Familial hypertrophic cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes.

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the ß-cardiac myosin heavy chain (ß-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy.

Unequal allelic expression of wild-type and mutated ß-myosin in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the ß-myosin heavy chain (ß-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of ß-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC.

Preventing ventricular dysfunction in pacemaker patients without advanced heart failure: results from a multicentre international randomized trial (PREVENT-HF).

AIMS: Previous experimental and clinical studies have consistently suggested that right ventricular (RV) apical pacing has important adverse effects. Ventricular pacing (VP), however, is required, and cannot be reduced in many patients with atrioventricular (AV) block. The PREVENT-HF study was an international randomized trial that explored differences in left ventricular (LV) remodelling during RV apical vs. biventricular (BIV) pacing in patients with AV block. METHODS AND RESULTS: Patients with an expected VP prevalence ≥80% were assigned to RV apical or BIV pacing. The primary endpoint was the change in LV end-diastolic volume (EDV) >12 months. Secondary endpoints were LV end-systolic volume (ESV), LV ejection fraction (EF), mitral regurgitation (MR), and a combination of heart failure (HF) events and cardiovascular hospitalizations. Overall, 108 patients were randomized (RV: 58; BIV: 50). Intention to treat and on-treatment analyses revealed no significant differences in any of the outcomes. Analysis of covariance (ANCOVA) difference for treatment according to randomization (in mL): LVEDV -3.92 (-18.71 to 10.85), P= 0.6; LVESV -1.38 (-12.07 to 9.31), P= 0.80; LVEF 2.47 (-3.00 to 7.94), P= 0.37. Analysis of covariance difference for the on-treatment analysis: LVEDV -4.90 (-20.02 to 10.22, PP= 0.52; LVESV -6.45 (-17.28 to 4.38), P= 0.24, LVEF 2.18 (-3.37 to 7.73), P= 0.44. Furthermore, secondary endpoints did not differ significantly. CONCLUSION: This study did not demonstrate significant LV volume differences >12 months between RV apical and BIV pacing for AV block. Thus, BIV pacing cannot be recommended as a routine treatment for AV block in these patients. However, the results encourage and inform the design of subsequent larger trials with higher power for detecting small volume changes. ClinicalTrials.gov Identifier: NCT00170326.

The left and right ventricle of a patient with a R723G mutation of the beta-myosin heavy chain and severe hypertrophic cardiomyopathy show no differences in the expression of myosin mRNA.

BACKGROUND: In familial hypertrophic cardiomyopathy (FHC), asymmetric left ventricular (LV) hypertrophy has been considered to be the predominant phenotypic expression, whereas right ventricular (RV) involvement is still ambiguous. In most cases, the right ventricle remains unaffected until secondary pulmonary hypertension develops. Several FHC-causing mutations of genes encoding sarcomere-related proteins have been identified which are transmitted in an autosomal-dominant manner. METHODS: We report the case of a 61 year old member of a Catalan family with a Arg723Gly missense mutation of the ß-myosin heavy chain (ß-MHC), that is associated with a malignant phenotype characterized by sudden cardiac death and heart failure. Because of progressive systolic LV dysfunction, the patient received a heart transplant in 2003. RESULTS: Molecular analysis of the myocardial tissue of the explanted heart, taken from the left and right ventricle, showed a similar deviation of the ratio of mutant vs wild type mRNA of the ß-MHC of 71.8 ± 5% and 68.5 ± 3%, respectively. This finding was confirmed for LV biopsies of this patient on protein level, showing a similar proportion of mutated ß-myosin. But since the patient is heterozygous for the ß-MHC mutation and the mutation is located in a coding region, the relative increase of the expression of the mutant allele is unexpected. It has been demonstrated before by our group for several ß-MHC mutations that the relative abundance of mutated mRNA/protein correlates with the clinical severity of the disease. But since the right ventricle shows no (or only minor) manifestation in terms of hypertrophy or dysfunction, the level of mRNA and protein expression is not the only factor responsible for the development of the phenotype of FHC. CONCLUSIONS: Several mechanisms through which cardiac stresses may incite maladaptive cardiac remodeling primarily of the left ventricle that result in myocardial hypertrophy and heart failure are proposed. One of those triggers could be the enhanced work load of the left ventricle, especially if a LV outflow tract gradient is present, in contrast to the lesser demands to the right ventricle which is adapted to the low pressure system of the pulmonary circulation. Further studies are needed to confirm the results of this case, as well as functional studies involving both ventricles.

