AQUA Mutant Protein Quantification of Endomyocardial Biopsy-Sized Samples From a Patient With Hypertrophic Cardiomyopathy.

In genetic diseases like hypertrophic cardiomyopathy, reliable quantification of the expression level of mutant protein can play an important role in disease research, diagnosis, treatment and prognosis. For heterozygous ß-myosin heavy chain (ß-MyHC) mutations it has been shown that disease severity is related to the fraction of mutant protein in the myocardium. Yet, heart tissue from patients with genetically characterized diseases is scarce. Here we asked, if even in the case of small endomyocardial biopsies, single quantifications produce reliable results. Myocardial samples were taken from four different regions of an explanted heart of a patient with hypertrophic cardiomyopathy carrying point mutation p.Gly716Arg in ß-MyHC. From both, large samples (15 mg) and small, endomyocardial biopsy-sized samples (≤ 1 mg) myosin was extracted and enzymatically digested to yield a specific peptide of interest that allowed to distinguish mutant and wild-type ß-MyHC. Absolute quantification by mass spectrometry (AQUA) of the peptide of interest was performed repeatedly for both sample sizes to determine the fraction of mutant ß-MyHC. Fractions of mutant ß-MyHC (32% on average) showed only small differences between the four cardiac regions and for large and small samples. The standard deviations were smaller than five percentage points for all cardiac regions. The two quantification methods (large and small sample size) produce results with comparable accuracy and precision. Consequently, with our method even small endomyocardial biopsies allow reliable protein quantification for potential diagnostic purposes.

Burst-Like Transcription of Mutant and Wildtype MYH7-Alleles as Possible Origin of Cell-to-Cell Contractile Imbalance in Hypertrophic Cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (ß-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype ß-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either ß-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant ß-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant ß-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in ß-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.

Intrinsic MYH7 expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy.

HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the ß-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.

Optimized pacing mode for hypertrophic cardiomyopathy: Impact of ECG fusion during pacing.

BACKGROUND: Electrocardiographic (ECG) fusion with intrinsic QRS could reduce the benefit of atrial synchronous biventricular pacing (AS-BiVP) in patients with hypertrophic obstructive cardiomyopathy (HOCM). OBJECTIVES: The purpose of this study was to assess the benefit of AS-BiVP and the influence of ECG fusion for reduction of left ventricular outflow tract gradient (LVOTG) in these patients. METHODS: Twenty-one symptomatic HOCM patients with severe LVOTG were included. Twelve patients were evaluated retrospectively for the prevalence of fusion and its influence on outcomes after AS-BiVP. Eleven patients (2 of the first population were also evaluated retrospectively) were prospectively included to evaluate the benefit of performing atrioventricular node ablation (AVNA) to achieve full ventricular capture if fusion was present during AS-BiVP. RESULTS: Seven of the first 12 patients (58%) had ECG fusion. After 54 ± 24 months of AS-BiVP, the presence of fusion was associated with lower values for reduction of resting, dynamic LVOTG and New York Heart Association (NYHA) class. In the prospectively evaluated patients, after 12 months of follow-up, resting LVOTG decreased from 98 ± 39 to 39 ± 24 mm Hg (P = .008); dynamic LVOTG decreased from 112 ± 38 to 60 ± 24 mm Hg (P = .013); NYHA class decreased from 2.8 ± 0.4 to 1.7 ± 0.6 (P = .014); endurance time during constant work rate cycling exercise (80% of peak oxygen consumption) increased from 399 ± 148 to 691 ± 249 seconds (P = .046); quality of life improved from 46 ± 22 to 22 ± 20 points (P = .02); and brain natriuretic peptide levels decreased from 318 ± 238 to 152 ± 118 pg/mL (P = .09). Eight of the 11 prospectively evaluated patients (73%) needed AVNA, which further decreased LVOTG from 108 ± 40 mm Hg at baseline to 89 ± 29 mm Hg after BiVP to 54 ± 22 mm Hg after AVNA (P = .003). CONCLUSION: As-BiVP that ensures no ECG fusion, by means of AVNA when needed, appears to be the optimal pacing mode in HOCM patients.

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.

