Yukiomi Tsuji

Pioglitazone, a peroxisome proliferator-activated receptor-gamma activator, attenuates atrial fibrosis and atrial fibrillation promotion in rabbits with congestive heart failure

BACKGROUND The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activator pioglitazone antagonizes angiotensin II actions and possesses anti-inflammatory and antioxidant properties in vitro. There is evidence that pioglitazone improves ventricular remodeling in some experimental models. OBJECTIVE The purpose of this study was to assess the effects of pioglitazone on arrhythmogenic atrial structural remodeling versus the effects of the angiotensin II type 1 receptor blocker candesartan in a rabbit model of congestive heart failure. METHODS Rabbits subjected to ventricular tachypacing at 380 to 400 bpm for 4 weeks in the absence and presence of treatment with pioglitazone, candesartan, and combined pioglitazone and candesartan were assessed by electrophysiologic study, atrial fibrosis measurements, and cytokine expression analyses. RESULTS Atrial fibrillation (AF) lasting longer than 2 seconds was induced in no nonpaced controls but in all ventricular tachypacing-only rabbits (mean duration of AF: 8.0 +/- 1.4 seconds). Pioglitazone reduced the duration of AF (3.5 +/- 0.2 seconds, P <.05) and attenuated atrial structural remodeling, with significant reductions in interatrial activation time (50 +/- 2 ms vs 41 +/- 2 ms, P <.05) and atrial fibrosis (16.8% +/- 0.8% vs 10.9% +/- 0.7%, P <.05; control 1.6% +/- 0.2%), effects comparable to those of candesartan (duration of AF: 3.0 +/- 0.2 seconds; activation time 44 +/- 2 ms; fibrosis: 9.4% +/- 0.6%). Both pioglitazone and candesartan reduced transforming growth factor-beta1, tumor necrosis factor-alpha, and activated extracellular signal-regulated kinase expression similarly, but neither affected p38-kinase or c-Jun N-terminal kinase activation. The effects of combined pioglitazone and candesartan therapy were not significantly different from the effects of pioglitazone or candesartan alone. CONCLUSION Pioglitazone can attenuate congestive heart failure-induced atrial structural remodeling and AF promotion, with effects similar to those of candesartan. PPAR-gamma may be a potential therapeutic target for human AF.

Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle

OBJECTIVE Heart failure in patients and in animal models is associated with action potential prolongation of the ventricular myocytes. Changes in several membrane currents have been already demonstrated to underlie this prolongation. However, information on the two components (I(Kr) and I(Ks)) of the delayed rectifier potassium current (I(K)) in rapid pacing induced heart failure is lacking. METHODS AND RESULTS Action potentials and whole-cell currents, I(K), I(to1), I(K1), and I(Ca-L) were recorded in apical myocytes of left ventricle from 10 rabbits subjected to left ventricular pacing at 350-380 beats/min for 3-4 weeks and 10 controls with sham operation. Action potential duration at 90% repolarization (APD(90)) was prolonged in myocytes from failing hearts compared to controls at both cycle lengths of 333 and 1000 ms. Both E-4031-sensitive and -resistant components of I(K) (I(Kr), I(Ks)) in myocytes from failing hearts were significantly less than those of control hearts; tail current densities of I(Kr) and I(Ks) following depolarization to +50 mV were 0.62+/-0.05 vs. 0.96+/-0.12 pA/pF (P<0.05), and 0.27+/-0.08 vs. 0.52+/-0.08 pA/pF (P<0.05), respectively. There was no significant difference between control and failing myocytes in the voltage- and time-dependence of activation of total I(K), I(Kr) and I(Ks). The peak of L-type Ca(2+) current (I(Ca-L)) was significantly reduced in myocytes from failing hearts (at +10 mV, -9.29+/-0.52 vs. -12.28+/-1.63 pA/pF, P<0.05), as was the Ca(2+)-independent transient outward current (I(to1); at +40 mV, 4.8+/-0.9 vs. 9.6+/-1.3 pA/pF, P<0.05). Steady state I-V curve for I(K1) was similar in myocytes from failing and control hearts. CONCLUSIONS Decrease of I(K) (both I(Kr) and I(Ks)) in addition to reduced I(to1), may underly action potential prolongation at physiological cycle length and thereby contribute to arrhythmogenesis in heart failure.

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents

OBJECTIVE To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. METHODS Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. RESULTS At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. CONCLUSIONS The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.

Potassium Channel Subunit Remodeling in Rabbits Exposed to Long-Term Bradycardia or Tachycardia Discrete Arrhythmogenic Consequences Related to Differential Delayed-Rectifier Changes

Background— Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits. Methods and Results— We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by ≈50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits. Conclusions— Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.

