Ca(2+)/calmodulin-dependent protein kinase II contributes to intracellular pH recovery from acidosis via Na(+)/H(+) exchanger activation

Abstract

The Na(+)/H(+) exchanger (NHE-1) plays a key role in pH(i) recovery from acidosis and is regulated by pH(i) and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH(i) indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH(i) recovery from the acid load (dpH(i)/dt)-an index of NHE-1 activity in HEPES buffer or in NaHCO(3) buffer in the presence of inhibition of anion transporters-was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH(i) recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing beta-galactosidase myocytes (dpH(i)/dt: 0.195+/-0.04 vs. 0.045+/-0.010 min(-)(1), respectively, n=8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.

Figure 1. CaMKII-inhibition decreased the activity of NHE-1 during acute acidosis

Representative superimposed recordings of pH_i and overall results of adult rat isolated myocytes loaded with SNARF-1/AM and subjected to acute intracellular acidosis induced by: (A) transient exposure to 20 mM NH₄Cl in HEPES buffer; (B) substitution of HEPES by NaHCO₃ buffer and (C) hypercapnia at constant pH_o in NaHCO₃ buffer in the presence of SITS. Protocols were performed either in the absence of drug (Control, Cont.) or in the presence of 1 μM KN93, its inert analogue KN92 or 3 μM of HOE642 (n=5 cells per group). NHE-1 activity was estimated from the rate of change in pH_i (dpH_i/dt) during the recovery at an identical pH_i of 6.8. In B and C, note that after the continuous pH_i recording, measurements of pH_i were performed every 2 min. In this and the following Figures overall results are expressed as mean ± SE. * p<0.05 vs Cont.

Figure 2. CaMKII-inhibition decreased the activity of NHE-1 during sustained acidosis

Representative superimposed recordings of pH_i (A) and overall results (B) of adult rat isolated myocytes subjected to sustained intracellular acidosis induced by exposure to 20 mM NH₄Cl followed by its washout with a Na⁺-free solution. Normal [Na⁺]_o was then reintroduced to release NHE-1 blockade and permit pH_i recovery. The protocol was performed either in the absence of drugs (Cont., n=6) or in the presence of 1 μM KN93 (n=5) or AIP (n=4). NHE-1 activity was estimated as in Figure 1. (C) Immunoblots and overall results of the effects of acidosis on the phosphorylation of CaMKII and its substrate Thr17 of phospholamban (PLN) (n=4-8 hearts per group). * p<0.05 vs Cont.

Figure 3. Overexpression of CaMKII enhanced the rate of pH_i recovery in isolated myocytes submitted to an acid load

(A) After 48 h of infection, fluorescent myocytes indicated the successful expression of β-galactosidase and CaMKIIδC genes. (B) Representative blots and overall results of phospho-CaMKII and GAPDH confirmed the overexpression of CaMKII in Ad.CaMKII vs. Ad.βgal infected cells (n=6 in both groups). (C) In CaMKII overexpressing cells, the recovery of pH_i was faster than in βgal overexpressing cells. Inhibition of NHE-1 by HOE, completely blocked the pH_i recovery in both, CaMKII and βgal overexpressing cells. Black and grey traces depict CaMKII and βgal-overexpressing myocytes respectively in the absence and presence of the NHE-1 inhibitor. Data of 6 independent experiments from 6 hearts. *p<0.05 vs. Ad.βgal.

Figure 4. Chronic inhibition of CaMKII decreased the rate of pHi recovery from an acid load

(A) Analysis of phospholamban (PLN) expression and basal phosphorylation of Ser16 and Thr17 sites of PLN, in AC3-I, AC3-C and WT mice. (B) Representative superimposed recordings of pH_i and overall results obtained in myocytes isolated from adult mice with chronic cardiac CaMKII inhibition (AC3-I) and their transgenic controls AC3-C. Data from 10 and 6 cells in AC3-C and AC3-I, respectively. *p<0.05 vs. AC3-C.

Figure 5. CaMKII-dependent activation of NHE-1 is independent of the mitogen-activated protein kinase pathway

(A) Immunoblots and overall results of sustained acidosis-induced increase in ERK1/2 phosphorylation. ERK1/2 phosphorylation was studied in the absence and the presence of the CaMKII inhibitors, KN93 or AIP (1 μM), MEK inhibitor PD98059 (30 μM, PD) and the combination PD+KN93 (n=4-17 hearts per group). (B) Superimposed typical pH_i records (C) and overall results (n=4-6 cells per group) of the experiments described in (A). H⁺ efflux (JH⁺) as an index of NHE-1 activity was calculated as product of the recovery rate (dpHi/dt) and the intrinsic buffering capacity (β_i).* p<0.05 with respect to control. # p<0.05 with to PD or KN-93.

Figure 6. In vitro phosphorylation of the carboxyl terminal of NHE-1

Phosphorylation of wild type (WT) GST-NHE1(625-815) fusion protein or GST-NHE1 fusion protein in which Ser648/703/796 were replaced by Ala (triple mutant, TM) by preactivated CaMKII or protein kinase Bα (PKBα) detected by ³²P incorporation and autoradiography (top panel). Equal protein loading was confirmed by Coomassie staining (bottom panel). Similar results were obtained in two other experiments.