Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats

Abstract

Aim: To investigate the role of matrix metalloproteinases (MMPs) in the responses of rats to a prolonged doxorubicin (DOX) treatment.

Methods: Male Wistar rats were used. DOX was administered by intraperitoneal injections of seven doses (cumulative dose was 15 mg/kg). Control animals were treated with saline. Tissue or plasma samples were collected at four and eight weeks after the application of the last dose. Protein levels were determined by immunoblot assay, and MMP activities were measured by gelatin zymography. Superoxide content was analyzed using a lucigenin chemiluminescence assay and superoxide dismutase (SOD) activities with a SOD assay kit. Qualitative structural alterations of the heart were characterized by transmission electron microscopy.

Results: Systolic blood pressure was higher in DOX-treated rats as compared with the control rats at 8 weeks after treatment. In contrast, there were no differences in the heart rate between the control and DOX-treated rats. DOX treatment caused marked heterogeneous subcellular alterations of cardiomyocytes and structural disorganizations of the cardiac extracellular space. The effects of DOX were linked to a stimulation of plasma MMP-2 and MMP-9 activities that had already increased by 4 weeks after the end of the treatment. In the left ventricle, however, DOX only led to increased MMP-2 activation at 8 weeks after the end of treatment. These changes in tissue MMP-2 were connected with stimulation of Akt kinase activation, inhibition of SOD, an increase in superoxide levels, induction of iNOS protein expression and caspase-3 activation.

Conclusion: Our results show that MMPs are involved in the chronic cardiotoxicity of DOX in rats. The data also suggest that reactive oxygen species (superoxide), NO production (iNOS) and the Akt kinase pathway can modulate MMP-2 activities in rat hearts influenced by DOX.

Figure 1

Effect of doxorubicin treatment on the systolic blood pressure (A) and heart rate (B). These parameters were measured by the non-invasive method of tail cuff plethysmography in both control and DOX-treated groups of rats. The measurements were performed before the first DOX or saline application and 4 or 8 weeks after end of their application. In each experimental group, eight animals were used for blood pressure and heart rate measurements and bars represent mean±SD from these independent measurements. C- control saline-treated rats; DOX- doxorubicin-treated rats; 0- rats before start of treatment; 4 weeks- rats 4 weeks after end of treatment; 8 weeks- rats 8 weeks after end of treatment, ^bP< 0.05 vs corresponding control value.

Figure 2

Representative electron microscopic images showing changes in ultrastructure of rat hearts 8 weeks after the application of the last dose of saline or doxorubicin. (A) Electron micrograph of control heart showing normal architecture of cardiomyocytes and without changes in extracellular space. CM – cardiomyocytes; EC – extracellular space. Bar: 1 μm. (B) Electron micrograph of the myocardium affected with doxorubicin demonstrating injury of cardiomyocytes and dense extracellular space. E- capilary endothelial cells; ECS – extracellular space; CM – cardiomyocytes. Bar: 1 μm. (C) Extracellular space (ECS) of the myocardium affected with DOX is filled with irregularly distributed dense material, mitochondria (Mi), vacuoles and myelinized structures (arrows) and extravased red cells (R). E – endothelial cells of capilary; CM – cardiomyocytes. Bar: 1 μm. (D) Subcellular injury of capillary endothelial cell (E) manifested with disrupted plasmalemma (arrow), reduced cytoplasm density and clumping of chromatin. N – nucleus; ECS – extracellular space; CM – cardiomyocytes, L – lumen. Bar: 1 μm.

Figure 3

Effect of doxorubicin treatment on tissue matrix metalloproteinases and TIMP-2. The MMPs and TIMP-2 were analyzed in protein fractions isolated from the left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks (C-8, DOX-8) at the end of the treatment. Tissue samples obtained from six animals in each experimental group were used for the analysis. (A) Records showing the influence of DOX-treatment on gelatinolytic activity of tissue 72 kDa MMP-2. The MMP activities were analyzed by zymography in polyacrylamide gels containing gelatin as a substrate and enzymatic gelatinolytic activities of 72 kDa MMP-2 were detected predominantly in samples from left ventricle. (B) Quantitative analysis of MMP-2 activities. Data are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent tissue samples per group. ^bP<0.05 vs corresponding control value. (C) Effect of DOX treatment on protein levels of MMP-2, TIMP-2, and GAPDH. The levels of proteins were analyzed by Western blot analysis using specific antibodies. An antibody specific for MMP-2 reacted with both 72 kDa and 63 kDa forms of MMP-2. Protein loading was confirmed by using GAPDH.

