Calcium-calmodulin kinase II mediates digitalis-induced arrhythmias.
BACKGROUND
Digitalis-induced Na(+) accumulation results in an increase in Ca(2+)(i) via the Na(+)/Ca(2+) exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca(2+) load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca(2+)(i) could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved.


RESULTS
In paced rat ventricular myocytes (0.5 Hz), 50 μmol/L ouabain increased contraction amplitude by 160 ± 5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca(2+) waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/L KN93 but not by 2.5 μmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 μmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca(2+) content and Ca(2+) spark frequency, indicative of enhanced SR Ca(2+) leak. KN93 suppressed the ouabain-induced increase in Ca(2+) spark frequency without affecting SR Ca(2+) content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice.


CONCLUSIONS
These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca(2+) leak from the SR, is the underlying mechanism involved.
Centro de Investigaciones Cardiovasculares, Conicet La Plata, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina.
Luis A
LA
Gonano
Marisa
M
Sepúlveda
Yanina
Y
Rico
Marcia
M
Kaetzel
Carlos A
CA
Valverde
John
J
Dedman
Alicia
A
Mattiazzi
Martin
M
Vila Petroff
D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D001145:Arrhythmias, Cardiac / Q000139:chemically induced* / Q000175:diagnosis / Q000503:physiopathology / Q000517:prevention & control; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002304:Cardiac Pacing, Artificial; D002316:Cardiotonic Agents / Q000633:toxicity*; D002478:Cells, Cultured; D004562:Electrocardiography; D004789:Enzyme Activation; D006339:Heart Rate / Q000187:drug effects*; D006352:Heart Ventricles / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology; D010042:Ouabain / Q000633:toxicity*; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D000099211:Phospholamban; D000096926:Benzenesulfonamides
D000889:Anti-Arrhythmia Agents; D001596:Benzylamines; D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002316:Cardiotonic Agents; D010042:Ouabain; D010455:Peptides; D047428:Protein Kinase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D013449:Sulfonamides; D000099211:Phospholamban; C503464:CaMKII inhibitor AIP; C072105:KN 93; D000096926:Benzenesulfonamides