Ionic Mechanisms of Acquired QT Prolongation and Torsades de Pointes in Rabbits With Chronic Complete Atrioventricular Block

Background—The ionic basis of acquired QT prolongation and torsade de pointes (TdP) unrelated to drugs is not fully understood. Methods and Results—We created a rabbit model with chronic complete atrioventricular block (AVB) (n=34), which showed prominent QT prolongation (by 120%), high incidence of spontaneous TdP (71%), and cardiac hypertrophy. Patch-clamp experiments were performed in left ventricular myocytes from 9 rabbits (8 with TdP, 1 without TdP) at ≈21 days of AVB and from 8 sham-operated controls with sinus rhythm. Action potential duration was prolonged in AVB myocytes compared with control (+61% at 0.5 Hz, +21% at 3 Hz). Both rapidly and slowly activating components of the delayed rectifier K+ current (IKr and IKs) in AVB myocytes were significantly smaller than in control by 50% and 55%, respectively. There was no significant difference in Ca2+-independent transient outward current (Ito1). L-type Ca2+ current (ICa,L) in control and AVB myocytes was similar in peak amplitude, but the half voltage for activation was shifted to the negative direction (5.9 mV) in AVB myocytes. Voltage dependence of ICa,L inactivation was not different in control and AVB myocytes. The inward rectifier K+ current (IK1) significantly increased in AVB myocytes compared with control. Conclusions—In the rabbit, chronic AVB leads to prominent QT prolongation and high incidence of spontaneous TdP. Downregulation of both IKr and IKs in association with altered ICa,L activation kinetics may underlie the arrhythmogenic ventricular remodeling.

Novel Calmodulin Mutations Associated With Congenital Arrhythmia Susceptibility

Background—Genetic predisposition to life-threatening cardiac arrhythmias such as congenital long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) represent treatable causes of sudden cardiac death in young adults and children. Recently, mutations in calmodulin (CALM1, CALM2) have been associated with severe forms of LQTS and CPVT, with life-threatening arrhythmias occurring very early in life. Additional mutation-positive cases are needed to discern genotype–phenotype correlations associated with calmodulin mutations. Methods and Results—We used conventional and next-generation sequencing approaches, including exome analysis, in genotype-negative LQTS probands. We identified 5 novel de novo missense mutations in CALM2 in 3 subjects with LQTS (p.N98S, p.N98I, p.D134H) and 2 subjects with clinical features of both LQTS and CPVT (p.D132E, p.Q136P). Age of onset of major symptoms (syncope or cardiac arrest) ranged from 1 to 9 years. Three of 5 probands had cardiac arrest and 1 of these subjects did not survive. The clinical severity among subjects in this series was generally less than that originally reported for CALM1 and CALM2 associated with recurrent cardiac arrest during infancy. Four of 5 probands responded to &bgr;-blocker therapy, whereas 1 subject with mutation p.Q136P died suddenly during exertion despite this treatment. Mutations affect conserved residues located within Ca2+-binding loops III (p.N98S, p.N98I) or IV (p.D132E, p.D134H, p.Q136P) and caused reduced Ca2+-binding affinity. Conclusions—CALM2 mutations can be associated with LQTS and with overlapping features of LQTS and CPVT.

Reactive oxidative metabolites are associated with atrial conduction disturbance in patients with atrial fibrillation

BACKGROUND Oxidative stress is associated with atrial fibrillation (AF). However, little is known about the relationship between serum markers of oxidation and electrical activity in patients with AF. OBJECTIVE The purpose of this study was to investigate the possible association between serum markers of reactive oxidative metabolism and atrial remodeling in paroxysmal and persistent AF. METHODS Derivatives of reactive oxidative metabolites (DROM), an index of oxidative stress, were measured in 306 consecutive patients with AF (225 paroxysmal, 81 persistent) undergoing radiofrequency (RF) catheter ablation. Filtered P-wave duration by P-wave signal-averaged ECG and levels of high-sensitivity C-reactive protein (CRP) as an inflammatory marker also were measured. RESULTS Patients were followed up for 1.2 +/- 0.8 years. DROM levels in patients with persistent AF were significantly higher than in patients with paroxysmal AF (341.6 +/- 85.5 Carratelli [Carr] units vs 305.0 +/- 77.7 Carr units, P <.001). DROM levels showed a tighter, positive correlation with filtered P-wave duration in persistent AF patients (r = 0.56, P <.001) than in all AF patients (r = 0.13, P <.05). DROM levels also showed a weaker but significant correlation with high-sensitivity CRP in patients with AF. Kaplan-Meier analysis revealed that the highest quartile of basal DROM levels exhibited a significantly higher AF recurrence rate after RF catheter ablation in patients with paroxysmal AF (P <.01). CONCLUSION Serum markers of oxidative stress reflect atrial conduction disturbance and predict AF recurrence after RF catheter ablation in paroxysmal AF patients. DROM could serve as a biomarker for predicting risk of AF recurrence after RF catheter ablation.

