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Introduction: Increased activation of the renin-angiotensin-aldosterone (RAA) system in heart failure is associated with an increased risk for sudden death. However, the mechanism whereby this signaling pathway contributes to arrhythmic risk is unknown. To evaluate the relationship, we used a cardiac-restricted angiotensin converting enzyme (ACE) overexpression model. We examined the electrophysiological consequences of homozygous and heterozygous ACE overexpression with and without a functional second somatic ACE allele.
Methods and Results: Surface electrocardiographic atrial activity was generally undetectable, and the QRS voltage was reduced by 2.4 fold at 6 weeks compared to control animals, suggesting an effect on current generation or conduction. Intracardiac electrocardiograms demonstrated reduced atrial electrogram voltage and the presence of various degrees of AV nodal block in ACE homozygous mice only. By electrophysiology (EP) studies, using programmed stimulation and burst pacing, all five ACE mice tested demonstrated sustained ventricular tachycardia whereas wild type (WT) (n=7) and heterozygote (HZ) (n=7) mice were noninducible. The atrioventricular (AV) and atrio-Hisian (AH) intervals were prolonged in ACE versus WT (AV p<.00005 and AH p<.0002) and HZ mice (AV p<.00004 and AH p<.0002). Another strain of mice that was created, called ACE 1/8, has a less extreme ACE overexpression. The 1/8 mice showed conduction intervals that were prolonged compared to WT and HZ, but still shorter than the original ACE mice. The conduction changes in the homozygous mice were associated with reductions in sodium channel (Scn5a) and connexin 40 (Cx40) mRNA abundances both in the atria and ventricles, and connexin 43 (Cx43) mRNA was reduced only in the atria. Connexin 45, L-type calcium (Cacna1) and transient outward potassium (Kv4.2) channel mRNAs were unchanged, however, suggesting specificity of the effect. HZ models showed a subtle physiological phenotype and intermediate levels of Cx and Na+ channel expression.
Conclusion: Increased cardiac ACE expression was associated with reduced surface electrocardiographic voltage, atrioventricular (AV) nodal block, and increased susceptibility to induced ventricular arrhythmia that could be explained, in part, by lowered sodium channel and connexin expression. While the molecular changes showed a gene dose effect, homozygous mice showed the most dramatic physiological phenotype, unlike heterozygous mice, consistent with a predicted threshold effect of ion channel changes on conduction. These observations may help explain the observed reduction in sudden death with the use of ACE inhibitors for congestive heart failure.
The renin-angiotensin-aldosterone system (RAA) is a key signaling pathway in the cardiovascular system. Activation of this system is associated with progression of atherosclerosis, hypertrophy and heart failure. A critical component of this system is ACE, which produces the eight amino acid peptide Ang II, a central effector molecule of the RAA system. In humans, increased Ang II levels, as seen in heart failure, are associated with an increased arrhythmic risk. This risk is reduced by ACE inhibitors. Recently, we described a cardiac-restricted ACE overexpression mouse (called ACE 8/8) that showed an increased risk of sudden death in the absence of heart failure. One prominent feature of this genetically altered mouse was a reduction in the surface electrocardiographic voltage without evidence of heart failure, pulmonary edema, or pericardial effusion, suggesting an underlying defect in cardiac current generation or conduction. Here, we investigate further the electrophysiological abnormalities mediated by cardiac-specific ACE overexpression by intracardiac EP studies and correlate findings with induced ion channel changes.
Two principal proteins involved in cardiac conduction and arrhythmogenesis are ion channels and connexins. Voltage-gated sodium channels are responsible for the initial depolarization phase of the cardiac action potential and are the main source of current for conduction. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, Scn5a, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing,) atrioventricular conduction delay, and ventricular fibrillation. Disruption of the mouse cardiac sodium channel gene causes atrioventricular conduction blocks, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. The L-type calcium (Cacna1) and transient outward potassium (Kv4.2) channel have also been implicated in conduction abnormalities and arrhythmogenesis.
Gap junctions, assembled from dodecamers of transmembrane proteins called connexins (Cx), form cell-to-cell pathways for the propagation of precisely orchestrated patterns of current flow that govern the regular rhythm of a healthy heart. Three Cxs, Cx43, Cx40 and Cx45, are expressed in the heart. In the adult mouse heart, Cx40 expression is restricted to the atria and ventricular conduction system. Cx43 is the most ubiquitous, being expressed in all the working myocytes and most of the conductive myocytes. Cx45 is weakly expressed in all conductive myocytes, including those of the nodal tissues.
