Preserved heart function after left ventricular pressure overload in adult mice subjected to neonatal cardiac hypoplasia.

Intrauterine growth restriction in animal models reduces heart size and cardiomyocyte number at birth. Such incomplete cardiomyocyte endowment is believed to increase susceptibility toward cardiovascular disease in adulthood, a phenomenon referred to as developmental programming. We have previously described a mouse model of impaired myocardial development leading to a 25% reduction of cardiomyocyte number in neonates. This study investigated the response of these hypoplastic hearts to pressure overload in adulthood, applied by abdominal aortic constriction (AAC). Echocardiography revealed a similar hypertrophic response in hypoplastic hearts compared with controls over the first 2 weeks. Subsequently, control mice develop mild left ventricular (LV) dilation, wall thinning and contractile dysfunction 4 weeks after AAC, whereas hypoplastic hearts fully maintain LV dimensions, wall thickness and contractility. At the cellular level, controls exhibit increased cardiomyocyte cross-sectional area after 4 weeks pressure overload compared with sham operated animals, but this hypertrophic response is markedly attenuated in hypoplastic hearts. AAC mediated induction of fibrosis, apoptosis or cell cycle activity was not different between groups. Expression of fetal genes, indicative of pathological conditions, was similar in hypoplastic and control hearts after AAC. Among various signaling pathways involved in cardiac hypertrophy, pressure overload induces p38 MAP-kinase activity in hypoplastic hearts but not controls compared with the respective sham operated animals. In summary, based on the mouse model used in this study, our data indicates that adult hearts after neonatal cardiac hypoplasia show an altered growth response to pressure overload, eventually resulting in better functional outcome compared with controls.

Improvement of fractional flow reserve and collateral flow by treatment with external counterpulsation (Art.Net.-2 Trial).

BACKGROUND: Arteriogenesis (collateral artery growth) is nature's most efficient rescue mechanism to overcome the fatal consequences of arterial occlusion or stenosis. The goal of this trial was to investigate the effect of external counterpulsation (ECP) on coronary collateral artery growth. MATERIALS AND METHODS: A total of 23 patients (age 61 +/- 2.5 years) with stable coronary artery disease and at least one haemodynamic significant stenosis eligible for percutaneous coronary intervention were prospectively recruited into the two study groups in a 2 : 1 manner (ECP : control). One group (ECP group, n = 16) underwent 35 1-h sessions of ECP in 7 weeks. In the control group (n = 7), the natural course of collateral circulation over 7 weeks was evaluated. All patients underwent a cardiac catheterization at baseline and after 7 weeks, with invasive measurements of the pressure-derived collateral flow index (CFIp, primary endpoint) and fractional flow reserve (FFR). RESULTS: In the ECP group, the CFIp (from 0.08 +/- 0.01 to 0.15 +/- 0.02; P < 0.001) and FFR (from 0.68 +/- 0.03 to 0.79 +/- 0.03; P = 0.001) improved significantly, while in the control group no change was observed. Only the ECP group showed a reduction of the Canadian Cardiovascular Society (CCS, P = 0.008) and New York Heart Association (NYHA, P < 0.001) classification. CONCLUSION: In this study, we provide direct functional evidence for the stimulation of coronary arteriogenesis via ECP in patients with stable coronary artery disease. These data might open a novel noninvasive and preventive treatment avenue for patients with non-acute vascular stenotic disease.

