Observing Compensatory Effects of RyR2, NCX and SERCA Down‐regulation in Neonatal Cardiomyocytes Using siRNA

There are three genes primarily responsible for the release and resequestration of calcium (Ca+2) during excitation-contraction coupling in cardiac muscle cells: the ryanodine receptor (RyR2), Sarcoplasmic reticulum calcium ATPase (SERCA), and the sodium-calcium exchanger (NCX). The RyR receptor releases stored calcium from the sarcoplasmic reticulum (SR) into the cytosol of the cell, while SERCA causes the resequestration of Ca+2 by SR, and NCX lowers cytosolic Ca+2 by exchanging it for Na+ across the cell membrane. Using inhibitory RNA gene silencing (siRNa) techniques, we observed that the down-regulation of each of the three primary genes involved produces compensatory changes of the other two. Quantification of mRNA levels was done using reverse transcription (qRT-PCR). Calcium transients (fluctuation in the concentration in cytosolic calcium) of 1 to 3 day old neonatal cardiomyocytes were measured using a Leica TC SP2 laser scanning confocal microscope; line scans were recorded at 200 Hz for 30 sec. Contractility recordings of paced cells were obtained using a video camera mounted on an inverted phase contrast microscope. Videos were analyzed using a Fourier analysis-based digital image processing technique. Observations showed that down-regulating RyR caused an under-expression of SERCA and an over-expression of NCX. Down-regulation of NCX causes no significant change in the expression of RyR but an over-expression of SERCA. Lastly, down-regulation of SERCA causes an over-expression of NCX, but an under-expression of RyR.