Ambulatory electrocardiography (AECG) is a primary diagnostic tool in patients with potential arrhythmic disorders. To study the pathophysiological mechanisms of arrhythmic disorders, mouse models are widely implemented. The use of a technique similar to AECG for mice is thus of great relevance. We have optimized a protocol which allows qualitative, long-term ECG data recording in conscious, freely moving mice. Automated algorithms were developed to efficiently process the large amount of data and calculate the average heart rate (HR), the mean peak-to-peak interval and heart rate variability (HRV) based on peak detection. Ectopic beats are automatically detected based on aberrant peak intervals. As we have incorporated a multiple lead configuration in our ECG set-up, the nature and origin of the suggested ectopic beats can be analyzed in detail. The protocol and analysis tools presented here are promising tools for studies which require detailed, long-term ECG characterization in mouse models with potential arrhythmic disorders.

Patients with Marfan syndrome (MFS), a connective tissue disorder caused by pathogenic variants in the gene encoding the extracellular matrix protein fibrillin-1, have an increased prevalence of primary cardiomyopathy, arrhythmias, and sudden cardiac death. We have performed an in-depth in vivo and ex vivo study of the cardiac phenotype of Fbn1^mgR/mgR mice, an established mouse model of MFS with a severely reduced expression of fibrillin-1. Using ultrasound measurements, we confirmed the presence of aortic dilatation and observed cardiac diastolic dysfunction in male Fbn1^mgR/mgR mice. Upon post-mortem examination, we discovered that the mutant mice consistently presented myocardial lesions at the level of the right ventricular free wall, which we characterized as spontaneous pseudoaneurysms. Histological investigation demonstrated a decrease in myocardial compaction in the MFS mouse model. Furthermore, continuous 24 h electrocardiographic analysis showed a decreased heart rate variability and an increased prevalence of extrasystolic arrhythmic events in Fbn1^mgR/mgR mice compared to wild-type littermates. Taken together, in this paper we document a previously unreported cardiac phenotype in the Fbn1^mgR/mgR MFS mouse model and provide a detailed characterization of the cardiac dysfunction and rhythm disorders which are caused by fibrillin-1 deficiency. These findings highlight the wide spectrum of cardiac manifestations of MFS, which might have implications for patient care.

AIM More than one third of patients with epilepsy continue to have seizures despite treatment with anti-epileptic drugs. In this study we evaluate whether selective silencing of excitatory hippocampal neurons using the inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM4Di leads to suppression of spontaneous hippocampal seizures in the intrahippocampal kainic acid (IHKA) mouse model for TLE. METHODS Mice (n=60) were injected with kainic acid in the right hippocampus (200ng/50nl, AP -2mm ML +1.5mm DV -1.8mm relative to bregma). Three weeks later, animals were injected in the KA-lesioned hippocampus with 500nl adeno-associated-viral vector carrying genes encoding hM4Di-mCherry fusion protein (2.7E+13 GC/ml, DREADD group, n = 43) or mCherry only (2.1E+13 GC/ml, non-DREADD group, n=13) under transcriptional control of CamKIIalpha promotor, specific for excitatory neurons. Subsequently a bipolar recording electrode was implanted at the injection site. Mice with most frequent hippocampal seizures were selected for further treatment. DREADD animals were treated with three subclinical single doses of clozapine (0.01, 0.03 and 0.1mg/kg ; n=8, 9 and 10) and with repeated doses (every 8 hours for 3 days , n=5, 4 and 5). Non-DREADD animals (n=4) received a single 0.1mg/kg clozapine injection. The fraction of time an animal spent in seizures (FTS) was used as a measure for epileptic activity. RESULTS Administration of 0.1mg/kg clozapine resulted in a strong inhibition of epileptic activity only in DREADD-expressing animals (baseline (BL) FTS values of 16 ± 2 % and 21 ± 4 %, post treatment FTS values of 6 ± 1 % and 20 ± 2 % in DREADD and non-DREADD animals respectively). Both groups were statistically different from each other for at least 18 hours after treatment. Acute administration of 0.1mg/kg lead to a stronger seizure suppression in comparison to 0.03 and 0.01mg/kg injection (BL FTS values of 15 ± 2 % and 18 ± 3 % reduced to 10 ± 1 % and 11 ± 1 % post treatment with 0.03 and 0.01mg/kg respectively). Similarly, sustained treatment for three consecutive days resulted into a dose-dependent suppression of epileptic activity (0.1mg/kg resulted in a more potent suppression than 0.03 and 0.01mg/kg; 0.03 mg/kg resulted in a more potent suppression than 0.01mg/kg, BL FTS values of 15 ± 2 %, 21 ± 2 % and 25 ± 2% reduced to 3 ± 2%, 6 ± 2% and 11 ± 2% during chronic treatment with 0.1, 0.03 and 0.01mg/kg respectively). CONCLUSION This study shows the potency of chemogenetics to robustly and chronically suppress spontaneous epileptic seizures. Moreover, this indicates that excitatory hippocampal neurons are essential in the seizure generation process in the IHKA mouse model. Additionally we show that different subclinical doses of clozapine can be used to activate DREADDs and that 0.1mg/kg clozapine is the most potent dose to suppress spontaneous seizures. This could lead to epilepsy therapy where a systemically administered drug very selectively modulates specific neurons of the seizure network, resulting in a very potent seizure suppression.