Circadian Rhythms in Cardiovascular Metabolism.

Energetic demand and nutrient supply fluctuate as a function of time-of-day, in alignment with sleep-wake and fasting-feeding cycles. These daily rhythms are mirrored by 24-hour oscillations in numerous cardiovascular functional parameters, including blood pressure, heart rate, and myocardial contractility. It is, therefore, not surprising that metabolic processes also fluctuate over the course of the day, to ensure temporal needs for ATP, building blocks, and metabolism-based signaling molecules are met. What has become increasingly clear is that in addition to classic signal-response coupling (termed reactionary mechanisms), cardiovascular-relevant cells use autonomous circadian clocks to temporally orchestrate metabolic pathways in preparation for predicted stimuli/stresses (termed anticipatory mechanisms). Here, we review current knowledge regarding circadian regulation of metabolism, how metabolic rhythms are synchronized with cardiovascular function, and whether circadian misalignment/disruption of metabolic processes contribute toward the pathogenesis of cardiovascular disease.

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.

Sustained Increases in Cardiomyocyte Protein O-Linked ß-N-Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment.

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.

GSK-3 at the heart of cardiometabolic diseases: Isoform-specific targeting is critical to therapeutic benefit.

Glycogen synthase kinase-3 (GSK-3) is a family of serine/threonine kinases. The GSK-3 family has 2 isoforms, GSK-3α and GSK-3ß. The GSK-3 isoforms have been shown to play overlapping as well as isoform-specific-unique roles in both, organ homeostasis and the pathogenesis of multiple diseases. In the present review, we will particularly focus on expanding the isoform-specific role of GSK-3 in the pathophysiology of cardiometabolic disorders. We will highlight recent data from our lab that demonstrated the critical role of cardiac fibroblast (CF) GSK-3α in promoting injury-induced myofibroblast transformation, adverse fibrotic remodeling, and deterioration of cardiac function. We will also discuss studies that found the exact opposite role of CF-GSK-3ß in cardiac fibrosis. We will review emerging studies with inducible cardiomyocyte (CM)-specific as well as global isoform-specific GSK-3 KOs that demonstrated inhibition of both GSK-3 isoforms provides benefits against obesity-associated cardiometabolic pathologies. The underlying molecular interactions and crosstalk among GSK-3 and other signaling pathways will be discussed. We will briefly review the specificity and limitations of the available small molecule inhibitors targeting GSK-3 and their potential applications to treat metabolic disorders. Finally, we will summarize these findings and offer our perspective on envisioning GSK-3 as a therapeutic target for the management of cardiometabolic diseases.

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.

Isoform-Specific Role of GSK-3 in High Fat Diet Induced Obesity and Glucose Intolerance.

Obesity-associated metabolic disorders are rising to pandemic proportions; hence, there is an urgent need to identify underlying molecular mechanisms. Glycogen synthase kinase-3 (GSK-3) signaling is highly implicated in metabolic diseases. Furthermore, GSK-3 expression and activity are increased in Type 2 diabetes patients. However, the isoform-specific role of GSK-3 in obesity and glucose intolerance is unclear. Pharmacological GSK-3 inhibitors are not isoform-specific, and tissue-specific genetic models are of limited value to predict the clinical outcome of systemic inhibiion. To overcome these limitations, we created novel mouse models of ROSA26CreERT2-driven, tamoxifen-inducible conditional deletion of GSK-3 that allowed us to delete the gene globally in an isoform-specific and temporal manner. Isoform-specific GSK-3 KOs and littermate controls were subjected to a 16-week high-fat diet (HFD) protocol. On an HFD, GSK-3α KO mice had a significantly lower body weight and modest improvement in glucose tolerance compared to their littermate controls. In contrast, GSK-3ß-deletion-mediated improved glucose tolerance was evident much earlier in the timeline and extended up to 12 weeks post-HFD. However, this protective effect weakened after chronic HFD (16 weeks) when GSK-3ß KO mice had a significantly higher body weight compared to controls. Importantly, GSK-3ß KO mice on a control diet maintained significant improvement in glucose tolerance even after 16 weeks. In summary, our novel mouse models allowed us to delineate the isoform-specific role of GSK-3 in obesity and glucose tolerance. From a translational perspective, our findings underscore the importance of maintaining a healthy weight in patients receiving lithium therapy, which is thought to work by GSK-3 inhibition mechanisms.

