Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein.

A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding-impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the beta-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans.

DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model.

Myotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK-/- mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A-V) block. Our results demonstrate that the A-V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK+/- mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Comparison of two murine models of familial hypertrophic cardiomyopathy.

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

Ventricular arrhythmia vulnerability in cardiomyopathic mice with homozygous mutant Myosin-binding protein C gene.

BACKGROUND: Homozygous mutant mice expressing a truncated form of myosin-binding protein C (MyBP-C(t/t)) develop severe dilated cardiomyopathy, whereas the heterozygous mutation (MyBP-C(t/+)) causes mild hypertrophic cardiomyopathy. Adult male MyBP-C(t/t) and MyBP-C(t/+) mice were evaluated for arrhythmia vulnerability with an in vivo electrophysiology study. METHODS AND RESULTS: Surface ECGs were obtained for heart rate, rhythm, and conduction intervals. Atrial, atrioventricular, and ventricular conduction parameters and refractoriness were assessed in 22 MyBP-C(t/t), 10 MyBP-C(t/+), and 17 wild-type MyBP-C(+/+) mice with endocardial pacing and intracardiac electrogram recording. Arrhythmia induction was attempted with standardized programmed stimulation at baseline and with isoproterenol. Heart rate variability and ambient arrhythmia activity were assessed with telemetric ECG monitors. Quantitative histological characterization was performed on serial sections of excised hearts. MyBP-C(t/t) and MyBP-C(t/+) mice have normal ECG intervals and sinus node, atrial, and ventricular conduction and refractoriness. Ventricular tachycardia was reproducibly inducible in 14 of 22 MyBP-C(t/t) mice (64%) during programmed stimulation, compared with 2 of 10 MyBP-C(t/+) mice (20%) and 0 of 17 wild-type controls (P<0.001). Ventricular ectopy was present only in MyBP-C(t/t) mice during ambulatory ECG recordings. There were no differences in heart rate variability parameters. Interstitial fibrosis correlated with genotype but did not predict arrhythmia susceptibility within the MyBP-C(t/t) group. CONCLUSIONS: MyBP-C(t/t) mice, despite prominent histopathology and ventricular dysfunction, exhibit normal conduction and refractoriness, yet are vulnerable to ventricular arrhythmias. Somatic influences between genetically identical mutant mice most likely account for variability in arrhythmia susceptibility. A sarcomeric protein gene mutation leads to a dilated cardiomyopathy and ventricular arrhythmia vulnerability phenotype.

Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model.

OBJECTIVES: We sought to study the role of I(KACh) in atrial fibrillation (AF) and the potential electrophysiologic effects of a specific I(KACh) antagonist. BACKGROUND: I(KACh) mediates much of the cardiac responses to vagal stimulation. Vagal stimulation predisposes to AF, but the specific role of I(KACh) in the generation of AF and the electrophysiologic effects of specific I(KACh) blockade have not been studied. METHODS: Adult wild-type (WT) and I(KACh)-deficient knockout (KO) mice were studied in the absence and presence of the muscarinic receptor agonist carbachol. The electrophysiologic features of KO mice were compared with those of WT mice to assess the potential effects of a specific I(KACh) antagonist. RESULTS: Atrial fibrillation lasting for a mean of 5.7+/-11 min was initiated in 10 of 14 WT mice in the presence of carbachol, but not in the absence of carbachol. Atrial arrhythmia could not be induced in KO mice. Ventricular tachyarrhythmia could not be induced in either type of mouse. Sinus node recovery times after carbachol and sinus cycle lengths were shorter and ventricular effective refractory periods were greater in KO mice than in WT mice. There was no significant difference between KO and WT mice in AV node function. CONCLUSIONS: Activation of I(KACh) predisposed to AF and lack of I(KACh) prevented AF. It is likely that I(KACh) plays a crucial role in the generation of AF in mice. Specific I(KACh) blockers might be useful for the treatment of AF without significant adverse effects on the atrioventricular node or the ventricles.

Induction of atrial tachycardia and fibrillation in the mouse heart.

