Computational approaches identify a transcriptomic fingerprint of drug-induced structural cardiotoxicity.

Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to improve the mechanistic understanding of SCT. First, we combined machine learning methods with a modified calcium transient assay in human-induced pluripotent stem cell-derived cardiomyocytes to identify nine parameters that could predict SCT. Next, we applied transcriptomic profiling to human cardiac microtissues exposed to structural and non-structural cardiotoxins. Fifty-two genes expressed across the three main cell types in the heart (cardiomyocytes, endothelial cells, and fibroblasts) were prioritised in differential expression and network clustering analyses and could be linked to known mechanisms of SCT. This transcriptomic fingerprint may prove useful for generating strategies to mitigate SCT risk in early drug discovery.

Myofiber directs macrophages IL-10-Vav1-Rac1 efferocytosis pathway in inflamed muscle following CTX myoinjury by activating the intrinsic TGF-ß signaling.

BACKGROUND: To explore the role of skeletal muscle specific TGF-ß signaling on macrophages efferocytosis in inflamed muscle caused by Cardiotoxin (CTX) injection. METHODS: CTX myoinjury was manipulated in TGF-ßr2flox/flox (control) mice or transgenic mice with TGF-ß receptor 2 (TGF-ßr2) being specifically deleted in skeletal muscle (SM TGF-ßr2-/-). Gene levels of TGF-ß signal molecules, special inflammatory mediators in damaged muscle or in cultured and differentiated myogenic precursor cells (MPC-myotubes) were monitored by transcriptome microarray or qRT-PCR. TGF-ß pathway molecules, myokines and embryonic myosin heavy chain in regenerating myofibers, the phenotype and efferocytosis of macrophages were evaluated by immunofluorescence, immunoblotting, Luminex, or FACS analysis. In vitro apoptotic cells were prepared by UV-irradiation. RESULTS: In control mice, TGF-ß-Smad2/3 signaling were significantly up-regulated in regenerating centronuclear myofibers after CTX-myoinjury. More severe muscle inflammation was caused by the deficiency of muscle TGF-ß signaling, with the increased number of M1, but the decreased number of M2 macrophages. Notably, the deficiency of TGF-ß signaling in myofibers dramatically affected on the ability of macrophages to conduct efferocytosis, marked by the decreased number of Annexin-V-F4/80+Tunel+ macrophages in inflamed muscle, and the impaired uptake of macrophages to PKH67+ apoptotic cells transferred into damaged muscle. Further, our study suggested that, the intrinsic TGF-ß signaling directed IL-10-Vav1-Rac1 efferocytosis signaling in muscle macrophages. CONCLUSIONS: Our data demonstrate that muscle inflammation can be suppressed potentially by activating the intrinsic TGF-ß signaling in myofibers to promote IL-10 dependent-macrophages efferocytosis. Video Abstract.

Clinical Trial in a Dish: Using Patient-Derived Induced Pluripotent Stem Cells to Identify Risks of Drug-Induced Cardiotoxicity.

Drug-induced cardiotoxicity is a significant clinical issue, with many drugs in the market being labeled with warnings on cardiovascular adverse effects. Treatments are often prematurely halted when cardiotoxicity is observed, which limits their therapeutic potential. Moreover, cardiotoxicity is a major reason for abandonment during drug development, reducing available treatment options for diseases and creating a significant financial burden and disincentive for drug developers. Thus, it is important to minimize the cardiotoxic effects of medications that are in use or in development. To this end, identifying patients at a higher risk of developing cardiovascular adverse effects for the drug of interest may be an effective strategy. The discovery of human induced pluripotent stem cells has enabled researchers to generate relevant cell types that retain a patient's own genome and examine patient-specific disease mechanisms, paving the way for precision medicine. Combined with the rapid development of pharmacogenomic analysis, the ability of induced pluripotent stem cell-derivatives to recapitulate patient-specific drug responses provides a powerful platform to identify subsets of patients who are particularly vulnerable to drug-induced cardiotoxicity. In this review, we will discuss the current use of patient-specific induced pluripotent stem cells in identifying populations who are at risk to drug-induced cardiotoxicity and their potential applications in future precision medicine practice. Graphic Abstract: A graphic abstract is available for this article.

