Post-mortem genetic testing in sudden cardiac death and genetic screening of relatives at risk: lessons learned from a Czech pilot multidisciplinary study.

Sudden cardiac death (SCD) might have an inherited cardiac condition background. Genetic testing supports post-mortem diagnosis and screening of relatives at risk. Our aim is to determine the feasibility of a Czech national collaboration group and to establish the clinical importance of molecular autopsy and family screening. From 2016 to 2021, we have evaluated 100 unrelated SCD cases (71.0% males, age: 33.3 (12.8) years). Genetic testing was performed by next-generation sequencing utilizing a panel of 100 genes related to inherited cardiac/aortic conditions and/or whole exome sequencing. According to autopsy, cases were divided into cardiomyopathies, sudden arrhythmic death syndrome, sudden unexplained death syndrome, and sudden aortic death. We identified pathogenic/likely pathogenic variants following ACMG/AMP recommendations in 22/100 (22.0%) of cases. Since poor DNA quality, we have performed indirect DNA testing in affected relatives or in healthy parents reaching a diagnostic genetic yield of 11/24 (45.8%) and 1/10 (10.0%), respectively. Cardiological and genetic screening disclose 83/301 (27.6%) relatives at risk of SCD. Genetic testing in affected relatives as starting material leads to a high diagnostic yield offering a valuable alternative when suitable material is not available. This is the first multidisciplinary/multicenter molecular autopsy study in the Czech Republic which supports the establishment of this type of diagnostic tests. A central coordinator and proper communication among centers are crucial for the success of a collaboration at a national level.

Acquisition and expression of fat conditioned flavor preferences following dopamine D1, opioid and NMDA receptor antagonism in C57BL/6 mice.

Objectives: Inbred mouse strains differ in the pharmacology mediating sugar and fat intake and conditioned flavor preferences (CFP). C57BL/6, BALB/c and SWR inbred mice are differentially sensitive to dopamine (DA) D1, opioid and muscarinic receptor antagonism of sucrose, saccharin or fat intake, and to DA, opioid, muscarinic and N-methyl-D-aspartate (NMDA) receptor antagonism of acquisition of sucrose-CFP. DA D1, opioid and NMDA receptor antagonists differentially alter fat (Intralipid)-CFP in BALB/c and SWR mice. The present study examined whether naltrexone, SCH23390 or MK-801 altered acquisition and expression of Intralipid-CFP in C57BL/6 mice.Methods: In acquisition, groups of male food-restricted C57BL/6 mice received vehicle, naltrexone (1, 5 mg/kg), SCH23390 (50, 200 nmol/kg) or MK-801 (100, 200 µg/kg) before 10 training sessions in which mice alternately consumed two novel-flavored 5% (CS+) and 0.5% (CS-) Intralipid solutions. Six two-bottle CS choice tests followed with both flavors mixed in 0.5% Intralipid without injections. In expression, C57BL/6 mice underwent the 10 training sessions without injections followed by two-bottle CS choice tests 30 min following vehicle, naltrexone (1, 5 mg/kg), SCH23390 (200, 800 nmol/kg) or MK-801 (100, 200 µg/kg).Results: Fat-CFP acquisition in C57BL/6 mice was significantly though marginally reduced following naltrexone, SCH23390 and MK-801. Fat-CFP expression was similarly reduced by naltrexone, SCH23390 and MK-801 in C57BL/6 mice. Discussion: C57BL/6 mice were more sensitive to DA D1, opioid and NMDA antagonists in the expression of fat-CFP relative to sugar-CFP, but were less sensitive to DA D1 and NMDA antagonists in the acquisition of fat-CFP relative to sugar-CFP.

Acquisition and expression of sucrose conditioned flavor preferences following dopamine D1, opioid and NMDA receptor antagonism in C57BL/6 mice.

