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D-penicillamine (D-pen) is a copper-chelating drug and has immune-modulatory properties. D-pen is used to treat rheumatoid arthritis, Wilson's disease, and kidney stones (cystinuria). However, associated adverse events (AEs) of D-pen treatment are frequent and often serious. Therefore, a comprehensive assessment of the safety profile of D-pen is urgently needed.We identified and analyzed AEs associated with D-pen between April-1970 to July-2020 from the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) databases and calculated the reported odds ratio (ROR) with 95% confidence intervals (CI) using the disproportionality analysis.A total of 9,150,234 AEs related to drugs were reported in the FAERS database, of which 542 were related to D-Pen. We report that D-pen was associated with dystonia (ROR: 20.52; 95%CI: 12.46-33.80), drug hypersensitivity (ROR: 5.42; 95%CI: 3.72-7.90), pancytopenia (ROR: 10.20; 95%CI: 5.61-18.56), joint swelling (ROR: 9.07; 95%CI: 5.51-14.94), renal-impairment (ROR: 6.68; 95%CI: 3.67-12.15), dysphagia (ROR: 5.05; 95%CI: 2.76-8.89), aggravation of condition (ROR: 4.16; 95%CI: 2.60-6.67), congestive cardiac failure (ROR: 4.04; 95%CI: 2.22-7.35), peripheral edema (ROR: 3.77; 95%CI: 2.17-6.55), tremor (ROR: 3.46; 95%CI: 2.00-6.01), pyrexia (ROR: 3.46; 95%CI: 2.00-6.01), and gait disturbance (ROR: 2.41; 95%CI: 1.29-4.52).Patients taking D-pen require close monitoring of renal function, blood counts, immunity, liver, cardiac function, and neurological function. D-pen suppresses immune system which maximizes the risk of infection.

Background: Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) for chronic myelogenous leukemia (CML) treatment. Of note, ponatinib is the only treatment option for CML patients with T315I (gatekeeper) mutation. Unexpected clinical cardiotoxicity, including fatal myocardial infarction and congestive heart failure, has hampered its clinical use. Herein, we aimed to investigate the cardiotoxic mechanism of ponatinib and strategies to prevent the cardiotoxic manifestations. Methods: We employed wild-type C57BL/6, cardiovascular (CV) comorbidity models e.g., transverse aortic constriction (TAC)-pressure overload (cardiac comorbidity) and high-fat diet fed ApoE -/- (vascular comorbidity), to investigate the cardiotoxic mechanism of ponatinib. Echocardiography was performed to assess cardiac function. Comprehensive immune profiling was performed to identify ponatinib-induced immune dynamics using flow cytometry analysis. Results: Echocardiographic assessment of ponatinib treated high-fat diet fed ApoE -/- and pressure overload (PO) murine model showed significant decline in cardiac function, suggesting the key role of CV-comorbidities in ponatinib-induced cardiomyopathy. An unbiased RNA-Seq analysis identified the enrichment of dysregulated inflammatory genes, including a multi-fold upregulation of alarmins S100A8/A9 as a top hit in ponatinib-treated hearts. A combination of in vitro and in vivo mechanistic analysis, identified that ponatinib activates the S100A8/9-TLR4-NLRP3-IL-1β signaling pathway in cardiac and systemic myeloid cells (monocytes and neutrophils), thereby leading to excessive myocardial and systemic inflammation. Finally, we demonstrate that ponatinib-induced excessive inflammation is central to the cardiac pathology because a broad immunosuppressive agent dexamethasone abolished the adverse cardiac remodeling and dysfunction of ponatinib treated hearts. Conclusions: These findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. Our results provide critical preclinical data and rationale for clinical investigation into immunosuppressive interventions to mitigate ponatinib-induced cardiotoxicity.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic neuronal growth and survival factor that is indispensable in the brain, as well as in multiple other tissues and organs, including the cardiovascular system. In approximately 30% of the general population, BDNF harbors a nonsynonymous single nucleotide polymorphism that may be associated with cardiometabolic disorders, coronary artery disease, and Duchenne muscular dystrophy cardiomyopathy. We recently showed that transgenic mice with the human BDNF rs6265 polymorphism (Val66Met) exhibit altered cardiac function, and that cardiomyocytes isolated from these mice are also less contractile. To identify the underlying mechanisms involved, we compared cardiac function by echocardiography and performed deep sequencing of RNA extracted from whole hearts of all three genotypes (Val/Val, Val/Met, and Met/Met) of both male and female Val66Met mice. We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including increased systolic (26.8%, p = 0.047) and diastolic diameters (14.9%, p = 0.022), increased systolic (57.9%, p = 0.039) and diastolic volumes (32.7%, p = 0.026), and increased stroke volume (25.9%, p = 0.033), with preserved ejection fraction and fractional shortening. Both males and females exhibited lower heart rates, but this change was more pronounced in female mice than in males. Consistent with phenotypic observations, the gene encoding SERCA2 (Atp2a2) was reduced in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin (Tcap) and upregulation of BNP (Nppb), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets.