Figure 4

Effect of doxorubicin treatment on plasma matrix metalloproteinases. The MMPs released into circulation were analyzed in plasma samples prepared from whole artery blood of control (C) and doxorubicin-treated (DOX) rats obtained 4 weeks (C-4, DOX-4) or 8 weeks (C-8, DOX-8) after the end of the treatment. Blood plasma samples obtained from six animals in each experimental group were used for the analysis. (A) Records showing the influence of DOX-treatment on gelatinolytic activities of MMPs. Zymographic analysis of blood plasma samples revealed several bands corresponding to the gelatinolytic activities of several proteinases and using positive controls the activities of 72 kDa MMP-2 and 92 kDa MMP-9 were identified. (B) The records showing protein levels of MMP-2 determined in plasma samples by Western blot analysis using a specific antibody which detected predominantly the presence of 72 kDa form of MMP-2 in plasma. (C) Quantitative analysis of MMP-2 activities. Data are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent plasma samples per group. ^bP<0.05 vs corresponding control value. (D) Quantitative analysis of MMP-9 activities. The data are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent plasma samples per group. ^bP<0.05 vs corresponding control value.

Figure 5

Effect of doxorubicin treatment on levels of superoxide anion (O₂^–), activities of SOD and protein levels of SOD-1. Left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks (C-8, DOX-8) at the end of the treatment was used for the analysis. Tissue of left ventricle obtained from six animals in each experimental group was used for the analysis. (A) Doxorubicin treatment induced increase in levels of superoxide (O₂^–) 8 weeks after the end of the DOX application. On the other hand, superoxide levels were not changed 4 weeks after DOX treatment. The superoxide levels were evaluated using Lucigenin Enhanced Chemiluminiscence assay and the obtained data were expressed as relative luminiscence unit per mg of tissue (RLU·mg^-1 tissue). Each bar represents mean±SD of six independent measurements. ^bP<0.05 vs corresponding control value. (B) Doxorubicin treatment induced inhibition of enzymatic activities of superoxide dismutase (SOD) 8 weeks after the end of the drug application. The SOD activities in left ventricle were analyzed using the SOD Assay kit (Fluka) and are expressed in U·mg^-1 of protein. Each bar represents mean±SD of six independent measurements. ^bP<0.05 vs corresponding control value. (C) The record showing the effects of DOX treatment on protein levels of SOD-1. The protein levels of SOD-1 were analyzed by Western blot analysis. (D) Quantitative analysis of SOD-1 protein levels. The data are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent tissue samples per group. ^bP<0.05 vs corresponding control value.

Figure 6

Effect of chronic DOX treatment on protein levels iNOS and cleaved caspase-3. For the analysis protein fractions isolated from the left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks (C-8, DOX-8) at the end of the treatment were used. Tissue samples obtained from six animals in each experimental group were used. (A) Western blot record showing the influence of saline and DOX on iNOS protein levels induction. The protein levels of iNOS were analyzed by Western blot analysis. (B) Quantitative analysis of iNOS protein levels. The data are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent tissue samples per group. ^bP<0.05 vs corresponding control value. (C) Western blot record showing the influence of saline and DOX on caspase-3 activation. Levels of fragment of activated caspase-3 resulting from cleavage adjacent to Asp175 were determined using a specific antibody. (D) Quantification of cleaved caspase-3 content in the left ventricular tissue after DOX treatment. The data were obtained from Western blot records and are expressed as a percentage of value for corresponding control. Each bar represents mean±SD of 6 independent tissue samples per group. ^bP<0.05 vscorresponding control value.

Figure 7

Effect of chronic DOX treatment on protein levels and activation of Akt kinase. For the analysis protein fractions isolated from the left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks (C-8, DOX-8) at the end of the treatment were used. Tissue samples obtained from six animals in each experimental group were used for the analysis. (A) Western blot record showing the influence of saline and DOX on protein levels and specific phosphorylation of Akt kinase. Levels of Akt kinase were determined by Western blot analysis using a specific antibody. The changes in the specific phosphorylation (activation) of Akt kinase were determined by Western blot analysis using an antibody which reacts with Akt kinase phosphorylated specifically on Ser473. (B) The quantification of Akt kinase phosphorylation and activation. The data were obtained from Western blot records and are expressed as a ratio of content of phosphorylated Akt kinase to total Akt kinase. Each bar represents mean±SD of 6 independent tissue samples per group. ^bP<0.05 vs corresponding control value.