Ross T. Murphy

Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations

Restrictive cardiomyopathy (RCM) is an uncommon heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic. We recognized a large family in which individuals were affected by either idiopathic RCM or hypertrophic cardiomyopathy (HCM). Linkage analysis to selected sarcomeric contractile protein genes identified cardiac troponin I (TNNI3) as the likely disease gene. Subsequent mutation analysis revealed a novel missense mutation, which cosegregated with the disease in the family (lod score: 4.8). To determine if idiopathic RCM is part of the clinical expression of TNNI3 mutations, genetic investigations of the gene were performed in an additional nine unrelated RCM patients with restrictive filling patterns, bi-atrial dilatation, normal systolic function, and normal wall thickness. TNNI3 mutations were identified in six of these nine RCM patients. Two of the mutations identified in young individuals were de novo mutations. All mutations appeared in conserved and functionally important domains of the gene. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Mutations in the muscle LIM protein and α-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), while mutations in multiple genes cause autosomal dominant DCM. Muscle LIM protein (MLP) is a member of the cysteine-rich protein (CRP) family and has been implicated in both myogenesis and sarcomere assembly. In the latter role, it binds zyxin and alpha-actinin, both of which are involved in actin organization. An MLP-deficient mouse has been described; these mice develop dilated cardiomyopathy and heart failure. Based upon these data, and the recent descriptions of mutations in MLP in patients with DCM or hypertrophic cardiomyopathy, we screened patients for mutations in the MLP and alpha-actinin-2 genes. We identified a patient with DCM and EFE, having a mutation in MLP with the residue lysine 69 substituted by arginine (K69R). This is within a highly conserved region adjacent to the first LIM domain involved in alpha-actinin binding. Analysis in cell culture systems demonstrated that the mutation abolishes the interaction between MLP and alpha-actinin-2 and the cellular localization of MLP was altered. In another individual with DCM, a W4R mutation was identified. However, this mutation did not segregate with disease in this family. In another patient with DCM, a Q9R mutation was identified in alpha-actinin-2. This mutation also disrupted the interaction with MLP and appeared to inhibit alpha-actinin function in cultured cells, in respect to the nuclear localization of actinin and the initiation of cellular differentiation.

Subclinical anthracycline- and trastuzumab-induced cardiotoxicity in the long-term follow-up of asymptomatic breast cancer survivors: a speckle tracking echocardiographic study

Objective To examine the long-term effects of standard chemotherapy on myocardial function in asymptomatic breast cancer survivors using two-dimensional speckle tracking echocardiography. Methods Seventy women (chemotherapy group) aged 54±8 years who had received anthracycline treatment with (n=19) or without (n=51) adjuvant trastuzumab up to 6 years previously, and 50 female controls were studied. Left ventricular systolic (ejection fraction (EF%), peak systolic myocardial excursion, (Sm)) and diastolic (peak mitral E and A velocities, six-point average of mitral annular E′ velocities) function, 2D global and regional longitudinal and radial strain were determined using standard 2D Doppler and tissue Doppler echocardiographic methods and speckle tracking software. Results Despite normal EF% (62±4% vs 60±3%, p=0.051) the chemotherapy group had reduced E/A ratios (0.9±0.3 vs 1.1±0.3, p=0.003), global E′ (10.2±2 vs 11.2±2.3, p=0.036), global Sm (9.0±1.3 vs 9.6±1.3, p=0.029) and global longitudinal 2D strain (−18.1±2.2 vs −19.6±1.8, p=0.0001) in comparison with controls. In 18 (26%) of the chemotherapy group, global longitudinal strain was below the lower limit of the control group. Cigarette smoking was a negative predictor of longitudinal strain, but only in the chemotherapy group. Radial strain did not differ significantly between the two groups. There were no significant differences in EF%, global Sm and longitudinal strain between trastuzumab-treated individuals and controls. Conclusions Subclinical systolic and diastolic myocardial abnormalities were present in asymptomatic breast cancer survivors up to 6 years after standard chemotherapy. Cigarette smoking had a negative effect on longitudinal strain in these individuals. Adjuvant trastuzumab treatment did not appear to have an additive adverse impact on myocardial function in the medium–long term.

