concentration was used because we observed in preliminary experiments that 1 mM of clofibric acid reduced cell viability in almost 90% in our cultures (data not shown). After several tests, it was verified that the 0.1 mM concentration only reduced the cell viability by 20%. The pH was carefully controlled (pH 7.2 to 7.5).

Morphology

Cultured rat myocardiocytes (CRMs) were evaluated after 24 h of exposure to clofibric acid under the light microscope with a 40× lens.

Mitochondrial Membrane Damage

In situ mitochondrial injury was assessed by the reduction of tetrazolium salts (Mosmann 1983). Intact, undamaged mitochondria in cultured heart cells allow limited penetration of reactants such as NBT and phenazine methosulfate. However, if the mitochondria have been damaged by toxic agents, the membrane becomes permeable to tetrazolium salts, allowing bioreduction by succinate dehydrogenase to their respective formazans. NBT-formazans were measured spectrophotometrically.

Assays were carried out following the kit’s instructions (Cell Growth Determination Kit/MTT based; Sigma), the results were expressed as a percentage of the absorbance of the samples in comparison to control.

Processing CRM Extracts for SDS-PAGE

CRM were processed for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to Lewis, Perillo, and Gonzalez (1988). After 24 h of treatment, the medium was decanted and the cells washed 3 times with 2 ml 40 mM NaCl at 4°C. The CRMs were homogenized in 1 ml buffer A (containing 0.1% Triton X-100) in a Potter-Elvehjem Tissue Homogenizer in crushed ice (4°C) for 30s at 60 strokes/min. Protein content was determined by the method of Bradford (1976). The suspension was centrifuged at 13, 000 × g for 5 min. Pellet fraction (Triton X-100 insoluble fractions) from the centrifugation CRM extracts were washed 5 times in buffer A and centrifuged at 13, 000 × g for 5 min at room temperature. The pellets were resuspended in Laemmli sample buffer placed in a boiling water bath for 3 min for SDS-PAGE. The Triton X-100 insoluble fractions (pellets) (25 μg) were electrophoretically separated on 12% SDS-PAGE and stained with Coomassie brilliant blue R-250 (Laemmli 1970).

Western Blotting Analysis

After electrophoretic separation, the proteins were transferred onto nitrocellulose membranes (0 to 2 μm) according to the method described in Burnette (1981). The membranes were blocked with 10%(w/v) nonfat dry milk in TBS (20 mM Tris-Cl, 0.9% NaCl, pH 7.5) at 25°C for 24 h. Membranes were probed with anti-serum, anti-desmin, or anti-vimentin monoclonal antibodies at a titer of 1:200 for 2 h at room temperature. Following three washes with TTBS (0.5% Tween 20 in TBS, pH 7.5), the membranes were incubated with the secondary antibody, anti-mouse IgG–AP conjugate (1:15,000). The blots were developed with BCIP. The relative amounts of desmin or vimentin detected on Western blots were quantified by scanning densitometry (Appraise Junior Densitometer, Beckman) (Burnette 1981).

Immunofluorescence

Cells grown to confluence in 25-cm² culture flasks were washed twice with phosphate-buffered saline (PBS) (with 10 mM EDTA and 2 mM MnCl₂, pH 8.2). Cells were then fixed per 15 min of incubation with 4.0% formaldehyde and 0.05% Triton X-100. Methanol:acetic acid (3:1) was then added and the flask was gently shaken for 5 min. Finally, fixed cells were washed with PBS.

Antibodies, anti-vimentin or anti-desmin, diluted 1:100 in PBS, were added to the fixed CRMs and incubated at 37°C for 25 min and then washed with PBS containing 10 mM EDTA, 2 mM MnCl₂, and 0.1% Tween 20, pH 8.2 (PBST). CRMs were then incubated with the secondary antibody, anti-mouse IgG–FITC diluted at 1:50 dilution for 1 h at 37°C. Finally, cells were thoroughly washed in PBST. Nuclei were contrast stained with propidium iodine. Cells were visualized under an Axiophot ZEISS epifluorescent microscope equipped with a triple filter. Cells were photographed on Fuji roll Handle ASA100 film with an exposure time of 30 s.

Densitometric Analysis

Gels were photographed with plastic film and the bands were analyzed by densitometry (Appraise Junior Densitometer, Beckman) at a wavelength of 540 nm.

Statistical Analysis

The Student’s t test was used to compare the means of the protein bands in rats treated with clofibric acid and untreated. A p value less than .05 was considered significant.

RESULTS

Primary Cell Culture

The primary cultures of rat myocardiocytes grew well. Cells began to adhere to the substrate within 24 h and had acquired the triangular form that is the typical morphology for these cells. By 48 to 72 h, cells were growing confluently and by 8 days, a uniform monolayer had formed. Figure 1A shows confluent CRM without treatment. The average length and width of the myocardiocytes was 15 and 3 μm, respectively, although some cells adopted an irregular form and were as long as 30 μm. The majority of the cells had only one nucleus; however, some presented two or three nuclei. Figure 1B shows the striking morphological changes observed in the presence of 0.1 mM clofibric acid. Extensive granulation of the cytoplasm and irregular membrane conformation were also apparent after 24 h of exposure.

