kn-93 and Acidosis

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.

Topics: Acidosis; Animals; Benzopyrans; Benzylamines; beta-Galactosidase; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cytoplasm; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Naphthols; Phosphorylation; Rats; Rats, Wistar; Rhodamines; Sodium-Hydrogen Exchangers; Sulfonamides

The multifunctional signal molecule calmodulin kinase II (CaMKII) has been associated with cardiac arrhythmogenesis under conditions where its activity is chronically elevated. Recent studies report that its activity is also acutely elevated during acidosis. We test a hypothesis implicating CaMKII in the arrhythmogenesis accompanying metabolic acidification.. We obtained monophasic action potential recordings from Langendorff-perfused whole heart preparations and single cell action potentials (AP) using whole-cell patch-clamped ventricular myocytes. Spontaneous sarcoplasmic reticular (SR) Ca(2+)release events during metabolic acidification were investigated using confocal microscope imaging of Fluo-4-loaded ventricular myocytes.. In Langendorff-perfused murine hearts, introduction of lactic acid into the Krebs-Henseleit perfusate resulted in abnormal electrical activity and ventricular tachycardia. The CaMKII inhibitor, KN-93 (2 microm), reversibly suppressed this spontaneous arrhythmogenesis during intrinsic rhythm and regular 8 Hz pacing. However, it failed to suppress arrhythmia evoked by programmed electrical stimulation. These findings paralleled a CaMKII-independent reduction in the transmural repolarization gradients during acidosis, which previously has been associated with the re-entrant substrate under other conditions. Similar acidification produced spontaneous AP firing and membrane potential oscillations in patch-clamped isolated ventricular myocytes when pipette solutions permitted cytosolic Ca(2+) to increase following acidification. However, these were abolished by both KN-93 and use of pipette solutions that held cytosolic Ca(2+) constant during acidosis. Acidosis also induced spontaneous Ca(2+) waves in isolated intact Fluo-4-loaded myocytes studied using confocal microscopy that were abolished by KN-93.. These findings together implicate CaMKII-dependent SR Ca(2+) waves in spontaneous arrhythmic events during metabolic acidification.

Topics: Acidosis; Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Female; Heart Ventricles; In Vitro Techniques; Male; Mice; Myocytes, Cardiac; Protein Kinase Inhibitors; Second Messenger Systems; Sulfonamides

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Topics: Acidosis; Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dantrolene; Disease Models, Animal; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Peptides; Phosphorylation; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Time Factors; Ventricular Function, Left; Ventricular Pressure

To assess the time course of phosphorylation of phospholamban residues, the underlying mechanisms determining these phosphorylations, and their functional impact on the mechanical recovery during acidosis.. Langendorff perfused rat hearts were submitted to 30 min of hypercapnic acidosis. Contractility, relaxation, and phosphorylation of phospholamban residues, immunodetected by specific antibodies, were determined.. Acidosis produced a mechanical impairment followed by a spontaneous recovery, most of which occurred within the first 3 min of acidosis (early recovery). During this period, contractility and relaxation recovered by 67+/-9% and 77+/-11%, respectively, from its maximal depression, together with an increase in the Ca(2+)-calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of Thr(17). The CaMKII inhibitor KN-93, at 1, 5 and 10 microM, decreased Thr(17) phosphorylation to basal levels and produced a similar impairment of the early relaxation recovery (50%). However, only 5 and 10 microM KN-93 inhibited the early contractile recovery and completely blunted the late mechanical recovery. Inhibition of the reverse mode of the Na(+)/Ca(2+) exchanger by KB-R7943 decreased Thr(17) phosphorylation but accelerated the early contractile recovery.. CaMKII-dependent Thr(17) phosphorylation significantly increased at the beginning of acidosis, is responsible for 50% of the early relaxation recovery, and is linked to the activation of the reverse Na(+)/Ca(2+) mode. The early contractile recovery and the late mechanical recovery are dependent on CaMKII but independent of the phosphorylation of the Thr(17) residue of phospholamban. The reverse Na(+)/Ca(2+) mode has an additional negative effect that opposes the early mechanical recovery.

