Calcium/calmodulin-dependent protein kinase II contributes to cardiac arrhythmogenesis in heart failure

Abstract

Background: Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo.

Methods and results: Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05).

Conclusions: We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Figure 1

CaMKII overexpression increases incidence of afterdepolarizations. A, Original patch-clamp recordings showing increased AP duration and EADs in a myocyte from a TG CaMKII mouse. B, Mean data for EAD occurrence at 0.5 Hz. CaMKII inhibition (0.1 μmol/L AIP) decreases EAD occurrence. C, ISO treatment (10⁻⁸ M ISO) induces DADs and spontaneous APs in TG myocytes. D, Average data for DAD and spontaneous AP occurrence. Statistical analysis was performed using Fisher 2-sided exact test.

Figure 2

Effects of ISO on intracellular Ca transients, relaxation time 80% (RT_80%), and diastolic Ca. A, Original Ca tracings showing the clear positive inotropic effects of ISO in WT and the diminished effects in TG. Average values for intracellular Ca transient amplitudes (B), relaxation (C), and diastolic Ca (D). *Significant different values versus baseline using Student-Newman-Keuls post hoc test. #Significant difference versus corresponding value of WT using ANOVA.

Figure 3

ISO increases SR Ca leak. A, Original line-scan images showing increased Ca spark frequency and decreased Ca spark occurrence in the presence of CaMKII inhibition (1 μmol/L KN-93) and in a myocyte specifically lacking CaMKIIδ isoform (left column). B, Original Ca spark tracings (middle column). C, Average values of Ca spark frequency. D, Average Ca spark amplitude. E, Calculated SR Ca leak (Ca spark frequency×amplitude×duration). Student t test was performed for C and D.

Figure 4

ISO increases SR Ca content in TG CaMKII cells (1 μmol/L KN-92 and KN-93). A, Original SR Ca content tracings measured by caffeine contractures (10 mmol/L). Average values for SR Ca content (B) and Ca transient amplitude (C). P<0.05 indicates significance using Student t test.

Figure 5

NSE of Ca transients and contractions. A, Original recordings showing ENSE and LNSE for Ca transients and shortening. B, Mean values for ENSE and LNSE showing increased incidence for TG. Decreased incidence of ENSE (C) and LNSE (D) after KN-93 treatment (1 μmol/L) and in cells from a mouse model specifically lacking CaMKIIδ isoform. E, Original recording showing sustained NSE and inhibition of these (F) using KN-93. No SNSEs were seen in KO cells. Statistical analysis was performed using Fisher 2-sided exact tests.

Figure 6

A, Original ECG recordings in one representative mouse showing ISO-induced arrhythmias and inhibition of these using (20 μmol/L/kg body weight KN-93). B, Detailed tracings for respective arrhythmic events. C, Mean data showing similar effects of ISO on heart rate in all mice as calculated using Student t test. D, Mean data showing decreased arrhythmias in vivo by inhibition of CaMKII using Fisher 2-sided exact test.