Mitochondrial Membrane Damage (MTT Activity Assays)

The viability of treated CRM cultures after exposure to clofibric acid (0.1 mM) was compared to that of untreated control CRM cultures. Cells exposed to 0.1 mM clofibric acid exhibited 80.4% cell viability compared to untreated controls. As a negative control, cells exposed to 1% Triton X-100 showed 1.17% cell viability compared to untreated controls (Table 1).

In Vitro SDS-PAGE, Western Blotting Analysis, and Densitometric Analysis

The results of in vitro SDS-PAGE and Western blotting analyses are shown in Figure 2A and B . Observe that the bands of proteins detected with anti-vimentin and anti-desmin in cells treated with 0.1 mM clofibric acid are less dense than those observed in untreated controls. In order to determine the observed change semiquantitatively, the mean values were compared and significant differences were found (p < .01). Myocardiocytes cell culture exposed to clofibric acid showed a decreased amounts of vimentin (26.3%) and desmin (42.1%) (Table 2).

Immunochemistry

Confluent cultures were examined under a fluorescent microscope. We observed a difference in the organization of the labelled proteins vimentin and desmin in 0.1 mM clofibric acid–treated cells compared to the untreated control (Figure 3A and B ). Note the change in the shape of treated cells, the filament assembly displayed a distinct pattern that the control. Intermediate filaments were uniform in diameter and length in control cells. Treated cells appeared to have a more condensed network of desmin and vimentin in the perinuclear zone. Furthermore nuclear staining showed a normal nuclear pattern in control cells and nuclear fragmentation in cells treated with clofibric acid

DISCUSSION

In recent years, prescriptions for medications that diminish the concentration of serum lipids have increased greater than 10-fold, totaling more than 26 million in 1992, in the United States (Newman and Hulley 1996) and over 100 million worldwide (Schroder-Bernhardi and Dietlein 2002). Early primary prevention studies of atherosclerosis using the fibric acid derivative clofibrate showed only modest effects on atherosclerosis and an alarming increase in mortality in the intervention group (Krakoff, Vela, and Briton 2000).

On the base of the previous studies (Morales-Aguilera and Sampayo-Reyes 1993; Sampayo-Reyes et al. 1997), we suspected that a possible explanation for the undesirable cardiac effects could be related to effects of fibric acid on cardiac cell proteins and of clofibric acid on the fundamental cytoskeletal proteins in myocardiocytes in vitro.

The decreased amounts of vimentin and desmin suggest a direct alteration in the proteins or in their synthesis, as has been described previously for the cytoskeletal proteins of erythrocytes of rats treated with clofibrate (Morales-Aguilera and Sampayo-Reyes 1993).

The exposure of myocardiocytes to clofibric acid induced a depletion of vimentin (26.3%) and desmin (42.1%). These results are in agreement with the reported observation of 13% and 4% decreases of vimentin and desmin, respectively, in chicken myocardiocytes exposed to fenofibrate (González et al. 1991). Using other fibric acid derivatives, those authors also observed 12% and 19% increases of vimentin and desmin, respectively, in the cytoplasmic fraction of chicken myocardiocytes treated with gemfibrozil, whereas bezafibrate had no effect on vimentin content but caused a 17% increase in desmin compared to control (González et al. 1991).

It is important to mention that the 0.1 mM concentration of clofibric acid was selected for the in vitro studies because therapeutics doses of clofibrate give a plasma concentration of clofibric acid between 162 and 200 μg/ml (IARC 1972). This concentration is in the order of 1 mM of clofibric acid. However, preliminary experiments showed that 1 mM of clofibric acid reduces cell viability in almost 90% (mortality) in our cultures. Therefore we choose 0.1 mM as the test concentration (20% reduction in cell viability; see Table 1). This concentration is equivalent to 21.4 μg/ml (approximately 80 times lower than the clofibric acid concentration produced by therapeutics doses in humans (IARC 1972).

Differences in the organization assembly were also observed. The effect of the clofibric acid on the cytoskeleton may be due to an alteration of the structure of vimentin and desmin. It has been observed that vimentin may participate in the mechanical support of the nucleus and the maintenance of its position within the cell and desmin filaments of muscle cells organize the array of myotubules (D’Amati et al. 1992).

The present study shows that under the experimental conditions described, fibric acid cause a decrease in the quantity of vimentin and desmin and an alteration of the normal structure of the cytoskeleton of myocardiocytes. Considering the role that intermediate filaments play in the dynamic structure of the cytoskeleton, it is likely that an alteration in the homeostasis of vimentin and desmin will directly effect the cytoskeletal organization of myocardiocytes. This likely contributes to the mechanism of their toxic action.

CONCLUSION

Our findings show that the concentration of vimentin and desmin in myocardiocytes of rat diminishes significantly after treatment with clofibric acid. Because the intermediate filaments act to anchor intracellular arrays to the plasma membrane and are actively involved in the muscular contraction, we suggest that alterations in the cytoskeleton could affect the functionality of heart fibres. In our laboratory, we have found changes in the erythrocytic membrane protein profile caused by fenofibrate, bezafibrate, and gemfibrozil (unpublished data). We suggest that fibric acids are able to modify the organization and the content of cytoskeletal proteins in rat myocardiocytes and that these alterations may be related to the adverse secondary effects. Further studies need to be done to understand the effect of clofibric acid toxicity.