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.

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.

Animal Models of Exercise From Rodents to Pythons.

Acute and chronic animal models of exercise are commonly used in research. Acute exercise testing is used, often in combination with genetic, pharmacological, or other manipulations, to study the impact of these manipulations on the cardiovascular response to exercise and to detect impairments or improvements in cardiovascular function that may not be evident at rest. Chronic exercise conditioning models are used to study the cardiac phenotypic response to regular exercise training and as a platform for discovery of novel pathways mediating cardiovascular benefits conferred by exercise conditioning that could be exploited therapeutically. The cardiovascular benefits of exercise are well established, and, frequently, molecular manipulations that mimic the pathway changes induced by exercise recapitulate at least some of its benefits. This review discusses approaches for assessing cardiovascular function during an acute exercise challenge in rodents, as well as practical and conceptual considerations in the use of common rodent exercise conditioning models. The case for studying feeding in the Burmese python as a model for exercise-like physiological adaptation is also explored.

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation.

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.

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.

Acid-functionalized single-walled carbon nanotubes alter epithelial tight junctions and enhance paracellular permeability.

Due to their unique properties, carbon nanotubes (CNTs) are being widely explored for industrial and medical applications. This has necessitated a thorough assessment of the effect of CNTs on human and animal physiology and health. Impact of CNTs on epithelial tight junctions has not been evaluated in the context of their toxic effects in many biological systems. In the present study, we examined the effect of acid functionalized single-walled carbon nanotubes (AF-SWCNTs) on the function and expression of two tight junction proteins (ZO-1 and occludin) in the Madin-Darby canine kidney (MDCK) cell line. Treatment of MDCK cells with AF-SWCNT resulted in a downregulation of tight junction proteins, decreased trans-epithelial electrical resistance (TER), increased paracellular permeability, and disruption of tight junctions. Taken together, our data demonstrate that AF-SWCNT disrupts tight junction barrier by downregulating tight junction proteins in MDCK epithelial cells.

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.

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.

Geometric constraints alter cell arrangements within curved epithelial tissues.

Organ and tissue formation are complex three-dimensional processes involving cell division, growth, migration, and rearrangement, all of which occur within physically constrained regions. However, analyzing such processes in three dimensions in vivo is challenging. Here, we focus on the process of cellularization in the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometric constraints. Using microfluidics combined with fluorescence microscopy, we extract quantitative information on the three-dimensional epithelial cell morphology. We observed a cellular membrane rearrangement in which cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges were observed more frequently in the anterior pole than in the embryo trunk. Furthermore, cells within the anterior pole skewed toward the trunk along their long axis relative to the embryo surface, with maximum skew on the ventral side. We constructed a vertex model for cells in a curved environment. We could reproduce the observed cellular skew in both wild-type embryos and embryos with distorted morphology. Further, such modeling showed that cell rearrangements were more likely in ellipsoidal, compared with cylindrical, geometry. Overall, we demonstrate that geometric constraints can influence three-dimensional cell morphology and packing within epithelial tissues.

3D Protein Dynamics in the Cell Nucleus.

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.

Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

Applications of imaging fluorescence correlation spectroscopy.

Imaging fluorescence correlation spectroscopy (imaging FCS), or the acquisition of fluorescence correlation functions at contiguous points in an imaging format, is a recent addition to quantitative bioimaging. Imaging FCS has been implemented in various modalities. These techniques provide excellent time resolution, have single molecules sensitivity and can be combined with super-resolution techniques, thus combining high spatial and temporal resolution. Although still at its beginning it has been applied in different forms to biological problems. This review looks at applications of imaging FCS in the last two years with the aim to give the reader an overview of the capabilities of these new techniques.

Neurocognition in first-degree healthy relatives (siblings) of bipolar affective disorder patients.

AIM: Cognitive deficits have been presupposed to be endophenotypic markers in bipolar disorder, but few studies have ascertained the cognitive deficits in healthy relatives of bipolar disorder patients. The aim of the present study was to assess the cognitive functions of first-degree relatives of patients with bipolar disorder and compare them with healthy controls. METHODS: Ten first-degree apparently healthy relatives of patients with bipolar disorder were compared with 10 age- and education-matched control subjects on computer-based cognitive tests. RESULTS: As compared to the control group, the relatives group performed significantly poorly on tests for executive function and vigilance, while on the test for working memory the performance was not significantly different on most of the parameters. CONCLUSIONS: Executive functioning and vigilance could be potential markers of the endophenotype in bipolar patients.

Matching avrami indices achieves similar hardnesses in palm oil-based fats.

This study demonstrates how chemically interesterified hydrogenated palm oil (IHPO) and partially hydrogenated palm oil (PHPO) can be structured to have similar mechanical properties. Crystallization of IHPO at 30 degrees C for 24 h yielded a fat with a solid fat content (SFC) of 45% and a yield force of 51.5 N. On the other hand, PHPO had a SFC of 50% and a yield force of 44 N when crystallized under the same conditions. The result was opposite from what would be expected from the SFC point of view, thus suggesting that the microstructure of the fat plays a key role in determining mechanical properties. By matching crystallization behavior using the Avrami index as a guide, microstructures and material hardness were successfully matched. These results suggest that the dynamics of structure formation was a key factor influencing the macroscopic mechanical properties of palm oil-based fats.