Curcumin-coated gold nanoparticles attenuate doxorubicin-induced cardiotoxicity via regulating apoptosis in a mouse model.

Doxorubicin (DOX) is one of the most widely used chemotherapy agents; however, its nonselective effect causes cardiotoxicity. Curcumin (Cur), a well known dietary polyphenol, could exert a significant cardioprotective effect, but the biological application of this substance is limited by its chemical insolubility. To overcome this limitation, in this study, we synthesised gold nanoparticles based on Cur (Cur-AuNPs). Ultraviolet-visible (UV-Vis) absorbance spectroscopy and transmission electron microscopy (TEM) were performed for the characterisation of synthesised NPs, and Fourier transform infrared (FTIR) spectroscopy were applied to detect Cur on the surface of AuNPs. Its cytotoxicity effect on H9c2 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The biological efficacy of Cur-AuNPs was assessed after acute cardiotoxicity induction in BALB/c mice with DOX injection. The serum biomarkers, myocardial histological changes, and cardiomyocyte apoptosis were then measured. The results revealed that the heart protection by Cur-AuNPs is more effective than Cur alone. Heart protective effect of Cur-AuNPs was evident both in the short-term (24 hours) and long-term (14 days) study. The results of Cur-AuNPs400 after 24 hours of toxicity induction displayed the reduction of the cardiac injury serum biomarkers (LDH, CK-MB, cTnI, ADT, and ALT) and apoptotic proteins (Bax and Caspase-3), as well as increase of Bcl-2 anti-apoptotic proteins without any sign of interfibrillar haemorrhage and intercellular spaces in the heart tissue microscopic images. Our long-term study signifies that Cur-AuNPs400 in DOX-intoxicated mice could successfully inhibit body and heart weight loss in comparison to DOX group.

Skeletal Muscle Regeneration in Cardiotoxin-Induced Muscle Injury Models.

Skeletal muscle injuries occur frequently in daily life and exercise. Understanding the mechanisms of regeneration is critical for accelerating the repair and regeneration of muscle. Therefore, this article reviews knowledge on the mechanisms of skeletal muscle regeneration after cardiotoxin-induced injury. The process of regeneration is similar in different mouse strains and is inhibited by aging, obesity, and diabetes. Exercise, microcurrent electrical neuromuscular stimulation, and mechanical loading improve regeneration. The mechanisms of regeneration are complex and strain-dependent, and changes in functional proteins involved in the processes of necrotic fiber debris clearance, M1 to M2 macrophage conversion, SC activation, myoblast proliferation, differentiation and fusion, and fibrosis and calcification influence the final outcome of the regenerative activity.

Mitochondrial dynamics imbalance and mitochondrial dysfunction contribute to the molecular cardiotoxic effects of lenvatinib.

Lenvatinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of resistant differentiated thyroid cancer, advanced renal cell carcinoma, unresectable hepatocellular carcinoma, and endometrial carcinoma. Although it is successful in cancer treatment, it can cause life-threatening side effects such as cardiotoxicity. The molecular mechanism of cardiotoxicity caused by lenvatinib is not fully known. In this study, the molecular mechanism of lenvatinib's cardiotoxicity was investigated focusing on mitochondrial toxicity in the H9c2 cardiomyoblastic cell line. Lenvatinib inhibited cell viability at 48 and 72 h exposure with three selected concentrations (1.25 µM, 5 µM and 10 µM); and inhibited intracellular ATP after 72 h exposure compared to the control group. Mitochondrial membrane potential was decreased after 48 h and did not show significant changes after 72 h exposure. Evaluated with real-time PCR, mitochondrial dynamics (Mfn1, Mfn2, OPA1, DRP1, Fis1) expression levels after lenvatinib treatment significantly changed. Lenvatinib triggered the tendency from fusion to fission in mitochondria after 48 h exposure, and increased both fusion and fission after 72 h. The mtDNA ratio increased after 48 h and decreased after 72 h. ASK1, JNK and AMPKα2 increased. UCP2 showed downregulation, SOD2 level showed upregulation and Cat levels decreased after drug treatment. Nrf1 and Nrf2 also changed concentration-dependently. Protein carbonyl levels increased significantly after lenvatinib treatments indicating oxidative stress. The protein levels of the electron transport chain complexes, LONP1, UCP2, and P21 showed significant differences after lenvatinib treatment. The outcome of our study is expected to be a contribution to the understanding of the molecular mechanisms of TKI-induced cardiotoxicity.