Novel mutation in cardiac troponin I in recessive idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy is a common cause of heart failure. Half of cases are believed to be hereditary, and mutations in cardiac sarcomeric contractile protein genes have been reported with autosomal dominant inheritance. We used mutation analysis suitable for identification of both dominant and recessive mutations to investigate the sarcomeric gene for cardiac troponin I (TNNI3) in 235 patients with dilated cardiomyopathy. We identified a novel TNNI3 mutation in a family with recessive disease. Functional studies showed impairment of troponin interactions that could lead to diminished myocardial contractility. TNNI3 is the first recessive gene identified for this condition, and we suggest that other such genes could be pinpointed by mutation analyses designed to identify homozygous mutations.

Prospective Familial Assessment in Dilated Cardiomyopathy Cardiac Autoantibodies Predict Disease Development in Asymptomatic Relatives

Background— In autoimmune disorders, circulating autoantibodies identify healthy relatives at risk years before clinical presentation. Healthy relatives of patients with dilated cardiomyopathy (DCM) who have echocardiographic changes, including left ventricular enlargement or depressed fractional shortening at baseline, have increased medium-term risk for DCM development. Approximately one third of relatives have serum anti-heart autoantibodies (AHAs) at baseline; we intended to assess their potential role in predicting DCM development. Methods and Results— Baseline evaluation, including electrocardiography, echocardiography, and AHA, was performed in 592 asymptomatic relatives of 169 consecutive DCM patients (291 males and 301 females; mean age 36±16 years). Relatives were classified in accordance with published echocardiographic criteria; those who did not have DCM were followed up (median of 58 months). DCM among relatives was diagnosed by echocardiography at follow-up. Of the 592 individuals evaluated, 77% were assessed as normal, 4.4% as having DCM, and 19% as possibly affected on the basis of depressed fractional shortening without ventricular dilatation in 17 and left ventricular enlargement without systolic dysfunction in 94. Five-year follow-up of 311 relatives revealed that 26 had progressed (13 to DCM, 11 to left ventricular enlargement, and 2 to depressed fractional shortening). Relatives who developed DCM were more frequently AHA-positive than those who did not (69% versus 37%, P=0.02). Five-year probability of progression to DCM, among normal or possibly affected relatives, was higher in AHA-positive cases (P=0.03). By Cox regression, positive AHAs at baseline were independent predictors of progression (RR 2.26, CI 1 to 5.1, P=0.03). Conclusions— Among healthy relatives of DCM patients, AHAs are independent predictors of disease development within 5 years.

Risk stratification in unstable angina and non-Q wave myocardial infarction using soluble cell adhesion molecules

OBJECTIVE To assess prospectively the prognostic value of soluble cellular adhesion molecules (CAMs) in patients with unstable angina and non-Q wave myocardial infarction and to compare their prognostic accuracy with that of C reactive protein (CRP). DESIGN AND SETTING Prospective observational study of patients presenting acutely with unstable angina and non-Q wave myocardial infarction to a single south Dublin hospital. METHODS Patients with Braunwald IIIA unstable angina and non-Q wave myocardial infarction had serum samples taken at presentation before initiation of antithrombotic treatment and were followed for six months. The primary end point was the occurrence of major adverse cardiovascular events (recurrent unstable angina, non-fatal myocardial infarction, and cardiovascular death) at six months. Concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble endothelial selectin, and soluble platelet selectin were measured using an enzyme linked immunosorbent assay technique. CRP was measured with an immunophelometric assay. RESULTS 91 patients (73 men and 18 women, mean (SD) age 61 (11) years) were studied; 27 patients (30%) had major adverse cardiac events during the six months of follow up. Concentration of CRP were significantly raised in patients who had an ischaemic event (mean (SEM) 11.5 (6.4) mg/l v5.4 (2.5) mg/l, p < 0.001). Concentrations of sVCAM-1 were also significantly raised in the ischaemic event group (979 (30) ng/mlv 729 (22) ng/ml, p < 0.001). Both sVCAM-1 and CRP concentrations correlated strongly with the occurrence of an adverse event. The sensitivity of CRP > 3 mg/l and sVCAM-1 > 780 ng/ml for predicting future events was > 90%. There was no difference in concentrations of sICAM-1, soluble endothelin selectin, or soluble platelet selectin between event and non-event groups. CONCLUSION Raised concentrations of sVCAM-1 and CRP are predictive of an increased risk of major adverse cardiovascular events six months after presentation with unstable angina and non-Q wave myocardial infarction. These findings suggest that the intensity of the vascular inflammatory process at the time of presentation is a determinant of clinical outcome in unstable coronary artery disease.

Echocardiographic Evaluation in Asymptomatic Relatives of Patients with Dilated Cardiomyopathy Reveals Preclinical Disease

Context Idiopathic dilated cardiomyopathy is familial in 25% of cases. However, identifying asymptomatic affected relatives is difficult because there is no specific molecular marker for the disease and penetrance of the genetic defect is incomplete. The natural history of asymptomatic disease is poorly defined. Contribution Clinical evaluation of asymptomatic relatives of patients with dilated cardiomyopathy showed that those with left ventricular enlargement or depressed fractional shortening were nearly 8 times as likely to progress to dilated cardiomyopathy as those with normal findings. Implications Screening of asymptomatic relatives of patients with dilated cardiomyopathy seems useful since early treatment may improve prognosis in affected individuals. The Editors Idiopathic dilated cardiomyopathy frequently presents at an advanced stage as severe heart failure or a catastrophic event, such as sudden death or stroke (1). The preclinical stage of dilated cardiomyopathy may last several years, even when the eventual clinical syndrome is acute. Although advances in heart failure management have improved the prognosis for patients presenting late, additional benefit might result from treatment before the onset of symptoms, making the identification of at-risk individuals a major focus of current research in heart failure. Recent studies have demonstrated that dilated cardiomyopathy is familial in at least 25% of cases (2-5). Most families exhibit an autosomal dominant mode of inheritance. Genetic analyses in extended kindreds have defined several dilated cardiomyopathy disease loci and genes (6-16). However, in most families with dilated cardiomyopathy, disease penetrance is incomplete and age-related, hampering attempts to identify the responsible gene defects. Without molecular markers, the diagnosis of familial dilated cardiomyopathy and the detection of early asymptomatic disease depend on clinical evaluation. Initial echocardiographic studies of the family members of patients with dilated cardiomyopathy noted an unusually high incidence of asymptomatic minor abnormalities of the left ventricle (3-5). These abnormalities may represent preclinical disease or formes frustes of dilated cardiomyopathy. We prospectively examined a large series of unselected patients with dilated cardiomyopathy and their family members to determine the prevalence and natural history of echocardiographic abnormalities among asymptomatic, apparently healthy relatives. Methods The human research committee at St. Georges Hospital Medical School, London, United Kingdom, reviewed and approved the entire study protocol. We obtained informed consent from each patient to contact family members and subsequently from each family member who agreed to participate in the study. Echocardiographic Criteria Experienced operators, who were unaware of the clinical context, performed all study echocardiography. We obtained chamber measurements at the mitral valve tip level from 2-dimensional guided M-mode or short-axis view recordings. We corrected dimensions for age and body surface area according to the formula of Henry and colleagues (17): percentage of predicted left ventricular end-diastolic dimension = measured left ventricular diastolic dimension/predicted left ventricular diastolic dimension 100; and predicted left ventricular diastolic dimension = (45.3 body surface area0.3) (0.03 age) 7.2. We classified individuals in accordance with earlier reports as follows. Dilated cardiomyopathy was defined as percentage of predicted left ventricular diastolic dimension of 112% or greater and fractional shortening less than 25%; left ventricular enlargement was defined as percentage of predicted left ventricular diastolic dimension of 112% or greater and fractional shortening of 25% or greater; depressed fractional shortening was defined as percentage of predicted left ventricular diastolic dimension less than 112% and fractional shortening less than 25%; and healthy was defined as percentage of predicted left ventricular diastolic dimension less than 112% and fractional shortening of 25% or greater. Proband Enrollment We recruited probands from 370 consecutive patients with dilated cardiomyopathy who were referred to the heart failure clinic at St. Georges Hospital. All patients underwent a comprehensive evaluation, including clinical history, physical examination, 12-lead electrocardiography, 2-dimensional echocardiography, 24-hour Holter monitoring, symptom-limited metabolic exercise testing, and laboratory investigation (including creatine kinase level measurement), to screen for asymptomatic skeletal muscle disease and iron studies to exclude hemochromatosis. We performed coronary angiography to exclude atherosclerotic heart disease in those older than 40 years of age or in those with features suggestive of coronary disease. We excluded individuals with documented coronary