Abstract P082: Cardiac Calcium Handling and Protein Phosphatase 2A Activity Are Differentially Regulated by JNK1 and JNK2 MAP Kinases
We previously identified the JNK-B56α-PP2A signaling axis as a major target in anthrax lethal toxin (LT)-induced cardiac dysfunction. Thus, we further tested whether LT-mediated loss in cardiac function is a consequence of dysregulated calcium handling resulting from JNK inactivation. To biochemically recapitulate the signaling effects of LT, we infected NRVM with HA-B56α adenovirus and determined PP2A activity as well as Ca2+i measurements. Over-expression of HA-B56α in NRVM did not induce a significant increase in cellular PP2A activity, however, it did induce a significant increase (p<0.01) in Ca2+i compared to virus control. Furthermore, PLB, PP2Ac and Akt protein co-immunoprecipitated with HA-B56α, and immunostaining revealed colocalization of B56α with PLB at the SR. Since B56α over-expression alone was not sufficient to induce PP2A activity or PP2A-mediated Ca2+i dysregulation, we hypothesized that JNK may serve as a functional regulator of Ca2+i handling through PP2A activation as well as B56α protein levels. Adenoviral-mediated over-expression of MEK7 in NRVM resulted in a significant reduction in LT-mediated Ca2+i dysregulation compared to virus control (p<0.01). To further determine whether the protection of MEK7 is mediated by JNK1 or JNK2, gain-of-function/loss-of-function experiments were performed utilizing adenoviral constructs for CA-MEK7, DN-JNK1 and DN-JNK2. Results confirmed that the protective effect of active MEK7 over-expression on Ca2+i was significantly lost with knock-down of JNK1 compared to JNK2 (p<0.05), suggesting that JNK1 plays a more substantial role in regulating PP2A activity than JNK2. Furthermore, the selective knock-down of JNK1 also increased Ca2+i levels (p<0.001) compared to MEK7 during LT treatment, which reveals the importance of JNK1 in PP2A-mediated Ca2+i dysregulation. Interestingly, immunoblotting of PLB did not reveal a JNK-dependent difference in PLB phosphorylation at Ser16, however, loss of JNK2 almost completely inhibited p-PLB-Thr17. Thus, our results suggest that JNK1 and JNK2 may differentially regulate Ca2+i through PP2A activation and PLB-Thr17 phosphorylation, respectively.
- © 2011 by American Heart Association, Inc.