The study of inbred mouse strains is a useful animal model to assess differences in ingestive behavior responses, including conditioned flavor preferences (CFP). C57BL/6, BALB/c and SWR inbred mice display differential sensitivity to dopamine (DA) D1, opioid and muscarinic cholinergic receptor antagonism of sucrose or saccharin intake as well as to muscarinic cholinergic antagonism of acquisition (learning) of sucrose-CFP. Given that DA D1, opioid and N-methyl-D-aspartate (NMDA) receptor antagonists differentially alter sucrose-CFP in BALB/c and SWR inbred mice, the present study examined whether systemic administration of naltrexone, SCH23390 or MK-801 altered acquisition and expression of sucrose-CFP in C57BL/6 mice. In acquisition experiments, male food-restricted C57BL/6 mice were treated with vehicle, naltrexone (1, 5 mg/kg), SCH23390 (50, 200 nmol/kg) or MK-801 (100, 200 µg/kg) 30 min prior to each of ten daily sessions in which they alternately consumed a flavored (CS+, e.g. cherry) 16% sucrose solution and a differently-flavored (CS-, e.g. grape) 0.05% saccharin solution followed by six two-bottle CS choice tests mixed in 0.2% saccharin without injections. SCH23390 and MK-801, but not naltrexone eliminated sucrose-CFP acquisition in food-restricted C57BL/6 mice. In expression experiments, food-restricted C57BL/6 mice underwent the ten training sessions without injections followed by two-bottle CS choice tests 30 min following vehicle, naltrexone (1, 5 mg/kg), SCH23390 (200, 800 nmol/kg) or MK-801 (100, 200 µg/kg). SCH23390 more effectively reduced the magnitude of sucrose-CFP expression than naltrexone or MK-801 in food-restricted C57BL/6 mice. Thus, dopamine D1 and NMDA receptor signaling is essential for learning of sucrose-CFP in C57BL/6 mice.

Strain differences in muscarinic cholinergic receptor antagonism of fat intake and acquisition and expression of fat-conditioned flavor preferences in male BALB/c, C57BL/6 and SWR mice.

Murine strain differences occur for both intakes of and preferences for sugars and fats. Previous studies demonstrated that the muscarinic cholinergic receptor antagonist, scopolamine (SCOP) more potently reduced sucrose and saccharin intakes in inbred C57BL/6 and BALB/c than SWR mice, sucrose-conditioned flavor preferences (CFP) expression in BALB/c, but not C57BL/6 or SWR mice, and sucrose-CFP acquisition in BALB/c relative to SWR and C57BL/6 mice. Although fat intake and fat-CFP are observed in all three strains, strain-specific effects were previously observed following dopamine D1, opiate and NMDA receptor antagonism of sweet and fat intake and CFP. The present study investigated whether muscarinic receptor antagonism differentially affected fat (Intralipid) intake and preferences in these strains by examining whether SCOP altered fat (Intralipid) intake and fat-CFP expression and acquisition in BALB/c, C57BL/6 and SWR mice. SCOP (0.1-10 mg/kg) significantly reduced Intralipid (5%) intake in all three strains across 2 h. In fat-CFP expression experiments, food-restricted mice consumed one flavored (conditioned stimulus (CS)+, 5 sessions) Intralipid (5%) solution and a differently-flavored (CS-, 5 sessions) Intralipid (0.5%) solution. Two-bottle CS choice tests with the two flavors mixed in 0.5% Intralipid occurred following vehicle and two SCOP doses (1, 5 mg/kg). SCOP elicited small, but significant reductions in fat-CFP expression in BALB/c and C57BL/6, but not SWR mice. In fat-CFP acquisition experiments, separate groups of BALB/c, C57BL/6 and SWR mice were treated prior to the ten acquisition training sessions with vehicle or two SCOP (2.5, 5 mg/kg) doses followed by six two-bottle choice tests without injections. SCOP eliminated fat-CFP acquisition in all three strains. Thus, muscarinic receptor signaling mediates learning, and to a lesser degree maintenance of fat-CFP while maximally inhibiting fat intake in the three strains.