disease, systemic arterial hypertension, primary valvular disease, pericardial disease, cor pulmonale, or a history of excess alcohol consumption (regular intake >40 g/d for women and >80 g/d for men). Family Member Enrollment We offered family screening to all patients with dilated cardiomyopathy, regardless of an overt family history, and we based inclusion on the willingness to participate and geographic availability. Of the patients, 189 probands and their families fulfilled inclusion criteria, resided in the United Kingdom, and were willing to participate. A trained research nurse constructed extended pedigrees based on each proband by using Cyrillic 2.1 (Cherwell Scientific Ltd., Oxford, United Kingdom), and we contacted at-risk first- and second-degree relatives and offered them noninvasive evaluation. Assessment of asymptomatic relatives who agreed to participate included medical history, clinical examination, 12-lead electrocardiography, and 2-dimensional echocardiography. Relatives with abnormalities proceeded to a full evaluation as described for probands. We reviewed medical records and autopsy findings to confirm or refute the diagnosis of dilated cardiomyopathy for all deceased relatives. Definition of Familial Dilated Cardiomyopathy We classified dilated cardiomyopathy as familial if at least 1 relative (in addition to the proband) had documented disease during life or at postmortem or if there was a history of unexplained sudden cardiac death before the age of 30 years. Follow-up of Asymptomatic Relatives All relatives with left ventricular enlargement and depressed fractional shortening (but not initially healthy relatives) underwent prospective annual evaluation. They were asymptomatic and did not receive treatment unless criteria for dilated cardiomyopathy developed. After initial observation of progression to dilated cardiomyopathy (approximately 9% over 40 months) among this group, we determined that initially healthy individuals should also be reevaluated to verify that left ventricular enlargement and depressed fractional shortening were risk markers for development of dilated cardiomyopathy. Accordingly, a nurse who had not been involved in the original screening project and who did not know the families randomly contacted 238 relatives whose initial echocardiograms were entirely normal from a list of all initially healthy relatives and invited them for reevaluation at a median of 57 months from their initial assessment. Follow-up evaluations consisted of repeated history and physical examination, 12-lead electrocardiography, and 2-dimensional echocardiography. Statistical Analysis To determine the number of initially healthy relatives requiring follow-up, we based power calculations for analysis of progression on the 9% rate of progression over 40 months observed among 140 study participants with left ventricular enlargement and depressed fractional shortening, a hypothesized rate of progression of 2% or less among those relatives with initially normal echocardiograms, and an level of 0.05. We determined the requirement of follow-up of 235 relatives or more who were initially classified as healthy at a power of 0.85 (that is, total population of 375 patients, with 140 patients with left ventricular enlargement and depressed fractional shortening and 235 healthy patients). We confirmed all quantitative variables to be normally distributed by using a 1-sample KolmogorovSmirnoff test and expressed them as means and SDs. For comparisons of continuous and discrete variables, we used the Student t-test, chi-square test, or Fisher exact test as appropriate. To account for the nesting of individuals within families, we estimated 95% CIs for prevalence of dilated cardiomyopathy, left ventricular enlargement, and depressed fractional shortening among relatives by fitting a hierarchical model for each condition using WinBUGS 1.4 (Imperial College and Medical Research Council, London, United Kingdom). We estimated prediction intervals from the posterior distribution for mean disease prevalence and presented them as 95% CIs. We used the KaplanMeier method to construct survival curves for risk for progression for relatives with left ventricular enlargement and depressed fractional shortening and healthy relatives. We fitted a Cox proportional hazards regression model to perform further analysis of progression to dilated cardiomyopathy. We used the Efron approximation to deal with ties. Covariates included in the initial model were initial diagnosis and age. A backward stepwise procedure converged upon a model, which only included initial diagnosis. To account for the clustering of related individuals within families, we used frailty models. A frailty model is a random-effects model that accounts for a familial effect in the analysis. These models allow for the lack of independence between events observed in members of the same family. Henderson and Oman (18) have discussed their use in more detail. The frailty was introduced into the model as an unobserved, no