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.

Aims Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. Methods and results To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. Conclusion Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans., Graphical Abstract

Clostridioides difficile is a spore-forming anaerobic bacterium and the leading cause of antibiotic-associated colitis. With few therapeutic options and high rates of disease recurrence, the need to develop new treatment options is urgent. Prior studies utilizing a repurposing approach identified three nonantibiotic Food and Drug Administration-approved drugs, amoxapine, doxapram, and trifluoperazine, with efficacy against a broad range of human pathogens; however, the protective mechanisms remained unknown. Here, we identified mechanisms leading to drug efficacy in a murine model of lethal C. difficile infection (CDI), advancing our understanding of the role of these drugs in infectious disease pathogenesis that center on host immune responses to C. difficile. Overall, these studies highlight the crucial involvement of innate immune responses, as well as the importance of immunomodulation as a potential therapeutic option to combat CDI., Frequent and excessive use of antibiotics primes patients to Clostridioides difficile infection (CDI), which leads to fatal pseudomembranous colitis, with limited treatment options. In earlier reports, we used a drug repurposing strategy and identified amoxapine (an antidepressant), doxapram (a breathing stimulant), and trifluoperazine (an antipsychotic), which provided significant protection to mice against lethal infections with several pathogens, including C. difficile. However, the mechanisms of action of these drugs were not known. Here, we provide evidence that all three drugs offered protection against experimental CDI by reducing bacterial burden and toxin levels, although the drugs were neither bacteriostatic nor bactericidal in nature and had minimal impact on the composition of the microbiota. Drug-mediated protection was dependent on the presence of the microbiota, implicating its role in evoking host defenses that promoted protective immunity. By utilizing transcriptome sequencing (RNA-seq), we identified that each drug increased expression of several innate immune response-related genes, including those involved in the recruitment of neutrophils, the production of interleukin 33 (IL-33), and the IL-22 signaling pathway. The RNA-seq data on selected genes were confirmed by quantitative real-time PCR (qRT-PCR) and protein assays. Focusing on amoxapine, which had the best anti-CDI outcome, we demonstrated that neutralization of IL-33 or depletion of neutrophils resulted in loss of drug efficacy. Overall, our lead drugs promote disease alleviation and survival in the murine model through activation of IL-33 and by clearing the pathogen through host defense mechanisms that critically include an early influx of neutrophils.

Background Muscular dystrophy ( MD ) causes a progressive cardiomyopathy characterized by diffuse fibrosis, arrhythmia, heart failure, and early death. Activation of the thromboxane‐prostanoid receptor ( TP r) increases calcium transients in cardiomyocytes and is proarrhythmic and profibrotic. We hypothesized that TP r activation contributes to the cardiac phenotype of MD , and that TP r antagonism would improve cardiac fibrosis and function in preclinical models of MD . Methods and Results Three different mouse models of MD (mdx/utrn double knockout, second generation mdx/ mTR double knockout, and delta‐sarcoglycan knockout) were given normal drinking water or water containing 25 mg/kg per day of the TP r antagonist ifetroban, beginning at weaning. After 6 months (10 weeks for mdx/utrn double knockout), mice were evaluated for cardiac and skeletal muscle function before euthanization. There was a 100% survival rate of ifetroban‐treated mice to the predetermined end point, compared with 60%, 43%, and 90% for mdx/utrn double knockout, mdx/mTR double knockout, and delta‐sarcoglycan knockout mice, respectively. TP r antagonism improved cardiac output in mdx/utrn double knockout and mdx/ mTR mice, and normalized fractional shortening, ejection fraction, and other parameters in delta‐sarcoglycan knockout mice. Cardiac fibrosis in delta‐sarcoglycan knockout was reduced with TP r antagonism, which also normalized cardiac expression of claudin‐5 and neuronal nitric oxide synthase proteins and multiple signature genes of Duchenne MD. Conclusions TP r antagonism reduced cardiomyopathy and spontaneous death in mouse models of Duchenne and limb‐girdle MD . Based on these studies, ifetroban and other TP r antagonists could be novel therapeutics for treatment of the cardiac phenotype in patients with MD .

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.

Background Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, which codes for the dystrophin protein. While progress has been made in defining the molecular basis and pathogenesis of DMD, major gaps remain in understanding mechanisms that contribute to the marked delay in cardiac compared to skeletal muscle dysfunction. Methods To address this question, we analyzed cardiac and skeletal muscle tissue microarrays from golden retriever muscular dystrophy (GRMD) dogs, a genetically and clinically homologous model for DMD. A total of 15 dogs, 3 each GRMD and controls at 6 and 12 months plus 3 older (47–93 months) GRMD dogs, were assessed. Results GRMD dogs exhibited tissue- and age-specific transcriptional profiles and enriched functions in skeletal but not cardiac muscle, consistent with a “metabolic crisis” seen with DMD microarray studies. Most notably, dozens of energy production-associated molecules, including all of the TCA cycle enzymes and multiple electron transport components, were down regulated. Glycolytic and glycolysis shunt pathway-associated enzymes, such as those of the anabolic pentose phosphate pathway, were also altered, in keeping with gene expression in other forms of muscle atrophy. On the other hand, GRMD cardiac muscle genes were enriched in nucleotide metabolism and pathways that are critical for neuromuscular junction maintenance, synaptic function and conduction. Conclusions These findings suggest differential metabolic dysfunction may contribute to distinct pathological phenotypes in skeletal and cardiac muscle. Electronic supplementary material The online version of this article (doi:10.1186/s12920-017-0257-2) contains supplementary material, which is available to authorized users.

We previously identified brain-derived neurotrophic factor (BDNF) as a putative cardiac-specific biomarker for preserved cardiac function in Duchenne Muscular Dystrophy (DMD). BDNF has cardioprotective qualities that may attenuate dilated cardiomyopathy, and a common, functional BDNF single nucleotide polymorphism rs6265 (Val66-Met) may be correlated with increased risk of cardiovascular events. We hypothesized that BDNF is protective in the setting of DMD and that BDNF’s beneficial effects are diminished in rs6265 allelic carriers. We compared circulating BDNF levels with cardiovascular and skeletal muscle functional parameters in DMD patients stratified by carrier status. Positive association of circulating BDNF with left ventricular ejection fraction in DMD patients was restricted to non-carriers of the rs6265 allele, whereas carriers exhibited worse cardiac function with increased BDNF, more fibrosis and worse skeletal muscle function. To more directly assess association of rs6265 with cardiac function, we used mice with knock-in of BDNF polymorphic Val66Met allele. RNASeq showed higher expression of extracellular matrix genes in Val66Met hearts, and there was a negative correlation of cardiac BDNF protein levels with fibrosis. Additionally, there were baseline echocardiographic and ECG differences, and isolated cardiomyocytes had decreased contractility. This overall cardiac phenotype was substantially enhanced when Val66Met mice were crossed with muscular dystrophy mice. Considered together, our results indicate that BDNF plays a protective role in the dystrophic heart and may represent a novel therapeutic candidate for DMD cardiomyopathy.