Cardiac magnetic field map topology quantified by Kullback-Leibler entropy identifies patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common primary inherited cardiac muscle disorder, defined clinically by the presence of unexplained left ventricular hypertrophy. The detection of affected patients remains challenging. Genetic testing is limited because only in 50%-60% of all HCM diagnoses an underlying mutation can be found. Furthermore, the disease has a varied clinical course and outcome, with many patients having little or no discernible cardiovascular symptoms, whereas others develop profound exercise limitation and recurrent arrhythmias or sudden cardiac death. Therefore prospective screening of HCM family members is strongly recommended. According to the current guidelines this includes serial echocardiographic and electrocardiographic examinations. In this study we investigated the capability of cardiac magnetic field mapping (CMFM) to detect patients suffering from HCM. We introduce for the first time a combined diagnostic approach based on map topology quantification using Kullback-Leibler (KL) entropy and regional magnetic field strength parameters. The cardiac magnetic field was recorded over the anterior chest wall using a multichannel-LT-SQUID system. CMFM was calculated based on a regular 36 point grid. We analyzed CMFM in patients with confirmed diagnosis of HCM (HCM, n=33, 43.8+/-13 years, 13 women, 20 men), a control group of healthy subjects (NORMAL, n=57, 39.6+/-8.9 years; 22 women and 35 men), and patients with confirmed cardiac hypertrophy due to arterial hypertension (HYP, n=42, 49.7+/-7.9 years, 15 women and 27 men). A subgroup analysis was performed between HCM patients suffering from the obstructive (HOCM, n=19) and nonobstructive (HNCM, n=14) form of the disease. KL entropy based map topology quantification alone identified HCM patients with a sensitivity of 78.8% and specificity of 86.9% (overall classification rate 84.8%). The combination of the KL parameters with a regional field strength parameter improved the overall classification rate to 87.9% (sensitivity: 84.8%, specificity: 88.9%, area under ROC curve: 0.94). KL measures applied to discriminate between HOCM and HNCM patients showed a correct classification of 78.8%. The combination of one KL and one regional parameter again improved the overall classification rate to 97%. A preliminary prospective analysis in two HCM families showed the feasibility of this diagnostic approach with a correct diagnosis of all 22 screened family members (1 HOCM, 4 HNCM, 17 normal). In conclusion, Cardiac Magnetic Field Mapping including KL entropy based topology quantifications is a suitable tool for HCM screening.

[Molecular genetic diagnosis in cardiology]. / Molekulargenetische Diagnostik in der Kardiologie.

Within the next few years the "book of life" will be written, i.e., the sequence of the human genome will be completely assembled. This knowledge will open new avenues for the understanding and treatment of diseases. Causal relationships between mutated genes and disease states have been established for a number of single gene disorders. Although molecular genetic tests are not yet feasible for routine clinical practice in most cases, faster and, eventually, less expensive technologies for the identification of mutations are on the horizon. Clinical guidelines also apply to molecular genetics with diagnostics test being performed only in those individuals who may benefit from it.

Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C.

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.

Mutation detection by cycle sequencing.

Candidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat-stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P-labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.

[Genetic screening of cardiomyopathies]. / Genetisches Screening bei Kardiomyopathien.

Cardiomyopathies comprise a heterogeneous group of primary heart muscle disorders with a strong genetic component. Nearly all cases of hypertrophic cardiomyopathy and at least 20-30% of cases with dilated cardiomyopathy are due to autosomal dominant mutations. The extent of genetic factors for arrhythmogenic right ventricular and restrictive cardiomyopathy is less clear. Recent studies have demonstrated that genetic causes of all cardiomyopathies are highly heterogeneous with more than 25 disease gene loci. Although the ability to diagnose cardiomyopathies at the molecular level has advanced, our understanding of disease pathways and the knowledge of individual diseases causing mutations has had little impact on the clinical management of patients. Once current technical limitations for large-scale mutation analysis are overcome, broad genotype/phenotype correlation studies may answer important clinical issues such as the precise relation between distinct mutations and the risk of sudden death, course of the disease and treatment of patients.

[Genetic aspects of the etiology of arrhythmia]. / Genetische Aspekte der Arrhythmieentstehung.

Cardiac arrhythmias are common causes of morbidity and mortality in clinical medicine. Much has been learned about cellular mechanisms of arrhythmogenesis in the past but genetic components have only recently been recognized for some heritable forms of arrhythmias. The long QT syndrome and the Brugada syndrome are both caused by molecular defects in ion channel proteins. Cardiac arrhythmias can also be associated with structural heart diseases. For example, sinus node dysfunction or AV-block can precede some forms of inherited dilated cardiomyopathy. A distinct genetic form of hypertrophic cardiomyopathy is associated with the Wolff-Parkinson-White syndrome and maps to chromosome 7q35. Arrhythmogenic right ventricular cardiomyopathy has a strong genetic basis and often manifests with ventricular tachycardia. Atrial fibrillation can also occur as familial disease and may be allelic with dilated cardiomyopathy as both diseases can be closely linked to chromosome 10q2.