Cardiac-restricted ACE overexpression mice and mouse genotyping
The generation of chimeric mutant ACE mice was performed by gene targeting. The promoter region of the somatic ACE gene was substituted by the a-MHC promoter, resulting in cardiac specific ACE overexpression. Chimeric mice were mated to C57BL/6 mice to generate F1 mice. Heterozygous F1 mice were bred to create F2 offspring of wild-type (WT), heterozygous (HZ), and homozygous (HM), cardiac-restricted ACE overexpression mice (8/8). All studies were performed on F2 or F3 generation litters produced by breeding heterozygous animals. In order to confirm the mouse genotype, genomic DNA was obtained through tail clipping. A 450-bp fragment was amplified for the wild-type allele and a 742-bp fragment was amplified for the mutant allele.
Surface ECG monitoring and intracardiac electrophysiology
Surface ECG was obtained by applying adhesive skin electrodes at all the four limbs and on the anterior (V1) and lateral (V6) chest wall. Intracardiac EP studies, including burst pacing, were performed using published methods (Bevilacqua, L.M. et al. J Interv. Card Electrophysiol. 4, 459-467: 2000). The data was stored and analyzed using a GE Prucka Cathlab 4.0 system (GE Healthcare, Milwaukee, WI) after positioning a Millar EPR800 (Millar Instruments Inc. Houston TX) 1.1 French octapolar catheter in the right ventricle through a right jugular vein cutdown. Voltage amplitudes were also measured from the electrogram; values from three leads (high right atrium, one from the His bundle, and right ventricle) were averaged for each mouse. Burst pacing of atria and ventricles was done separately using a Grass SD9 stimulator (Astro-Med Inc., W.Warwick, RI) at incrementally decreasing cycle lengths between 50-150 msec applied over a period of 1-15 secs and inducible tachycardia or fibrillation (>5 consecutive beats) was noted.
Quantitative mRNA assay
To determine the abundances of different cardiac genes including scn5a, Cacna1, kv4.2, Nkx2.5, Cx40, Cx43 and Cx45, total RNA from wild type, heterozygous and ACE homozygous mice was isolated and reverse transcription was carried out at 42°C for 30 min with iScript reverse transcriptase of iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) and 1.0 mg total RNA. The first strand cDNA was used as template for subsequent SYBR real time PCR. All amplifications were performed in triplicate and consisted of 40 cycles of 30 s at 95°C, 30 s at 60°C, and 20 s at 72°C in a BioRed thermocycler iCycler (Hercules, CA). b-Actin was used as an internal reference when making quantitative comparison.
Immunoblot analysis of connexin protein
To determine if protein levels corresponded to the changes seen in the mRNA abundances, total atrial and ventricular lysates from WT and ACE hearts from 6-8 weeks of age were studied by western blotting. Immunoblots were blocked and followed by incubation with anti-Cx43 primary antibody (Chemicon International, Temecula, CA). HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was then applied, followed by chemiluminescent processing (ECL; Amersham Pharmacia Biotech, Buckinghamshire, England) and autoradiography. Connexin band intensities were compared to endogenous GAPDH.
Statistical Analysis
All continuous variables were compared between ACE mice and gender- and age-matched controls. Values are presented as mean ± 1 SEM. The two-tailed Student’s t-test and ANOVA where appropriate were used, and a value of P < 0.05 was considered statistically significant.
• Cardiac ACE overexpression causes reduced surface and electrogram voltages, poor atrial and ventricular conduction, and AV nodal block
• Homozygous ACE overexpression results in susceptibility to ventricular arrhythmias.
• Decreases in expression of sodium channels, connexin 40, and connexin 43 may explain the electrophysiological phenotype.
This study was supported by the Howard Hughes Medical Institute (EAW), National Institutes of Health (NIH) grant HL64828 (SCD), a Department of Veterans Affairs merit grant (SCD), an American Heart Association Established Investigator Award (SCD), a research fellowship from the National Institutes of Health NRSA-1F32 HL078115-01 (VSK), and a research fellowship from the Georgia affiliate of the National Kidney Foundation (HDX). The authors would like to acknowledge Matthew Clutts for help with this study.
The genes of these mice were altered so they have a lot of a specific enzyme. When they have a lot of this enzyme (known as ACE), they have heart problems. We were investigating these problems by taking recordings from outside and inside the heart to show that the hearts of these mice are not beating or conducting electricity properly. These mice serve as a model for human heart disease, and this study can help explain why the current heart medications (ACE inhibitors) work they way that they do.
electrophysiological studies (burst pacing, programmed stimulation)
electrophysiology, angiotensin converting enzyme, RAA path, overexpression, cardiac
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