Final Report Summary - ADAM-15 AND AF (Evaluation of ADAM-15 in atrial fibrillation)
Summary Description of the Project objectives
Atrial fibrillation (AF) is the most common arrhythmia in clinical practice, affecting more than 6 million Europeans, and is responsible for significant morbidity and mortality. AF is a major public health burden as it is associated with a five-fold increased stroke risk, a doubling in dementia risk, a tripling in heart failure risk, and a nearly two-fold increase in mortality.
The pathophysiologic basis of AF is complex and incompletely understood. A contemporary paradigm based on both animal and clinical observations acknowledges complexity in the development of AF, and recognizes the role of susceptible atrial substrate, which may foster reentry, as well focal electrophysiologic triggers and modulating factors.
In the past years there have been increasing data supporting a genetic component to AF. While familial forms of AF have long been reported, a genetic predisposition for more common forms of AF has only recently been recognized. Two studies consisting of more than 5.000 individuals, demonstrated an increased risk for AF among the first-degree relatives of those with AF. Further evidence of the heritability of AF was demonstrated in subjects with lone AF or AF in the absence of structural heart disease. In nearly 38% of individuals with lone AF at least one relative with the arrhythmia could be identified, and a substantial number had multiple affected relatives. Collectively, the familial aggregation observed in AF is consistent with an underlying genetic mechanism for this arrhythmia.
In recent years, genome-wide association studies (GWAS) have uncovered common variants underlying risk for AF. The Cohorts for Heart and Aging Research in Genomics Epidemiology (CHARGE)-AF consortium is an international consortium of investigators from 18 studies with over 8.000 subjects with AF and more than 86.000 subjects without AF. Despite the benefits of GWAS, challenges remain. In most cases, the SNPs identified by GWAS do not necessarily have a direct pathogenic role in the development of the condition, and there may be many potential candidate genes at a disease locus to consider. Identification of the disease causing variants typically requires refinement of the locus with fine mapping, deep re-sequencing, and/or correlation between SNPs and transcription of candidate genes at the locus (expression quantitative trait loci or eQTL). Ultimately it is necessary to demonstrate that causal alleles have differential functional effects on transcription or protein function. These challenges are exacerbated when the SNPs associated with disease are located in intergenic regions, at times hundreds of thousands of base pairs from the closest gene.
In recent work an eQTL between a single nucleotide polymorphism (SNP) at the chromosome 1q21 locus for AF and the gene ADAM-15 has been identified suggesting a potential role of ADAM-15 in AF pathophysiology. ADAMs (A Disintegrin And Metalloproteinase) are a large family of membrane-bound glycoproteins implicated in essential cellular processes like cell adhesion, cell fusion, proteolysis and signal transduction. ADAMs have been shown to play a role in normal development, cancer, neovascularisation, immune response and atherosclerosis. It has been shown that ADAMs are upregulated in the heart of patients with dilated cardiomyopathy and atrial fibrillation suggesting a role in structural remodelling.
Taken together, preliminary data suggest a regulatory effect of a SNP strongly associated with AF susceptibility on ADAM-15 gene expression. Thus, while ADAM-15 is a plausible candidate gene, more study is warranted. Therefore, the hypothesis of the proposed project is that ADAM-15 is involved in AF pathobiology by regulating structural remodelling, thus, leading to an arrhythmogenic substrate for AF. To test this hypothesis the following specific research objective will be addressed:
Main research objective: To characterize the cardiac phenotype of an ADAM-15 knockout mouse
Aim 1: Determination of electrical remodeling of an ADAM-15 knockout mouse
Aim 2: Assessment of structural remodeling of an ADAM-15 knockout mouse
Description of the work performed since the beginning of the project
During his outgoing phase Dr. Clauss performed all the work that was proposed in the application. In brief, he learned all the techniques during the initial training period, then he practiced and finally performed the experiments on mice. The work included general handling of mice (e.g. ear tagging, tail snipping, tissue harvesting, blood collection, mouse anesthesia), mouse colony management (e.g. genotyping, breeding), electrocardiography (ECG), echocardiography, blood pressure measurement, invasive hemodynamics, invasive electrophysiologic studies (EP studies), optical mapping, and implantation of loop recorders. Besides the practical part of these experiments, Dr. Clauss also learned to analyze all the acquired data, and to perform statistical analysis.
During the return phase he received the LMU Junior researcher fund that allowed him to purchase equipment necessary for invasive EP studies and telemetry recordings in mice. He received an animal ethics approval from the local authority in Munich and could perform a first pilot study in the laboratory in Munich.