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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Mechanisms of Fibroblast Activation and Myocardial Fibrosis: Lessons Learned from FB-Specific Conditional Mouse Models.

Heart failure (HF) is a leading cause of morbidity and mortality across the world. Cardiac fibrosis is associated with HF progression. Fibrosis is characterized by the excessive accumulation of extracellular matrix components. This is a physiological response to tissue injury. However, uncontrolled fibrosis leads to adverse cardiac remodeling and contributes significantly to cardiac dysfunction. Fibroblasts (FBs) are the primary drivers of myocardial fibrosis. However, until recently, FBs were thought to play a secondary role in cardiac pathophysiology. This review article will present the evolving story of fibroblast biology and fibrosis in cardiac diseases, emphasizing their recent shift from a supporting to a leading role in our understanding of the pathogenesis of cardiac diseases. Indeed, this story only became possible because of the emergence of FB-specific mouse models. This study includes an update on the advancements in the generation of FB-specific mouse models. Regarding the underlying mechanisms of myocardial fibrosis, we will focus on the pathways that have been validated using FB-specific, in vivo mouse models. These pathways include the TGF-ß/SMAD3, p38 MAPK, Wnt/ß-Catenin, G-protein-coupled receptor kinase (GRK), and Hippo signaling. A better understanding of the mechanisms underlying fibroblast activation and fibrosis may provide a novel therapeutic target for the management of adverse fibrotic remodeling in the diseased heart.

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.

A Pharmacovigilance Study of Hydroxychloroquine Cardiac Safety Profile: Potential Implication in COVID-19 Mitigation.

In light of the favorable outcomes of few small, non-randomized clinical studies, the Food and Drug Administration (FDA) has issued an Emergency Use Authorization (EUA) to Hydroxychloroquine (HCQ) for hospitalized coronavirus disease 2019 (COVID-19) patients. In fact, subsequent clinical studies with COVID-19 and HCQ have reported limited efficacy and poor clinical benefits. Unfortunately, a robust clinical trial for its effectiveness is not feasible at this emergency. Additionally, HCQ was suspected of causing cardiovascular adverse reactions (CV-AEs), but it has never been directly investigated. The objective of this pharmacovigilance analysis was to determine and characterize HCQ-associated cardiovascular adverse events (CV-AEs). We performed a disproportionality analysis of HCQ-associated CV-AEs using the FDA adverse event reporting system (FAERS) database. The FAERS database, comprising more than 11,901,836 datasets and 10,668,655 patient records with drug-adverse reactions, was analyzed. The disproportionality analysis was used to calculate the reporting odds ratios (ROR) with 95% confidence intervals (CI) to predict HCQ-associated CV-AEs. HCQ was associated with higher reporting of right ventricular hypertrophy (ROR: 6.68; 95% CI: 4.02 to 11.17), left ventricular hypertrophy (ROR: 3.81; 95% CI: 2.57 to 5.66), diastolic dysfunction (ROR: 3.54; 95% CI: 2.19 to 5.71), pericarditis (ROR: 3.09; 95% CI: 2.27 to 4.23), torsades de pointes (TdP) (ROR: 3.05; 95% CI: 2.30 to 4.10), congestive cardiomyopathy (ROR: 2.98; 95% CI: 2.01 to 4.42), ejection fraction decreased (ROR: 2.41; 95% CI: 1.80 to 3.22), right ventricular failure (ROR: 2.40; 95% CI: 1.64 to 3.50), atrioventricular block complete (ROR: 2.30; 95% CI: 1.55 to 3.41) and QT prolongation (ROR: 2.09; 95% CI: 1.74 to 2.52). QT prolongation and TdP are most relevant to the COVID-19 treatment regimen of high doses for a comparatively short period and represent the most common HCQ-associated AEs. The patients receiving HCQ are at higher risk of various cardiac AEs, including QT prolongation and TdP. These findings highlight the urgent need for prospective, randomized, controlled studies to assess the risk/benefit ratio of HCQ in the COVID-19 setting before its widespread adoption as therapy.

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.

Cardiotoxicity of the BCR-ABL1 tyrosine kinase inhibitors: Emphasis on ponatinib.