Ca2+-Related Signaling and Protein Phosphorylation Abnormalities Play Central Roles in a New Experimental Model of Electrical Storm

Background— Electrical storm (ES), characterized by recurrent ventricular tachycardia/fibrillation, typically occurs in implantable cardioverter-defibrillator patients and adversely affects prognosis. However, the underlying molecular basis is poorly understood. In the present study, we report a new experimental model featuring repetitive episodes of implantable cardioverter-defibrillator firing for recurrent ventricular fibrillation (VF), in which we assessed involvement of Ca2+-related protein alterations in ES. Methods and Results— We studied 37 rabbits with complete atrioventricular block for ≈80 days, all with implantable cardioverter-defibrillator implantation. All rabbits showed long-QT and VF episodes. Fifty-three percent of rabbits developed ES (≥3 VF episodes per 24-hour period; 103±23 VF episodes per rabbit). Expression/phosphorylation of Ca2+-handling proteins was assessed in left ventricular tissues from rabbits with the following: ES; VF episodes but not ES (non-ES); and controls. Left ventricular end-diastolic diameter increased comparably in ES and non-ES rabbits, but contractile dysfunction was significantly greater in ES than in non-ES rabbits. ES rabbits showed striking hyperphosphorylation of Ca2+/calmodulin-dependent protein kinase II, prominent phospholamban dephosphorylation, and increased protein phosphatase 1 and 2A expression versus control and non-ES rabbits. Ryanodine receptors were similarly hyperphosphorylated at Ser2815 in ES and non-ES rabbits, but ryanodine receptor Ser2809 and L-type Ca2+ channel &agr;-subunit hyperphosphorylation were significantly greater in ES versus non-ES rabbits. To examine direct effects of repeated VF/defibrillation, VF was induced 10 times in control rabbits. Repeated VF tissues showed autophosphorylated Ca2+/calmodulin-dependent protein kinase II upregulation and phospholamban dephosphorylation like those of ES rabbit hearts. Continuous infusion of a calmodulin antagonist (W-7) to ES rabbits reduced Ca2+/calmodulin-dependent protein kinase II hyperphosphorylation, suppressed ventricular tachycardia/fibrillation, and rescued left ventricular dysfunction. Conclusions— ES causes Ca2+/calmodulin-dependent protein kinase II activation and phospholamban dephosphorylation, which can explain the vicious cycle of arrhythmia promotion and mechanical dysfunction that characterizes ES.

The Calcium/Calmodulin/Kinase System and Arrhythmogenic Afterdepolarizations in Bradycardia-Related Acquired Long-QT Syndrome

Background—Sustained bradycardia is associated with long-QT syndrome in human beings and causes spontaneous torsades de pointes in rabbits with chronic atrioventricular block (CAVB), at least partly by downregulating delayed-rectifier K+-current to cause action potential (AP) prolongation. We addressed the importance of altered Ca2+ handling, studying underlying mechanisms and consequences. Methods and Results—We measured ventricular cardiomyocyte [Ca2+]i (Indo1-AM), L-type Ca2+-current (ICaL) and APs (whole-cell perforated-patch), and Ca2+-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca2+]i transients, and caffeine-induced [Ca2+]i release, and CAVB cells showed spontaneous early afterdepolarizations (EADs). ICaL density was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone for ICaL window current. Ca2+-calmodulin–dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca2+]i-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase as a contributor to enhanced SR Ca2+ loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca2+]i and ICaL inactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-induced ICaL inactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide prevented ICaL inactivation voltage shifts. The SR Ca2+-uptake inhibitor thapsigargin and the SR Ca2+ release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca2+ loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca2+]i required 2 weeks, paralleling the EAD time course. Conclusions—CAVB-induced remodeling enhances [Ca2+]i load and activates the Ca2+-calmodulin-CaMKII system, producing [Ca2+]i-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.

Eicosapentaenoic acid prevents atrial fibrillation associated with heart failure in a rabbit model

Atrial fibrillation (AF) is associated with morbidity and mortality of heart failure. Eicosapentaenoic acid (EPA), which is contained in fish oil, was shown to reduce the risk of cardiovascular diseases. We investigated the effects of EPA on AF associated with heart failure in a rabbit model. Rabbits were subjected to ventricular tachypacing (VTP) for 4 wk with or without EPA treatment. Continuous VTP induced heart failure status in these rabbits. The duration of AF (DAF) induced by burst pacing was analyzed by electrophysiological studies. VTP resulted in increased DAF following burst pacing. EPA treatment attenuated increased DAF. Atrial fibrosis increased in response to VTP, accompanied by extracellular signal-regulated kinase (ERK) phosphorylation and transforming growth factor-β1 (TGF-β1) expression in the atrium. Treatment with EPA attenuated atrial fibrosis, ERK phosphorylation, and TGF-β1 expression in response to VTP. EPA treatment increased adiponectin as an anti-inflammatory adipokine and decreased tumor necrosis factor-α as a proinflammatory adipokine in the atrium and epicardial adipose tissues. EPA attenuated VTP-induced AF promotion and atrial remodeling, which was accompanied by modulating the profiles of adipokine production from epicardial adipose tissue. EPA may be useful for prevention and treatment of AF associated with heart failure.