Proarrhythmic defects in Timothy syndrome require calmodulin kinase II.
BACKGROUND
Timothy syndrome (TS) is a disease of excessive cellular Ca(2+) entry and life-threatening arrhythmias caused by a mutation in the primary cardiac L-type Ca(2+) channel (Ca(V)1.2). The TS mutation causes loss of normal voltage-dependent inactivation of Ca(V)1.2 current (I(Ca)). During cellular Ca(2+) overload, the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS.


RESULTS
We developed an adult rat ventricular myocyte model of TS (G406R) by lentivirus-mediated transfer of wild-type and TS Ca(V)1.2. The exogenous Ca(V)1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous Ca(V)1.2 with nifedipine and maintain peak I(Ca) at control levels in infected cells. TS Ca(V)1.2-infected ventricular myocytes exhibited the signature voltage-dependent inactivation loss under Ca(2+) buffering conditions, not permissive for CaMKII activation. In physiological Ca(2+) solutions, TS Ca(V)1.2-expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased I(Ca) facilitation, and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in I(Ca) facilitation, normalized the action potential, and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation.


CONCLUSIONS
In TS, the loss of voltage-dependent inactivation is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.
Thiel
William H
WH
Vanderbilt University, Nashville, TN, USA.
Chen
Biyi
B
Hund
Thomas J
TJ
Koval
Olha M
OM
Purohit
Anil
A
Song
Long-Sheng
LS
Mohler
Peter J
PJ
Anderson
Mark E
ME
D000328:Adult; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000201:enzymology / Q000209:etiology / Q000235:genetics*; D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000235:genetics*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D006321:Heart / Q000503:physiopathology; D006352:Heart Ventricles / Q000503:physiopathology; D006801:Humans; D007668:Kidney / Q000196:embryology; D008297:Male; D009154:Mutation; D032383:Myocytes, Cardiac / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013577:Syndrome
C425903:CACNA2D1 protein, human; D015220:Calcium Channels; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium