Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Mechanistic Insights into the Adsorption of Monoclonal Antibodies at the Water/Vapor Interface.

Monoclonal antibodies (mAbs) are active components of therapeutic formulations that interact with the water-vapor interface during manufacturing, storage, and administration. Surface adsorption has been demonstrated to mediate antibody aggregation, which leads to a loss of therapeutic efficacy. Controlling mAb adsorption at interfaces requires a deep understanding of the microscopic processes that lead to adsorption and identification of the protein regions that drive mAb surface activity. Here, we report all-atom molecular dynamics (MD) simulations of the adsorption behavior of a full IgG1-type antibody at the water/vapor interface. We demonstrate that small local changes in the protein structure play a crucial role in promoting adsorption. Also, interfacial adsorption triggers structural changes in the antibody, potentially contributing to the further enhancement of surface activity. Moreover, we identify key amino acid sequences that determine the adsorption of antibodies at the water-air interface and outline strategies to control the surface activity of these important therapeutic proteins.

Cardiac fibroblast GSK-3α aggravates ischemic cardiac injury by promoting fibrosis, inflammation, and impairing angiogenesis.

Myocardial infarction (MI) is the leading cause of death worldwide. Glycogen synthase kinase-3 (GSK-3) has been considered to be a promising therapeutic target for cardiovascular diseases. GSK-3 is a family of ubiquitously expressed serine/threonine kinases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Previously, our group identified that cardiac fibroblast (FB) GSK-3ß acts as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of FB-GSK-3α in MI pathology is not defined. To determine the role of FB-GSK-3α in MI-induced adverse cardiac remodeling, GSK-3α was deleted specifically in the residential fibroblast or myofibroblast (MyoFB) using tamoxifen (TAM) inducible Tcf21 or Periostin (Postn) promoter-driven Cre recombinase, respectively. Echocardiographic analysis revealed that FB- or MyoFB-specific GSK-3α deletion prevented the development of dilative remodeling and cardiac dysfunction. Morphometrics and histology studies confirmed improvement in capillary density and a remarkable reduction in hypertrophy and fibrosis in the KO group. We harvested the hearts at 4 weeks post-MI and analyzed signature genes of adverse remodeling. Specifically, qPCR analysis was performed to examine the gene panels of inflammation (TNFα, IL-6, IL-1ß), fibrosis (COL1A1, COL3A1, COMP, Fibronectin-1, Latent TGF-ß binding protein 2), and hypertrophy (ANP, BNP, MYH7). These molecular markers were essentially normalized due to FB-specific GSK-3α deletion. Further molecular studies confirmed that FB-GSK-3α could regulate NF-kB activation and expression of angiogenesis-related proteins. Our findings suggest that FB-GSK-3α plays a critical role in the pathological cardiac remodeling of ischemic hearts, therefore, it could be therapeutically targeted.

Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1ß Mediated Inflammation.

BACKGROUND: The tyrosine kinase inhibitor ponatinib is the only treatment option for chronic myelogenous leukemia patients with T315I (gatekeeper) mutation. Pharmacovigilance analysis of Food and Drug Administration and World Health Organization datasets has revealed that ponatinib is the most cardiotoxic agent among all Food and Drug Administration-approved tyrosine kinase inhibitors in a real-world scenario. However, the mechanism of ponatinib-induced cardiotoxicity is unknown. METHODS: The lack of well-optimized mouse models has hampered the in vivo cardio-oncology studies. Here, we show that cardiovascular comorbidity mouse models evidence a robust cardiac pathological phenotype upon ponatinib treatment. A combination of multiple in vitro and in vivo models was employed to delineate the underlying molecular mechanisms. RESULTS: An unbiased RNA sequencing analysis identified the enrichment of dysregulated inflammatory genes, including a multifold upregulation of alarmins S100A8/A9, as a top hit in ponatinib-treated hearts. Mechanistically, we demonstrate that ponatinib activates the S100A8/A9-TLR4 (Toll-like receptor 4)-NLRP3 (NLR family pyrin domain-containing 3)-IL (interleukin)-1ß signaling pathway in cardiac and systemic myeloid cells, in vitro and in vivo, thereby leading to excessive myocardial and systemic inflammation. Excessive inflammation was central to the cardiac pathology because interventions with broad-spectrum immunosuppressive glucocorticoid dexamethasone or specific inhibitors of NLRP3 (CY-09) or S100A9 (paquinimod) nearly abolished the ponatinib-induced cardiac dysfunction. CONCLUSIONS: Taken together, these findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. From a translational perspective, our results provide critical preclinical data and rationale for a clinical investigation into immunosuppressive interventions for managing ponatinib-induced cardiotoxicity.

Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression.

Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CM UPRT mice) and tested our hypothesis that CM-derived miRNAs (CM miRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PB sEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PB sEV of CM UPRT mice 6 h after 4TUc injection. In PB sEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a (CM miR-208a) levels peaked 12 h after experimentally induced MI in PB sEV and 24 h after MI in the lung. Notably, miR-208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PB sEV from mice that underwent MI (MI-PB sEV) or sham surgery (Sham-PB sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-κB pathway, was lower in the lungs of MI-PB sEV-treated animals than the lungs of animals treated with Sham-PB sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, CM UPRT mice enables us to track PB sEV-mediated transport of CM miRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

Cardiac natriuretic peptide deficiency sensitizes the heart to stress-induced ventricular arrhythmias via impaired CREB signalling.

AIMS: The cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. METHODS AND RESULTS: We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP-deficient (Nppa-/-) or BNP-deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human-derived induced pluripotent stem cell cardiomyocytes (iPS-CMs). In the unstressed state, both ANP- and BNP-deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodelling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress-induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild-type hearts to stress-induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen-activated protein kinase (p38 MAPK) signalling cascade. CONCLUSIONS: Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signalling which sensitizes the heart to stress-induced ventricular arrhythmias.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

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Repurposing Nintedanib for pathological cardiac remodeling and dysfunction.

Heart Failure (HF) is the leading cause of death worldwide. Myocardial fibrosis, one of the clinical manifestations implicated in almost every form of heart disease, contributes significantly to HF development. However, there is no approved drug specifically designed to target cardiac fibrosis. Nintedanib (NTB) is an FDA approved tyrosine kinase inhibitor for idiopathic pulmonary fibrosis (IPF) and chronic fibrosing interstitial lung diseases (ILD). The favorable clinical outcome of NTB in IPF patients is well established. Furthermore, NTB is well tolerated in IPF patients irrespective of cardiovascular comorbidities. However, there is a lack of direct evidence to support the therapeutic efficacy and safety of NTB in cardiac diseases. In this study we examined the effects of NTB treatment on cardiac fibrosis and dysfunction using a murine model of HF. Specifically, 10 weeks old C57BL/6J male mice were subjected to Transverse Aortic Constriction (TAC) surgery. NTB was administered once daily by oral gavage (50 mg/kg) till 16 weeks post-TAC. Cardiac function was monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 16 weeks post-TAC. In the control group, systolic dysfunction started developing from 4 weeks post-surgery and progressed till 16 weeks. However, NTB treatment prevented TAC-induced cardiac functional decline. In another experiment, NTB treatment was stopped at 8 weeks, and animals were followed till 16 weeks post-TAC. Surprisingly, NTB's beneficial effect on cardiac function was maintained even after treatment interruption. NTB treatment remarkably reduced cardiac fibrosis as confirmed by Masson's trichrome staining and decreased expression of collagen genes (COL1A1, COL3A1). Compared to the TAC group, NTB treated mice showed a lower HW/TL ratio and cardiomyocyte cross-sectional area. NTB treatment reduced myocardial and systemic inflammation by inhibiting pro-inflammatory subsets and promoting regulatory T cells (Tregs). Our in vitro studies demonstrated that NTB prevents myofibroblast transformation, TGFß1-induced SMAD3 phosphorylation, and the production of fibrogenic proteins (Fibronectin-1, α-SMA). However, NTB promoted immunosuppressive phenotype in Tregs, and altered vital signaling pathways in isolated cardiac fibroblast and cardiomyocytes, suggesting that its biological effect and underlying cardiac protection mechanisms are not limited to fibroblast and fibrosis alone. Our findings provide a proof of concept for repurposing NTB to combat adverse myocardial fibrosis and encourage the need for further validation in large animal models and subsequent clinical development for HF patients.

Targeting 5-HT2B Receptor Signaling Prevents Border Zone Expansion and Improves Microstructural Remodeling After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) induces an intense injury response that ultimately generates a collagen-dominated scar. Although required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process after MI. Serotonin 2B receptor (5-HT2B) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI. METHODS: We used 2 pharmacological antagonists to explore the effect of 5-HT2B inhibition on outcomes after MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible 5-HT2B ablation driven by Tcf21MCM and PostnMCM was used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT2B, respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. RESULTS: 5-HT2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT2B antagonism resulted in collagen fiber redistribution to thinner collagen fibers that were more anisotropic, enhancing left ventricular contractility, whereas fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT2B from Tcf21-lineage resident cardiac fibroblasts and saw similar improvements to the pharmacological approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT2B after onset of injury in Postn-lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation. CONCLUSIONS: Together, our findings illustrate that 5-HT2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

Deletion of Cardiomyocyte Glycogen Synthase Kinase-3 Beta (GSK-3ß) Improves Systemic Glucose Tolerance with Maintained Heart Function in Established Obesity.

Obesity is an independent risk factor for cardiovascular diseases (CVD), including heart failure. Thus, there is an urgent need to understand the molecular mechanism of obesity-associated cardiac dysfunction. We recently reported the critical role of cardiomyocyte (CM) Glycogen Synthase Kinase-3 beta (GSK-3ß) in cardiac dysfunction associated with a developing obesity model (deletion of CM-GSK-3ß prior to obesity). In the present study, we investigated the role of CM-GSK-3ß in a clinically more relevant model of established obesity (deletion of CM-GSK-3ß after established obesity). CM-GSK-3ß knockout (GSK-3ßfl/flCre+/-) and controls (GSK-3ßfl/flCre-/-) mice were subjected to a high-fat diet (HFD) in order to establish obesity. After 12 weeks of HFD treatment, all mice received tamoxifen injections for five consecutive days to delete GSK-3ß specifically in CMs and continued on the HFD for a total period of 55 weeks. To our complete surprise, CM-GSK-3ß knockout (KO) animals exhibited a globally improved glucose tolerance and maintained normal cardiac function. Mechanistically, in stark contrast to the developing obesity model, deleting CM-GSK-3ß in obese animals did not adversely affect the GSK-3αS21 phosphorylation (activity) and maintained canonical ß-catenin degradation pathway and cardiac function. As several GSK-3 inhibitors are in the trial to treat various chronic conditions, including metabolic diseases, these findings have important clinical implications. Specifically, our results provide critical pre-clinical data regarding the safety of GSK-3 inhibition in obese patients.

IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction.

Acute myocardial infarction (MI) provokes an inflammatory response in the heart that removes damaged tissues to facilitate tissue repair/regeneration. However, overactive and prolonged inflammation compromises healing, which may be counteracted by antiinflammatory mechanisms. A key regulatory factor in an inflammatory response is the antiinflammatory cytokine IL-10, which can be produced by a number of immune cells, including subsets of B lymphocytes. Here, we investigated IL-10-producing B cells in pericardial adipose tissues (PATs) and their role in the healing process following acute MI in mice. We found that IL-10-producing B cells were enriched in PATs compared to other adipose depots throughout the body, with the majority of them bearing a surface phenotype consistent with CD5+ B-1a cells (CD5+ B cells). These cells were detected early in life, maintained a steady presence during adulthood, and resided in fat-associated lymphoid clusters. The cytokine IL-33 and the chemokine CXCL13 were preferentially expressed in PATs and contributed to the enrichment of IL-10-producing CD5+ B cells. Following acute MI, the pool of CD5+ B cells was expanded in PATs. These cells accumulated in the infarcted heart during the resolution of MI-induced inflammation. B cell-specific deletion of IL-10 worsened cardiac function, exacerbated myocardial injury, and delayed resolution of inflammation following acute MI. These results revealed enrichment of IL-10-producing B cells in PATs and a significant contribution of these cells to the antiinflammatory processes that terminate MI-induced inflammation. Together, these findings have identified IL-10-producing B cells as therapeutic targets to improve the outcome of MI.

Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction.

Over one million Americans experience myocardial infarction (MI) annually, and the resulting scar and subsequent cardiac fibrosis gives rise to heart failure. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not been studied in myocardium after MI. MI was induced by ligation of the left anterior descending artery in mice with either heterozygous or homozygous knockout of CDH11, wild-type mice receiving bone marrow transplants from Cdh11-deficient animals, and wild-type mice treated with a functional blocking antibody against CDH11 (SYN0012). Flow cytometry revealed significant CDH11 expression in noncardiomyocyte cells after MI. Animals given SYN0012 had improved cardiac function, as measured by echocardiogram, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 reduced bone marrow-derived myeloid cells and increased proangiogenic cells in the heart 3 days after MI. Cardiac fibroblast and macrophage interactions increased IL-6 secretion in vitro. Our findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI and that targeting CDH11 with a blocking antibody improves outcomes by altering recruitment of bone marrow-derived cells, limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Cardiomyocyte-GSK-3α promotes mPTP opening and heart failure in mice with chronic pressure overload.

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.

Cardiomyocyte SMAD4-Dependent TGF-ß Signaling is Essential to Maintain Adult Heart Homeostasis.

The role of the transforming growth factor (TGF)-ß pathway in myocardial fibrosis is well recognized. However, the precise role of this signaling axis in cardiomyocyte (CM) biology is not defined. In TGF-ß signaling, SMAD4 acts as the central intracellular mediator. To investigate the role of TGF-ß signaling in CM biology, the authors deleted SMAD4 in adult mouse CMs. We demonstrate that CM-SMAD4-dependent TGF-ß signaling is critical for maintaining cardiac function, sarcomere kinetics, ion-channel gene expression, and cardiomyocyte survival. Thus, our findings raise a significant concern regarding the therapeutic approaches that rely on systemic inhibition of the TGF-ß pathway for the management of myocardial fibrosis.

Generation of Nppa-tagBFP reporter knock-in mouse line for studying cardiac chamber specification.

Nppa is a cardiac hormone which plays critical roles in regulating salt-water balance. Its expression is restricted to the atria of the healthy post-natal heart. During heart development, spatio-temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re-expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking-in a tagBFP reporter cassette into 3'-UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa-tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa-tagBFP reporter knock-in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.

Ponatinib-induced cardiotoxicity: delineating the signalling mechanisms and potential rescue strategies.

AIMS: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukaemia (CML). However, cardiotoxicity of these agents remains a serious concern. The underlying mechanism of these adverse cardiac effects is largely unknown. Delineation of the underlying mechanisms of TKIs associated cardiac dysfunction could guide potential prevention strategies, rescue approaches, and future drug design. This study aimed to determine the cardiotoxic potential of approved CML TKIs, define the associated signalling mechanism and identify potential alternatives. METHODS AND RESULTS: In this study, we employed a zebrafish transgenic BNP reporter line that expresses luciferase under control of the nppb promoter (nppb:F-Luciferase) to assess the cardiotoxicity of all approved CML TKIs. Our in vivo screen identified ponatinib as the most cardiotoxic agent among the approved CML TKIs. Then using a combination of zebrafish and isolated neonatal rat cardiomyocytes, we delineated the signalling mechanism of ponatinib-induced cardiotoxicity by demonstrating that ponatinib inhibits cardiac prosurvival signalling pathways AKT and extra-cellular-signal-regulated kinase (ERK), and induces cardiomyocyte apoptosis. As a proof of concept, we augmented AKT and ERK signalling by administration of Neuregulin-1ß (NRG-1ß), and this prevented ponatinib-induced cardiomyocyte apoptosis. We also demonstrate that ponatinib-induced cardiotoxicity is not mediated by inhibition of fibroblast growth factor signalling, a well-known target of ponatinib. Finally, our comparative profiling for the cardiotoxic potential of CML approved TKIs, identified asciminib (ABL001) as a potentially much less cardiotoxic treatment option for CML patients with the T315I mutation. CONCLUSION: Herein, we used a combination of in vivo and in vitro methods to systematically screen CML TKIs for cardiotoxicity, identify novel molecular mechanisms for TKI cardiotoxicity, and identify less cardiotoxic alternatives.

