Phospholipase Cε hydrolyzes perinuclear phosphatidylinositol 4-phosphate to regulate cardiac hypertrophy.

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.

Implementing comprehensive pharmacogenomics in a community hospital-associated primary care setting.

BACKGROUND: Pharmacogenomics (PGx) is an emerging field. Many drug-gene interactions are known but not yet routinely addressed in clinical practice. Therefore, there is a significant gap in care, necessitating development of implementation strategies. OBJECTIVE: The objective of the study was to assess the impact of implementing a PGx practice model which incorporates comprehensive pharmacogenomic risk evaluation, testing and medication optimization administered by 7 PGx-certified ambulatory care pharmacists embedded across 30 primary care clinic sites. METHODS: Pharmacogenomic services were implemented in 30 primary care clinics within the Cincinnati, Ohio area. Patients are identified for pharmacogenomic testing using a clinical decision support tool (CDST) that is fully integrated in the electronic medical record (EMR) or by provider designation (e.g., psychotropic drug failure). Pharmacogenomic testing is performed via buccal swab using standardized clinic processes. Discrete data results are returned directly into the EMR/CDST for review by PGx-certified ambulatory care pharmacists. Recommendations and prescriptive changes are then discussed and implemented as a collaborative effort between pharmacist, primary care provider, specialists, and patient. RESULTS: A total of 422 unique interactions were assessed by the embedded ambulatory care PGx pharmacists (N = 7) during this interim analysis. About half (213) were pharmacogenomic interactions, and of these, 124 were actionable. When an intervention was actionable, 82% of the time a change in medication was recommended. The underlying reasons for recommending therapy alterations were most commonly ineffective therapy (43%), adverse drug reaction prevented (34%), or adverse drug reaction observed (13%). CONCLUSION: Variations in drug metabolism, response, and tolerability can negatively impact patient outcomes across many disease states and treatment specialties. Incorporation of pharmacogenomic testing with accessible clinical decision support into the team-based care model allows for a truly comprehensive review and optimization of medications. Our initial analysis suggests that comprehensive PGx testing should be considered to enhance medication safety and efficacy in at-risk patients.

Comparison of targeted vs. expanded pharmacogenomic testing: What are we missing?

BACKGROUND: Pharmacogenomics (PGx) is used as a medication management strategy by a small but growing number of institutions. PGx allows prescribers to individually treat patients concordant with their genes. Recent litigation for preventable PGx-mediated adverse events highlights the need to accelerate PGx implementation for patient safety. Genetic variations cause drug metabolism, transport, and target changes, affecting medication response and tolerability. PGx testing often consists of targeted testing aimed at specific gene-drug pairs or disease states. Conversely, expanded panel testing can evaluate all known actionable gene-drug interactions, enhancing proactive clarity regarding patient response. OBJECTIVES: Evaluate the divergence of targeted PGx testing with a single gene-drug pair test (cardiac), a two-gene panel, and a focused psychiatric panel compared to expanded PGx testing. METHODS: An expanded PGx panel (≥25 genes) was compared to a single gene-drug pair test of CYP2C19/clopidogrel, a dual gene test of CYP2C19/CYP2D6, a 7-gene psychiatric list, and a 14-gene psychiatric panel to inform specific depression and pain management drugs. The expanded panel provided a baseline to evaluate total PGx variations compared to those possibly missed by targeted testing. RESULTS: Targeted testing did not identify up to 95% of total PGx gene-drug interactions discovered. The expanded panel reported all gene-drug interactions for any medication with Clinical Pharmacogenomics Implementation Consortium (CPIC) guidance or U.S. Food and Drug Administration (FDA) labeling for that gene. Single gene CYP2C19/clopidogrel testing missed or did not report on ∼95% of total interactions, CYP2C19/CYP2D6 testing missed or did not report ∼89%, and the 14-gene panel missed or did not report on ∼73%. The 7-gene list missed ∼20% of discovered potential PGx interactions but was not designed to identify gene-drug interactions. CONCLUSIONS: Targeted PGx testing for limited genes or by specialty may miss or not report significant portions of PGx gene-drug interactions. This can lead to potential patient harm from the missed interactions and subsequent failed therapies and/or adverse reactions.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Research Priorities for Heart Failure With Preserved Ejection Fraction: National Heart, Lung, and Blood Institute Working Group Summary.

Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.

cellHarmony: cell-level matching and holistic comparison of single-cell transcriptomes.