Cardiac toxicity from bisphenol A exposure in human-induced pluripotent stem cell-derived cardiomyocytes.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical that is widely used in a variety of products, including plastics, medical equipment and receipts. Hence, most people are exposed to BPA through the skin, via inhalation and via the digestive system, and such exposure has been linked to cardiovascular diseases including coronary artery disease, hypertension, atherosclerosis, and myocardial infarction. However, the underlying mechanisms of cardiac dysfunction caused by BPA remain poorly understood. In this study, we found that BPA exposure altered cardiac function in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Acute BPA exposure in hiPSC-CMs resulted in reduced field potential, as measured by multielectrode array (MEA). Furthermore, we observed that BPA dose-dependently inhibited ICa, INa or IKr channels. In addition, BPA exposure dose-dependently inhibited calcium transients and contraction in hiPSC-CMs. Our findings suggest that BPA exposure leads to cardiac dysfunction and cardiac risk factors such as arrhythmia.

Workshop Report: FDA Workshop on Improving Cardiotoxicity Assessment With Human-Relevant Platforms.

Given that cardiovascular safety concerns remain the leading cause of drug attrition at the preclinical drug development stage, the National Center for Toxicological Research of the US Food and Drug Administration hosted a workshop to discuss current gaps and challenges in translating preclinical cardiovascular safety data to humans. This white paper summarizes the topics presented by speakers from academia, industry, and government intended to address the theme of improving cardiotoxicity assessment in drug development. The main conclusion is that to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Potential regulatory applications of human-derived cardiomyocytes and future directions in employing human-relevant platforms to fill the gaps and overcome barriers and challenges in preclinical cardiovascular safety assessment were discussed. This paper is intended to serve as an initial step in a public-private collaborative development program for human-relevant cardiotoxicity tools, particularly for cardiotoxicities characterized by contractile dysfunction or structural injury.

SNX17 protects the heart from doxorubicin-induced cardiotoxicity by modulating LMOD2 degradation.

Anthracyclines including doxorubicin (DOX) are still the most widely used and efficacious antitumor drugs, although their cardiotoxicity is a significant cause of heart failure. Despite considerable efforts being made to minimize anthracycline-induced cardiac adverse effects, little progress has been achieved. In this study, we aimed to explore the role and underlying mechanism of SNX17 in DOX-induced cardiotoxicity. We found that SNX17 was downregulated in cardiomyocytes treated with DOX both in vitro and in vivo. DOX treatment combined with SNX17 interference worsened the damage to neonatal rat ventricular myocytes (NRVMs). Furthermore, the rats with SNX17 deficiency manifested increased susceptibility to DOX-induced cardiotoxicity (myocardial damage and fibrosis, impaired contractility and cardiac death). Mechanistic investigation revealed that SNX17 interacted with leiomodin-2 (LMOD2), a key regulator of the thin filament length in muscles, via its C-TERM domain and SNX17 deficiency exacerbated DOX-induced cardiac systolic dysfunction by promoting aberrant LMOD2 degradation through lysosomal pathway. In conclusion, these findings highlight that SNX17 plays a protective role in DOX-induced cardiotoxicity, which provides an attractive target for the prevention and treatment of anthracycline induced cardiotoxicity.