Topics: Acidosis; Animals; Benzylamines; Blotting, Western; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Depression, Chemical; Electrophoresis, Polyacrylamide Gel; Hypercapnia; Male; Myocardial Contraction; Perfusion; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Wistar; Sodium-Calcium Exchanger; Sulfonamides; Thiourea; Threonine; Time Factors

Initially during acidosis, Ca transient amplitude (Delta[Ca]i) and the rate constant of [Ca]i decline (k(Ca)) are decreased, but later during acidosis Delta[Ca]i and k(Ca) partially recover. This recovery in rat myocytes could be inhibited by KN-93 suggesting that CaMKII-dependent protein phosphorylation (and enhanced SR Ca uptake) may be responsible. To test whether phospholamban (PLB) is required for the Delta[Ca]i and k(Ca) recovery during acidosis, we used isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. [Ca]i was measured using fluo-3. During the initial phase of acidosis (1-4 min), Delta[Ca]i decreased in WT myocytes (n = 8) from 1.75 +/- 0.19 to 1.10 +/- 0.13 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 3.20 +/- 0.22 to 2.38 +/- 0.18 s(-1) (P < 0.05). Later during acidosis (6-12 min), Delta[Ca]i partially recovered to 1.41 +/- 0.18 DeltaF/F0 and k(Ca) to 2.78 +/- 0.22 s(-1) (i.e. both recovered by approximately 50%). CaMKII inhibition using KN-93 completely prevented this recovery of Delta[Ca]i and k(Ca) during late acidosis in WT myocytes. In PLB-KO myocytes (n = 11) Delta[Ca]i decreased during early acidosis from 2.92 +/- 0.31 to 1.33 +/- 0.17 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 10.45 +/- 0.56 to 7.58 +/- 0.68 s(-1) (P < 0.05). However, Delta[Ca]i did not recover during late acidosis and k(Ca) decreased even more (6.59 +/- 0.65 s(-1)). Parallel results were seen for contractile parameters. We conclude that PLB is crucial to the recovery of Delta[Ca]i and k(Ca) during acidosis. Moreover, PLB phosphorylation by CaMKII plays an important role in limiting the decline in Ca transients (and contraction) during acidosis.

Topics: Acidosis; Animals; Benzylamines; Calcium; Calcium Signaling; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Gene Deletion; Guanidines; Kinetics; Mice; Mice, Knockout; Myocytes, Cardiac; Sarcoplasmic Reticulum; Sodium-Hydrogen Exchangers; Sulfonamides; Sulfones

It has been suggested that the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) increases during acidosis in cardiac muscle. Thus we have investigated the role of CaMKII during acidosis by monitoring intracellular Ca2+ (using fura-2) and ICa (using the perforated patch clamp technique) during acidosis, in the absence and presence of the CaMKII inhibitor KN-93, in rat isolated ventricular myocytes. In the absence of KN-93, acidosis (pH 6.5) increased the amplitude of the fura-2 transient and prolonged its decay, but in the presence of KN-93 acidosis did not alter the amplitude and prolonged the decay more. In the absence of KN-93, acidosis increased the amplitude of the caffeine-induced fura-2 transient but did not alter its amplitude in the presence of KN-93. ICa did not change significantly during acidosis in the absence of KN-93 but decreased during acidosis in the presence of KN-93. These results suggest that activation of CaMKII during acidosis helps to compensate for the direct inhibitory effects of acidosis on sarcoplasmic reticular Ca2+ uptake and ICa.

Topics: Acidosis; Animals; Benzylamines; Caffeine; Calcium; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Heart Ventricles; Hydrogen-Ion Concentration; Male; Muscle Fibers, Skeletal; Myocardium; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Sulfonamides