To understand the molecular pathogenesis of human disease, precision analyses to define alterations within and between disease-associated cell populations are desperately needed. Single-cell genomics represents an ideal platform to enable the identification and comparison of normal and diseased transcriptional cell populations. We created cellHarmony, an integrated solution for the unsupervised analysis, classification, and comparison of cell types from diverse single-cell RNA-Seq datasets. cellHarmony efficiently and accurately matches single-cell transcriptomes using a community-clustering and alignment strategy to compute differences in cell-type specific gene expression over potentially dozens of cell populations. Such transcriptional differences are used to automatically identify distinct and shared gene programs among cell-types and identify impacted pathways and transcriptional regulatory networks to understand the impact of perturbations at a systems level. cellHarmony is implemented as a python package and as an integrated workflow within the software AltAnalyze. We demonstrate that cellHarmony has improved or equivalent performance to alternative label projection methods, is able to identify the likely cellular origins of malignant states, stratify patients into clinical disease subtypes from identified gene programs, resolve discrete disease networks impacting specific cell-types, and illuminate therapeutic mechanisms. Thus, this approach holds tremendous promise in revealing the molecular and cellular origins of complex disease.

Myofibroblast-Specific TGFß 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 (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. OBJECTIVE: TGFß (transforming growth factor-ß) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFß signaling during chronic Mybpc340kDa expression. METHODS AND RESULTS: To specifically block TGFß signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFß receptor II (TßRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. CONCLUSIONS: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFß signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.

Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure.

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

Inhibition of fibronectin polymerization alleviates kidney injury due to ischemia-reperfusion.

Fibrosis is a common feature of chronic kidney disease; however, no clinical therapies effectively target the progression of fibrosis. Inhibition of fibronectin polymerization with the small peptide pUR4 attenuates fibrosis in the liver and heart. Here, we show that pUR4 decreases renal fibrosis and tissue remodeling using a clinically relevant model of kidney injury, unilateral ischemia-reperfusion. This work highlights the benefits of inhibiting matrix polymerization, alone or in conjunction with cell-based therapies, as a novel approach to diminish the maladaptive responses to ischemic kidney injury that lead to chronic renal failure.

Diagnostic performance of quantitative magnetic resonance imaging biomarkers for predicting portal hypertension in children and young adults with autoimmune liver disease.

BACKGROUND: Primary sclerosing cholangitis, autoimmune hepatitis and autoimmune sclerosing cholangitis are forms of chronic, progressive autoimmune liver disease (AILD) that can affect the pediatric population. OBJECTIVE: To determine whether quantitative MRI- and laboratory-based biomarkers are associated with conventional imaging findings of portal hypertension (radiologic portal hypertension) in children and young adults with AILD. MATERIALS AND METHODS: Forty-four patients with AILD enrolled in an institutional registry underwent a research abdominal MRI examination at 1.5 tesla (T). Five quantitative MRI techniques were performed: liver MR elastography, spleen MR elastography, liver iron-corrected T1 mapping, liver T2 mapping, and liver diffusion-weighted imaging (DWI, quantified as apparent diffusion coefficients). Two anatomical sequences were used to document splenomegaly, varices and ascites. We calculated aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores - laboratory-based biomarkers of liver fibrosis. We used receiver operating characteristic (ROC) curve analyses to establish the diagnostic performance of quantitative MRI and laboratory biomarkers for indicating the presence of radiologic portal hypertension. RESULTS: Twenty-three (52%) patients were male; mean age was 15.2±4.0 years. Thirteen (30%) patients had radiologic portal hypertension. Liver and spleen stiffness demonstrated the greatest diagnostic performance for indicating the presence of portal hypertension (area-under-the-ROC-curve [AUROC]=0.98 and 0.96, respectively). The APRI and FIB-4 scores also demonstrated good diagnostic performance (AUROC=0.87 and 0.88, respectively). CONCLUSION: MRI-derived measures of liver and spleen stiffness as well as laboratory-based APRI and FIB-4 scores are highly associated with imaging findings of portal hypertension in children and young adults with AILD and thus might be useful for predicting portal hypertension impending onset and directing personalized patient management.

Cardiac Fibrosis: The Fibroblast Awakens.

Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however, upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of the cardiac fibroblast impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis.

G Protein-Coupled Receptor-G-Protein ßγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein ßγ-subunit (Gßγ), GPCR-kinase 2, and ß-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gßγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gßγ signaling and ET system expression. Notably, systemic pharmacologic Gßγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gßγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gßγ in AKI and suggest GPCR-Gßγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.

Targeting GPCR-Gßγ-GRK2 signaling as a novel strategy for treating cardiorenal pathologies.

The pathologic crosstalk between the heart and kidney is known as cardiorenal syndrome (CRS). While the specific mechanisms underlying this crosstalk remain poorly understood, CRS is associated with exacerbated dysfunction of either or both organs and reduced survival. Maladaptive fibrotic remodeling is a key component of both heart and kidney failure pathogenesis and progression. G-protein coupled receptor (GPCR) signaling is a crucial regulator of cardiovascular and renal function. Chronic/pathologic GPCR signaling elicits the interaction of the G-protein Gßγ subunit with GPCR kinase 2 (GRK2), targeting the receptor for internalization, scaffolding to pathologic signals, and receptor degradation. Targeting this pathologic Gßγ-GRK2 interaction has been suggested as a possible strategy for the treatment of HF. In the current review, we discuss recent updates in understanding the role of GPCR-Gßγ-GRK2 signaling as a crucial mediator of maladaptive organ remodeling detected in HF and kidney dysfunction, with specific attention to small molecule-mediated inhibition of pathologic Gßγ-GRK2 interactions. Further, we explore the potential of GPCR-Gßγ-GRK2 signaling as a possible therapeutic target for cardiorenal pathologies.

