A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway.

The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

Rationale: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process.

Loss of ADAMTS19 causes progressive non-syndromic heart valve disease.

Valvular heart disease is observed in approximately 2% of the general population. Although the initial observation is often localized (for example, to the aortic or mitral valve), disease manifestations are regularly observed in the other valves and patients frequently require surgery. Despite the high frequency of heart valve disease, only a handful of genes have so far been identified as the monogenic causes of disease.

Myofibroblast-Specific TGFβ Receptor II Signaling in the Fibrotic Response to Cardiac Myosin Binding Protein C-Induced Cardiomyopathy.

Rationale: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc3).

Cardiac Dysfunction in the Sigma 1 Receptor Knockout Mouse Associated With Impaired Mitochondrial Dynamics and Bioenergetics.

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown.

Cardiac Fibrosis in Proteotoxic Cardiac Disease is Dependent Upon Myofibroblast TGF -β Signaling.

Background Transforming growth factor beta ( TGF -β) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte-specific expression of a mutant αB-crystallin (Cry AB), which causes human desmin-related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so-called myofibroblast state and TGF -β binding mediates an essential signaling pathway underlying this process.

In Vivo Remodeling of an Extracellular Matrix Cardiac Patch in an Ovine Model.

Lack of an ideal patch material for cardiac repairs continues to challenge congenital heart surgeons. The current materials are unable to grow and result in scarring, contraction, and arrhythmias. An acellular extracellular matrix (ECM) patch derived from porcine small intestinal submucosa has demonstrated remodeling potential when used to repair various tissues.

Activation of Autophagy Ameliorates Cardiomyopathy in -Targeted Knockin Mice.

Background: Alterations in autophagy have been reported in hypertrophic cardiomyopathy (HCM) caused by Danon disease, Vici syndrome, or LEOPARD syndrome, but not in HCM caused by mutations in genes encoding sarcomeric proteins, which account for most of HCM cases. , encoding cMyBP-C (cardiac myosin-binding protein C), is the most frequently mutated HCM gene.

Methods And Results: We evaluated autophagy in patients with HCM carrying mutations and in a -targeted knockin HCM mouse model, as well as the effect of autophagy modulators on the development of cardiomyopathy in knockin mice.

MMI-0100 Inhibits Cardiac Fibrosis in a Mouse Model Overexpressing Cardiac Myosin Binding Protein C.

Background: Cardiac stress can trigger production of a 40-kDa peptide fragment derived from the amino terminus of the cardiac myosin-binding protein C. Cardiac stress, as well as cMyBP-C mutations, can trigger production of 1 such truncated protein fragment, a 40-kDa peptide fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40-kDa fragment in mouse cardiomyocytes (cMyBP-C) leads to cardiac disease, fibrosis, and death within the first year.

Inhibition of Mutant αB Crystallin-Induced Protein Aggregation by a Molecular Tweezer.

Background: Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid β-protein, tau, and α-synuclein.

In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region.

Sickle cell anemia mice develop a unique cardiomyopathy with restrictive physiology.

Cardiopulmonary complications are the leading cause of mortality in sickle cell anemia (SCA). Elevated tricuspid regurgitant jet velocity, pulmonary hypertension, diastolic, and autonomic dysfunction have all been described, but a unifying pathophysiology and mechanism explaining the poor prognosis and propensity to sudden death has been elusive. Herein, SCA mice underwent a longitudinal comprehensive cardiac analysis, combining state-of-the-art cardiac imaging with electrocardiography, histopathology, and molecular analysis to determine the basis of cardiac dysfunction.

Alterations in Multi-Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein-C.

Background: The geometric organization of myocytes in the ventricular wall comprises the structural underpinnings of cardiac mechanical function. Cardiac myosin binding protein-C (MYBPC3) is a sarcomeric protein, for which phosphorylation modulates myofilament binding, sarcomere morphology, and myocyte alignment in the ventricular wall. To elucidate the mechanisms by which MYBPC3 phospho-regulation affects cardiac tissue organization, we studied ventricular myoarchitecture using generalized Q-space imaging (GQI).