Fine-structure mapping of the hereditary inclusion body myopathy locus.

The gene responsible for a recessive form of hereditary inclusion body myopathy (HIBM) has previously been mapped to a 10-cM interval on chromosome 9p1-q1. We report the results of further mapping studies using two-point linkage analyses and linkage disequilibrium analyses with 20 HIBM families. We demonstrate that the HIBM gene (HGMW-approved symbol IBM2) lies between loci D9S1791 and D9S50, which are about 1 Mb apart. Genetic analyses in 56 affected individuals of Persian, Afghani, and Iraqi Jewish descent demonstrated a common haplotype at these loci, indicating that a founding mutation accounts for disease in these related ethnic groups. beta-Tropomyosin, an abundant skeletal muscle protein that maps within 1 cM of D9S1791, was excluded as the disease gene because an intragenic polymorphism did not exhibit linkage disequilibrium in HIBM probands. We conclude that the disease gene resides in a 1-Mb interval on chromosome 9 and speculate that a novel muscle protein encoded there is mutated in HIBM.

Risk stratification of sudden cardiac death and malignant ventricular arrhythmias in right ventricular dysplasia-cardiomyopathy.

UNLABELLED: Arrhythmogenic right ventricular dysplasia-cardiomyopathy is in most cases a benign cause of ventricular arrhythmias in young patients. The major reason of mortality is sudden arrhythmic death with an annual rate of 2-3% as the first manifestation of the disease in most cases. Little is known about risk factors of sudden arrhythmic death so far. The purpose of the retrospective study was to classify risk factors from invasive and non-invasive examinations. METHODS: In a cohort of 121 consecutive patients sampled from 1986 to 1998 the value of right ventricular dilatation, left ventricular involvement analysed by angiocardiography or echocardiography and standard ECG parameters such as precordial T wave inversions, right precordial ST elevation, precordial QRS dispersion, left precordial JT interval prolongation and complete right bundle branch block were determined. The whole cohort of patients were divided into two groups with high arrhythmic risk (aborted or non-aborted sudden death, recurrent ventricular tachycardia despite medical treatment, recurrent syncopes) and low risk (frequent ventricular premature beats, non sustained ventricular tachycardia, uneventful course under medical therapy). RESULTS: From angiocardiography or echocardiography in a quantitative approach right ventricular dilatation (p<0.0001) and additional left ventricular abnormalities (p<0.0001) could be identified as major risk factors. From an ECG point of view increased precordial QRS dispersion > or =50 ms (p<0.01) with complete right bundle branch block and right ventricular dilatation in most cases and precordial T wave inversions beyond V3 (p<0.0001) and the phenomenon of left precordial JT interval prolongation (JT dispersion > or =30 ms) in cases of additional left ventricular abnormalities represented non-invasive predictors of recurrent arrhythmic events. Right precordial ST segment elevation could be excluded as risk factor of sudden arrhythmic death. CONCLUSIONS: Right ventricular dilatation with ECG depolarisation abnormalities and additional left ventricular involvement with striking ECG repolarisation abnormalities could be identified as strong risk factors of recurrent arrhythmic events in ARVD with unfavorable prognosis.

Heart failure in arrhythmogenic right ventricular dysplasia-cardiomyopathy.

