Pierluigi Carullo

Genome-wide analysis of histone marks identifying an epigenetic signature of promoters and enhancers underlying cardiac hypertrophy

Significance Cardiac failure is a leading cause of mortality worldwide and a major financial burden for healthcare systems. New tools for understanding cardiovascular disease and developing better therapeutic approaches are therefore needed. To this end, transcriptional regulation has been extensively studied in cardiac hypertrophy and failure, but there is still a lack of understanding of the epigenetic framework in which transcription factors act. Our report adds significant knowledge to the field because we demonstrate, in vivo, that a complex and specific epigenetic signature regulates gene expression by modulating promoters and enhancers, a large number of which have been described here. These findings advance our understanding of the mechanisms underlying this pathology. Cardiac hypertrophy, initially an adaptive response of the myocardium to stress, can progress to heart failure. The epigenetic signature underlying this phenomenon is poorly understood. Here, we report on the genome-wide distribution of seven histone modifications in adult mouse cardiomyocytes subjected to a prohypertrophy stimulus in vivo. We found a set of promoters with an epigenetic pattern that distinguishes specific functional classes of genes regulated in hypertrophy and identified 9,207 candidate active enhancers whose activity was modulated. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. We propose that the epigenetic landscape is a key determinant of gene expression reprogramming in cardiac hypertrophy and provide a basis for understanding the role of chromatin in regulating this phenomenon.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

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.

Distinct Effects of Leukocyte and Cardiac Phosphoinositide 3-Kinase γ Activity in Pressure Overload–Induced Cardiac Failure

Background— Signaling from phosphoinositide 3-kinase &ggr; (PI3K&ggr;) is crucial for leukocyte recruitment and inflammation but also contributes to cardiac maladaptive remodeling. To better understand the translational potential of these findings, this study investigates the role of PI3K&ggr; activity in pressure overload–induced heart failure, addressing the distinct contributions of bone marrow–derived and cardiac cells. Methods and Results— After transverse aortic constriction, mice knock-in for a catalytically inactive PI3K&ggr; (PI3K&ggr; KD) showed reduced fibrosis and normalized cardiac function up to 16 weeks. Accordingly, treatment with a selective PI3K&ggr; inhibitor prevented transverse aortic constriction–induced fibrosis. To define the cell types involved in this protection, bone marrow chimeras, lacking kinase activity in the immune system or the heart, were studied after transverse aortic constriction. Bone marrow–derived cells from PI3K&ggr; KD mice were not recruited to wild-type hearts, thus preventing fibrosis and preserving diastolic function. After prolonged pressure overload, chimeras with PI3K&ggr; KD bone marrow–derived cells showed slower development of left ventricular dilation and higher fractional shortening than controls. Conversely, in the presence of a wild-type immune system, KD hearts displayed bone marrow–derived cell infiltration and fibrosis at early stages but reduced left ventricular dilation and preserved contractile function at later time points. Conclusions— Together, these data demonstrate that, in response to transverse aortic constriction, PI3K&ggr; contributes to maladaptive remodeling at multiple levels by modulating both cardiac and immune cell functions.

DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy

Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)—5-mCs oxidation product—in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease.

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction Cascade

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.

The Circulating Level of FABP3 Is an Indirect Biomarker of MicroRNA-1

OBJECTIVES This study sought to identify proteins from the cardiomyocyte (CM) secretome that are directly targeted by the muscle-specific microRNA-1 (miR-1), and thus reflect the pathophysiological state of the CM. BACKGROUND MicroRNAs play critical regulatory roles during myocardial remodeling and progression to heart failure. However, it remains unknown whether secreted microRNA-targeted proteins can be used as indicators of myocardial microRNA expression and function. METHODS A proteomic analysis based on multidimensional protein identification technology was performed on supernatants from cultured CMs overexpressing miR-1. Biochemical assays and an inducible cardiac-specific transgenic mouse model overexpressing miR-1 were used to demonstrate that heart-type fatty acid-binding protein-3 (FABP3) is a target of miR-1. Levels of miR-1 and FABP3 in cardiac tissue and plasma samples from mouse models as well as human patients were quantified by quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The study included wild-type mice subjected to ventricular pressure overload or fasting, as well as patients diagnosed with ventricular hypertrophy due to valvular aortic stenosis, acromegaly, or growth hormone deficiency, conditions associated with altered miR-1 expression. RESULTS An inverse relationship between myocardial expression of miR-1 and circulating levels of FABP3 was found both in vitro and in vivo under various pathological conditions. CONCLUSIONS Assessment of FABP3 plasma levels in human patients might be used for indirectly measuring cardiac miR-1 activity.

Bioinspired negatively charged calcium phosphate nanocarriers for cardiac delivery of MicroRNAs

AIM To develop biocompatible and bioresorbable negatively charged calcium phosphate nanoparticles (CaP-NPs) as an innovative therapeutic system for the delivery of bioactive molecules to the heart. MATERIALS & METHODS CaP-NPs were synthesized via a straightforward one-pot biomineralization-inspired protocol employing citrate as a stabilizing agent and regulator of crystal growth. CaP-NPs were administered to cardiac cells in vitro and effects of treatments were assessed. CaP-NPs were administered in vivo and delivery of microRNAs was evaluated. RESULTS CaP-NPs efficiently internalized into cardiomyocytes without promoting toxicity or interfering with any functional properties. CaP-NPs successfully encapsulated synthetic microRNAs, which were efficiently delivered into cardiac cells in vitro and in vivo. CONCLUSION CaP-NPs are a safe and efficient drug-delivery system for potential therapeutic treatments of polarized cells such as cardiomyocytes.

T cell costimulation blockade blunts pressure overload-induced heart failure

Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF.

Peptidomimetic Targeting of Cavβ2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function

Background: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. Methods: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cav&bgr;2 chaperone regulates channel density at the plasma membrane. Results: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cav&agr;1.2 and the Akt-dependent phosphorylation status of Cav&bgr;2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cav&bgr;2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cav&agr;1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cav&agr;1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cav&bgr;2, thus facilitating the chaperoning of Cav&agr;1.2; and promotion of Cav&agr;1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cav&bgr;2 to the nucleus, where it limits the transcription of Cav&agr;1.2 through recruitment of the heterochromatin protein 1&ggr; epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cav&bgr;2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. Conclusions: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cav&bgr;2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.