| Abstract |
The cellular mechanisms governing cardiac pacemaker activity are not clear. The discovery of common genes such as tinman in all organisms with a heart, has provided new prospects in the study of cardiac function, development and pathology. In this concept, Drosophila heart has proven itself to be an excellent model for human cardiac development and function.
In this study, we measured the heart rate in Drosophila third instar larvae of OR-R line in certain conditions, using an extracellular recording electrode attached on the posterior cardiac region. The use of a peristaltic pump allowed us to change the ionic and pharmacological environment of the experiment, while the constant recycling of saline retained the proper oxygenation of our preparations at 25oC, keeping them in normal cardiac functioning for over an hour. In order to investigate the possible roles of several ions and ionic channels we used three approaches and in each of them we measured the heart rate. Firstly, we conducted a series of experiments in which calcium or sodium were ablated from the saline immersing our preparations. Secondly, we used pharmacological blockers of calcium channels and specifically several concentrations of Ni+ that block T-type and L-type calcium channels in vertebrates, the L-type calcium channel blockers, verapamil and niphedipine in concentrations 30 and 5 μΜ respectively, and finally cyclopiazonic acid, an inhibitor of SR Ca2+-ATPase at a concentration of 100 μΜ. Thirdly, to further analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied the conditional mutation in Drosophila SERCA ( Sarco-endoplasmic Reticulum Calcium-ATPase), kum, in which SERCA deactivates after five minutes of heat-shock at 40oC.
We observed in wild type larvae, that calcium-free saline containing 0,2mM EGTA, abrupts cardiac activity, in about 1-2 minutes, while very low concentration (0,1mM) significantly reduces the heart rate until it totally quits, at about 6-7 minutes. Additionally, sodium-free saline treatment for 5 minutes, reduces heart rate and transduces continues cardiac pumping into a discontinuous bursting activity. Moreover, treatment with 1mM nickel-containing saline, ceases cardiac activity in contrast to low concentration of 50-100 μΜ, that had no apparent effect in cardiac function. The specific blockers of L-type calcium channels verapamil and niphedipin had quite similar inhibitory effects, namely the significant reduction of cardiac contraction rate, notable bradycardia and arrhythmia in wild type animals. Besides treatment with cyclopiazonic acid, reduces heart rate and creates a characteristic pattern of cardiac activity in both wild type and unheated kum170 animals, but had a minor effect in heat-shocked kum170 animals.
These data indicate that calcium plays a major role in the electrical properties of cardiac cells in Drosophila third instar larvae, and that there must be calcium channels, similar to the L-type and/or T-type calcium channels found in vertebrates, controlling cardiac contraction. In addition, SERCA malfunction or inhibition is responsible for a dramatic distortion of normal heart beating phenotype, that could be similar to pathological conditions observed in humans.
|
Collections
