Gabriele Giacomo Schiattarella

Inhibition of Hypertrophy Is a Good Therapeutic Strategy in Ventricular Pressure Overload

Hypertrophic growth of ventricular myocytes is a hallmark feature of numerous forms of cardiovascular disease.1 The hypertrophic process is complex, involving a vast array of structural, signaling, transcriptional, electrophysiological, metabolic, and functional events within the growing cell.2,3 Other cellular elements within the ventricle, fibroblasts, vascular smooth muscle cells, and endothelium, also manifest intricate stress responses, resulting in fibrosis, inflammatory cell infiltration, endothelial dysfunction, and vascular stiffness. Current thinking holds that these events, the reaction of the heart to a host of pathological stresses, provide short-term benefit. However, if disease-related stress remains unchecked, these remodeling events become maladaptive and predispose to cardiovascular morbidity and mortality. Response by Crozatier and Ventura-Clapier on p 1447 Among the risks conferred by disease-related ventricular hypertrophy are ventricular tachyarrhythmia, predisposing to sudden cardiac death, and transition to heart failure. Ultimately, these events derive from wholesale reprogramming and relative dedifferentiation of the cardiac myocyte, coupled with similar events in other cell types. Conventional thinking holds that hypertrophic growth of the myocardium is a compensatory response of the heart to increases in workload demand, serving to minimize wall stress and maintain contractile function. However, several lines of evidence, preclinical and epidemiological, highlight the maladaptive features of chronic ventricular hypertrophy. Indeed, in many instances, suppression of load-induced growth is well tolerated.4 Furthermore, left ventricular hypertrophy is among the most robust markers of increased risk for developing chronic heart failure.5 Therefore, we submit that suppression of load-induced ventricular hypertrophy warrants careful consideration as a therapeutic strategy. Although evidence suggests that a small fraction of cells within the ventricle are capable of re-entering the cell cycle,6–8 the majority of cardiomyocytes are postmitotic and, hence, do not retain the ability to divide. Rather, they respond to stress by growing, shrinking, or dying. In …

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction CascadeNovelty and Significance

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate &bgr;-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of &bgr;-adrenergic receptors leads to impaired cardiac function, and &bgr;-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. Objective: To determine whether miR-133 affects &bgr;-adrenergic receptor signaling during progression to heart failure. Methods and Results: Based on bioinformatic analysis, &bgr;1-adrenergic receptor (&bgr;1AR) and other components of the &bgr;1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective &bgr;1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic &bgr;1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. Conclusions: miR-133 controls multiple components of the &bgr;1AR transduction cascade and is cardioprotective during heart failure.

AKAP121 downregulation impairs protective cAMP signals, promotes mitochondrial dysfunction, and increases oxidative stress

AIMS The aim of the present study was to determine the function and the role of the scaffold protein AKAP121, tethering cAMP dependent protein kinase A to the outer wall of mitochondria, in neonatal ventricular myocytes and the heart. METHODS AND RESULTS Competitive peptides displacing AKAP121 from mitochondria in the tissue and in the cells were used to investigate the role of AKAP121 in mitochondrial function, reactive oxygen species (ROS) generation, and cell survival. Displacement of AKAP121 from mitochondria by synthetic peptides triggers the death program in cardiomyocytes. Under pathological conditions in vivo, in a rat model of cardiac hypertrophy induced by ascending aorta banding, the levels of AKAP121 are significantly down-regulated. Disappearance of AKAP121 is associated with mitochondrial dysfunction, high oxidative stress, and apoptosis. In vivo delocalization of AKAP121 by competitive peptides replicates some of the molecular signatures induced by pressure overload: mitochondrial dysfunction, increased mitochondrial ROS, and apoptosis. CONCLUSION These data suggest that AKAP121 regulates the response to stress in cardiomyocytes, and therefore AKAP121 downregulation might represent an important event contributing to the development of cardiac dysfunction.

Therapeutic targeting of autophagy in cardiovascular disease

Autophagy is an evolutionarily ancient process of intracellular catabolism necessary to preserve cellular homeostasis in response to a wide variety of stresses. In the case of post-mitotic cells, where cell replacement is not an option, finely tuned quality control of cytoplasmic constituents and organelles is especially critical. And due to the ubiquitous and critical role of autophagic flux in the maintenance of cell health, it comes as little surprise that perturbation of the autophagic process is observed in multiple disease processes. A large body of preclinical evidence suggests that autophagy is a double-edged sword in cardiovascular disease, acting in either beneficial or maladaptive ways, depending on the context. In light of this, the autophagic machinery in cardiomyocytes and other cardiovascular cell types has been proposed as a potential therapeutic target. Here, we summarize current knowledge regarding the dual functions of autophagy in cardiovascular disease. We go on to analyze recent evidence suggesting that titration of autophagic flux holds potential as a novel treatment strategy.

