Beatrice Musumeci

Angiotensin II AT2 receptor subtype: an uprising frontier in cardiovascular disease?

The renin–angiotensin system (RAS) plays a pivotal role in the regulation of fluid, electrolyte balance and blood pressure, and is a modulator of cellular growth and proliferation. Biological actions of RAS are linked to the binding of the effector molecule, angiotensin II (AngII), to specific membrane receptors, mostly the AT1 subtype and, to a lesser extent, other subtypes. Following the identification and characterization of the AT2 subtype receptor, it has been proposed that a complex interaction between AngII and its receptors may play an important role in the effects of RAS. In this paper current information on AngII subtype receptors – their structure, regulation and intracellular signalling – are reviewed, with a particular emphasis on the potential relevance for cardiovascular pathophysiology. In addition, we discuss modulation of expression of the AT2 receptor and its interaction with the AT1 receptor subtype, as well as the potential effects of this receptor on blood pressure regulation. A better understanding of the integrated effects of the AngII subtype receptors may help to elucidate the function of the RAS, as well as their participation in the mechanisms of cardiovascular disease and attendant therapeutic implications.

Isoleucyl-tRNA synthetase levels modulate the penetrance of a homoplasmic m.4277T>C mitochondrial tRNA Ile mutation causing hypertrophic cardiomyopathy

The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patients clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.

Functional cross-talk between angiotensin II and epidermal growth factor receptors in NIH3T3 fibroblasts.

Background The main angiotensin (Ang) II subtype receptors (AT1R and AT2R) are involved in cellular growth processes and exert functionally antagonistic effects. Objective To characterize the mechanisms by which Ang II receptors influence growth, by investigating the interactions between Ang II subtype receptors and epidermal growth factor (EGF) receptors on mitogen-activated protein kinase (MAPK) pathway activation. Design and methods The experiments were performed using a mouse fibroblast cell line, NIH3T3, by transient co-transfection with rat AT1R or AT2R expression vectors, or both. Extracellular-signal-regulated kinase (ERK)1/2 phosphorylation was analysed by western blot and the ERK activity was evaluated using PathDetect, an in-vivo signal transduction pathway trans-reporting system. Selective Ang II receptor antagonists (losartan for AT1R and PD123319 for AT2R) were used to investigate the contributions of each receptor to the response observed. Results Our data show that, in this cellular model, both Ang II receptors phosphorylate ERK1/2. However, in the cells expressing AT1R, the EGF-induced MAPK pathway was enhanced in the presence of Ang II in a synergistic fashion. In contrast, a reduction of EGF-induced MAPK activation was observed in the cells expressing AT2R. In cells expressing both Ang II subtype receptors, Ang II promoted an enhancement of EGF-induced MAPK activation. However, in the presence of the AT1R antagonist, losartan, the effect of EGF was reduced. Conclusion These data indicate the existence of an opposite cross-talk of AT1R and AT2R with EGF receptors, and suggest a complex functional interaction between these pathways in the regulation of cellular growth processes.

Patterns of Left Ventricular Longitudinal Strain and Strain Rate in Olympic Athletes

BACKGROUND Two-dimensional speckle-tracking echocardiography is an emerging modality for the assessment of systolic and diastolic myocardial deformation in a broad variety of clinical scenarios. However, normal values and physiologic limits of left ventricular strain and strain rate in trained athletes are largely undefined. METHODS Two hundred consecutive Olympic athletes (grouped into skill, power, mixed, and endurance disciplines) and 50 untrained controls were evaluated by two-dimensional speckle-tracking echocardiography. Left ventricular global systolic longitudinal strain (GLS), systolic strain rate, early diastolic strain rate (SRE) and late diastolic strain rate (SRA) were calculated. RESULTS GLS was normal, although mildly lower, in athletes compared with controls (-18.1 ± 2.2% vs -19.4 ± 2.3%, P < .001), without differences related to type of sport. Systolic strain rate was also lower in athletes (-1.00 ± 0.15 vs -1.11 ± 0.15 sec(-1), P < .001), with the lowest value in endurance disciplines (-0.96 ± 0.13 sec(-1), P < .001). No difference existed for SRE (1.45 ± 0.32 vs 1.51 ± 0.35 sec(-1), P = .277), while SRA was lower in athletes (0.67 ± 0.25 vs 0.81 ± 0.20 sec(-1), P < .001). Both SRE (1.37 ± 0.30 sec(-1), P < .001) and SRA (0.62 ± 0.23 sec(-1), P < .001) showed the lowest values in endurance disciplines. The fifth and 95th percentiles calculated as reference values in athletes were as follows: for GLS, -15% and -22%; for systolic strain rate, -0.8 and -1.2 sec(-1); for SRE, 1.00 and 2.00 sec(-1); and for SRA, 0.30 and 1.20 sec(-1). CONCLUSION The present study shows that highly trained athletes have normal GLS and strain rate parameters of the left ventricle, despite mild differences compared with untrained controls. These data may be implemented as reference values for the clinical assessment of the athletes and to support the diagnosis of physiologic cardiac adaptations in borderline cases.

Unexpectedly Low Mutation Rates in Beta-Myosin Heavy Chain and Cardiac Myosin Binding Protein Genes in Italian Patients With Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere‐related genes have been implicated in HCM etiology, those encoding β‐myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high‐performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. J. Cell. Physiol. 226: 2894–2900, 2011.