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-1β (NRG-1β), 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.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Background In Duchenne muscular dystrophy (DMD), abnormal cardiac function is typically preceded by a decade of skeletal muscle disease. Molecular reasons for differences in onset and progression of these muscle groups are unknown. Human biomarkers are lacking. Methods We analyzed cardiac and skeletal muscle microarrays from normal and golden retriever muscular dystrophy (GRMD) dogs (ages 6, 12, or 47+ months) to gain insight into muscle dysfunction and to identify putative DMD biomarkers. These biomarkers were then measured using human DMD blood samples. Results We identified GRMD candidate genes that might contribute to the disparity between cardiac and skeletal muscle disease, focusing on brain-derived neurotropic factor (BDNF) and osteopontin (OPN/SPP1). BDNF was elevated in cardiac muscle of younger GRMD but was unaltered in skeletal muscle, while SPP1 was increased only in GRMD skeletal muscle. In human DMD, circulating levels of BDNF were inversely correlated with ventricular function and fibrosis, while SPP1 levels correlated with skeletal muscle function. Conclusion These results highlight gene expression patterns that could account for differences in cardiac and skeletal disease in GRMD. Most notably, animal model-derived data were translated to DMD and support use of BDNF and SPP1 as biomarkers for cardiac and skeletal muscle involvement, respectively.

Visual Abstract, Highlights • SMAD4 is the central intracellular mediator of TGF-β pathway. • CM-specific loss of SMAD4 causes cardiac dysfunction independent of fibrotic remodeling. • Deletion CM-SMAD4 affects CM survival. • CM-SMAD4 loss leads to down-regulation of several ion channels’ genes, resulting in cardiac conduction abnormalities. • CM-SMAD4 deletion alters sarcomere shortening kinetics, in parallel with reduction in cardiac myosin-binding protein C levels. • These results demonstrate a fundamental role for CM-SMAD4–dependent TGF-β signaling in adult heart homeostasis., Summary The role of the transforming growth factor (TGF)-β pathway in myocardial fibrosis is well recognized. However, the precise role of this signaling axis in cardiomyocyte (CM) biology is not defined. In TGF-β signaling, SMAD4 acts as the central intracellular mediator. To investigate the role of TGF-β signaling in CM biology, the authors deleted SMAD4 in adult mouse CMs. We demonstrate that CM-SMAD4–dependent TGF-β 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-β pathway for the management of myocardial fibrosis.

Background: Ponatinib, a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) holds significant promise in the treatment of chronic myelogenous leukemia (CML), although the potential cardiotoxicity of this agent remains a concern. In order to overcome ponatinib associated cardiotoxicity, delineation of linked cardiotoxic mechanisms and potential rescue methods are urgently warranted. Objectives: The objectives of the present study were to delineate the signaling mechanisms responsible for ponatinib-induced cardiotoxicity and to identify the potential rescue strategies. Methods: In this study, we employed direct in vivo drug screening in zebrafish for the prediction of cardiotoxicity. We also exploited neonatal rat ventricular myocytes (NRVMs) to explore cardiotoxic mechanism associated with ponatinib. Results: We observed that ponatinib leads to cardiomyocyte apoptosis, elevation in BNP level, decrease in heart rate and fractional shortening leading to cardiac dysfunction in Zebrafish. Consistently, in cultured rat cardiomyocytes, ponatinib was found most cytotoxic drug among all the approved CML TKIs. Mechanistically, ponatinib inhibits the essential prosurvival AKT and ERK signaling pathway, resulting to cardiomyocyte apoptosis. Interestingly, we evaluated the cardioprotective effects of Neuregulin-1β to limit ponatinib toxicity. Neuregulin-1β treatment significantly protects ponatinib-induced cardiotoxicity by supplementing the essential cardiomyocyte prosurvival AKT/ERK signaling pathways. Additionally, the allosteric tyrosine kinase inhibitor asciminib depicts less pronounced cardiotoxic effects than ponatinib in similar settings. Conclusions: This study will advance our mechanistic understanding of ponatinib-induced cardiotoxicity, accelerate the discovery of cardioprotective strategies to prevent and rescue the ponatinib-induced cardiotoxicity. Furthermore, this study suggests asciminib as an alternate treatment option than ponatinib for patients suffering from CML with T315I “gatekeeper” mutation.

