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Objectives The increased abnormal activity in heart failure is one of the major mechanisms of arrhythmogenesis which contributes directly to the sudden cardiac death. The current study is to explore the mechanisms underlying the increased abnormal impulses in heart failure (HF). Methods Using the whole-cell patch clamp technique in current-clamp configuration, we studied freshly dissociated ventricular myocytes from a model of pressure-overload heart failure. In addition, we employed a unique coupling-clamp technique to electrically couple two isolated myocytes with a controlled value of coupling conductance (Gc). This method was also used to couple one cell to an invariable voltage source (-10mV) via various conductances (Gsac) designed to mimic the stretch-induced membrane depolarisation. Results Action potential (AP) morphology was significantly altered in ventricular myocytes from failing heart compared with sham-operated controls, with significantly elevated plateau levels and prolonged APD 30 and APD 90 . Increased pacing frequency (3Hz) significantly prolonged both APD 30 and APD 90 in failing myocytes but not in control myocytes. With rapid pacing, the critical Gc required for AP propagation decreased from 3.3 ± 0.1 to 2.7 ± 0.1 nS (n = 10, p Conclusions These results demonstrate facilitation of electrical conduction and arrhythmia propagation in failing myocytes. Thus, in addition to its role in enhancing automaticity, AP remodelling contributes to arrhythmia development and antagonises diminished coupling that stems from fibrosis and down-regulated gap junctions.

Objectives Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic mechanism in ventricular myocytes allowing a faster ventricular relaxation (and diastolic filling) at fast heart rates. Previous studies suggest that CaMKII activity is required for FDAR but the molecular targets remain elusive. We propose that CaMKII regulates FDAR by a mechanism that involves CaMKII-dependent alteration of myofilament sensitivity to Ca 2+ . Methods [Ca 2+ ] i and sarcomere length were measured by IonOptix Ca 2+ image system. Myofilament sensitivity to Ca 2+ was assessed by measuring the gradient of cell length-fura2 trajectory during contraction and late relaxation. Western blot was used to detect the target proteins. Results Increasing pacing rate from 0.5Hz to 4Hz in left ventricular (LV) myocytes accelerated Ca 2+ decline and sarcomere relaxation time constants (from 152 ± 13ms to 60 ± 5ms and from 36 ± 2ms to 18 ± 1ms, respectively, p 50 increased from 1.62 ± 0.06 to 1.84 ± 0.06, p 2+ . Inhibition of PKA (H89, 1 μM) or PKC (CHE, 1 μM) had no effect on myofilament Ca 2+ desensitisation and FDAR, whereas CaMKII inhibitor KN93 (1 μM) abolished frequency-dependent myofilament desensitisation to Ca 2+ and FDAR. Because cardiac troponin I (Tn-I) is the major regulator for myofilament sensitivity to Ca 2+ and both PKA and PKC share the same phosphorylation sites Ser23/24, we determined the Ser23/24 phosphorylation in ventricular myocytes and found that Ser23/24 phosphorylation was largely reduced by PKA and PKC inhibitors but not by CaMKII inhibition. However, a phospho-Ser-antibody showed that CaMKII inhibitor KN93 significantly reduced Tn-I phosphorylation in the Tn-I immunoprecipitates, indicating that CaMKII phosphorylates Tn-I at sites different from the PKA and PKC sites. Indeed, a co-immunoprecipitation of CaMKII and Tn-I has been detected. Conclusions Our results suggest that FDAR is regulated by a frequency-dependent desensitisation of myofilament sensitivity to Ca 2+ , in which CaMKII-dependent Tn-I phosphorylation plays a major role.

Objectives CaMKII activation is pro-arrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. Methods Action potentials (APs) were recorded by whole-cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch-activated-channel (SAC) current. Results SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after depolarizations (EADs) and automaticity in KO myocytes in which an increase in L-type calcium channel (LTCC) current (I Ca ) and a reduction of sarcoplasmic reticulum (SR) Ca 2+ leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I Ca in promoting stretch-induced abnormal impulses. In addition, APD prolongation by LTCC opener S(-)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K + channel blocker 4-aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. Conclusions I Ca activation plays a central role in stretch-induced abnormal impulses and APD prolongation is arrhythmogenic only when I Ca is highly activated. At increased I Ca activation, CaMKII inhibition cannot suppress abnormal impulse induction.