A common variant of the ABO gene protects against hypertension in a Spanish population.

The objective of this study was to establish whether genetic polymorphisms that could be related to angiotensin-converting enzyme (ACE) levels are associated with hypertension. A total of 10 haplotype-tagging single-nucleotide polymorphisms in ACE, the ACE I/D polymorphism, and 2 polymorphisms in the ABO (rs495828 and rs8176746) were investigated for association with hypertension in 269 hypertensive patients and 254 healthy controls. All analyses were adjusted for age and body mass index, and corrected for multiple testing. Only one polymorphism of the ABO gene (rs495828) presented nominal pointwise P<0.05 values (odds ratio = 0.33, 95% CI 0.19-0.58, P = 6 × 10(-5)) and achieved P<3.8 × 10(-3), the nominal P-value considered significant after Bonferroni correction. Analysis of the genotype frequencies showed that the model that correctly explained the observed association was the recessive model (odds ratio = 0.03, 95% CI 0.01-0.15, P = 1 × 10(-6)). These results indicate that genetic variants that could be related to ACE activity are good predictors of hypertension, and identify ABO as a good candidate gene for genetic studies of hypertension risk. Further studies are required to confirm this association.

Pharmacogenetic predictors of angiotensin-converting enzyme inhibitor-induced cough: the role of ACE, ABO, and BDKRB2 genes.

BACKGROUND AND OBJECTIVE: Dry cough is the most common reason for stopping angiotensin-converting enzyme inhibitors (ACEi) therapy. The role of ACE in the metabolism of bradykinin has been proposed as a pathogenic mechanism. This study included a complete analysis of the variability of the genes involved in bradykinin metabolism (ACE and XPNPEP2) and bradykinin receptors (BDKRB2). We included two polymorphisms in the ABO (related to ACE levels); two polymorphisms in the AGTR1, and one polymorphism in the BKRB1 (related to ACEi response). METHODS: A total of 281 patients who had been treated with ACEi were retrospectively recruited [102 patients were considered as cases (cough) and 179 patients were considered as controls (no cough)], and 56 polymorphisms were tested for association. RESULTS: We found that genetic polymorphisms in BDKRB2 [rs8016905; P=0.003; odds ratio (OR)=2.21] and ABO (rs495828; P=0.001; OR=2.45) are associated with ACEi-induced cough after correction for multiple testing. The effect of polymorphisms in ABO was sex specific (female patients; P=0.0006; OR=3.26). When we analyzed the subgroup of patients homozygous GG for rs4343, two polymorphisms in the ACE were found to have protective properties (rs4459610 and rs4267385; P=0.005 and 0.004; OR=0.25). We also found a strong interaction between the ABO polymorphisms, rs495828 and rs8176746 (P<0.0001; OR=3.7). CONCLUSION: These results highlight the importance of genetic determinants of ACE levels as good predictors of the ACEi response, and provide ABO as a good candidate gene for pharmacogenetic studies of ACEi-related cough. Moreover, our results also confirm the importance of bradykinin in the pathogenesis of this adverse effect.

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.

Biventricular pacing in hypertrophic obstructive cardiomyopathy: a pilot study.

BACKGROUND: Right ventricular apex pacing for gradient reduction in hypertrophic obstructive cardiomyopathy (HOCM) with severe left ventricular (LV) obstruction has yielded conflicting results. OBJECTIVE: The purpose of this study was to assess the feasibility and effectiveness of biventricular pacing in HOCM. METHODS: Transvenous biventricular pacing was attempted in 12 severely symptomatic HOCM patients. Optimal intervals were programmed after implant. Echocardiographic LV pressure gradient and synchrony were assessed. LV lead implantation was successful in 9 patients. Optimal pacing mode was biventricular in 6 patients, left ventricular only in 2 patients, and right ventricular only in 1 patient. RESULTS: Functional capacity and quality of life progressively improved. New York Heart Association functional class decreased from 3.2 ± 0.4 at baseline to 1.9 ± 0.3 at 3 months and to 1.4 ± 0.5 at 1 year (P <.05); 6-minute walk test increased from 349 ± 116 m at baseline to 454 ± 144 m at 3 months and to 517 ± 206 m (P <.05); and quality of life increased from 54 ± 16 points at baseline to 28 ± 13 points at 3 months and 27 ± 15 points at 1 year (P <.05). There was also a progressive reduction in LV gradient from 74 ± 23 mmHg at baseline to 50 ± 27 mmHg acutely, 40 ± 26 mmHg at 3 months, and 28 ± 17 mmHg at 1 year (P <.05). Gradient reduction was associated with diminished peak longitudinal displacement of the LV septum and earlier displacement of the lateral wall. A progressive reduction of LV mass was observed, from 356 ± 110 g at baseline to 315 ± 70 g at 3 months (P = .13) and to 284 ± 42 g at 1 year (P <.05). CONCLUSION: Biventricular pacing is feasible and usually the best configuration for gradient reduction in HOCM. Biventricular pacing reduces LV hypertrophy.