BACKGROUND: Atrial tachycardia and fibrillation in humans may be partly consequent to vagal stimulation. Induction of fibrillation in the small heart is considered to be impossible due to lack of a critical mass of > 100-200 mm2. Even with the recent progression of the technology of in vivo and in vitro mouse electrophysiological studies, few reports describe atrial tachycardia or fibrillation in mice. The purpose of this study was to attempt provocation of atrial tachyarrhythmia in mice using transvenous pacing following cholinergic stimulation. METHODS AND RESULTS: In vivo electrophysiology studies were performed in 14 normal mice. A six-lead ECG was recorded from surface limb leads, and an octapolar electrode catheter was inserted via jugular vein cutdown approach for simultaneous atrial and ventricular endocardial recording and pacing. Atrial tachycardia and fibrillation were inducible in one mouse at baseline electrophysiology study and eleven of fourteen mice after carbamyl choline injection. The mean duration of atrial tachycardia was 126 +/- 384 s. The longest episode lasted 35 min and only terminated after atropine injection. Reinduction of atrial tachycardia after administration of atropine was not possible. CONCLUSION: Despite the small mass of the normal mouse atria, sustained atrial tachycardia and fibrillation can be easily and reproducibly inducible with endocardial pacing after cholinergic agonist administration. This finding may contribute to our understanding of the classical theories of arrhythmogenesis and critical substrates necessary for sustaining microreentrant circuits. The techniques of transcatheter parasympathetic agonist-mediated atrial tachycardia induction may be valuable in further murine electrophysiological studies, especially mutant models with potential atrial arrhythmia phenotypes.

Progressive atrioventricular conduction block in a mouse myotonic dystrophy model.

INTRODUCTION: Myotonic dystrophy is caused by expansion of a CTG trinucleotide repeat on human chromosome 19, and leads to progressive skeletal myopathy and atrioventricular conduction disturbances. A murine model of myotonic dystrophy has been designed by targeted disruption of the myotonic dystrophy protein kinase (DMPK) gene. The DMPK-deficient mice display abnormalities in A-V conduction characteristics, similar to the human cardiac phenotype. The purpose of this study was to determine whether age-related progression of A-V block occurs in a mouse model of DMPK-deficiency. METHODS AND RESULTS: Surface ECGs and intracardiac electrophysiology (EP) studies were performed in 60 immature and 90 adult homozygous (DMPK-/-), heterozygous (DMPK+/-), and wild-type (WT) DMPK+/+ control mice. Complete studies were obtained on 141 of 150 mice. The RR, PR, QRS, and QT intervals were measured on ECG. Sinus node recovery time, AV refractory periods, paced AV Wenckebach and 2:1 block cycle lengths, atrial and ventricular effective refractory periods were compared between genotypes and age groups. There were no differences in ECG intervals or EP findings in the young mutant mice, but progressive PR prolongation in older mice. The A-V conduction defects are also sensitive to DMPK gene dosage. Adult DMPK-/- mice develop 1 degrees, 2 degrees and 3 degrees A-V block, whereas DMPK+/- mice develop only 1 degrees heart block. CONCLUSION: These data demonstrate that both age and DMPK dose are important factors regulating cardiac conduction in myotonic dystrophy. This mouse model of DM is remarkably similar to the human phenotype, with age-related progression in atrioventricular conduction defects.

A targeted disruption in connexin40 leads to distinct atrioventricular conduction defects.

INTRODUCTION: Gap junctions consist of connexin (Cx) proteins that enable electrical coupling of adjacent cells and propagation of action potentials. Cx40 is solely expressed in the atrium and His-Purkinje system. The purpose of this study was to evaluate atrioventricular (AV) conduction in mice with a homozygous deletion of Connexin40 (Cx40(-/-)). METHODS: Surface ECGs, intracardiac electrophysiology (EP) studies, and ambulatory telemetry were performed in Cx40(-/-) mutant mice and wild-type (WT) controls. Atrioventricular (AV) conduction parameters and arrhythmia inducibility were evaluated using programmed stimulation. Analysis of heart rate variability was based on results of ambulatory monitoring. RESULTS: Significant findings included prolonged measures of AV refractoriness and conduction in connexin40-deficient mice, including longer PR, AH, and HV intervals, increased AV refractory periods, and increased AV Wenckebach and 2:1 block cycle lengths. Connexin40-deficient mice also had an increased incidence of inducible ventricular tachycardia, decreased basal heart rates, and increased heart rate variability. CONCLUSION: A homozygous disruption of Cx40 results in prolonged AV conduction parameters due to abnormal electrical coupling in the specialized conduction system, which may also predispose to arrhythmia vulnerability.

Familial hypertrophic cardiomyopathy mice display gender differences in electrophysiological abnormalities.

