Giuseppe Rengo

Analysis of AAV Serotypes 1–9 Mediated Gene Expression and Tropism in Mice After Systemic Injection

This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.

Adrenergic Nervous System in Heart Failure Pathophysiology and Therapy

Heart failure (HF), the leading cause of death in the western world, develops when a cardiac injury or insult impairs the ability of the heart to pump blood and maintain tissue perfusion. It is characterized by a complex interplay of several neurohormonal mechanisms that become activated in the syndrome to try and sustain cardiac output in the face of decompensating function. Perhaps the most prominent among these neurohormonal mechanisms is the adrenergic (or sympathetic) nervous system (ANS), whose activity and outflow are enormously elevated in HF. Acutely, and if the heart works properly, this activation of the ANS will promptly restore cardiac function. However, if the cardiac insult persists over time, chances are the ANS will not be able to maintain cardiac function, the heart will progress into a state of chronic decompensated HF, and the hyperactive ANS will continue to push the heart to work at a level much higher than the cardiac muscle can handle. From that point on, ANS hyperactivity becomes a major problem in HF, conferring significant toxicity to the failing heart and markedly increasing its morbidity and mortality. The present review discusses the role of the ANS in cardiac physiology and in HF pathophysiology, the mechanisms of regulation of ANS activity and how they go awry in chronic HF, methods of measuring ANS activity in HF, the molecular alterations in heart physiology that occur in HF, along with their pharmacological and therapeutic implications, and, finally, drugs and other therapeutic modalities used in HF treatment that target or affect the ANS and its effects on the failing heart.

G Protein–Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure

Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated &bgr;AR with a GRK2-derived peptide that binds G&bgr;&ggr; (&bgr;ARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and &bgr;ARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (&agr;MHC-Cre×GRK2 fl/fl) or only after administration of tamoxifen (&agr;MHC-MerCreMer×GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved &bgr;AR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring &bgr;AR signaling by other means to improve outcomes in heart failure.

Myocardial Adeno-Associated Virus Serotype 6–βARKct Gene Therapy Improves Cardiac Function and Normalizes the Neurohormonal Axis in Chronic Heart Failure

Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. The peptide &bgr;ARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve &bgr;AR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term &bgr;ARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results— In HF rats, we delivered &bgr;ARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the &bgr;-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. &bgr;ARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac &bgr;AR signaling. Addition of metoprolol neither enhanced nor decreased &bgr;ARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-&bgr;ARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, &bgr;ARKct alone improves outcomes more than a &bgr;-blocker alone, whereas both treatments are compatible. These findings show that &bgr;ARKct gene therapy can be of long-term therapeutic value in HF.

Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of β-adrenergic receptor (βAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting α2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial α2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein–coupled receptor kinase 2 (GRK2). Adrenal gland–specific GRK2 inhibition reversed α2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac βAR signaling and function, and increased sympatholytic efficacy of a α2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Stable Myocardial-Specific AAV6-S100A1 Gene Therapy Results in Chronic Functional Heart Failure Rescue

Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca2+)-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over &bgr;-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

Natriuretic Peptide-Guided Therapy in Chronic Heart Failure: A Meta-Analysis of 2,686 Patients in 12 Randomized Trials

Background The role of cardiac natriuretic peptides in the management of patients with chronic heart failure (HF) remains uncertain. The purpose of this study was to evaluate whether natriuretic peptide-guided therapy, compared to clinically-guided therapy, improves mortality and hospitalization rate in patients with chronic HF. Methodology/Principal Findings MEDLINE, Cochrane, ISI Web of Science and SCOPUS databases were searched for articles reporting natriuretic peptide-guided therapy in HF until August 2012. All randomized trials reporting clinical end-points (all-cause mortality and/or HF-related hospitalization and/or all-cause hospitalization) were included. Meta-analysis was performed to assess the influence of treatment on outcomes. Sensitivity analysis was performed to test the influence of potential effect modifiers and of each trial included in meta-analysis on results. Twelve trials enrolling 2,686 participants were included. Natriuretic peptide-guided therapy (either B-type natriuretic peptide [BNP]- or N-terminal pro-B-type natriuretic peptide [NT-proBNP]-guided therapy) significantly reduced all-cause mortality (Odds Ratio [OR]:0.738; 95% Confidence Interval [CI]:0.596 to 0.913; p = 0.005) and HF-related hospitalization (OR:0.554; CI:0.399 to 0.769; p = 0.000), but not all-cause hospitalization (OR:0.803; CI:0.629 to 1.024; p = 0.077). When separately assessed, NT-proBNP-guided therapy significantly reduced all-cause mortality (OR:0.717; CI:0.563 to 0.914; p = 0.007) and HF-related hospitalization (OR:0.531; CI:0.347 to 0.811; p = 0.003), but not all-cause hospitalization (OR:0.779; CI:0.414 to 1.465; p = 0.438), whereas BNP-guided therapy did not significantly reduce all-cause mortality (OR:0.814; CI:0.518 to 1.279; p = 0.371), HF-related hospitalization (OR:0.599; CI:0.303 to 1.187; p = 0.142) or all-cause hospitalization (OR:0.726; CI:0.609 to 0.964; p = 0.077). Conclusions/Significance Use of cardiac peptides to guide pharmacologic therapy significantly reduces mortality and HF related hospitalization in patients with chronic HF. In particular, NT-proBNP-guided therapy reduced all-cause mortality and HF-related hospitalization but not all-cause hospitalization, whereas BNP-guided therapy did not significantly reduce both mortality and morbidity.