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.

Toward an implantable wireless cardiac monitoring platform integrated with an FDA-approved cardiovascular stent.

Continuous monitoring of blood pressure from a minimally invasive device in the pulmonary artery can serve as a diagnostic and early warning system for cardiac health. The foremost challenge in such a device is the wireless transfer of data and power from within the blood vessel to an external device while maintaining unrestricted flow through the artery. We present a miniaturized system, which is attached to the outer surface of a regular or drug-eluting FDA-approved stent. When expanded, the stent maintains a patent vessel lumen while allowing contact between the electronic sensors and the blood supply. The stent-based antenna can be used for both wireless telemetry and power transfer for the implanted electronics. Using the stent platform as both a radiating antenna and a structural support allows us to take advantage of an FDA-approved device whose safety and surgical procedure are well established. The electronics package has been reduced to an area of less than 1 mm(2), with a thickness under 300 mum. A minimally invasive implantation procedure allows the delivery of the stent-based implant in nearly any major vessel of the body. This article describes an initial prototype with two stents configured as a single dipole, a 2.4-GHz transmitter microchip, and a battery, and validates transcutaneous transmission through ex vivo and in vivo porcine studies. The results demonstrate the feasibility of a stent-based wireless platform for continuous monitoring of blood pressure.

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.

Desfibrilador automático implantado en pacientes con miocardiopatía hipertrófica: criterios de selección, evolución y predictores de terapia apropiada / Automatic defibrillator implanted in patients with hypertrophic myocardiopathy: selection criteria, evolution and appropriate therapy predictors

Introducción y objetivos: la miocardiopatía hipertrófica es una enfermedad de origen genético con prevalencia de 1 porciento al 2 porciento. La mitad de los pacientes fallecen por muerte súbita cardiaca, la mayoría por arritmias ventriculares. Todavía no está claro a qué pacientes se les debe implantar un desfibrilador automático. El objetivo de este trabajo es describir una serie de pacientes con implante, los criterios empleados y los resultados obtenidos, así como analizar los predictores de terapia apropiada por el desfibrilador.Métodos: se incluyeron 20 pacientes que recibieron un desfibrilador de tercera generación. En todos se realizó estudio electrofisiológico y seguimiento prospectivo con registro de eventos. En 18 (90 porciento) se hizo estudio genético.

Introduction and objectives: hypertrophic myocardiopathy is a genetic entity with 1% to 2% prevalence. Half patients die of sudden cardiac death, most due to ventricular arrhythmias. There is still no clarity with regard to the patients to whom an automatic defibrillator has to be implanted. The objective of this work is to describe a series of patients with implant, the criteria used and the results obtained, as well as to analyze the predictors of appropriate therapy with the defibrillator. Methods: 20 patients that received a third generation defibrillator were included. Electrophysiological study and prospective follow-up with register of events was performed in all. Genetic study was done in 18 (90%). Results: 55% were men with mean age 40 (11-78) years. Six (30%) received implant for secondary prevention and 14 (70%) for primary prevention; the last ones because of several risk factors. A sustained arrhythmia was induced in 15 (75%) and in 3 (15%) monomorphic sustained ventricular tachycardia. At 22 months of follow-up, 4 (20%) underwent appropriate therapy and 2 (10%) died. Clinical monomorphic ventricular tachycardia (p=0.03) and the induced one (p<0.01) were significant therapy predictors. In 10 (56%) a mutation was identified; in 8 (44%) in the b-myosin gene. Conclusions: monomorphic sustained clinical ventricular tachycardia and the induced one were predictors of the appropriate defibrillator therapy in this series. The stratification based on the risk factors addition is actually a good option for primary prevention. Mutations in the heavy b-myosin chain are also the most frequent in our population.

[Clinical and echocardiographic follow-up of patients with hypertrophic obstructive cardiomyopathy treated by percutaneous septal ablation]. / Seguimiento clínico y ecocardiográfico de pacientes con miocardiopatía hipertrófica obstructiva tratados con ablación septal percutánea.

INTRODUCTION AND OBJECTIVES: Alcohol septal ablation is a therapeutic option for patients with hypertrophic obstructive cardiomyopathy who remain symptomatic despite medical treatment. Our aim was to monitor clinical and echocardiographic progression in patients with hypertrophic obstructive cardiomyopathy treated by septal ablation at our center. METHODS: Thirty-five septal ablations were performed in 34 patients (79% male) who had symptomatic hypertrophic obstructive cardiomyopathy despite optimum medical treatment. The procedure was successful in 32 (i.e., the reduction in left ventricular outflow tract pressure gradient, or LVOTPG, was >50%). During clinical and echocardiographic follow-up, New York Heart Association (NYHA) functional class and LVOTPG were monitored. RESULTS: The patients' mean age was 63 (12) years. The mean follow-up period was 9 (3) months. Immediately after septal ablation, LVOTPG decreased significantly, from 74.2 (25.3) mm Hg to 26 (25) mm Hg (P<.001), and remained low throughout follow-up. Moreover, echocardiography showed that the interventricular septum thickness also decreased during follow-up, from 19 (3) mm to 15 (2) mm (P<.0001). A significant improvement in NYHA functional class (from 93% in class III-IV to 84% in class I-II) was also observed. Two deaths occurred within 48 hours after the procedure. The most frequent complication was complete heart block (20%; n=6). CONCLUSIONS: Alcohol septal ablation is effective in patients with hypertrophic obstructive cardiomyopathy who remain symptomatic despite medical treatment. However, the procedure is associated with a significant rate of complications and should, therefore, be reserved for selected patients, in particular for elderly patients and those with comorbid conditions.

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.

[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.

Utility of ambulatory 24-hour esophageal pH and motility monitoring in noncardiac chest pain: report of 90 patients and review of the literature.

It is unclear whether prolonged motility monitoring improves the diagnostic yield of standard esophageal tests in patients with noncardiac chest pain. Our aim was to assess the diagnostic value of ambulatory 24-hr pH and pressure monitoring in patients with noncardiac chest pain. Stationary manometry, edrophonium testing, and ambulatory pH and motility studies were performed in 90 consecutive patients with recurrent chest pain and normal coronary angiograms. Normality limits of ambulatory 24-hr motility were established in 30 healthy controls. The diagnoses of specific esophageal motility disorders (nutcracker esophagus and diffuse esophageal spasm) by stationary and ambulatory manometry were discordant in 48% of the patients. Edrophonium testing was positive in 9 patients, but correlated poorly with esophageal diagnoses. During ambulatory studies, 144 chest pain events occurred in 42 patients, and 72 (50%) were related to esophageal dysfunction. Strict temporal associations of events with esophageal dysfunction in relation to ambulatory 24-hr pH/motility scores permitted four patient categorizations: true positives (event-related and abnormal tests), N = 15; true negatives (event-unrelated and abnormal tests), N = 10; reduced esophageal pain threshold (event-related and normal tests), N = 4; and indeterminate origin (event-unrelated and normal tests), N = 13. Overall, 19 patients (21%) had a probable esophageal cause for chest pain (14 esophageal motility disorder, 4 acid reflux, 1 both). In conclusion, ambulatory manometry increases the diagnostic yield of standard esophageal testing in noncardiac chest pain, but the gain is small. Causes of chest pain other than high esophageal pressures and acid reflux must still be sought in most patients with chest pain of unknown origin after a negative cardiac work-up.