We have previously shown that the administration of the β2-adrenergic receptor agonist, clenbuterol, induces both apoptotic (Burniston et al. 2002b) and necrotic (Burniston et al. 2002a) cell death in the heart and soleus muscle of the rat. Here we demonstrate that these two death pathways occur sequentially with a substantial amount of co-localisation.
Male Wistar rats (Rattus norvegicus) (287 ± 20 g) were administered (S.C.) 5 mg kg-1 of clenbuterol (experimental groups) or the saline vehicle only (controls). Before being humanely killed at specific time points (2-24 h), hearts and soleus muscles were rapidly excised, snap frozen and serial cryosections (5 µm) cut. Apoptosis was detected using an anti-caspase 3 antibody (Ab; R&D systems). For the detection of necrotic myocytes, all animals received an injection (I.P.) of an anti-myosin Ab 1 h prior to the clenbuterol challenge. This Ab is too large to be admitted through the membrane of viable myocytes, but can enter and bind to the sarcomeric myosin of necrotic myocytes in vivo. Primary Ab binding was then amplified using secondary immunoperoxidase techniques and visualised with Nova Red (Vector Laboratories). Cell death was quantified in the subendocardial region of the heart and mid-belly of each soleus using image analysis. Results are expressed as percentage area, or percentage number of damaged fibres for the heart and soleus, respectively.
No cell death was found in the hearts or solei of animals receiving only the saline vehicle (zero time point). In contrast, administration of clenbuterol induced both apoptotic and necrotic cell death in both striated muscles (Fig. 1). Apoptosis was first observed after 2 h, and necrotic myocyte death at 4 h following clenbuterol administration. The clearance rate of apoptotic cell death also occurred earlier than that of necrosis with no apoptosis evident in either tissue after 24 h.
The sequential nature of the myocyte apoptosis and necrosis suggests that some of the necrotic cells may have been apoptotic cells which have undergone secondary necrosis. This was confirmed by double immunofluorescence labelling, which showed co-localisation of apoptotic and necrotic cells in both the cardiac and soleus muscles.
This work was supported by the British Heart Foundation