Multidimensional Prognostic Index Based on a Comprehensive Geriatric Assessment Predicts Short-Term Mortality in Older Patients With Heart Failure

Background—Multidimensional impairment of older patients may influence the clinical outcome of diseases. The aim of this study was to evaluate whether a Multidimensional Prognostic Index (MPI) based on a comprehensive geriatric assessment predicts short-term mortality in older patients with heart failure. Methods and Results—In this prospective study with a 1-month follow-up, 376 patients aged 65 and older with a diagnosis of heart failure were enrolled. A standardized comprehensive geriatric assessment that included information on functional (activities of daily living and instrumental activities of daily living), cognitive (Short Portable Mental Status Questionnaire), and nutritional status (Mini Nutritional Assessment), as well as on risk of pressure sore (Exton-Smith Scale), comorbidities (Cumulative Illness Rating Scale Index), medications, and social support network, was used to calculate the MPI for mortality using a previously validated algorithm. The New York Heart Association, the Enhanced Feedback for Effective Cardiac Treatment, and the Acute Decompensated Heart Failure National Registry regression model scores were also calculated. Higher MPI values were significantly associated with higher 30-day mortality, both in men (MPI-1, 2.8%; MPI-2, 15.3%; MPI-3, 47.4%; P=0.000) and women (MPI-1, 0%; MPI-2, 6.5%; MPI-3, 14.6%; P=0.011). The discrimination of the MPI was also good, with areas under the receiver operating characteristic curves (men: 0.83; 95% CI, 0.75 to 0.90; women: 0.80; 95% CI, 0.71 to 0.89) greater than receiver operating characteristic areas of New York Heart Association (men: 0.63; 95% CI, 0.57 to 0.69; P=0.015; women: 0.65; 95% CI, 0.55 to 0.75; P=0.064), Enhanced Feedback for Effective Cardiac Treatment (men: 0.69; 95% CI, 0.58 to 0.79; P=0.045; women: 0.71; 95% CI, 0.55 to 0.87; P=0.443), and Acute Decompensated Heart Failure National Registry scores (men: 0.65; 95% CI, 0.52 to 0.78; P=0.023; women: 0.67; 95% CI, 0.49 to 0.83, P=0.171). Conclusions—The MPI, calculated from information collected in a standardized comprehensive geriatric assessment, is useful to estimate the risk of 1-month mortality in older patients with heart failure.

The emerging role of microRNAs in Alzheimer's disease

MicroRNAs (miRNAs) are small non-coding RNA which have been shown to regulate gene expression. The alteration ofmiRNAs expression has been associated with several pathological processes, including neurodegeneration. In the search for easily accessible and non-invasive biomarkers for Alzheimers disease (AD) diagnosis and prognosis, circulating miRNAs are among the most promising candidates. Some of them have been consistently identified as AD-specific miRNAs and their targets also seem implicated in pathophysiological processes underlying AD. Here, we review the emerging role for miRNA in AD, giving an overview on general miRNAs biology, their implications in AD pathophysiology and their potential role as future biomarkers.

G Protein–Coupled Receptor Kinase 2 Activity Impairs Cardiac Glucose Uptake and Promotes Insulin Resistance After Myocardial Ischemia

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the &bgr;-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through &bgr;-adrenergic receptors and metabolism through the insulin receptor.Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile.