A study of organelle Ca2+ dynamics in cardiac muscle

Organelle Ca2+ dynamics of cardiac muscle were studied using fluorescent indicators and a bioluminescent Ca2+ probe, namely aequorin that was targeted to SR. Enzymatically dissociated adult cardiac myocytes were subjected to prolonged incubation with acetoxymethyl (AM) derivatives of Fura-2, FuraFF and MgFura-2 which each have different Ca2+ affinities. This prolonged incubation allowed indicator loading into sub-cellular compartments. Permeabilisation with saponin and subsequent washout removed cytosolic component of the indicator. Results from the fluorescent indicator studies can be summarised as follows: 1. Studies with Fura-2-AM loaded cardiomyocytes revealed the existence of a sub-cellular compartment or compartments, which exchanges Ca2+ passively with a very slow time course. The identity of this is unclear, although likely candidates include mitochondria and nuclear envelope. 2. The signal from FuraFF-AM loaded cardiomyocytes appears to be almost exclusively from mitochondria with no contribution by SR. Parallel studies were done with SR vesicles and mitochondria prepared from cardiac tissue. Mitochondrial signals were very similar to that from permeabilised cells. However, SR vesicles loaded with FuraFF-AM did not appear to be responsive to changes in external [Ca2+]. The inability of AM loading to introduce dye with Ca2+ sensitivity into SR vesicles may be a consequence of low esterase activity. This may explain the lack of a clear SR signal in measurements from permeabilised cells. An adenovirus vector was developed to allow over-expression of the bioluminescent Ca2+ sensitive protein aequorin specifically targeted to SR (Ad-CSQ-Aeq). Experiments were carried with these over-expressing cells in the presence of 5μM ruthenium red and therefore absence of RyRa Ca2+ flux. [Ca2+]CYT was clamped at 162nM and 380nM. Under these conditions [Ca2+]SR was measured as 5.20 +/- 0.230 x 10-4 M ([Ca2+]CYT =162nM, n=6) and 1.21 +/- 0.180 X 10-3 M ([Ca2+]CYT =380nM. n=14). The relationship between [Ca2+]SR and rate of Ca2+ uptake in the absence of RyR2 flux, revealed a non-linear lumenal dependence. High [Ca2+]SR has a negative feedback effect upon SERCA2a resulting in a slower rate of uptake. The non-linearity of the lumenal dependence suggests a mechanism other than a simple trans-SR gradient. Analysis of leak in the presence of 25μM thapsigargin and 6muM ruthenium red indicates a simple linear relationship between [Ca2+]SR and the rate of Ca2+ leak. The kinetics of this relationship suggests a simple leak 'channel' or pathway, the identity of which is unclear. It is unlikely to be via RyR2 or reverse mode of SERCA2a due to presence of inhibitors. On the basis of a mean uptake and leak uptake curve an estimate of the equilibrium value of [Ca2+]SR in absence of RyR2 activity is approximately 960?M