Cardiopulmonary exercise test and sudden cardiac death risk in hypertrophic cardiomyopathy

Background In hypertrophic cardiomyopathy (HCM), most of the factors associated with the risk of sudden cardiac death (SCD) are also involved in the pathophysiology of exercise limitation. The present multicentre study investigated possible ability of cardiopulmonary exercise test in improving contemporary strategies for SCD risk stratification. Methods A total of 623 consecutive outpatients with HCM, from five tertiary Italian HCM centres, were recruited and prospectively followed, between September 2007 and April 2015. The study composite end point was SCD, aborted SCD and appropriate implantable cardioverter defibrillator (ICD) interventions. Results During a median follow-up of 3.7 years (25th–75th centile: 2.2–5.1 years), 25 patients reached the end point at 5 years (3 SCD, 4 aborted SCD, 18 appropriate ICD interventions). At multivariate analysis, ventilation versus carbon dioxide relation during exercise (VE/VCO2 slope) remains independently associated to the study end point either when challenged with the 2011 American College of Cardiology Foundation/American Heart Association guidelines-derived score (C index 0.748) or with the 2014 European Society of Cardiology guidelines-derived score (C index 0.750). A VE/VCO2 slope cut-off value of 31 showed the best accuracy in predicting the SCD end point within the entire HCM study cohort (sensitivity 64%, specificity 72%, area under the curve 0.72). Conclusions Our data suggest that the VE/VCO2 slope might improve SCD risk stratification, particularly in those HCM categories classified at low-intermediate SCD risk according to contemporary guidelines. There is a need for further larger studies, possibly on independent cohorts, to confirm our preliminary findings.

Congenital Accessory Mitral Valve Tissue Anomaly in a Patient with Genetically Confirmed Hypertrophic Cardiomyopathy

Accessory mitral valve tissue is a rare congenital cardiac anomaly that was initially described in children in association with other cardiac congenital abnormalities and, more recently, has also been reported in adults. The authors report a patient with genetically confirmed hypertrophic cardiomyopathy who also had a highly mobile, free-floating membrane-like structure in contiguity with the ventricular side of the anterior mitral valve leaflet, a feature consistent with the diagnosis of accessory mitral valve tissue.

Heart Failure Progression in Hypertrophic Cardiomyopathy – Possible Insights From Cardiopulmonary Exercise Testing –

BACKGROUND Heart failure (HF) progression and its complications represent major emergent concerns in hypertrophic cardiomyopathy (HCM). We investigated the possible adjunctive role of cardiopulmonary exercise testing (CPET) in predicting HF-related events. An exercise-derived risk model, theHYPertrophicExercise-derivedRiskHF(HYPERHF), has been developed. METHODSANDRESULTS A multicenter cohort of 620 consecutive HCM outpatients was recruited and followed (2007 to 2015). The endpoint was death from HF, cardiac transplantation, NYHA III-IV class progression, severe functional deterioration leading to hospitalization for septal reduction, and hospitalization for HF worsening. During a median follow-up of 3.8 years (25-75th centile: 2.3-5.3 years), 84 patients reached the endpoint. Peak circulatory power (peak oxygen consumption * peak systolic blood pressure), ventilatory efficiency and left atrial diameter were independently associated with the endpoint and, accordingly, integrated into the HYPERHFmodel (C index: 0.849; best cutoff value equal to 15%). CONCLUSIONS CPET is useful in the evaluation of HCM patients. In this context, the HYPERHFscore might allow early identification of those patients at high risk of HF progression and its complications. (Circ J 2016; 80: 2204-2211).

Genetic susceptibility to cerebrovascular accidents.

Summary: Cerebrovascular accidents are the third leading cause of death after myocardial infarction and cancer in all Western societies. A more complete understanding of the pathogenetic determinants of stroke is required in order to achieve a better prevention and treatment of this common disease. Recently, based on convincing epidemiological and experimental evidence, the concept of stroke as a complex, multifactorial, polygenic disease has been well assessed. Thus, together with known modifiable determinants, such as smoking, obesity, hypertension, cardiac diseases and diabetes, specific hereditary factors for stroke are now taken into account when analysing the pathogenesis of cerebrovascular accidents. In particular, there have recently been important findings related to the genetic basis of stroke in suitable animal models and in humans, thus representing the promise of a more thorough understanding of the pathogenesis of stroke in the future and of a more specific preventive and therapeutic approach to this common pathological condition.

A Next-Generation Sequencing Approach to Identify Gene Mutations in Early- and Late-Onset Hypertrophic Cardiomyopathy Patients of an Italian Cohort

Sequencing of sarcomere protein genes in patients fulfilling the clinical diagnostic criteria for hypertrophic cardiomyopathy (HCM) identifies a disease-causing mutation in 35% to 60% of cases. Age at diagnosis and family history may increase the yield of mutations screening. In order to assess whether Next-Generation Sequencing (NGS) may fulfil the molecular diagnostic needs in HCM, we included 17 HCM-related genes in a sequencing panel run on PGM IonTorrent. We selected 70 HCM patients, 35 with early (≤25 years) and 35 with late (≥65 years) diagnosis of disease onset. All samples had a 98.6% average of target regions, with coverage higher than 20× (mean coverage 620×). We identified 41 different mutations (seven of them novel) in nine genes: MYBPC3 (17/41 = 41%); MYH7 (10/41 = 24%); TNNT2, CAV3 and MYH6 (3/41 = 7.5% each); TNNI3 (2/41 = 5%); GLA, MYL2, and MYL3 (1/41=2.5% each). Mutation detection rate was 30/35 (85.7%) in early-onset and 8/35 (22.9%) in late-onset HCM patients, respectively (p < 0.0001). The overall detection rate for patients with positive family history was 84%, and 90.5% in patients with early disease onset. In our study NGS revealed higher mutations yield in patients with early onset and with a family history of HCM. Appropriate patient selection can increase the yield of genetic testing and make diagnostic testing cost-effective.