Cardiomyocyte-specific deletion of GSK-3ß leads to cardiac dysfunction in a diet induced obesity model.

BACKGROUND AND RATIONALE: Obesity, an independent risk factor for the development of myocardial diseases is a growing healthcare problem worldwide. It's well established that GSK-3ß is critical to cardiac pathophysiology. However, the role cardiomyocyte (CM) GSK-3ß in diet-induced cardiac dysfunction is unknown. METHODS: CM-specific GSK-3ß knockout (CM-GSK-3ß-KO) and littermate controls (WT) mice were fed either a control diet (CD) or high-fat diet (HFD) for 55weeks. Cardiac function was assessed by transthoracic echocardiography. RESULTS: At baseline, body weights and cardiac function were comparable between the WT and CM-GSK-3ß-KOs. However, HFD-fed CM-GSK-3ß-KO mice developed severe cardiac dysfunction. Consistently, both heart weight/tibia length and lung weight/tibia length were significantly elevated in the HFD-fed CM-GSK-3ß-KO mice. The impaired cardiac function and adverse ventricular remodeling in the CM-GSK-3ß-KOs were independent of body weight or the lean/fat mass composition as HFD-fed CM-GSK-3ß-KO and controls demonstrated comparable body weight and body masses. At the molecular level, on a CD, CM-GSK-3α compensated for the loss of CM-GSK-3ß, as evident by significantly reduced GSK-3αs21 phosphorylation (activation) resulting in a preserved canonical ß-catenin ubiquitination pathway and cardiac function. However, this protective compensatory mechanism is lost with HFD, leading to excessive accumulation of ß-catenin in HFD-fed CM-GSK-3ß-KO hearts, resulting in adverse ventricular remodeling and cardiac dysfunction. CONCLUSION: In summary, these results suggest that cardiac GSK-3ß is crucial to protect against obesity-induced adverse ventricular remodeling and cardiac dysfunction.

ERBB signaling attenuates proinflammatory activation of nonclassical monocytes.

Immune activation in chronic systolic heart failure (HF) correlates with disease severity and prognosis. Recombinant neuregulin-1 (rNRG-1) is being developed as a possible therapy for HF, based on the activation of ERBB receptors in cardiac cells. Work in animal models of HF led us to hypothesize that there may be direct effects of NRG-1 on immune system activation and inflammation. We investigated the expression of ERBB receptors and the effect of rNRG-1 isoform glial growth factor 2 (GGF2) in subpopulations of peripheral blood mononuclear cells (PB MNCs) in subjects with HF. We found that human monocytes express both ERBB2 and ERBB3 receptors, with high interindividual variability among subjects. Monocyte surface ERBB3 and TNF-α mRNA expression were inversely correlated in subjects with HF but not in human subjects without HF. GGF2 activation of ERBB signaling ex vivo inhibited LPS-induced TNF-α production, specifically in the CD14lowCD16+ population of monocytes in a phosphoinositide 3-kinase-dependent manner. GGF2 suppression of TNF-α correlated directly with the expression of ERBB3. In vivo, a single dose of intravenous GGF2 reduced TNF-α expression in PB MNCs of HF subjects participating in a phase I safety study of GGF2. These results support a role for ERBB3 signaling in the regulation of TNF-α production from CD14lowCD16+ monocytes and a need for further investigation into the clinical significance of NRG-1/ERBB signaling as a modulator of immune system function.NEW & NOTEWORTHY This study identified a novel role of neuregulin-1 (NRG-1)/ERBB signaling in the control of proinflammatory activation of monocytes. These results further improve our fundamental understanding of cardioprotective effects of NRG-1 in patients with heart failure.