Main results achieved so far
During the outgoing phase Dr. Clauss characterized an ADAM15-knockout mouse model. The body weight, heart/body weight ratio, or heart weight/tibia length as measurements for cardiac hypertrophy were not different between groups. ECG parameters including heart rate, P wave duration, PR interval, QRS duration, and QTc duration did not differ significantly among groups. Blood pressure measurement showed no differences between knockouts and wildtypes regarding systolic, diastolic, or mean blood pressure. There was no significant difference between groups in left ventricular diameter (LVEDD, a parameter for dilatation), left atrial diameter (LA diameter, a parameter for atrial structural remodeling), wall thickness (septum and posterior wall thickness, parameters for hypertrophy), or in cardiac function (ejection fraction (EF)). Dr. Clauss also performed invasive EP studies in these mice including evaluation of sinus node, atrial, AV node, and ventricular electrical properties. No difference between groups was observed regarding sinus node recovery time (SNRT), atrial effective refractory period (AERP), Wenckebach Cylce length, 2:1 conduction cycle length, AV node refractory period (AVERP), retrograde Wenckebach cycle length, or ventricular refractory period (VERP). However, ADAM15 knockout mice showed a significantly increased vulnerability for atrial arrhythmias. Atrial arrhythmias could be induced more frequently (*p<0.05) the cumulative duration of atrial arrhythmias was significantly longer (*p<0.05) and the average duration of each induced arrhythmia episode was prolonged (*p<0.05).
During the return phase Dr. Clauss performed a pilot study on a total of 10 mice to establish a mouse EP facility in the laboratory in Munich. The study included basic parameters like body and heart weight, ECG, echocardiography and invasive EP studies. Technically, all the experiments were successful. Due to the low number of mice tested (n=5 per group) there are no significant differences so far. However, there is a trend towards a dysregulated sinus node function in the knockout mice. Therefore, Dr. Clauss will continue the work on this mouse model and will further be supported by a doctoral student and another young cardiology fellow.
Expected final results and their potential impact and use
During the outgoing phase Dr. Clauss observed a significant increased risk of developing arrhythmias in ADAM15 knockout mice compared to wildtype controls. The induced arrhythmia episodes were also significantly prolonged.
The results of the study will have major impact to the field. First, it is the proof that common genetic variants can have a functional effect on cardiac rhythm and may therefore – at least in part – explain the increased vulnerability for AF in patients harboring these variants. Second, as a proof-of-principle this will encourage further research on other GWAS risk loci. During his return phase Dr. Clauss started to work on another genetic risk locus identified by GWAS before that has no functional implications on the cardiovascular (patho-)physiology so far. Third, ADAM15 knockout mice could serve as a stable model system for atrial fibrillation in general that is not available so far. At the moment there is no mouse model for AF without enormous structural side effects (e.g. extreme atrial dilatation or hypertrophy) that allows evaluation of only very specific details. Having a stable AF mouse, however, would improve and accelerate research in this field.
Atrial fibrillation (AF) is the most common arrhythmia in clinical practice, affecting more than 6 million Europeans, and is responsible for significant morbidity and mortality. AF is a major public health burden as it is associated with a five-fold increased stroke risk, a doubling in dementia risk, a tripling in heart failure risk, and a nearly two-fold increase in mortality.
The pathophysiologic basis of AF is complex and incompletely understood. A contemporary paradigm based on both animal and clinical observations acknowledges complexity in the development of AF, and recognizes the role of susceptible atrial substrate, which may foster reentry, as well focal electrophysiologic triggers and modulating factors.
In the past years there have been increasing data supporting a genetic component to AF. While familial forms of AF have long been reported, a genetic predisposition for more common forms of AF has only recently been recognized. Two studies consisting of more than 5.000 individuals, demonstrated an increased risk for AF among the first-degree relatives of those with AF. Further evidence of the heritability of AF was demonstrated in subjects with lone AF or AF in the absence of structural heart disease. In nearly 38% of individuals with lone AF at least one relative with the arrhythmia could be identified, and a substantial number had multiple affected relatives. Collectively, the familial aggregation observed in AF is consistent with an underlying genetic mechanism for this arrhythmia.
In recent years, genome-wide association studies (GWAS) have uncovered common variants underlying risk for AF. The Cohorts for Heart and Aging Research in Genomics Epidemiology (CHARGE)-AF consortium is an international consortium of investigators from 18 studies with over 8.000 subjects with AF and more than 86.000 subjects without AF. Despite the benefits of GWAS, challenges remain. In most cases, the SNPs identified by GWAS do not necessarily have a direct pathogenic role in the development of the condition, and there may be many potential candidate genes at a disease locus to consider. Identification of the disease causing variants typically requires refinement of the locus with fine mapping, deep re-sequencing, and/or correlation between SNPs and transcription of candidate genes at the locus (expression quantitative trait loci or eQTL). Ultimately it is necessary to demonstrate that causal alleles have differential functional effects on transcription or protein function. These challenges are exacerbated when the SNPs associated with disease are located in intergenic regions, at times hundreds of thousands of base pairs from the closest gene.