Genetically-manipulated mice harboring an alpha-myosin heavy chain Arg403Gln missense mutation (alpha-MHC403/+) display a phenotype characteristic of familial hypertrophic cardiomyopathy (FHC). Male and female (30 +/- 8 week old) heterozygous alpha-MHC403/+ mice and litter-mate controls were evaluated using a surface electrocardiogram (ECG) and an in vivo cardiac electrophysiology study (EPS). Wild type animals had normal intracardiac electrophysiology, with no significant differences between male and female control mice during EPS. The female wild-type mice did have slower heart rates and longer ECG intervals than their male wild-type counterparts. The female alpha-MHC403/+ mice had similar ECG's, cardiac conduction times, and refractory periods compared with female wild-type mice. In contrast, male FHC mice had distinctive ECG and electrophysiologic abnormalities including right axis deviation, prolonged ventricular repolarization and prolonged sinus node recovery times. During programmed ventricular stimulation, 62% of male alpha-MHC403/+ mice and 28% of female alpha-MHC403/+ mice had inducible ventricular tachycardia. These studies identify gender-specific electrophysiologic abnormalities in alpha-MHC403/+ FHC mice, concordant with the histological and hemodynamic derangements previously reported.

Maturational atrioventricular nodal physiology in the mouse.

INTRODUCTION: Dual AV nodal physiology is characterized by discontinuous conduction from the atrium to His bundle during programmed atrial extrastimulus testing (A2V2 conduction curves), AV nodal echo beats, and induction of AV nodal reentry tachycardia (AVNRT). The purpose of this study was to characterize in vivo murine maturational AV nodal conduction properties and determine the frequency of dual AV nodal physiology and inducible AVNRT. METHODS AND RESULTS: A complete transvenous in vivo electrophysiologic study was performed on 30 immature and 19 mature mice. Assessment of AV nodal conduction included (1) surface ECG and intracardiac atrial and ventricular electrograms; (2) decremental atrial pacing to the point of Wenckebach block and 2:1 conduction; and (3) programmed premature atrial extrastimuli to determine AV effective refractory periods (AVERP), construct A2V2 conduction curves, and attempt arrhythmia induction. The mean Wenckebach block interval was 73 +/- 12 msec, 2:1 block pacing cycle length was 61 +/- 11 msec, and mean AVERP100 was 54 +/- 11 msec. The frequency of dual AV nodal physiology increased with chronologic age, with discontinuous A2V2 conduction curves or AV nodal echo beats in 27% of young mice < 8 weeks and 58% in adult mice (P = 0.03). CONCLUSION: These data suggest that mice, similar to humans, have maturation of AV nodal physiology, but they do not have inducible AVNRT. Characterization of murine electrophysiology may be of value in studying genetically modified animals with AV conduction abnormalities. Furthermore, extrapolation to humans may help explain the relative rarity of AVNRT in the younger pediatric population.

Phenotypic screening for heart rate variability in the mouse.

We developed a technology for heart rate (HR) variability (HRV) analysis in the mouse for characterization of HR dynamics, modulated by vagal and sympathetic activity. The mouse is the principal animal model for studying biological processes. Mouse strains are now available harboring gene mutations providing fundamental insights into molecular mechanisms underlying cardiac electrical diseases. Future progress depends on enhanced understanding of these fundamental mechanisms and the implementation of methods for the functional analysis of mouse cardiovascular physiology. By telemetric techniques, standard time and frequency-domain measures of HRV were computed with and without autonomic blockade, and baroreflex sensitivity testing was performed. HR modulation in the high-frequency component is predominantly mediated by the parasympathetic nervous system, whereas the low-frequency component is under the influence of both the parasympathetic and sympathetic systems. The presented technology and protocol allow for assessment of autonomic regulation of the murine HR. Phenotypic screening for HR regulation in mice will further enhance the value of the mouse as a model of heritable electrophysiological human disease.

QT dispersion in alpha-myosin heavy-chain familial hypertrophic cardiomyopathy mice.