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB) harbor cardioprotective qualities that may attenuate CM. In a small cohort of DMD patients we found that BDNF blood levels positively correlated with preserved ejection fraction (EF) and less fibrosis, and carriers of the common BDNF single nucleotide polymorphism rs6265 (Val66-Met) tended to exhibit earlier age of onset of fibrosis with subsequent progression to LV dysfunction, compared to non-carriers. We thus hypothesized that BDNF/TrkB signaling delays CM progression in DMD. To test this hypothesis, we administered the TrkB agonist 7,8-dihydroxyflavone (DHF) to mdx 4cv ; mTR KO mice in their drinking water for 26 weeks, beginning at 8 weeks of age. Based on echocardiography, DHF treatment preserved cardiac output compared to vehicle-treated controls. Conversely, mdx 4cv ; mTR KO mice injected intraperitoneally with the TrkB inhibitor (K252a) displayed bradycardia and PR interval prolongation, as measured by EKG, as well as acute (10-20 min) reduction in percent EF and fractional shortening, as measured by echocardiography. K252a also significantly reduced sarcomere shortening in isolated murine cardiomyocytes. BDNF might also contribute to cardiac repair. Using humanized BDNF polymorphic mice, which have the Val66-Met mutation, we found that post-myocardial infarction cardiac dysfunction was significantly exacerbated in Met/Met mice compared to Val/Val littermate controls. Finally, we evaluated the role of BDNF/TrkB in human cardiomyocytes differentiated from induced pluripotent cells obtained from DMD patients (DMD iPSC-CMs) and found that they highly express BDNF protein in lysates and supernatants. DMD iPSC-CMs also expressed a truncated version of TrkB that lacks the tyrosine kinase domain essential for canonical BDNF/TrkB signaling. Nonetheless, DMD iPSC-CMs responded to treatment with recombinant BDNF, including increased phosphorylation of GSK-3α, mTOR, AMPK, and MSK1/2. Considered together, our results indicate that BDNF plays a protective role in the dystrophic heart and might represent a novel therapeutic candidate for DMD cardiomyopathy.

Muscular dystrophy causes mechanical damage and increased membrane permeability of cardiomyocytes, leading to progressive cardiomyopathy and diffuse myocardial fibrosis that typically begins in the free wall of the LV. Cardiac dysfunction is a primary cause of death in Duchenne (DMD) and other muscular dystrophy (MD) patients. Activation of the thromboxane/prostanoid (TP) receptor increases calcium transients in cardiomyocytes, causes arrhythmia, and is pro-fibrotic. We thus hypothesized that TP receptor activation contributes to the cardiac phenotype of MD, and that blockade of the TP receptor would improve cardiac fibrosis and function in mouse MD models. We gave 3 different mouse models of MD either normal drinking water or water containing 25 mg/kg/day of the TP receptor antagonist ifetroban, from weaning to the predetermined endpoint. TP receptor antagonism improved 10-week survival from 60% to 100% in utrophin/dystrophin double knockout mice, a model of severe DMD, and increased cardiac output compared with surviving vehicle-treated mice. In the mdx/mTR mouse model of DMD, treatment with ifetroban likewise improved 6-month survival from 43% to 100% and increased cardiac output. Finally, we examined delta-sarcoglycan knockout (dSG KO) male mice, a model of limb-girdle muscular dystrophy (LGMD) that replicates the DMD cardiac phenotype with improved survival. TP receptor antagonism normalized fractional shortening, ejection fraction, and LVSP in dSG KO mice, and decreased plasma ANP. However, it had no effect on the contraction deficits of isolated cardiomyocytes other than to normalize the slowed relaxation of dSG KO. Ifetroban-treated mice had improved myocardial, but not skeletal muscle fibrosis. This was most noticeable in the LV free wall and occurred in conjunction with decreased TGF-beta activity and normalized plasma WBC. Typical of DMD cardiomyopathy, dSG KO hearts had reduced expression of neuronal nitric oxide synthase (nNOS) and claudin-5, which was improved with TPr antagonism. The results of our studies indicate that TP receptor activation may contribute to MD cardiomyopathy, and oral antagonism of the TP receptor may be a novel therapeutic for the cardiac phenotype in DMD and LGMD patients.

Several studies have established a relationship between hypertension and salt intake; however the mechanisms by which salt causes hypertension are poorly understood. There is also evidence that sodium (Na+) accumulates in the interstitium with aging and hypertension in concentrations exceeding the plasma. We tested the hypothesis that increased NaCl would convert human monocytes to an inflammatory phenotype and to define mechanisms involved. We exposed monocytes from 17 human volunteers to normal physiological NaCl (NS: 150 mM/L), elevated NaCl (HS: 190 mM/L), or an equiosmoloar concentration of mannitol. Exposure of human monocytes to high salt, but not mannitol, increased formation of immunogenic isolevuglandins (isoLG) (NS: 1688±384 vs Mann:1762±429 vs HS: 2381± 635 MFI p

Neuregulin-1β (NRG-1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG-1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure-associated cachexia, a severe co-morbidity characterized by a loss of muscle mass. We therefore assessed NRG-1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN - cimaglermin alfa), a version of NRG-1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG-1β bi-weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired-end RNA sequencing performed. NRG-1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as MYH3, MYHC, MYL6B, KY and HES1 . Importantly, NRG-1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down-regulating MyoD expression. Consistent with this, MyoD was increased in NRG-1β treated animals. In vitro experiments with myoblast cell lines confirmed that NRG-1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG-1β-mediated anti-atrophic, anti-cachexia effect that may provide additional benefits to this potential therapy in heart failure.

We hypothesized that gene expression profiles of mitral valve (MV) leaflets from patients with Barlow's disease (BD) are distinct from those with fibroelastic deficiency (FED).MVs were obtained from patients with BD (7 men, 3 women; 61.4 ± 12.7 years old) or FED (6 men, 5 women; 54.5 ± 6.0 years old) undergoing operations for severe mitral regurgitation (MR). Normal MVs were obtained from 6 donor hearts unmatched for transplant (3 men, 3 women; 58.3 ± 7.5 years old), and gene expression was assessed using cDNA microarrays. Select transcripts were validated by quantitative reverse-transcription polymerase chain reaction, followed by an assessment of protein levels by immunostaining.The global gene expression profile for BD was clearly distinct from normal and FED groups. A total of 4,684 genes were significantly differential (fold-difference1.5, p0.05) among the three groups, 1,363 of which were commonly altered in BD and FED compared with healthy individuals (eg TGFβ2 [transforming growth factor β2] and TGFβ3 were equally upregulated in BD and FED). Most interesting were 329 BD-specific genes, including ADAMTS5 (a disintegrin-like and metalloprotease domain with thrombospondin-type 5), which was uniquely downregulated in BD based on microarrays and quantitative reverse-transcription polymerase chain reaction. Consistent with this finding, the ADAMTS5 substrate versican was increased in BD and conversely lower in FED.MV leaflets in BD and FED exhibit distinct gene expression patterns, suggesting different pathophysiologic mechanisms are involved in leaflet remodeling. Moreover, downregulation of ADAMTS5 in BD, along with the accumulation of its substrate versican in the valvular extracellular matrix, might contribute to leaflet thickening and enlargement.

Background— Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM. Methods and Results— RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in KCNJ11 expression with ICM. KCNJ11 encodes the Kir6.2 subunit of the cardioprotective K ATP channel. Using wild-type mice and kcnj11 -deficient ( kcnj11 -null) mice, we examined the effect of kcnj11 expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in kcnj11 -null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in kcnj11 -null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in kcnj11 -null mice. Despite impaired relaxation, kcnj11 -null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca 2+ , thereby increasing sarcoendoplasmic reticulum Ca 2+ –mediated calcium reuptake. However, kcnj11 -null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca 2+ -ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca 2+ function, thereby contributing to diastolic dysfunction. Conclusions— KCNJ11 expression is decreased in human ICM. Lack of kcnj11 expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca 2+ -ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.