Quantification of mutant versus wild-type myosin in human muscle biopsies using nano-LC/ESI-MS.

A liquid chromatography/electrospray ionization mass spectrometry (nano-LC/ESI-MS) approach is described by which abundance of proteins (e.g., of beta-myosin heavy chain; MW 223 kDa) carrying a point mutation can be determined in tissue samples where the mutant protein is coexpressed with its wild-type forms. After enzymatic cleavage of the extracted parent protein, mutant and wild-type species of the peptide with the locus of the point mutation were quantified. Synthetic peptides, identical to wild-type and mutant peptides but labeled with stable isotopes ((13)C, (15)N), were added in known amounts as internal standards. The peak areas obtained by MS for the stable-isotope-labeled peptides and for the native peptides were used for quantification. To demonstrate the suitability of this approach we determined the relative abundance of beta-myosin with the Arg723Gly exchange in muscle biopsies of patients with Familial Hypertrophic Cardiomyopathy (HCM). For two such patients the fraction of mutated myosin was 62%, i.e., significantly different from 50%, which is quite unexpected for an autosomal dominant disease in heterozygous patients. Correlation between abundance of mutant myosin and clinical malignancy seen for several mutations in the myosin head domain emphasizes the relevance of such quantification. The approach for quantification described here is generally applicable for quantification of proteins with single point mutations even if only small amounts of tissue are available.

Correlation between insulin resistance surrogates and echocardiographic findings in asymptomatic patients with morbid obesity: a cross-sectional study.

OBJECTIVE: To assess the relationship between insulin resistance (IR) and left ventricular diastolic dysfunction (LVDD) in asymptomatic patients with morbid obesity (MO). METHODS: The study cohort consisted of 231 patients (165 women and 66 men) with MO (mean body mass index [BMI] of 46.0 kg/m2) and a control group of 93 age-and sex-matched apparently healthy control subjects (56 women and 37 men; mean BMI of 24.1 kg/m2). Tissue Doppler imaging echocardiography was used to provide measurements of ejection fraction, LVDD (peak early tissue Doppler velocity/peak late tissue Doppler velocity or Em/Am ratio), left ventricular mass (LVM), and left ventricular hypertrophy (LVH). Adiponectin levels, the homeostasis model assessment index, and the ratio of triglycerides to high-density lipoprotein cholesterol (TG/HDL) were used as surrogate markers of IR. RESULTS: The ejection fraction was normal and similar in the patient and control groups. LVDD (Em/Am ratio <1.0) and LVH prevalences were 52% and 30%, respectively, in the group with MO (significantly higher than in the control group; P<0.0005). The patients with MO displayed higher IR on the basis of all 3 surrogate markers (P<0.0005, respectively). Log-transformed adiponectin showed the strongest correlations with LVM and Em/Am ratios; log-transformed homeostasis model assessment index and TG/HDL ratio displayed less robust yet significant correlations. Stepwise multiple linear regression analysis identified hypertension and the TG/HDL ratio as independent predictors of 35.5% of the variance of LVDD. In contrast, LVM was mainly predicted by BMI, hypertension, and sex. CONCLUSION: LVH and LVDD are highly prevalent in asymptomatic patients with MO. IR is significantly correlated with both variables. Furthermore, LVDD is independently predicted by the presence of hypertension and the TG/HDL ratio. The prognostic implications of these findings warrant further studies.

Preventing ventricular dysfunction in pacemaker patients without advanced heart failure: rationale and design of the PREVENT-HF study.

AIMS: Right ventricular (RV) pacing has been shown to cause heart failure symptoms in patients with and without previous systolic left ventricular (LV) dysfunction. The aim here was to evaluate the preventive effect of biventricular pacing vs. RV apical pacing in patients with indication for permanent ventricular pacing. Methods PREVENT-HF is an ongoing multicentre randomized controlled pilot study designed to assess whether biventricular pacing is superior to RV pacing in patients receiving a bradycardia pacemaker for standard indications. Patients with Class I or IIa indication according to ACC/AHA guidelines for cardiac pacing judged likely to require high (>or=80%) ventricular pacing are randomized to receive either RV or biventricular stimulation. Patients are ineligible if younger than 18 years, have Class III or IV heart failure, or experienced a recent myocardial infarction or cardiac surgery. Echocardiographic parameters of LV function are assessed at baseline, 6 months, and 12 months. The primary endpoint is change in LV end diastolic volume. Secondary outcomes include LV ejection fraction, mortality, morbidity, and mitral regurgitation. In subsets of patients, NT-pro-BNP and oxygen uptake are analysed. Centres in Spain (five), Italy (four), and Germany (seven) will enrol 100 patients. CONCLUSION: PREVENT-HF will contribute to better define the role of chronic biventricular pacing for advanced atrioventricular block.

Hypertrophic cardiomyopathy-related beta-myosin mutations cause highly variable calcium sensitivity with functional imbalances among individual muscle cells.

Disease-causing mutations in cardiac myosin heavy chain (beta-MHC) are identified in about one-third of families with hypertrophic cardiomyopathy (HCM). The effect of myosin mutations on calcium sensitivity of the myofilaments, however, is largely unknown. Because normal and mutant cardiac MHC are also expressed in slow-twitch skeletal muscle, which is more easily accessible and less subject to the adaptive responses seen in myocardium, we compared the calcium sensitivity (pCa(50)) and the steepness of force-pCa relations (cooperativity) of single soleus muscle fibers from healthy individuals and from HCM patients of three families with selected myosin mutations. Fibers with the Arg723Gly and Arg719Trp mutations showed a decrease in mean pCa(50), whereas those with the Ile736Thr mutation showed slightly increased mean pCa(50) with higher active forces at low calcium concentrations and residual active force even under relaxing conditions. In addition, there was a marked variability in pCa(50) between individual fibers carrying the same mutation ranging from an almost normal response to highly significant differences that were not observed in controls. While changes in mean pCa(50) may suggest specific pharmacological treatment (e.g., calcium antagonists), the observed large functional variability among individual muscle cells might negate such selective treatment. More importantly, the variability in pCa(50) from fiber to fiber is likely to cause imbalances in force generation and be the primary cause for contractile dysfunction and development of disarray in the myocardium.

[Hypertrophic cardiomyopathy. Genetic basis and clinical implications]. / Miocardiopatía hipertrófica. Bases genéticas e implicaciones clínicas.

Thanks to advances in molecular biology during the last decade, the etiology of hypertrophic cardiomyopathy has been elucidated. Although more than 150 causal mutations of 9 genes that encode contractile proteins have been identified, many of the pathogenetic mechanisms remain unclear. In this review we discuss the current state of knowledge of the functional effects of some mutations, particularly two of the most lethal beta-myosin mutations -Arg403Gln and Arg723Gly (Barcelona mutation)- and their contributions to the pathogenesis of hypertrophy, sudden death and ischemia. Their potential roles in diagnostic and therapeutic strategies are emphasized.