Murine genetic variance in muscarinic cholinergic receptor antagonism of acquisition and expression of sucrose-conditioned flavor preferences in three inbred mouse strains.

Conditioned flavor preferences (CFP) are elicited by sucrose relative to saccharin in inbred mice with both the robustness of the preferences and sensitivity to pharmacological receptor antagonists sensitive to genetic variance. Dopamine, opioid and N-methyl-d-aspartate receptor antagonists differentially interfere with the acquisition (learning) and expression (maintenance) of sucrose-CFP in BALB/c and SWR inbred mice. Further, the muscarinic cholinergic receptor antagonist, scopolamine (SCOP) more potently reduces both sucrose and saccharin intake in BALB/c and C57BL/6 relative to SWR inbred mice. The present study examined whether SCOP altered the expression and acquisition of sucrose-CFP in BALB/c, C57BL/6 and SWR mice. In expression experiments, food-restricted mice alternately consumed a flavored (CS+, e.g., cherry, 5 sessions) 16% sucrose solution and a differently-flavored (CS-, e.g., grape, 5 sessions) 0.05% saccharin solution. Two-bottle CS choice tests with the two flavors mixed in 0.2% saccharin solutions occurred following vehicle or SCOP at doses of 1 or 5 mg/kg. SCOP significantly reduced the magnitude of the expression of sucrose-CFP in BALB/c, but not either C57BL/6 or SWR mice. In acquisition experiments, separate groups of BALB/c, C57BL/6 and SWR mice were treated prior to acquisition training sessions with vehicle or 2.5 or 5 mg/kg SCOP doses that was followed by six two-bottle CS choice tests without injections. SCOP dose-dependently reduced (1 mg/kg) and eliminated (2.5 mg/kg) the acquisition of sucrose-CFP in BALB/c mice, and reduced the magnitude of acquisition of sucrose-CFP in SWR mice. In contrast, neither SCOP dose affected the acquisition of sucrose-CFP in C57BL/6 mice. Thus, muscarinic cholinergic receptor signaling is essential for the learning of sucrose-CFP in BALB/c mice, to a lesser degree in SWR mice, but not in C57BL/6 mice. Murine genetic variance differentially modulates muscarinic cholinergic receptor control of sweet intake per se relative to learned conditioned flavor preferences of sweets.

[Arytmogenic ventricular cardiomyopathy]. / Arytmogenní ventrikulární kardiomyopatie.

Arrhythmogenic ventricular cardiomyopathy is considered to be a primary cardiomyopathy. Over the last few decades, although being a relatively rare disease with its prevalence 1:2000 - 1:5000, there were numerous studies performed with the aim to elucidate the underlaying causes, pathogenesis, diagnostical aspects and possible treatment options of the disease. Arrhythmogenic ventricular cardiomyopathy is genetically conditioned disease where proteins of the cell-cell junctions are involved. Mutations of the myocardial intercalated dics proteins, mainly desmosomal proteins (e.g.plakoglobin), are held to be responsible for electromechanical instability of the myocardium which causes regressive changes in cardiomyocytes in most cases of arrhythmogenic ventricular cardiomyopathy. Subsequent morphological changes include fibrofatty replacement and inflammation of the myocardium. The condition results in structural changes of the heart hence arrhytmias and other signs of heart disease. There are 3 variants of this cardiomyopathy: 'classical variant with predominant right ventricular involvement, biventricular and variant with left ventricular predominance. Clinical findings in patients with arrhythmogenic ventricular cardiomyopathy suggested the most appropriate means of the diagnostics and helped to create Task Force Criteria for in vivo diagnosis of the disease. The major pitfall and significance of arrhythmogenic ventricular cardiomyopathy lies in its common presentation as sudden cardiac death affecting mostly young adults.