Gottfried Greve

Molecular genetic analysis of long QT syndrome in Norway indicating a high prevalence of heterozygous mutation carriers

Mutations in the KCNQ1, HERG, SCN5A, minK and MiRP1 genes cause long QT syndrome (LQTS), of which there are two forms: the Romano Ward syndrome and the Jervell and Lange‐Nielsen syndrome. We have performed DNA sequencing of the LQTS‐associated genes in 169 unrelated patients referred for genetic testing with respect to Romano Ward syndrome and in 13 unrelated patients referred for genetic testing with respect to Jervell and Lange‐Nielsen syndrome. A total of 37 different mutations in the 5 genes, of which 20 were novel, were identified. Among patients with the most stringent clinical criteria of Romano Ward syndrome, a mutation was identified in 71 %. Twelve of the 13 unrelated patients referred for genetic testing with respect to Jervell and Lange‐Nielsen syndrome were provided with a molecular genetic diagnosis. Cascade genetic screening of 505 relatives of index patients with molecularly defined LQTS identified 251 mutation carriers. The observed penetrance was 41 %. Although caution must be exerted, the prevalence of heterozygotes for mutations in the LQTS‐associated genes in Norway could be in the range 1/100–1/300, based on the prevalence of patients with Jervell and Lange‐Nielsen syndrome.

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

SummaryWe have investigated the associations between β-tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at −150°C, cryosectioned at −70°C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti-β-tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the β-tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of β-tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti-β-tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific β-tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.

Heterotrisomy, a significant contributing factor to ventricular septal defect associated with Down syndrome?

Abstract. Down syndrome (DS; trisomy 21) is associated with a wide range of variable clinical features, one of the most common being congenital heart defects (CHD). We used molecular genetic techniques to study the inheritance of genes on chromosome 21 in children with DS and CHD. Polymorphic markers on the long arm of chromosome 21 were analysed in 99 families who had a child with DS. Of these, 60 children had a CHD and 39 children had no CHD. Heterotrisomy describes the inheritance of an allele from each of three different grandparents. In some cases heterotrisomy will involve the inheritance of three different alleles. Heterotrisomic regions were defined as those showing retention of non-disjoining parental heterozygosity at polymorphic loci in the non-disjoined chromosomes of children with DS. Using polymorphic non-coding markers, we identified a consistent 9.6-cM minimum region (D21S167-HMG14) of heterotrisomy in children with DS and ventricular septal defect (VSD). Comparing individuals with DS and VSD to all others with DS (those either with no CHD or with any other CHD combined) shows the individuals with DS and VSD to have significantly more non-reduction or heterotrisomy in this region (P=0.006, Fishers exact test, two-tailed). We postulate that heterotrisomy for a gene or genes in this region is a contributing factor to the pathogenesis of VSD in trisomy 21 either through the presence of three different specific alleles or through the presence of specific combinations of alleles.

Right Ventricular Myocardial Responses to Chronic Pulmonary Regurgitation in Lambs: Disturbances of Activation and Conduction

Patients after repair of tetralogy of Fallot are at increased risk of arrhythmic death. Clinical data suggest that pulmonary regurgitation predisposes to these arrhythmias, although the cellular electrophysiologic effects of pulmonary regurgitation are unknown. We induced pulmonary regurgitation in lambs, and 3 mo later, having quantified the pulmonary regurgitant (PR) fraction, studied right ventricular mechanical and electrophysiologic properties in vivo and in vitro. The PR fraction was greater in PR (75 ± 10%) than in sham-operated animals (8 ± 4%; p < 0.01). In vivo, monophasic action potential duration and activation time, at rest and during acute right ventricular stretch, were similar in both groups. However, the dispersion of activation time was greater in PR animals at rest (13 ± 1.1 versus 8 ± 1.1 ms; p < 0.05). Furthermore, the dispersion of activation increased during right ventricular stretch in PR, but not in sham-operated animals. In vitro, myocardial force-frequency responses were similar in both groups, indicating preserved systolic performance, but mechanical restitution studies showed a prolonged refractory period (447 ± 22 versus 370 ± 26 ms; p < 0.05) and a decreased recovery time constant (184 ± 19 versus 265 ± 20 ms; p < 0.001) in PR animals, indicating altered calcium cycling. Furthermore, the myocardial conduction velocity was reduced in PR animals (31 ± 3.58 versus 47.9 ± 5.1 cm/s; p < 0.01), resulting from a 2-fold increase in intracellular resistance (437.25 ± 125.93 versus 194 ± 43.27 Ω · cm; p = 0.025). Chronic PR leads to inhomogeneity of right ventricular activation, alters myocardial calcium cycling, reduces conduction velocity, and increases intracellular resistivity. These may contribute to the development of arrhythmias associated with PR, including those in patients after tetralogy repair.