Identification of the Ligands of Protein Interaction Domains through a Functional Approach

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1α, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

Genetic Deletion of Uncoupling Protein 3 Exaggerates Apoptotic Cell Death in the Ischemic Heart Leading to Heart Failure

Background Uncoupling protein 3 (ucp3) is a member of the mitochondrial anion carrier superfamily of proteins uncoupling mitochondrial respiration. In this study, we investigated the effects of ucp3 genetic deletion on mitochondrial function and cell survival under low oxygen conditions in vitro and in vivo. Methods and Results To test the effects of ucp3 deletion in vitro, murine embryonic fibroblasts and adult cardiomyocytes were isolated from wild‐type (WT, n=67) and ucp3 knockout mice (ucp3−/−, n=70). To test the effects of ucp3 genetic deletion in vivo, myocardial infarction (MI) was induced by permanent coronary artery ligation in WT and ucp3−/− mice. Compared with WT, ucp3−/− murine embryonic fibroblasts and cardiomyocytes exhibited mitochondrial dysfunction and increased mitochondrial reactive oxygen species generation and apoptotic cell death under hypoxic conditions in vitro (terminal deoxynucleotidyl transferase‐dUTP nick end labeling–positive nuclei: WT hypoxia, 70.3±1.2%; ucp3−/− hypoxia, 85.3±0.9%; P<0.05). After MI, despite similar areas at risk in the 2 groups, ucp3−/− hearts demonstrated a significantly larger infarct size compared with WT (infarct area/area at risk: WT, 48.2±3.7%; ucp3−/−, 65.0±2.9%; P<0.05). Eight weeks after MI, cardiac function was significantly decreased in ucp3−/− mice compared with WT (fractional shortening: WT MI, 42.7±3.1%; ucp3−/− MI, 24.4±2.9; P<0.05), and this was associated with heightened apoptotic cell death (terminal deoxynucleotidyl transferase‐dUTP nick end labeling–positive nuclei: WT MI, 0.7±0.04%; ucp3−/− MI, 1.1±0.09%, P<0.05). Conclusions Our data indicate that ucp3 levels regulate reactive oxygen species levels and cell survival during hypoxia, modulating infarct size in the ischemic heart.

A meta-analysis of the impact of pre-existing and new-onset atrial fibrillation on clinical outcomes in patients undergoing transcatheter aortic valve implantation.

AIMS Little is known about the prognostic role of pre-existing atrial fibrillation (AF) and new-onset AF (NOAF) in transcatheter aortic valve implantation (TAVI). Therefore, the aim of this meta-analysis was to compare the short- and long-term clinical outcomes of patients undergoing TAVI with and without pre-existing and new-onset AF. METHODS AND RESULTS Twenty-six studies, enrolling 14,078 patients undergoing TAVI, of whom 33.4% had pre-existing AF and 17.5% had NOAF, were analysed for early and long-term all-cause mortality, cardiovascular mortality and cerebrovascular events (CVE). In patients with pre-existing AF, 30-day all-cause mortality was similar to patients in sinus rhythm (SR). Conversely, long-term all-cause and cardiovascular mortality were significantly greater in pre-existing AF patients than in patients with SR (20 studies; 8,743 patients; HR: 1.68; p<0.00001, and three studies; 1,138 patients; HR: 2.07; p=0.01, respectively). Pre-existing AF was not a predictor of CVE at long-term follow-up. NOAF patients showed similar short- and long-term all-cause mortality when compared to patients in SR, whereas they experienced a significantly higher incidence of CVE at short-term follow-up (six studies; 2,025 patients; HR: 2.86; p<0.00001). A non-significant increase in the incidence of CVE was observed at long-term follow-up. CONCLUSIONS Pre-existing AF is a predictor of all-cause mortality in patients undergoing TAVI. NOAF is related to the occurrence of CVE at short-term follow-up. Similarly to surgical aortic valve replacement (SAVR), the optimal management and risk stratification of these patients should be further investigated.

Increased mortality after transcatheter aortic valve implantation (TAVI) in patients with severe aortic stenosis and low ejection fraction: A meta-analysis of 6898 patients