Miocardiopatía hipertrófica. Bases genéticas e implicaciones clínicas / Hypertrophic cardiomyopathy. Genetic basis and clinical implications

Gracias a los avances de la biología molecular de la última década, conocemos con cierto detalle la etiología de la miocardiopatía hipertrófica (MCH). Se han identificado ya más de 150 mutaciones causales en 9 proteínas contráctiles del sarcómero. Quedan sin embargo por explicar muchos aspectos de su patogenia. En esta revisión se analizan las consecuencias funcionales de algunas mutaciones, y en particular de 2 mutaciones de las más graves de la Beta-miosina, la Arg403Gln y la Arg723Gly (mutación Barcelona), y se discute su contribución al esclarecimiento de la patogenia de la hipertrofia, la muerte súbita y la isquemia. Se subrayan sus posibles implicaciones en el diagnóstico y el tratamiento (AU)

Thanks to advances in molecular biology during the last decade, the etiology of hypertrophic cardiomyopathy has been elucidated. Although more than 150 causal mutations of 9 genes that encode contractile proteins have been identified, many of the pathogenetic mechanisms remain unclear. In this review we discuss the current state of knowledge of the functional effects of some mutations, particularly two of the most lethal Beta-myosin mutations –Arg403Gln and Arg723Gly (Barcelona mutation)– and their contributions to the pathogenesis of hypertrophy, sudden death and ischemia. Their potential roles in diagnostic and therapeutic strategies are emphasized (AU)

[Late T-lymphocyte and monocyte activation in coronary restenosis. Evidence for a persistent inflammatory/immune mechanism?]. / Linfocitos T y monocitos activados en la reestenosis coronaria. ¿Reflejan la persistencia de un mecanismo inflamatorio?

AIMS: This study was made to determine if restenosis after percutaneous coronary angioplasty is associated with acute or chronic inflammatory/immunologic activity, and explored possible relationships with latent infection. PATIENTS AND METHOD: Forty-six consecutive patients underwent elective PTCA and 6 months of angiographic follow-up. Peripheral venous blood samples were obtained at baseline, 24-48 h, and 4-6 months post-intervention. Flow-cytometric methods were used to measure early and late circulating leukocyte activation status. Il-6 and TNF-alpha cytokines, and Il-2 soluble receptor concentrations were determined in all plasma samples. Chlamydia pneumoniae and Cytomegalovirus antibody assays were performed to detect infectious disease. RESULTS: Angiographic coronary stenosis developed in 27 out of 46 patients. At 6 months of follow-up, these patients showed a significant increase in circulating cytotoxic T-lymphocytes CD3+/CD56+ (18.8 7.1 vs 6.12 2.7%; p = 0.005) and activated monocytes (CD11b: 1,383 624 vs 990 484 MFI, p = 0.025; CD64: 76.0 28.7 vs 56.7 21.8 MFI; p = 0.014), with no apparent relation to increased cytokines or latent infectious disease. CONCLUSIONS: Restenosis appears to be associated to inflammatory and immunological activity that persists 6 months after coronary intervention. No relationship was found with the infections studied. The presence of inflammatory activity 4-6 months after PTCA suggess that pharmacological therapeutic interventions to prevent restenosis should be maintained for months.

Linfocitos T y monocitos activados en la reestenosis coronaria. ¿Reflejan la persistencia de un mecanismo inflamatorio? / Late T-Lymphocyte and monocyte activation in coronary restenosis. Evidence for a persistent inflammatory/immune mechanism?