Right Ventricular Distension Alters Monophasic Action Potential Duration During Pulmonary Arterial Occlusion in Anaesthetised Lambs: Evidence for Arrhythmogenic Right Ventricular Mechanoelectrical Feedback

Abnormal loading and distension of the right ventricle may induce arrhythmia through the process of mechanoelectrical feedback. Nonetheless, the electrophysiological effects of right ventricular distension are ill‐defined and the mechanisms which underpin mechanoelectrical feedback in the right ventricle are unknown. We examined the effects of changes in right ventricular load (complete occlusion of both caval veins or the main pulmonary artery) in 14 anaesthetised lambs, instrumented with right ventricular surface electrodes and strain gauges for recording monophasic action potential and segment length, and an integrated conductance and micromanometer‐tipped catheter for measurement of right ventricular pressure and volume. Caval occlusion did not alter right ventricular segment length and monophasic action potential duration. By contrast, pulmonary arterial occlusion increased the segment length and decreased the monophasic action potential duration at 25, 50 and 70% repolarisation by 29 ± 6, 22 ± 4 and 17 ± 3 ms, respectively (all P < 0.01). Of the 42 pulmonary arterial occlusions, 38 were associated with early afterdepolarisations (EADs) which increased progressively in magnitude as the occlusion was maintained until, in 32, overt arrhythmia was observed. By contrast, none of the four occlusions in which EADs were not observed resulted in arrhythmia. As a result, the proportion of occlusions which resulted in arrhythmia were greater in those associated with EADs than in those which were not (P = 0.002). Right ventricular distension alters the pattern of repolarisation, precipitates early afterdepolarisations and results in a variety of ventricular arrhythmia, including ventricular tachycardia.

Possible Common Aetiology behind Maternal Preeclampsia and Congenital Heart Defects in the Child: a Cardiovascular Diseases in Norway Project Study

BACKGROUND The aetiology of congenital heart defects (CHD) is mostly unknown, but maternal factors may modify the infant risk of CHD. We investigated the association between maternal preeclampsia and offspring risk of severe CHD in a nation-wide cohort study. METHODS Information on all births registered in the Medical Birth Registry of Norway, 1994-2009, was completed with information on CHD diagnoses from national health registries and the Cardiovascular Diseases in Norway Project (CVDNOR). RESULTS Among 914 703 singleton births without chromosomal abnormalities, 32 864 (3.6%) were born after a pregnancy with preeclampsia. The preeclampsia was diagnosed before the 34th week of pregnancy (early-onset preeclampsia) in 2618 (8.0% of preeclamptic pregnancies). CHDs were diagnosed in 10 691 infants; of these, 2473 had severe CHD. The risk of severe CHD was compared between births with and without maternal preeclampsia and estimated with binomial log-linear regression. When adjusting for year of birth, maternal age, parity, and pregestational diabetes, the risk ratio (RR) for severe CHD in offspring of mothers with any preeclampsia was 1.3 [95% confidence interval (CI) 1.1, 1.5], and in pregnancies with early-onset preeclampsia, the RR was 2.8 (95% CI 1.8, 4.4). The association between early-onset preeclampsia and specific types of severe CHD was stronger for atrioventricular septal defects (AVSD), with adjusted RR 13.5 (95% CI 6.8, 26.8). CONCLUSIONS Early-onset preeclampsia was strongly associated with infant risk of severe CHD, specifically; the risk of AVSD was 15-fold higher if the mother was diagnosed with early-onset preeclampsia, suggesting common aetiological factors for early-onset preeclampsia and erroneous fetal heart development.

Mortality and complications in 3495 children with isolated ventricular septal defects

Background Ventricular septal defects (VSDs) are the most common congenital heart defects (CHDs). Previous studies indicate an increased risk of endocarditis, aortic regurgitation, left ventricular outflow tract obstructions, pulmonary hypertension, arrhythmias and sudden death in patients with isolated VSDs. The present nationwide cohort study reports mortality and cardiac complications requiring hospitalisation or intervention in children with isolated VSDs. Methods and results Medical information concerning all 943 871 live births in Norway in 1994–2009 was retrieved from the Medical Birth Registry of Norway, the Cardiovascular Disease in Norway project, the Oslo University Hospitals Clinical Registry of Congenital Heart Defects and the Norwegian Cause of Death Registry. Isolated VSDs were identified in 3495 children without known chromosomal aberrations or extracardiac malformations. Surgical or catheter-based treatment of VSD was performed in 181 (5.2%) cases. Twelve (0.3%) children with VSDs died before 2013. There was no operative mortality, and no excess mortality in children with isolated VSDs compared with children without VSDs (adjusted HR 0.8 (0.5 to 1.4), p=0.48). The following conditions were recorded as possible cardiac complications of the VSDs: endocarditis in 3 children (0.9‰), aortic regurgitation in 12 children (3.4‰), left ventricular outflow tract obstructions in no children (0.0‰), pulmonary hypertension in 1 child (0.3‰) and arrhythmias in 16 children (4.6‰). Conclusions The entire group of children with isolated VSDs had a favourable prognosis without excess mortality. Cardiac complications requiring hospitalisation or intervention, including endocarditis, aortic regurgitation, left ventricular outflow tract obstructions, pulmonary hypertension and arrhythmias, were infrequent during childhood. Trial registration number NCT02026557.

