In freshly isolated rabbit SANC, Protein Phosphatase Inhibition (PPI) by the PP1/2A inhibitor, Calyculin A, (100-500 nM) reduced PP activity by 90%, and increased basal PLB phosphorylation at Thr17 and Ser16 by about 2.5-fold. PPI increased: the rate of spontaneous Ca2+ release of the LCR ensemble (measured via confocal fluo-4 imaging) by nearly four-fold in saponin-permeabilized SANC; the L-type Ca2+ current (ICaL) amplitude by 30% in voltage-clamped, single SANC; and the LCR size in spontaneously firing single, intact SANC. PPI also decreased the LCR period, and this reduction predicted a concurrent 25% reduction in the spontaneous AP cycle length. We found that PP are important regulators of PDE functioning: addition of PP inhibitors to the reaction buffer increased total PDE activity approximately 50 % in both SANC and VM lysates. We found that PP1 is present in SANC via Western Blot technique. A numerical model simulation of the effect of PPI on SANC firing rate, incorporating experimental observed changes in ICaL and PLB phosphorylation effects on SR Ca2+ pumping, closely predicted the experimental results. We measured expression of different types of PPs and PP1 inhibitors mRNAs as well as protein abundance in rabbit SANC, LV and RA cells. Conclusion: Basal PP activity modulates spontaneous SANC AP firing rate, in part at least, by modulating ICaL, PLB phosphorylation, and SR-generated LCRs. We identified transcripts coding PP1, PP2A, PP2B and PP1 inhibitors in VM, RA and SANC. We found that the level of all of these transcripts except PP2B was significantly lower in SANC compared to VM. Western blotting confirmed significantly lower abundance of PP1 in both SANC (48%) and RA (43%) compared to VM, whereas PP2B abundance was 5.6 fold higher in SANC and 4.7 fold higher in RA vs. VM. Although PP2A levels trended lower in both SANC and RA, the difference did not reach more stringent levels of significance. The abundance levels of these protein phosphatases were not significantly difference between SANC and RA. Protein abundance of endogenous protein phosphatase inhibitor I1 was also not different between the cell types; however, PP1 inhibitor DARPP-32 was found to be 2.6 fold higher in SANC and 2.9 fold higher in RA compared to VM. The PP1 inhibitor KEPI-1 was significantly lower in SANC compared to both LV (23%) and RA (30%). We employed genetic manipulation (siRNA targeting PP2B) in HL-1 cells to determine their effect on the spontaneous beating rate. On average (n= 7) PP2B mRNA expression level was reduced to 20 % of control in response to its mRNA silencing. The beating rate of HL-1 cells increased 2-2.5 fold in response to silencing of PP2B. We used FRET (Fluorescence resonance energy transfer) technology to detect the impact of PP2B on cAMP level in HL-1 cells. FRET detection of cytoplasmic cAMP in the single live beating cell revealed 1.30.2 fold increase (p<0.05, n=3) in response to IBMX treatment in cells transfected with Scrambled siRNA (Scr-siRNA), while cells with reduced PP2B expression demonstrated 2.40.3 fold increase (p<0.05, n=5). For plasma membrane cAMP we observed similar picture: 1.40.2 fold increase of basal level after IBMX treatment of Scr-siRNA transfected cells, while cells with reduced PP2B expression have demonstrated 2.20.3 fold increase (n=5), p<0.05. In order to see partitioning of different PPs in the total picture of cell ability to dephosphorylate proteins, we have modified Promega ProFluor Ser/Thr PPase assay, and now we are able to measure PP activity in cell lysates in the same conditions for PP1, PP2A, PP2B and PP2C. We found that PP2A is a dominant PP in SANC. In order to differentiate between PP1 and PP2A, we studied dose-dependent effects of Calyculin A and Okadaic acid at low nanomolar concentrations and found that PP2A is more sensitive to these inhibitors than PP1. We found that PP1 is present in SANC, but its relative activity is low. We tried to discern the PP1 partitioning by using I-1, the most specific PP1 inhibitor. Unfortunately, this approach did not help, mainly due to the high level of PP2A activity in cells, contamination in the activated I-1 and low level of PP1 (relatively to the total variability). Knowing that in cells PP are often located in different microenvironments (for example they can be bound to scaffolding proteins), now we are developing a new approach to investigate role of PPs in cell functioning. Currently we are developing methods of immunoprecipitation of the most important complexes in SANC and VM in order to detect via co-immunoprecipitation (with further Western blot and mass spectrometry analysis) which PPs are bound to them and are involved in their functioning. Initially, we took SERCA and mAKAP as our targets. This method is especially important because even low abundant phosphatase can have an important and specific role in cell regulation. Currently we continue our work to develop and improve co-immunoprecipitation techniques. The most recent experiments demonstrated that activity of PPs depends tremendously on cations present in phosphatase reaction. We have developed conditions in which PP2A activity is not so high but at the same time PP1 and PP2B activities appear much higher than previously. Using Calyculin A and Okadaic acid at low nanomolar concentrations under the new conditions helped us demonstrate a much clearer presence of PP1 and PP2B activities in SANC. These data also prove the importance of intracellular microenvironment cation balance in the activities of PPs.
| Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A et al. (2014) Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design. J Mol Cell Cardiol 66:106-15 |