Prevalence and clinical significance of systolic impairment in hypertrophic cardiomyopathy

Objectives: To determine the frequency of systolic impairment (SI) and its impact on the natural history of hypertrophic cardiomyopathy (HCM). Methods: 1080 patients (mean (SD) age 43 (15) years, 660 men) with HCM were evaluated. Initial assessment included history, examination, 48 hour Holter monitoring, cardiopulmonary exercise testing, and echocardiography; SI was defined as a fractional shortening (FS) ⩽ 25%. Survival data were collected at clinic visits or by direct communication with patients and their general practitioners. The results of serial echocardiography in 462 patients with normal FS at presentation are also reported. Results: 26 (2.4%) patients (49 (14) years, 18 men) had SI at the initial visit. During follow up (58 (49) months), nine (34.6%) died or underwent cardiac transplantation compared with 108 (10.2%) patients with normal FS (p  =  0.01). Five year survival from death (any cause) or transplantation was 90.1% (95% confidence interval (CI) 87.8 to 92.4) in patients with normal systolic function versus 52.4% (95% CI 25.2 to 79.6, p < 0.0001) in patients with SI. In patients who underwent serial echocardiography, 22 (4.8%, aged 41 (15) years) developed SI over 66 (40) months; the annual incidence of SI was 0.87% (95% CI 0.54 to 1.31). On initial evaluation patients who developed SI had a higher frequency of syncope (67 (15.2%) v 10 (45.5%) of those who did not develop SI, p  =  0.001), non-sustained ventricular tachycardia (91 (20.6%) v 11 (50%), p  =  0.002), and an abnormal blood pressure response on exercise (131 (29.7%) v 15 (68.2%), p  =  0.001). Patients with SI had greater wall thinning (p  =  0.001), left ventricular cavity enlargement (p < 0.0005), and deterioration in New York Heart Association functional class (p  =  0.001) during follow up. Thirteen (59.1%) patients who progressed to SI died or underwent transplantation compared with 38 (8.6%) patients who maintained normal systolic function. Conclusions: SI is an infrequent complication of HCM but, when present, is associated with a poor prognosis.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Evidence of prolonged inflammation in unstable angina and non–Q wave myocardial infarction☆

OBJECTIVES This study was designed to document the inflammatory response up to one year after acute presentation with unstable angina (UA) and non-Q wave infarction (NQMI) as reflected by the expression of soluble cell adhesion molecules (CAMs). BACKGROUND Coronary plaque inflammation is a key component in the pathogenesis of acute coronary syndromes. Cell adhesion molecules are critical mediators of the inflammatory process. Soluble forms of these molecules are detectable in serum and are elevated acutely in patients with UA and NQMI. METHODS Patients presenting with UA and NQMI had serum samples taken at presentation and then after three, six and 12 months. A control group of similar age and gender distribution was used for comparison. Levels of soluble inter-cellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial-selectin and platelet-selectin were measured using an ELISA technique. RESULTS We studied 91 patients (M/F = 73/18, mean age 62 +/- 11 years, 56 UA and 35 NQMI) and 24 controls (M/F = 18/6, mean age 56 +/- 12 years). Levels of all four soluble CAMs were significantly elevated in both UA and NQMI patients at presentation, three and six months in comparison with controls. Levels in UA and NQMI groups fell between six and 12 months after initial presentation. CONCLUSIONS The results suggest that the inflammatory stimulus triggering expression of CAMs is sustained for up to six months after presentation with either UA or NQMI and then returns toward control values over the following six months.