Neuregulin-1β (NRG-1β) is critical for cardiac development and repair, and recombinant forms are currently being assessed as possible therapeutics for systolic heart failure. We previously demonstrated that recombinant NRG-1β reduces cardiac fibrosis in an animal model of cardiac remodeling and heart failure, suggesting that there may be direct effects on cardiac fibroblasts. Here we show that NRG-1β receptors (ErbB2, ErbB3, and ErbB4) are expressed in normal human cardiac ventricular (NHCV) fibroblast cell lines. Treatment of NHCV fibroblasts with recombinant NRG-1β induced activation of the AKT pathway, which was phosphoinositide 3-kinase (PI3K)-dependent. Moreover, the NRG-1β-induced PI3K/AKT signaling in these cells required phosphorylation of both ErbB2 and ErbB3 receptors at tyrosine (Tyr)1248 and Tyr1289 respectively. RNASeq analysis of NRG-1β-treated cardiac fibroblasts obtained from three different individuals revealed a global gene expression signature consistent with cell growth and survival. We confirmed enhanced cellular proliferation and viability in NHCV fibroblasts in response to NRG-1β, which was abrogated by PI3K, ErbB2, and ErbB3 inhibitors. NRG-1β also induced production and secretion of cytokines (interleukin-1α and interferon-γ) and pro-reparative factors (angiopoietin-2, brain-derived neurotrophic factor, and crypto-1), suggesting a role in cardiac repair through the activation of paracrine signaling.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCachannels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCachannels, are promising targets. Specifically, BKCachannel agonists like NS1619 maintain DA patency even in the presence of O2and may be clinically useful.

NRG-1β (neuregulin-1β) serves multiple functions during embryonic heart development by signalling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1β induces cardiomyogenesis of mESCs (mouse embryonic stem cells) at the later stages of differen-tiation through ErbB4 receptor activation. In the present study we systematically examined NRG-1β induction of cardiac myocytes in mESCs and identified a novel time window, the first 48 h, for NRG-1β-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast with the later differentiation of mESCs in which NRG-1β induces cardiomyogenesis via the ErbB4 receptor, we found that knocking down ErbB3 or ErbB2 with siRNA during the early differentiation inhibited NRG-1β-induced cardiomyogenesis in mESCs. Microarray analysis of RNA expression at this early time point indicated that NRG-1β treatment in mESCs resulted in gene expression changes important to differentiation including up-regulation of components of PI3K (phosphoinositide 3-kinase), a known mediator of the NRG-1β/ErbB signalling pathway, as well as activation of CREB (cAMP-response-element-binding protein). Further study demonstrated that the NRG-1β-induced phosphorylation of CREB was required for cardiomyogenesis of mESCs. In summary, we report a previously unrecognized role for NRG-1β/ErbB3/CREB signalling at the pre-mesoderm stage for stem cell cardiac differentiation.

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.

Mitral valve (MV) leaflets in ischemic mitral regurgitation (IMR) undergo structural remodeling associated with adaptive changes. We hypothesized that the gene expression profiles of MV leaflets from patients with IMR are distinct from normal MVs. MVs were obtained from 7 patients (4M, age= 70.4±8

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Summary CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces; however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. Using cDNA microarray analysis, we characterized the transcriptional profile of the intracellular pathogen Brucella melitensis at 4 h (adaptational period) and 12 h (replicative phase) following HeLa cells infection. The intracellular pathogen transcriptome was determined using initially enriched and then amplified B. melitensis RNA from total RNA of B. melitensis-infected HeLa cells. Analysis of microarray results identified 161 and 115 pathogen genes differentially expressed at 4 and 12 h p.i., respectively. In concordance with phenotypic studies, most of the genes expressed were involved in pathogen growth and metabolism, and were down-regulated at the earliest time point (78%), but up-regulated at 12 h p.i. (75%). Further characterization of specific genes identified in this study will elucidate biological processes and pathways to help understand how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host.

Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on host cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a rigorous combinatorial approach with three separate statistical algorithms. A total of 36 genes met the criteria of upregulated expression in response to Stx1 treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were validated by real-time reverse transcriptase PCR for genes encoding early growth response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2; inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10 (regulation of mitogen-activated protein kinase signaling). Stx1-mediated signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to be involved in the increased expression and production of the proinflammatory mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1 to increase the expression of genes encoding phosphatases suggests that mechanisms to dampen the macrophage proinflammatory response may be built into host response to the toxins.