Objetivos. El objetivo de este trabajo ha sido estudiar la relación de la reestenosis coronaria postangioplastia con la actividad inflamatoria e inmunitaria en la sangre periférica, y explorar su posible asociación con infecciones latentes. Pacientes y método. Se estudiaron 46 pacientes consecutivos con angina a los que se practicó una angioplastia coronaria percutánea y un control angiográfico a los 6 meses. Se tomaron muestras de sangre venosa antes de la intervención, 24-48 h después y a los 6 meses. Se determinó la expresión de las moléculas de adhesión en los leucocitos mediante la técnica de la citometría de flujo. Se cuantificaron las concentraciones de las citocinas proinflamatorias interleucina (IL) 6 y factor de necrosis tumoral (TNF) alfa, así como los receptores solubles de la IL-2 en el plasma, y se utilizaron las reacciones antigénicas para detectar la infección por Chlamydia pneumoniae y Cytomegalovirus. Resultados. Los pacientes con reestenosis coronaria angiográfica (27 de 46) presentaron un aumento significativo de los linfocitos T citotóxicos CD3+/CD56+ (18,8 ñ 7,1 frente a 6,12 ñ 2,7 por ciento; p = 0,005) y de monocitos activados (CD11b: 1.383 ñ 624 frente a 990 ñ 484 MFI; p = 0,025; CD64: 76,0 ñ 28,7 frente a 56,7 ñ 21,8 MFI; p = 0,014) a los 6 meses, sin relación aparente con el aumento de las citocinas o la positividad de las reacciones antigénicas infecciosas. Conclusiones. La reestenosis coronaria se asocia a una reacción inflamatoria/inmunitaria persistente, que no parece estar relacionada con las infecciones latentes estudiadas. La persistencia de actividad inflamatoria a los 4-6 meses sugiere que las intervenciones destinadas a prevenir la reestenosis deberían mantenerse durante meses (AU)

[Genes and coronary heart disease]. / Bases genéticas de la enfermedad coronaria.

The causes of atherosclerotic cardiovascular disease have been intensely scrutinized for the last few decades. Since the classic risk factors have been found to be incomplete predictors of the disease, additional risk factors based on molecular genetics are now being sought. Polymorphisms are gene variations that have only modest effects on the function of coded proteins or enzymes. However, they are common and may be risk factors in the presence of environmental risk factors (cholesterol, stress, tobacco). Recent advances in molecular biology have made it possible to detect numerous polymorphisms that might have a detrimental effect on vascular biology, suggesting the hypothesis that multiple polymorphisms in the presence of environmental factors could act synergistically in the pathogenesis of atherosclerosis and coronary heart disease, which are typically polygenic and multifactorial diseases. In this review, the current status of our knowledge of polymorphisms and mutations potentially implicated in the mechanisms of coronary artery disease is discussed. Genotype/phenotype, gene-gene, and gene-environmental interactions related to lipid metabolism, the renin-angiotensin-aldosterone and adrenergic systems, insulin resistance, oxidative stress and endothelial function, inflammation and thrombosis are analyzed. Individual coronary risk might be related to the presence of a critical accumulation detrimental polymorphisms.

Bases genéticas de la enfermedad coronaria / Genes and coronary heart disease

La etiología de la enfermedad coronaria ha sido objeto de intenso estudio durante las últimas décadas. Dado que los factores de riesgo clásico sólo explican a lo sumo la mitad de los casos, se están buscando nuevos factores etiológicos en el ámbito de la genética molecular. Los polimorfismos son mutaciones del ADN, que apenas alteran la función de la proteína codificada, pero son frecuentes y pueden ser un factor de riesgo genético cuando el organismo se enfrenta a determinados factores de riesgo ambientales (colesterol, estrés, tabaco). Los recientes avances en biología molecular han facilitado la detección de numerosos polimorfismos que pueden tener un efecto patógeno, y han hecho pensar en la hipótesis de que la suma de polimorfismos desfavorables y un marco ambiental propicio puede facilitar la aparición de la aterosclerosis y de la enfermedad coronaria en particular, enfermedades típicamente poligénicas y multifactoriales. En esta revisión se analiza el estado actual de los conocimientos sobre los polimorfismos y mutaciones que pueden estar implicados en la fisiopatogenia de la enfermedad coronaria y sus complicaciones, como los relacionados con el metabolismo lipídico, el sistema renina-angiotensina-aldosterona y el sistema simpático adrenérgico, la resistencia a la insulina, el estrés oxidativo y la función endotelial, la inflamación y la trombosis. El riesgo coronario puede depender del número acumulado de polimorfismos desfavorables que porta el individuo (AU)