Patients with familial hypertrophic cardiomyopathy (FHC) are at risk for ventricular arrhythmias and sudden death. Regional variability in the QT interval [QT dispersion (QTd)] is significantly increased in humans with FHC and ventricular arrhythmias. A mouse model of FHC resulting from a mutation in the alpha-myosin heavy-chain (Arg403Gln) was used to study the electrophysiologic phenotype of this disease. Cardiac electrophysiology studies and surface ECGs were performed in FHC mice and wild-type controls to evaluate the feasibility and significance of QTd measurements in predicting the risk for ventricular arrhythmias. Atrial and ventricular pacing electrodes were placed by either a transvenous or epicardial approach. Standard pacing and extrastimulus protocols were followed. The QT interval was measured in six surface ECG leads. QTd was defined as the difference between the maximum and minimum measured QT intervals. Male FHC mice had greater QTd than wild-type controls (37.1 +/- 3.0 ms versus 23.9 +/- 1.9 ms, p = 0.001). There was also a significant gender difference in QTd within each genotype; female wild-type mice had greater QTd than male wild-type mice (37.4 +/- 5.3 ms versus 23.9 +/- 1.9 ms, p = 0.005), and male FHC mice had greater QTd than female FHC mice (37.1 +/- 3.0 ms versus 27.2 +/- 2.0 ms, p = 0.02). Twelve of 23 FHC mice had inducible ventricular arrhythmias, whereas only 2 of 32 wild-type mice were inducible (p = 0.004). Although a significantly increased number of FHC mice had arrhythmias compared with wild-type mice, QTd did not correlate with arrhythmia inducibility. The importance of this study is that it validates the mouse model for further investigation of arrhythmogenic risk and gender differences in the electrophysiologic phenotype in FHC. It also suggests that although gender- and genotype-specific QTd values are increased, they do not predict arrhythmia risk in FHC mice.

In vivo electrophysiologic studies in endothelial nitric oxide synthase (eNOS)-deficient mice.

INTRODUCTION: Endothelial nitric oxide synthase (eNOS) mediates attenuation of the L-type calcium channel and modulates myocyte contractility. Arrhythmogenic afterdepolarizations are seen in vitro in ouabain-treated isolated myocytes from eNOS-deficient mice. The aim of these studies was to characterize the baseline electrophysiologic (EP) phenotype of eNOS-deficient mice and their potential susceptibility to cardiac conduction abnormalities and inducible arrhythmias. METHODS AND RESULTS: Surface ECG and in vivo intracardiac EP studies were performed in 27 mice lacking the eNOS gene and 21 wild-type littermate control mice. Baseline studies were performed in 10 eNOS-deficient mice and 10 wild-type controls. Subsequently, 17 eNOS-deficient mice and 11 wild-type controls were pretreated with digoxin, and ECG and EP testing were repeated. Data analysis revealed no significant differences in ECG intervals or cardiac conduction parameters, except sinus cycle length was higher in eNOS-deficient mice than wild-type mice (P < 0.01). After digoxin pretreatment, 7 of 17 eNOS-deficient mice had inducible ventricular tachycardia and 2 others had frequent ventricular premature beats, compared with only 3 of 11 wild-type mice with inducible ventricular tachycardia. In addition, 2 digoxin-treated eNOS-deficient mice and 1 wild-type mouse had inducible nonsustained atrial fibrillation. CONCLUSION: Mice with a homozygous targeted disruption of the eNOS gene have slower heart rates but no other distinguishable EP characteristics under basal sedated conditions. Partial inhibition of the Na+/K+ ATPase pump with digoxin administration increases ventricular ectopic activity in eNOS-/- mice, a phenotype analogous to afterdepolarizations seen in vitro in this eNOS-deficient mouse model.

Electrophysiological characterization of murine myocardial ischemia and infarction.

BACKGROUND: Genetically altered mice will provide important insights into a wide variety of processes in cardiovascular physiology underlying myocardial infarction (MI). Comprehensive and accurate analyses of cardiac function in murine models require implementation of the most appropriate techniques and experimental protocols. OBJECTIVE: In this study we present in vivo, whole-animal techniques and experimental protocols for detailed electrophysiological characterization in a mouse model of myocardial ischemia and infarction. METHODS: FVB mice underwent open-chest surgery for ligation of the left anterior descending coronary artery or sham-operation. By means of echocardiographic imaging, electrocardiography, intracardiac electrophysiology study, and conscious telemetric ECG recording for heart rate variability (HRV) analysis, we evaluated ischemic and post-infarct cardiovascular morphology and function in mice. RESULTS: Coronary artery ligation resulted in antero-apical infarction of the left ventricular wall. MI mice showed decreased cardiac function by echocardiography, infarct-typical pattern on ECG, and increased arrhythmia vulnerability during electrophysiological study. Electrophysiological properties were determined comprehensively, but were not altered significantly as a consequence of MI. Autonomic nervous system function, measured by indices of HRV, did not appear altered in mice during ischemia or infarction. CONCLUSIONS: Cardiac conduction, refractoriness, and heart rate variability appear to remain preserved in a murine model of myocardial ischemia and infarction. Myocardial infarction may increase vulnerability to inducible ventricular tachycardia and atrial fibrillation, similarly to EPS findings in humans. These data may be of value as a reference for comparison with mutant murine models necessitating ischemia or scar to elicit an identifiable phenotype. The limitations of directly extrapolating murine cardiac electrophysiology data to conditions in humans need to be considered.