Skeletal Muscle for Endomyocardial Biopsy: Comparable Stress Response in Doxorubicin Cardio-myopathy

Abstract

In the present study, we compared the cell damage response in skeletal and cardiac muscle tissue when exposed to doxorubicin. This was carried out by means of a less invasive informative substitute to endomyocardiac biopsy based on Hsp70 immunodetection and a subcellular analysis of the nucleolus. Male Sprague Dawley rats (62 g body weight) were randomly distributed into 3 group, the control and doxorubicin I and doxorubicin II groups, in which 15 and 25 mg/kg body weight of doxorubicin (0.1 ml, i.v.) was administered, respectively. After 15, 30, 45 and 60 minutes, portions of the left and right ventricle wall and interventricle wall, together with skeletal muscle from the posterior and anterior member, were prepared for Hsp70 immunodetection by Western blot analysis and ultrastructural study using the thin cut technique. Differential cell response between the control and treated groups was observed in Hsp70 immunodetection and at the subcellular level. In the control group, the Hsp70 recognition levels and typical normal nucleolar morphology were similar, while the treated groups showed variable-dependent Hsp70 recognition and segregation of nucleolar components, forming ring-like figures of a variable-independent nature. Comparison of cardiac and skeletal muscle tissue cell response to doxorubicin toxic aggression revealed parallelism in terms of Hsp70 accumulation in certain regions of both tissues (15 mg/kg body weight of doxorubicin), which suggests that replacing endomyocardiac biopsy analysis with skeletal muscle analysis may be a safe option.

Keywords: Hsp70; doxorubicin; endomyocardiac biopsy; muscle skeletal biopsy; nucleolar segregation.

Fig. 1

Transmission electron micrographs of a cardiac muscle (A) and skeletal muscle cells (B) from the control group. Characteristic central and peripheral positions of the nuclelus and a typical distribution of nucleolar components (inset) was observed Nuclei (N), Nucleolus (n), nucleolar organisation region (white arrow head) and the granular component (black arrow head). Myofibrils (MF), mitochondria (m) and the perinuclear chromatin (C) were also revealed. Scale bars: 1 µm (Figs. 1A , 1B) and 0.12 µm (insets, taken at 75 kV).

Fig. 2

Transmission electron micrograph of cardiac muscle and skeletal muscle cell nucleoli with respect to different post-treatment times of a low dose of doxorubicin (DOX-I group: 15 mg/kg bw). The reorganisation of nucleolar components showed segregation of nucleolar components (A–D, E, H) and compact nucleorar structures (F–G). Cardiac muscle cells (A–D), skeletal muscle cells (E–H). Post-treatment times (in minutes): 15 (A, E), 30 (B, F), 45 (C, G), 60 (D, H). Nucleolar organisation region (white arrow head), dense fibrilar component (arrow), granular component (black arrow head); myofibrils (MF), mitochondria (m) and perinuclear chromatin (C). Scale bar: 0.12 µm, taken at 75 kV.

Fig. 3

Transmission electron micrograph of cardiac muscle and skeletal muscle cell nucleoli with respect to different post-treatment times of a high dose of doxorubicin (DOX-II group: 25 mg/kg bw). The reorganisation of nucleolar components showed segregation for nucleolar components (B–D, F, H), and compact nucleorar structures (A, G) were observed in all post-treatment times. Cardiac muscle cells (A–D), skeletal muscle cells (E–H). Post-treatment times (in minutes): 15 (A, E), 30 (B, F), 45 (C, G), 60 (D, H). Nucleolar organisation region (white arrow head), dense fibrilar component (arrow), granular component (black arrow head), and perinuclear chromatin (C). Scale bar: 0.12 µm, taken at 75 kV.

Fig. 4

Western blot analysis of Hsp 70 from the cardiac and skeletal muscle tissue of the CON and DOX groups at the post-administration times. Similar Hsp70 recognition between the LV, RV, IV and PM, slightly less recognition in the AM, were observed in the CON group. A greater amount of Hsp70 was recognized in the LV, RV and PM in the DOX-I group compared with the IV and AM, for all post-treatment times. In the DOX-II group, tissue and time independent Hsp70 recognition were observed in a uniform region for all time points except 60 min, with recognition in the LV and IV greater compared with the RV, AM and PM. A: Control group (sterile water: 0.1 ml; i.v). B: DOX-I group (15 mg/kg bw). C: DOX-II group (25 mg/kg bw. Post-treatment times (in minutes): 15, 30, 45 and 60. Tissues regions: left (LV) and right ventricular (RV) and interventricular walls (IV) and posterior (PM) and anterior member skeletal muscle (protein µg per lane: 3.6 µg).

Fig. 5

Densitometric analysis of the relative levels of Hsp70 accumulation in the cardiac and skeletal tissue regions in the DOX I (A: 15 mg/kg bw) and DOX II (B: 25 mg/kg bw) groups compared with the CON (sterile water, 0.1 ml i.v.) group for the different post-treatment times (in minutes). In CON group, the accumulation of Hsp70 was similar in the LV, RV, IV and PM, with recognition slightly less in the AM. By contrast, in the DOX I group, the relative levels of Hsp70 accumulation in the LV, RV and PM were greater than in the IV and AM for all post-treatment times; in the DOX-II group, the relative levels of Hsp70 accumulation were uniform in all regions and for all post-treatment times, except at 60 min, at which the levels were greater in the LV and IV compared with the RV, AM and PM. Tissues regions: left (LV), right ventricular (RV) and interventricular walls (IV) and posterior (PM) and anterior (AM) member skeletal muscle. Data are means ± S.E. of 3 animals in each group. P<0.05.