In recent work an eQTL between a single nucleotide polymorphism (SNP) at the chromosome 1q21 locus for AF and the gene ADAM-15 has been identified suggesting a potential role of ADAM-15 in AF pathophysiology. ADAMs (A Disintegrin And Metalloproteinase) are a large family of membrane-bound glycoproteins implicated in essential cellular processes like cell adhesion, cell fusion, proteolysis and signal transduction. ADAMs have been shown to play a role in normal development, cancer, neovascularisation, immune response and atherosclerosis. It has been shown that ADAMs are upregulated in the heart of patients with dilated cardiomyopathy and atrial fibrillation suggesting a role in structural remodelling.
Taken together, preliminary data suggest a regulatory effect of a SNP strongly associated with AF susceptibility on ADAM-15 gene expression. Thus, while ADAM-15 is a plausible candidate gene, more study is warranted. Therefore, the hypothesis of the proposed project is that ADAM-15 is involved in AF pathobiology by regulating structural remodelling, thus, leading to an arrhythmogenic substrate for AF. To test this hypothesis the following specific research objective will be addressed:
Main research objective: To characterize the cardiac phenotype of an ADAM-15 knockout mouse
Aim 1: Determination of electrical remodeling of an ADAM-15 knockout mouse
Aim 2: Assessment of structural remodeling of an ADAM-15 knockout mouse
Description of the work performed since the beginning of the project
During his outgoing phase Dr. Clauss performed all the work that was proposed in the application. In brief, he learned all the techniques during the initial training period, then he practiced and finally performed the experiments on mice. The work included general handling of mice (e.g. ear tagging, tail snipping, tissue harvesting, blood collection, mouse anesthesia), mouse colony management (e.g. genotyping, breeding), electrocardiography (ECG), echocardiography, blood pressure measurement, invasive hemodynamics, invasive electrophysiologic studies (EP studies), optical mapping, and implantation of loop recorders. Besides the practical part of these experiments, Dr. Clauss also learned to analyze all the acquired data, and to perform statistical analysis.
During the return phase he received the LMU Junior researcher fund that allowed him to purchase equipment necessary for invasive EP studies and telemetry recordings in mice. He received an animal ethics approval from the local authority in Munich and could perform a first pilot study in the laboratory in Munich.
Main results achieved so far
During the outgoing phase Dr. Clauss characterized an ADAM15-knockout mouse model. The body weight, heart/body weight ratio, or heart weight/tibia length as measurements for cardiac hypertrophy were not different between groups. ECG parameters including heart rate, P wave duration, PR interval, QRS duration, and QTc duration did not differ significantly among groups. Blood pressure measurement showed no differences between knockouts and wildtypes regarding systolic, diastolic, or mean blood pressure. There was no significant difference between groups in left ventricular diameter (LVEDD, a parameter for dilatation), left atrial diameter (LA diameter, a parameter for atrial structural remodeling), wall thickness (septum and posterior wall thickness, parameters for hypertrophy), or in cardiac function (ejection fraction (EF)). Dr. Clauss also performed invasive EP studies in these mice including evaluation of sinus node, atrial, AV node, and ventricular electrical properties. No difference between groups was observed regarding sinus node recovery time (SNRT), atrial effective refractory period (AERP), Wenckebach Cylce length, 2:1 conduction cycle length, AV node refractory period (AVERP), retrograde Wenckebach cycle length, or ventricular refractory period (VERP). However, ADAM15 knockout mice showed a significantly increased vulnerability for atrial arrhythmias. Atrial arrhythmias could be induced more frequently (*p<0.05) the cumulative duration of atrial arrhythmias was significantly longer (*p<0.05) and the average duration of each induced arrhythmia episode was prolonged (*p<0.05).
During the return phase Dr. Clauss performed a pilot study on a total of 10 mice to establish a mouse EP facility in the laboratory in Munich. The study included basic parameters like body and heart weight, ECG, echocardiography and invasive EP studies. Technically, all the experiments were successful. Due to the low number of mice tested (n=5 per group) there are no significant differences so far. However, there is a trend towards a dysregulated sinus node function in the knockout mice. Therefore, Dr. Clauss will continue the work on this mouse model and will further be supported by a doctoral student and another young cardiology fellow.
Expected final results and their potential impact and use
During the outgoing phase Dr. Clauss observed a significant increased risk of developing arrhythmias in ADAM15 knockout mice compared to wildtype controls. The induced arrhythmia episodes were also significantly prolonged.
The results of the study will have major impact to the field. First, it is the proof that common genetic variants can have a functional effect on cardiac rhythm and may therefore – at least in part – explain the increased vulnerability for AF in patients harboring these variants. Second, as a proof-of-principle this will encourage further research on other GWAS risk loci. During his return phase Dr. Clauss started to work on another genetic risk locus identified by GWAS before that has no functional implications on the cardiovascular (patho-)physiology so far. Third, ADAM15 knockout mice could serve as a stable model system for atrial fibrillation in general that is not available so far. At the moment there is no mouse model for AF without enormous structural side effects (e.g. extreme atrial dilatation or hypertrophy) that allows evaluation of only very specific details. Having a stable AF mouse, however, would improve and accelerate research in this field.