G Protein Coupled Receptor-mediated Transactivation of Extracellular Proteases.

G protein-coupled receptors (GPCRs) comprise the largest family of receptors in humans. Traditional activation of GPCRs involves binding of a ligand to the receptor, activation of heterotrimeric G proteins and induction of subsequent signaling molecules. It is now known that GPCR signaling occurs through G protein-independent pathways including signaling through ß-arrestin and transactivation of other receptor types. Generally, transactivation occurs when activation of one receptor leads to the activation of another receptor(s). GPCR-mediated transactivation is an essential component of GPCR signaling, as activation of other receptor types, such as receptor tyrosine kinases, allows GPCRs to expand their signal transduction and affect various cellular responses. Several mechanisms have been identified for receptor transactivation downstream of GPCRs, one of which involves activation of extracellular proteases, such as a disintegrin and metalloprotease, and matrix metalloproteases . These proteases cleave and release ligands that are then able to activate their respective receptors. A disintegrin and metalloprotease, and matrix metalloproteases can be activated via various mechanisms downstream of GPCR activation, including activation via second messenger, direct phosphorylation, or direct G protein interaction. Additional understanding of the mechanisms involved in GPCR-mediated protease activation and subsequent receptor transactivation could lead to identification of new therapeutic targets.

Extracellular matrix-mediated cellular communication in the heart.

The extracellular matrix (ECM) is a complex and dynamic scaffold that maintains tissue structure and dynamics. However, the view of the ECM as an inert architectural support has been increasingly challenged. The ECM is a vibrant meshwork, a crucial organizer of cellular microenvironments. It plays a direct role in cellular interactions regulating cell growth, survival, spreading, proliferation, differentiation and migration through the intricate relationship among cellular and acellular tissue components. This complex interrelationship preserves cardiac function during homeostasis; however it is also responsible for pathologic remodeling following myocardial injury. Therefore, enhancing our understanding of this cross-talk may provide mechanistic insights into the pathogenesis of heart failure and suggest new approaches to novel, targeted pharmacologic therapies. This review explores the implications of ECM-cell interactions in myocardial cell behavior and cardiac function at baseline and following myocardial injury.

From Bench to Bedside: New Approaches to Therapeutic Discovery for Heart Failure.

Heart failure is a significant global health problem, which is becoming worse as the population ages, and remains one of the biggest burdens on our economy. Despite significant advances in cardiovascular medicine, management and surgery, mortality rates remain high, with almost half of patients with heart failure dying within five years of diagnosis. As a multifactorial clinical syndrome, heart failure still represents an epidemic threat, highlighting the need for deeper insights into disease mechanisms and the development of innovative therapeutic strategies for both treatment and prevention. In this review, we discuss conventional heart failure therapies and highlight new pharmacological agents targeting pathophysiological features of the failing heart, for example, non-coding RNAs, angiotensin receptor-neprilysin inhibitors, cardiac myosin activators, BGP-15 and molecules targeting GRK2 including M119, gallein and paroxetine. Finally, we address the disparity between phase II and phase III clinical trials that prevent the translation of emerging HF therapies into new and approved therapies.

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. METHODS AND RESULTS: We generated cardiac myocyte-specific transgenic mice using a Tet-Off inducible system to permit controlled expression of the 40-kDa fragment in cardiomyocytes. When expression of the 40-kDa protein is induced by crossing the responder animals with tetracycline transactivator mice under conditions in which substantial quantities approximating those observed in diseased hearts are reached, the double-transgenic mice subsequently experience development of sarcomere dysgenesis and altered cardiac geometry, and the heart fails between 12 and 17 weeks of age. The induced double-transgenic mice had development of cardiac hypertrophy with myofibrillar disarray and fibrosis, in addition to activation of pathogenic MEK-ERK pathways. Inhibition of MEK-ERK signaling was achieved by injection of the mitogen-activated protein kinase (MAPK)/ERK inhibitor U0126. The drug effectively improved cardiac function, normalized heart size, and increased probability of survival. CONCLUSIONS: These results suggest that the 40-kDa cardiac myosin-binding protein C fragment, which is produced at elevated levels during human cardiac disease, is a pathogenic fragment that is sufficient to cause hypertrophic cardiomyopathy and heart failure.

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.

Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury.

Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (ß-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates ß-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.