LRTM1 promotes the differentiation of myoblast cells by negatively regulating the FGFR1 signaling pathway.

The proliferation and differentiation of myoblast cells are regulated by the fibroblast growth factor receptor (FGFR) signaling pathway. Although the regulation of FGFR signaling cascades has been widely investigated, the inhibitory mechanism that particularly function in skeletal muscle myogenesis remains obscure. In this study, we determined that LRTM1, an inhibitory regulator of the FGFR signaling pathway, negatively modulates the activation of ERK and promotes the differentiation of myoblast cells. LRTM1 is dynamically expressed during myoblast differentiation and skeletal muscle regeneration after injury. In mouse myoblast C2C12 cells, knockout (KO) of Lrtm1 significantly prevents the differentiation of myoblast cells; this effect is associated with the reduction of MyoD transcriptional activity and the overactivation of ERK kinase. Notably, further studies demonstrated that LRTM1 associates with p52Shc and inhibits the recruitment of p52Shc to FGFR1. Taken together, our findings identify a novel negative regulator of FGFR1, which plays an important role in regulating the differentiation of myoblast cells.

Taurine Has Potential Protective Effects against the Chronic Cardiotoxicity Induced by Doxorubicin in Mice.

Doxorubicin (DOX) is an effective anticancer anthracycline drug; however, the cardiotoxicity limits its application. The aim of the present study was to investigate the potential protective effect of taurine against DOX-induced chronic cardiotoxicity in mice. We found that exogenous supplementation of taurine can inhibit the weight loss of mice caused by DOX. The increased activity of myocardial enzymes creatine kinase (CK) and lactate dehydrogenase (LDH) in response to DOX treatment were significantly hampered. In addition, taurine supplementation alleviated the decrease in superoxide dismutase (SOD) activity, glutathione (GSH) content, glutathione peroxidase 4 (Gpx4) expression, and the increase in malondialdehyde (MDA) content caused by DOX. Besides, taurine alleviated myocardial myofibrillar disruption and mitochondrial edema. Furthermore, our results showed that taurine decreased the expressions of cleaved caspase-3 and Bax/Bcl2, thereby inhibiting apoptosis. These collective data demonstrated that exogenous taurine supplementation has a potentially protective effect against the myocardial damage caused by doxorubicin in mice by enhancing antioxidant capacity and reducing oxidative damage and apoptosis.

Treg cells limit IFN-γ production to control macrophage accrual and phenotype during skeletal muscle regeneration.

Skeletal muscle regeneration is a highly orchestrated process that depends on multiple immune-system cell types, notably macrophages (MFs) and Foxp3+CD4+ regulatory T (Treg) cells. This study addressed how Treg cells rein in MFs during regeneration of murine muscle after acute injury with cardiotoxin. We first delineated and characterized two subsets of MFs according to their expression of major histocompatibility complex class II (MHCII) molecules, i.e., their ability to present antigens. Then, we assessed the impact of Treg cells on these MF subsets by punctually depleting Foxp3+ cells during the regenerative process. Treg cells controlled both the accumulation and phenotype of the two types of MFs. Their absence after injury promoted IFN-γ production, primarily by NK and effector T cells, which ultimately resulted in MF dysregulation and increased inflammation and fibrosis, pointing to compromised muscle repair. Thus, we uncovered an IFN-γ-centered regulatory layer by which Treg cells keep MFs in check and dampen inflammation during regeneration of skeletal muscle.

Reduction of Superoxide Dismutase 1 Delays Regeneration of Cardiotoxin-Injured Skeletal Muscle in KK/Ta-Ins2Akita Mice with Progressive Diabetic Nephropathy.

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.

Ameliorative effect of rosiglitazone, a peroxisome proliferator gamma agonist on adriamycin-induced cardio toxicity via suppressing oxidative stress and apoptosis.

We investigated the rosiglitazone (RSG) effect on adriamycin (ADM)-induced cardio toxicity in experimental animals. Forty adult Wistar male rats were separated into four groups as follows: normal control; RSG (10 mg/kg)-treated; ADM (10 mg/kg)-administered; and ADM (10 mg/kg) + RSG (10 mg/kg)-treated. Serum lipid level, different biochemical biomarkers, histological analysis, and nuclear factor erythroid 2-related factor/heme oxygenase-1 (Nrf2/HO-1), Caspase 3, B-cell lymphoma 2 (Bcl-2), and Bax gene expression were assessed in serum and cardiac tissue samples. Our results show that RSG treatment in ADM-administered animals significantly diminished low-density lipoprotein cholesterol, triglyceride, and total cholesterol, and increases high-density lipoprotein cholesterol (HDL-c) in comparison with the ADM group. RSG treatment reduced the effect of ADM administration on cardiac dysfunction markers such as cardiac troponin T Creatine Kinase-MB, aspartate aminotransferase, and lactate dehydrogenase, showing the amelioration of cardio toxicity in ADM-administered rats. Additionally, RSG treatment significantly decreased the level of malondialdehyde and nitric oxide in cardiovascular tissue. RSG-treated rats in combination with ADM likewise showed a significant increase in reduced glutathione, superoxide dismutase, catalase content, and the activity of glutathione peroxidase (GPx) as compared with ADM group. Moreover, RSG treatment in ADM rats significantly increased an Nrf2 and HO-1 expression in comparison with ADM group. While in apoptosis parameters, RSG treatment in ADM rats significantly diminished a cleaved caspase-3 and Bax expression as well as expanded Bcl-2 expression when contrasted with ADM group of rats. In conclusion, RSG is capable of protecting heart toxicity in ADM-treated animals through defensive effects on oxidative stress and biochemical markers.

Fasting regulates EGR1 and protects from glucose- and dexamethasone-dependent sensitization to chemotherapy.

Fasting reduces glucose levels and protects mice against chemotoxicity, yet drugs that promote hyperglycemia are widely used in cancer treatment. Here, we show that dexamethasone (Dexa) and rapamycin (Rapa), commonly administered to cancer patients, elevate glucose and sensitize cardiomyocytes and mice to the cancer drug doxorubicin (DXR). Such toxicity can be reversed by reducing circulating glucose levels by fasting or insulin. Furthermore, glucose injections alone reversed the fasting-dependent protection against DXR in mice, indicating that elevated glucose mediates, at least in part, the sensitizing effects of rapamycin and dexamethasone. In yeast, glucose activates protein kinase A (PKA) to accelerate aging by inhibiting transcription factors Msn2/4. Here, we show that fasting or glucose restriction (GR) regulate PKA and AMP-activated protein kinase (AMPK) to protect against DXR in part by activating the mammalian Msn2/4 ortholog early growth response protein 1 (EGR1). Increased expression of the EGR1-regulated cardioprotective peptides atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in heart tissue may also contribute to DXR resistance. Our findings suggest the existence of a glucose-PKA pathway that inactivates conserved zinc finger stress-resistance transcription factors to sensitize cells to toxins conserved from yeast to mammals. Our findings also describe a toxic role for drugs widely used in cancer treatment that promote hyperglycemia and identify dietary interventions that reverse these effects.

Left Ventricular Hypertrophy Increases Susceptibility to Bupivacaine-induced Cardiotoxicity through Overexpression of Transient Receptor Potential Canonical Channels in Rats.