Accelerated cardiac remodeling in desmoplakin transgenic mice in response to endurance exercise is associated with perturbed Wnt/β-catenin signaling.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis.

Disturbance in Z-disk mechanosensitive proteins induced by a persistent mutant myopalladin causes familial restrictive cardiomyopathy.

Background: Familial restrictive cardiomyopathy (FRCM) has a poor prognosis due to diastolic dysfunction and restrictive physiology (RP). Myocardial stiffness, with or without fibrosis, underlie RP, but the mechanism(s) of restrictive remodeling is unclear. Myopalladin (MYPN) is a messenger molecule that links structural and gene regulatory molecules via translocation from the Z-disk and I-bands to the nucleus in cardiomyocytes.

Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryAB(R120G)), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes.

Asymmetric cell-matrix and biomechanical abnormalities in elastin insufficiency induced aortopathy.

Aortopathy is characterized by vascular smooth muscle cell (VSMC) abnormalities and elastic fiber fragmentation. Elastin insufficient (Eln (+/-)) mice demonstrate latent aortopathy similar to human disease. We hypothesized that aortopathy manifests primarily in the aorto-pulmonary septal (APS) side of the thoracic aorta due to asymmetric cardiac neural crest (CNC) distribution.

TGF-β mediates early angiogenesis and latent fibrosis in an Emilin1-deficient mouse model of aortic valve disease.

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation.

Cardiac-specific VLCAD deficiency induces dilated cardiomyopathy and cold intolerance.

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial β-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death.

Enhanced autophagy ameliorates cardiac proteinopathy.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein.

Functional dissection of myosin binding protein C phosphorylation.

Cardiac myosin binding protein C (cMyBP-C) phosphorylation is differentially regulated in the normal heart and during disease development. Our objective was to examine in detail three phosphorylatable sites (Ser-273, Ser-282, and Ser-302) present in the protein's cardiac-specific sequences, as these residues are differentially and reversibly phosphorylated during normal and abnormal cardiac function. Three transgenic lines were generated: DAA, which expressed cMyBP-C containing Asp-273, Ala-282, and Ala-302, in which a charged amino acid was placed at residue 273 and the remaining two sites rendered nonphosphorylatable by substituting alanines for the two serines; AAD containing Ala-273, Ala-282, and Asp-302, in which aspartate was placed at residue 302 and the remaining two sites rendered nonphosphorylatable; and SDS containing Ser-273, Asp-282, and Ser-302.

Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP.

The COP9 signalosome is required for autophagy, proteasome-mediated proteolysis, and cardiomyocyte survival in adult mice.

Background: The COP9 signalosome (CSN) is an evolutionarily conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8). We have recently discovered in perinatal mouse hearts that CSN regulates not only proteasome-mediated proteolysis but also macroautophagy. However, the physiological significance of CSN in a post-mitotic organ of adult vertebrates has not been determined.

An endogenously produced fragment of cardiac myosin-binding protein C is pathogenic and can lead to heart failure.

Rationale: A stable 40-kDa fragment is produced from cardiac myosin-binding protein C when the heart is stressed using a stimulus, such as ischemia-reperfusion injury. Elevated levels of the fragment can be detected in the diseased mouse and human heart, but its ability to interfere with normal cardiac function in the intact animal is unexplored.

Objective: To understand the potential pathogenicity of the 40-kDa fragment in vivo and to investigate the molecular pathways that could be targeted for potential therapeutic intervention.

Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves.

Bicuspid or bifoliate aortic valve (BAV) results in two rather than three cusps and occurs in 1-2% of the population placing them at higher risk of developing progressive aortic valve disease. Only NOTCH-1 has been linked to human BAV, and genetically modified mouse models of BAV are limited by low penetrance and additional malformations. Here we report that in the Adamts5(-/-) valves, collagen I, collagen III, and elastin were disrupted in the malformed hinge region that anchors the mature semilunar cusps and where the ADAMTS5 proteoglycan substrate versican, accumulates.