BACKGROUND There is conflicting evidence regarding the safety and efficacy of transcatheter aortic valve implantation (TAVI) procedures in patients with severe aortic stenosis and low left ventricular ejection fraction (EF). The primary aim of this study was to determine the impact of TAVI on short- and long-term mortality in patients with low EF (EF <50%); the secondary aim was to analyze the impact of TAVI procedure on EF recovery in the same setting of patients. METHODS AND RESULTS Twenty-six studies enrolling 6898 patients with severe aortic stenosis undergoing TAVI procedure were included in the meta-analysis and analyzed for 30-day, 6-month and 1-year all-cause and cardiovascular mortality; a further meta-analysis was also performed in patients with low EF to assess EF changes post TAVI. In low EF patients, both all-cause and cardiovascular short- and long-term mortality were significantly higher when compared to patients with normal EF (30-day-all-cause mortality: 0.13; 95% confidence interval [CI]: 0.01 to 0.25, I(2)=49.65, Q=21.85; 1-year-all-cause mortality: 0.25; 95% [CI]: 0.16 to 0.34, I(2)=25.57, Q=16.12; 30-day-cardiovascular mortality: 0.03; 95% [CI]: -0.31 to 0.36, I(2)=66.84, Q=6.03; 1-year-cardiovascular mortality: 0.29; 95% [CI]: 0.12 to 0.45, I(2)=0.00, Q=1.88). Nevertheless, in low EF patients TAVI was associated with a significant recovery of EF, which started at discharge and proceeded up to 1-year-follow-up. CONCLUSIONS Patients with low EF severe aortic stenosis have higher mortality following TAVI compared to normal EF patients, despite a significant and sustained improvement in EF.

Effects of successful percutaneous lower extremity revascularization on cardiovascular outcome in patients with peripheral arterial disease

BACKGROUND Lower extremity peripheral arterial disease (LE-PAD) reduces walking capacity and is associated with an increased cardiovascular risk. Endovascular revascularization of LE-PAD improves walking performance and quality of life. In the present study, we determined whether successful lower limbs revascularization also impacts cardiovascular outcome in LE-PAD patients. METHODS 479 consecutive LE-PAD patients at stage II of Fontaines classification, with ankle/brachial index ≤ 0.90 and one or more stenosis >50% in at least one leg artery, were enrolled in the study. According to the Trans-Atlantic Inter Society Consensus II recommendations, 264 (55.1%) underwent percutaneous lower extremity angioplasty (PTA group), while 215 (44.9%) were managed with conservative therapy (MT group). The incidence of major cardiovascular events (including cardiovascular death, myocardial infarction, ischemic stroke, coronary and carotid revascularizations) was prospectively analyzed by Kaplan-Meier curves. Crude and adjusted HRs (95% CI) of developing a cardiovascular event were calculated by Cox analysis. RESULTS No baseline differences were observed among the groups, except for a lower maximum walking distance in the PTA group. During a median follow-up of 21 months (12.0-29.0), the incidence of cardiovascular events was markedly lower in PTA compared to MT patients (6.4% vs. 16.3%; p=0.003), and patients in the MT group showed a 4.1-fold increased cardiovascular risk compared to patients in the PTA group, after adjustment for potential confounders (95% CI 1.22-13.57, p=0.023). CONCLUSIONS This study shows that successful revascularization of LE-PAD patients affected by intermittent claudication, in addition to improving functional status, reduces the occurrence of future major cardiovascular events.

Abdominal aortic aneurysm in patients affected by intermittent claudication: prevalence and clinical predictors

BackgroundAbdominal aortic aneurysm (AAA) is a frequent cause of death among elderly. Patients affected by lower extremity peripheral arterial disease (LE-PAD) seem to be particularly at high risk for AAA. We aimed this study at assessing the prevalence and the clinical predictors of the presence of AAA in a homogeneous cohort of LE-PAD patients affected by intermittent claudication.MethodsWe performed an abdominal ultrasound in 213 consecutive patients with documented LE-PAD (ankle/brachial index ≤0.90) attending our outpatient clinic for intermittent claudication. For each patient we registered cardiovascular risk factors and comorbidities, and measured neutrophil count.ResultsThe ultrasound was inconclusive in 3 patients (1.4%), thus 210 patients (169 males, 41 females, mean age 65.9 ± 9.8 yr) entered the study. Overall, AAA was present in 19 patients (9.0%), with a not significant higher prevalence in men than in women (10.1% vs 4.9%, p = 0.300). Patients with AAA were older (71.2 ± 7.0 vs 65.4 ± 9.9 years, p = 0.015), were more likely to have hypertension (94.7% vs 71.2%, p = 0.027), and greater neutrophil count (5.5 [4.5 – 6.2] vs 4.1 [3.2 – 5.5] x103/μL, p = 0.010). Importantly, the c-statistic for neutrophil count (0.73, 95% CI 0.60 – 0.86, p =0.010) was higher than that for age (0.67, CI 0.56–0.78, p = 0.017). The prevalence of AAA in claudicant patients with a neutrophil count ≥ 5.1 x103/μL (cut-off identified at ROC analysis) was as high as 29.0%.ConclusionsPrevalence of AAA in claudicant patients is much higher than that reported in the general population. Ultrasound screening should be considered in these patients, especially in those with an elevated neutrophil count.