BACKGROUND: Local anesthetics, particularly potent long acting ones such as bupivacaine, can cause cardiotoxicity by inhibiting sodium ion channels; however, the impact of left ventricular hypertrophy on the cardiotoxicity and the underlying mechanisms remain undetermined. Transient receptor potential canonical (TRPC) channels are upregulated in left ventricular hypertrophy. Some transient receptor potential channel subtypes have been reported to pass relatively large cations, including protonated local anesthetics; this is known as the "pore phenomenon." The authors hypothesized that bupivacaine-induced cardiotoxicity is more severe in left ventricular hypertrophy due to upregulated TRPC channels. METHODS: The authors used a modified transverse aortic constriction model as a left ventricular hypertrophy. Cardiotoxicity caused by bupivacaine was compared between sham and aortic constriction male rats, and the underlying mechanisms were investigated by recording sodium ion channel currents and immunocytochemistry of TRPC protein in cardiomyocytes. RESULTS: The time to cardiac arrest by bupivacaine was shorter in aortic constriction rats (n =11) than in sham rats (n = 12) (mean ± SD, 1,302 ± 324 s vs. 1,034 ± 211 s; P = 0.030), regardless of its lower plasma concentration. The half-maximal inhibitory concentrations of bupivacaine toward sodium ion currents were 4.5 and 4.3 µM, which decreased to 3.9 and 2.6 µM in sham and aortic constriction rats, respectively, upon coapplication of 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3 channel activator. In both groups, sodium ion currents were unaffected by QX-314, a positively charged lidocaine derivative, that hardly permeates the cell membrane, but was significantly decreased with QX-314 and 1-oleoyl-2-acetyl-sn-glycerol coapplication (sham: 79 ± 10% of control; P = 0.004; aortic constriction: 47± 27% of control; P = 0.020; n = 5 cells per group). Effects of 1-oleoyl-2-acetyl-sn-glycerol were antagonized by a specific TRPC3 channel inhibitor. CONCLUSIONS: Left ventricular hypertrophy exacerbated bupivacaine-induced cardiotoxicity, which could be a consequence of the "pore phenomenon" of TRPC3 channels upregulated in left ventricular hypertrophy.

Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Mesalazine Induces Oxidative Stress and Cytochrome c Release in Isolated Rat Heart Mitochondria: An Analysis of Cardiotoxic Effects.

Mesalazine is widely used in the management of inflammatory bowel disease. Previous studies reported that mesalazine-induced cardiotoxicity is a rare, potentially fatal complication. Mitochondria play an important role in myocardial tissue homeostasis. Deterioration in mitochondrial function will eventually lead to cardiomyocyte death and consequently cardiovascular dysfunction. The aim of the current study was to investigate the effects of mesalazine on rat heart mitochondria. Rat heart mitochondria were isolated by mechanical lysis and differential centrifugation. Parameters of mitochondrial toxicity including succinate dehydrogenase (SDH) activity, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release were evaluated. Results revealed that mesalazine induced a concentration- and time-dependent rise in mitochondrial ROS formation, inhibition of SDH, MMP collapse, mitochondrial swelling, and cytochrome c release in rat heart mitochondria. These results indicate that the cardiotoxic effects of mesalazine are most likely associated with mitochondrial dysfunction and ROS formation, which finally ends in cytochrome c release signaling and induction of apoptosis.

H19 controls reactivation of the imprinted gene network during muscle regeneration.

The H19 locus controls fetal growth by regulating expression of several genes from the imprinted gene network (IGN). H19 is fully repressed after birth, except in skeletal muscle. Using loss-of-function H19(Δ3) mice, we investigated the function of H19 in adult muscle. Mutant muscles display hypertrophy and hyperplasia, with increased Igf2 and decreased myostatin (Mstn) expression. Many imprinted genes are expressed in muscle stem cells or satellite cells. Unexpectedly, the number of satellite cells was reduced by 50% in H19(Δ3) muscle fibers. This reduction occurred after postnatal day 21, suggesting a link with their entry into quiescence. We investigated the biological function of these mutant satellite cells in vivo using a regeneration assay induced by multiple injections of cardiotoxin. Surprisingly, despite their reduced number, the self-renewal capacity of these cells is fully retained in the absence of H19. In addition, we observed a better regeneration potential of the mutant muscles, with enhanced expression of several IGN genes and genes from the IGF pathway.