The advent of tyrosine kinase inhibitors (TKIs) targeted therapy revolutionized the treatment of chronic myeloid leukemia (CML) patients. However, cardiotoxicity associated with these targeted therapies puts the cancer survivors at higher risk. Ponatinib is a third-generation TKI for the treatment of CML patients having gatekeeper mutation T315I, which is resistant to the first and second generation of TKIs, namely, imatinib, nilotinib, dasatinib, and bosutinib. Multiple unbiased screening from our lab and others have identified ponatinib as most cardiotoxic FDA approved TKI among the entire FDA approved TKI family (total 50+). Indeed, ponatinib is the only treatment option for CML patients with T315I mutation. This review focusses on the cardiovascular risks and mechanism/s associated with CML TKIs with a particular focus on ponatinib cardiotoxicity. We have summarized our recent findings with transgenic zebrafish line harboring BNP luciferase activity to demonstrate the cardiotoxic potential of ponatinib. Additionally, we will review the recent discoveries reported by our and other laboratories that ponatinib primarily exerts its cardiotoxicity via an off-target effect on cardiomyocyte prosurvival signaling pathways, AKT and ERK. Finally, we will shed light on future directions for minimizing the adverse sequelae associated with CML-TKIs.

Mouse Models of Heart Failure with Preserved or Reduced Ejection Fraction.

Heart failure (HF) is a chronic, complex condition with increasing incidence worldwide, necessitating the development of novel therapeutic strategies. This has led to the current clinical strategies, which only treat symptoms of HF without addressing the underlying causes. Multiple animal models have been developed in an attempt to recreate the chronic HF phenotype that arises following a variety of myocardial injuries. Although significant strides have been made in HF research, an understanding of more specific mechanisms will require distinguishing models that resemble HF with preserved ejection fraction (HFpEF) from those with reduced ejection fraction (HFrEF). Therefore, current mouse models of HF need to be re-assessed to determine which of them most closely recapitulate the specific etiology of HF being studied. This will allow for the development of therapies targeted specifically at HFpEF or HFrEF. This review will summarize the commonly used mouse models of HF and discuss which aspect of human HF each model replicates, focusing on whether HFpEF or HFrEF is induced, to allow better investigation into pathophysiological mechanisms and treatment strategies.

Neutrophil-Derived S100A8/A9 Amplify Granulopoiesis After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) triggers myelopoiesis, resulting in heightened production of neutrophils. However, the mechanisms that sustain their production and recruitment to the injured heart are unclear. METHODS: Using a mouse model of the permanent ligation of the left anterior descending artery and flow cytometry, we first characterized the temporal and spatial effects of MI on different myeloid cell types. We next performed global transcriptome analysis of different cardiac cell types within the infarct to identify the drivers of the acute inflammatory response and the underlying signaling pathways. Using a combination of genetic and pharmacological strategies, we identified the sequelae of events that led to MI-induced myelopoiesis. Cardiac function was assessed by echocardiography. The association of early indexes of neutrophilia with major adverse cardiovascular events was studied in a cohort of patients with acute MI. RESULTS: Induction of MI results in rapid recruitment of neutrophils to the infarct, where they release specific alarmins, S100A8 and S100A9. These alarmins bind to the Toll-like receptor 4 and prime the nod-like receptor family pyrin domain-containing 3 inflammasome in naïve neutrophils and promote interleukin-1ß secretion. The released interleukin-1ß interacts with its receptor (interleukin 1 receptor type 1) on hematopoietic stem and progenitor cells in the bone marrow and stimulates granulopoiesis in a cell-autonomous manner. Genetic or pharmacological strategies aimed at disruption of S100A8/A9 and their downstream signaling cascade suppress MI-induced granulopoiesis and improve cardiac function. Furthermore, in patients with acute coronary syndrome, higher neutrophil count on admission and after revascularization correlates positively with major adverse cardiovascular disease outcomes. CONCLUSIONS: Our study provides novel evidence for the primary role of neutrophil-derived alarmins (S100A8/A9) in dictating the nature of the ensuing inflammatory response after myocardial injury. Therapeutic strategies aimed at disruption of S100A8/A9 signaling or their downstream mediators (eg, nod-like receptor family pyrin domain-containing 3 inflammasome, interleukin-1ß) in neutrophils suppress granulopoiesis and may improve cardiac function in patients with acute coronary syndrome.

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.