Clenbuterol induces apoptosis on in vivo in rat heart and skeletal muscle
http://www.thebullmagazine.com/page....f=1&noupdate=1
by Karl Hoffman
Every organism requires a means to rid itself of cells that are no longer required, are damaged, or that may be harmful to the organism. Cell death is a tightly controlled process and may occur by apoptotic or non-apoptotic means (necrosis). Apoptosis is the regulated destruction of a cell and may be triggered by either external or internal signals. Unlike necrosis, apoptosis does not result in an inflammatory response from the surrounding cells and the morphological features are quite distinct. Apoptosis, or programmed cell death, involves complex signaling that results in a molecular cascade triggering a series of proteases known as caspases. Caspases are central to the process of apoptosis and are activated specifically in apoptotic cells. Caspases cleave a limited set of proteins within the cell which are critical for cell survival, resulting in cell death.
In rats, doses of clenbuterol on the order of 100 mcg (micrograms) per kg of body weight have been shown to induce necrosis of cardiac myocytes (muscle cells) (1). The same research group that examined necrosis recently showed that much smaller doses of clenbuterol, as low as 1 mcg/kg of body weight, could induce apoptosis in cardiac myocytes (2). Before looking at the mechanisms and implications of this, we must convert these figures to Human Equivalent Doses (HED) to see if there is any relevance to humans here. In some cases, when extrapolating animal doses to human doses a direct mg/kg to mg/kg conversion is adequate. Considering necrosis, using this conversion 100 mcg/kg would translate into 10,000 mcg for a 100 kg bodybuilder. It’s unlikely that anyone routinely consumes this much clenbuterol, so necrosis would not be an issue in humans. Apoptosis is a different story however. The 1 mcg/kg figure would amount to 100 mcg in a human. People routinely consume between 100 and 200 mcg of clenbuterol daily so apoptosis is a distinct possibility in humans.
For some drugs and routes of administration extrapolating from animals to humans works better if one takes body surface area into account:
http://www.fda.gov/cber/gdlns/dose.htm
In the case of rats in this study with an average weight of 289 grams, one multiplies the toxic dose in mg/kg by 0.15 to arrive at the HED in mg/kg. Using this algorithm for necrosis we arrive at an HED of 1500 mcg for the 100 kg bodybuilder. This is still far outside the range of clenbuterol doses used by people, so again necrosis is not relevant in the human heart. However, scaling this way for apoptosis gives us an HED of 16 mcg for or 100 kg subject. So regardless of how we calculate HED, clen doses commonly used by humans are well within the apoptotic range.
Clenbuterol is a relatively specific beta 2 receptor agonist, but it does show some degree of beta 1 binding at high doses. In addition to acting directly on beta receptors, clenbuterol facilitates release of norepinephrine (NE) from sympathetic nerve terminals by stimulating pre-synaptic beta 2 adrenoreceptors. (Ephedrine acts in a similar manner, both directly and indirectly by inducing NE release.) To test the mechanism of clenbuterol’s apoptotic action, the authors administered reserpine to deplete the NE releasing capacity of the sympathetic nerve terminals. This resulted in a 68% reduction in apoptosis. Similarly, prior administration of bisoprolol, a selective beta 1 antagonist resulted in a 98% reduction in apoptosis. Based on the above observations the authors concluded that NE is primarily responsible for cardiac apoptosis due to clenbuterol. Note that after administrtion of a single dose of clenbuterol the authors observed apoptosis at 4 hours. The long elimination time of clenbuterol allows for the buildup of high concentrations in the heart (3).
In the soleus muscle, 10 mcg/kg of clenbuterol was the minimum dose that induced apoptosis. In the soleus only beta 2 blockade was capable of reducing apoptosis. This latter observation implies that while NE is responsible for the cardiac toxicity of clenbuterol, the drug is acting directly on beta 2 receptors in the soleus to induce apoptosis.
An interesting possibility is that the clenbuterol induced cardiac apoptosis might be a protective response gone awry to the cardiac hypertrophy that accompanies chronic beta receptor stimulation, since both cardiac apoptosis and hypertrophy occur in humans with long term use of beta agonists :
"Accumulating evidence suggests that chronic stimulation of ß-adrenergic receptor (ß-AR) in patients causes progressive cardiac dysfunction, cell loss, and cardiac chamber remodeling. Consistent with this notion, it has been demonstrated that stimulation of the ß-AR causes hypertrophy and apoptosis in cardiac myocytes" (4).
Under physiologic conditions where the heart is exposed to normal levels of NE a balance is struck between hypertrophy and apoptosis. ICER (inducible cAMP early repressor) is upregulated by cAMP (cyclic adenosine monophosphate), which in turn is upregulated by exposure to beta agonists like NE. ICER initiates signaling that ultimately leads to apoptosis, as depicted in the diagram below, adapted from (5).
Schematic representation of ICER-mediated feedback signaling, with ICER-mediated effects italicized.
Evidently under exposure to chronic supraphysiological levels of beta agonists ICER leads to deleterious levels of apoptosis while at the same time being unable to offset beta agonist induced hypertrophy. Hence the combination of apoptosis and hypertrophy mentioned in the quote above from (4).
Paraphrasing a passage from (2), the take home message for both cardiac patients and bodybuilders based on the research discussed here might be the following:
Clenbuterol has recently been used as an adjunct to the implantation of left ventricular assist devices (The Harefield Protocol) as a bridge to recovery and has been shown to aid the reverse remodelling of the myocardium. These patients also receive 'combination therapy' that includes beta 1-AR blockade. It is likely therefore, that in this case the heart will be protected from the myotoxic effects of clenbuterol, as explained above. However, their skeletal musculature will remain vulnerable to beta 2-AR-induced myocyte death. The potential additional loss of skeletal muscle bulk in already severely ill patients, together with the effects on their protein metabolism and exercise capacity, warrants further investigation before the use of clenbuterol becomes widely accepted as a standard therapeutic intervention. With regard to the illicit use of clenbuterol the philosophy of "the more you take, the greater the benefit" must engender a cause for concern.
Perhaps a person would be wise to use alternatives to clenbuterol such as forskolin, green tea, and caffeine, at least until similar studies are carried out on these fat loss agents. At a minimum it would seem prudent to use the smallest dose of clenbuterol that one finds effective, and limit the time of exposure. The latter is the norm with most users anyway, who typically use clenbuterol for short periods of time (on the order of two weeks) due to the downregulation of beta receptors that accompanies clenbuterol use. One still wonders about the wisdom of cycling clenbuterol on a two-week on / two off schedule over long periods of time to offset receptor downregulation.
References
Burniston JG, Ng Y, Clark WA, Colyer J, Tan LB, Goldspink DF. Myotoxic effects of clenbuterol in the rat heart and soleus muscle. J Appl Physiol. 2002 Nov;93(5):1824-32.
Burniston JG, Tan LB, Goldspink DF. {beta}2-Adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle. J Appl Physiol. 2004 Dec 10; [Epub ahead of print]
Soma LR, Uboh CE, Guan F, Luo Y, Teleis D, Runbo L, Birks EK, Tsang DS, Tissue distribution of clenbuterol in the horse. J Vet Pharmacol Therap 27: 91- 98, 2004
Tomita H, Nazmy M, Kajimoto K, Yehia G, Molina CA, Sadoshima J. Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to beta-adrenergic receptor stimulation. Circ Res. 2003 Jul 11;93(1):12-22.
Sussman MA. ICER-capades: putting cardiac cyclic AMP signaling "on ice". Circ Res. 2003 Jul 11;93(1):6-8.
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