UNLABELLED: Sudden arrhythmic death and heart failure are essential factors influencing the prognosis of arrhythmogenic right ventricular dysplasia-cardiomyopathy. Heart failure is a rare, but often lethargic event although little is known about morphology, time course and non-invasive predictors. METHOD: In a retrospective study of a consecutive cohort of 121 patients with ARVD over a follow-up period of up to 12 years morphological features of heart failure, time course from the initial diagnosis and standard 12-lead ECG as a non-invasive predictor of developing heart failure were analysed. RESULTS: Heart failure occurred in 13 patients (11%) with isolated right ventricular dilatation and loss of function in 10 cases (77%) and biventricular failure in three cases (23%). Patients developed NYHA class IV in four cases, class III-IV in two cases and class II in seven cases in 4-8 years. In standard ECG of 12 patients (92%) complete right bundle branch block was present at the time of initial diagnosis (n=6) or in a time interval of 4 years (n=6). Morphological distinction of isolated right and biventricular heart failure could be achieved not only by imaging techniques such as echocardiography or cardioangiography, but also by standard ECG with right atrial hypertrophy and an increased mean precordial QRS dispersion of 47.1+/-18.9 ms in cases of right heart failure and biatrial hypertrophy and a reduced precordial QRS dispersion of 33.0+/-23.1 ms in cases of biventricular heart failure. CONCLUSIONS: Heart failure in ARVD consists of isolated right ventricular and biventricular dilatation and pump failure in a time course of 4-8 years after developing complete right bundle branch block as a strong non-invasive predictor from standard 12-lead ECG.

[Genetics of dilated cardiomyopathy]. / Genetik der dilatativen Kardiomyopathie.

Dilated cardiomyopathy (DCM) is a heart muscle disorder characterized by cardiac dilatation and impaired systolic function. In an increasing number of all DCM cases a specific etiology can be identified and in the remaining patients DCM is termed idiopathic. There is a wide variation of the clinical presentation in DCM. The majority of patients manifests classical disease, i.e. heart failure due to left (and right) ventricular systolic dysfunction. However, some cases may come to clinical attention because of supraventricular arrhythmias such as sinus node dysfunction, AV-block or atrial fibrillation. Although a multitude of etiologies may be responsible for DCM (e.g. viral, immunological, toxic), the disease is inherited as a single gene disorder in at least 20 to 35% of cases. Most genetic forms of DCM are caused by autosomal dominant gene defects. Six dominant disease loci on chromosomes 1p1-q1, 1q32, 3p22-p25, 6q23, 9q13 und 10q21-q23 have been identified but the corresponding disease genes are not yet known. X-linked DCM without skeletal muscle disease is a rare variety of adult DCM which can be caused by specific mutations in the dystrophin gene on chromosome Xp21.

A rapid protocol for cardiac troponin T gene mutation detection in familial hypertrophic cardiomyopathy.

Mutations in the human cardiac troponin T gene (TNNT2) are associated with familial hypertrophic cardiomyopathy (FHC) linked to chromosome 1q3 (CMH2). Mutation analyses of TNNT2 have been restricted to RNA-based screening methods because only the TNNT2 cDNA sequence was known. We characterized the genomic structure of 15 TNNT2 exons spliced into the adult isoform. A protocol for rapid mutation detection based on direct sequencing of large PCR-amplified genomic DNA fragments revealed a known TNNT2 mutation (Phe110Ile) in one of 30 FHC probands. Three polymorphic short tandem repeat elements (D1S477, D1S2622, and D1S1723), useful for FHC pedigree analyses at CMH2, were shown to be physically tightly linked to TNNT2.

Effects of two familial hypertrophic cardiomyopathy-causing mutations on alpha-tropomyosin structure and function.

Missense mutations in alpha-tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled tropomyosins differed in their response to myosin S1 binding to the actin-tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.

Clinical features of hypertrophic cardiomyopathy caused by mutation of a "hot spot" in the alpha-tropomyosin gene.

OBJECTIVES: We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the alpha-tropomyosin gene in affected subjects from three unrelated families. BACKGROUND: Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by alpha-tropomyosin mutations have been hampered by the rarity of these genetic defects. METHODS: The haplotypes of three kindreds with FHC due to an alpha-tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared. RESULTS: Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness +/- SD: 24 +/- 4.5 mm in anterior septum of Family DT; 15 +/- 2.7 mm in anterior septum and free wall of Family DB; 18 +/- 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable. CONCLUSIONS: Nucleotide residue 579 in the alpha-tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the alpha-tropomyosin mutation Asp175Asn.

Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disorder manifesting as cardiac hypertrophy with myocyte disarray and an increased risk of sudden death. Mutations in five different loci cause FHC and 3 disease genes have been identified: beta cardiac myosin heavy chain, alpha tropomyosin and cardiac troponin T. Because these genes encode contractile proteins, other FHC loci are predicted also to encode sarcomere components. Two further FHC loci have been mapped to chromosomes 11p13-q13 (CMH4, ref. 6) and 7q3 (ref. 7). The gene encoding the cardiac isoform of myosin binding protein-C (cardiac MyBP-C) has recently been assigned to chromosome 11p11.2 and proposed as a candidate FHC gene. Cardiac MyBP-C is arrayed transversely in sarcomere A-bands and binds myosin heavy chain in thick filaments and titin in elastic filaments. Phosphorylation of MyBP-C appears to modulate contraction. We report that cardiac MyBP-C is genetically linked to CMH4 and demonstrate a splice donor mutation in one family with FHC and a duplication mutation in a second. Both mutations are predicted to disrupt the high affinity, C-terminal, myosin-binding domain of cardiac MyBP-C. These findings define cardiac MyBP-C mutations as the cause of FHC on chromosome 11p and reaffirm that FHC is a disease of the sarcomere.

Familial Hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome maps to a locus on chromosome 7q3.

We have mapped a disease locus for Wolff-Parkinson-White syndrome (WPW) and familial hypertrophic cardiomyopathy (FHC) segregating in a large kindred to chromosome 7 band q3. Although WPW syndrome and FHC have been observed in members of the same family in prior studies, the relationship between these two diseases has remained enigmatic. A large family with 25 surviving individuals who are affected by one or both of these conditions was studied. The disease locus is closely linked to loci D7S688, D7S505, and D7S483 (maximum two point LOD score at D7S505 was 7.80 at theta = 0). While four different FHC loci have been described this is the first locus that can be mutated to cause both WPW and/or FHC.

Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy can be caused by mutations in the genes for beta cardiac myosin heavy chain, alpha-tropomyosin, or cardiac troponin T. It is not known how often the disease is caused by mutations in the tropomyosin and troponin genes, and the associated clinical phenotypes have not been carefully studied. METHODS: Linkage between polymorphisms of the alpha-tropomyosin gene or the cardiac troponin T gene and hypertrophic cardiomyopathy was assessed in 27 families. In addition, 100 probands were screened for mutations in the alpha-tropomyosin gene, and 26 were screened for mutations in the cardiac troponin T gene. Life expectancy, the incidence of sudden death, and the extent of left ventricular hypertrophy were compared in patients with different mutations. RESULTS: Genetic analyses identified only one alpha-tropomyosin mutation, identical to one previously described. Five novel mutations in cardiac troponin were identified, as well as a further example of a previously described mutation. The clinical phenotype of four troponin T mutations in seven unrelated families was similar and was characterized by a poor prognosis (life expectancy, approximately 35 years) and a high incidence of sudden death. The mean (+/- SD) maximal thickness of the left ventricular wall in subjects with cardiac troponin T mutations (16.7 +/- 5.5 mm) was significantly less than that in subjects with beta cardiac myosin heavy-chain mutations (23.7 +/- 7.7 mm, P < 0.001). CONCLUSIONS: Mutations in alpha-tropomyosin are a rare cause of familial hypertrophic cardiomyopathy, accounting for approximately 3 percent of cases. Mutations in cardiac troponin T account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy in this referral-center population. These mutations are characterized by relatively mild and sometimes subclinical hypertrophy but a high incidence of sudden death. Genetic testing may therefore be especially important in this group.

Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere.

We demonstrate that missense mutations (Asp175Asn; Glu180Gly) in the alpha-tropomyosin gene cause familial hypertrophic cardiomyopathy (FHC) linked to chromosome 15q2. These findings implicated components of the troponin complex as candidate genes at other FHC loci, particularly cardiac troponin T, which was mapped in this study to chromosome 1q. Missense mutations (Ile79Asn; Arg92Gln) and a mutation in the splice donor sequence of intron 15 of the cardiac troponin T gene are also shown to cause FHC. Because alpha-tropomyosin and cardiac troponin T as well as beta myosin heavy chain mutations cause the same phenotype, we conclude that FHC is a disease of the sarcomere. Further, because the splice site mutation is predicted to function as a null allele, we suggest that abnormal stoichiometry of sarcomeric proteins can cause cardiac hypertrophy.