Ultrastructural studies of intercalated disc separations in the rat heart during the calcium paradox.

SummaryThe ultrastructure of intercalated disc separations were studied in isolated rat hearts subjected to 5 min of coronary perfusion with small volumes of a calcium-free solution (i.e., 10.0 ml, 5.0 ml, and 2.5 ml). The same groups of hearts were studied after 15 min of calcium repletion. A semiquantitative examination shows that after calcium depletion 20%–45% of the intercalated discs (ID) were separated in the 2.5-ml group, 50%–75% in the 5.0-ml group, and 75%–90% in the 10.0-ml group. Readmission of calcium did not give any significant changes in the percentage of ID dehiscence in the two lowest volume groups, which indicates that ID separation has been irreversible during the first 15 min of calcium repletion. A semiquantitative analysis has also been performed of the percentages of severely damaged cells at each of the three volume groups after calcium repletion. It appears that in the two lowest volume groups, the percentage of widened discs tend to exceed the percentage of severely injured cells after calcium readmission. This suggests that ID separation not necessarily implies severe injuries to the implicated cells during calcium repletion. After calcium-free perfusion, cellular edema, cytoplasmic disintegration, and plasmalemmal fragmentation were present in the interdigitating cellular projections of the dissociated ID. Similar injuries did also occasionally occur outside the ID, usually situated in close proximity to a capillary.

Maternal Diabetes, Birth Weight, and Neonatal Risk of Congenital Heart Defects in Norway, 1994-2009.

OBJECTIVE: To investigate the association between pregestational or gestational diabetes and offspring risk of congenital heart defects and the association between large-for-gestational-age birth weight and risk of cardiac defects in offspring of diabetic women. METHODS: Information on pregestational and gestational diabetes, cardiac defects, and birth weight among all births in Norway in 1994–2009 was ascertained from the Medical Birth Registry of Norway, national health registries, and the Cardiovascular Disease in Norway project. The relative risk (RR) compared offspring risk of cardiac defects for maternal diabetes with offspring risk in nondiabetic mothers adjusted for year of birth, maternal age, and parity. RESULTS: Among 914,427 births (live births, stillbirths, terminated pregnancies), 5,618 (0.61%) were complicated by maternal pregestational diabetes and 9,726 (1.06%) by gestational diabetes. Congenital heart defects were identified in 10,575 offspring. The prevalence of cardiac defects differed between groups: 344 of 10,000 births to women with pregestational diabetes, 172 of 10,000 to women with gestational diabetes, and 114 of 10,000 in women without diabetes (adjusted RRs 2.92, 95% confidence interval [CI] 2.54–3.36 and 1.47, 95% CI 1.26–1.71). During the study period, the adjusted RRs for congenital heart defects did not change. The risk of cardiac defects in neonates very large for gestational age (birth weight greater than 3 standard deviations above the mean) was compared with neonates with birth weight appropriate for gestational age. For pregestational diabetes, the prevalences of offspring cardiac defects were 561 compared with 248 per 10,000 births (adjusted RR 2.23, 95% CI 1.39–3.59) and for gestational diabetes 388 compared with 132 per 10,000 (adjusted RR 2.73, 95% CI 1.53–4.85). CONCLUSION: The increased risk of having a child with a congenital heart defect has not changed for diabetic women in Norway since 1994. Among women with pregestational or gestational diabetes, having a large-for-gestational-age neonate was associated with a two- to threefold increased risk of cardiac defects compared with neonates with normal birth weight.

Regional activation of the immediate-early response gene c-fos in infarcted rat hearts

Regional infarction of the left ventricle is followed by hypertrophy of the viable myocardium. This compensatory growth of cardiac myocytes requires induction of gene transcription and synthesis of proteins. In this study, we examined the expression of the immediate‐early response gene c‐fos following ligation of the left coronary artery in rat hearts. RNase protection assay demonstrated a rapid increase in the c‐fos mRNA level in the ventricular myocardium. After two days of infarction, the c‐fos expression was attenuated and was comparable to that observed in sham‐operated control hearts. In situ tissue distribution of Fos protein‐like immunoreactivity revealed the appearance of positively stained cells adjacent to the lateral border of the ischaemic myocardium, in the left ventricular subendocardial areas, in the papillary muscles of the left ventricle, in the proximity of great transmural vessels, and focally in the normo‐perfused subepicardial myocardium. Double staining using antibodies recognizing the Fos protein and α‐actinin, confirmed that the accumulation of nuclear Fos protein‐like immunoreactivity was mainly seen in the cardiac myocytes. However, double staining of the Fos protein and Hoechst DNA labelling showed that detectable immunoreactivity occurred only in a limited proportion of the total nuclei present in these myocardial regions. Moreover, the regions showing c‐fos activation correspond to the areas in which the appearance of subsequent growth responses are most pronounced following myocardial infarction. The present results therefore indicate that an early and regional c‐fos activation is taking place in viable cardiac myocytes following left coronary artery ligation, and that c‐fos is a possible regulating factor of sequential events leading to altered pattern of gene expression and protein synthesis in the hypertrophying heart.