Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

Acetylation of PAX7 controls muscle stem cell self-renewal and differentiation potential in mice.

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.

Cardiotoxicity and pro-inflammatory effects of the immune checkpoint inhibitor Pembrolizumab associated to Trastuzumab.

BACKGROUND: The immunotherapy has revolutionized the world of oncology in the last decades with considerable advantages in terms of overall survival in cancer patients. The association of Pembrolizumab and Trastuzumab was recently proposed in clinical trials for the treatment of Trastuzumab-resistant advanced HER2-positive breast cancer. Although immunotherapies are frequently associated with a wide spectrum of immune-related adverse events, the cardiac toxicity has not been properly studied. PURPOSE: We studied, for the first time, the putative cardiotoxic and pro-inflammatory effects of Pembrolizumab associated to Trastuzumab. METHODS: Cell viability, intracellular calcium quantification and pro-inflammatory studies (analyses of the production of Interleukin 1ß, 6 and 8, the expression of NF-kB and Leukotriene B4) were performed in human fetal cardiomyocytes. Preclinical studies were also performed in C57BL6 mice by analyzing fibrosis and inflammation in heart tissues. RESULTS: The combination of Pembrolizumab and Trastuzumab leads to an increase of the intracellular calcium overload (of 3 times compared to untreated cells) and to a reduction of the cardiomyocytes viability (of 65 and 20-25%, compared to untreated and Pembrolizumab or Trastuzumab treated cells, respectively) indicating cardiotoxic effects. Notably, combination therapy increases the inflammation of cardiomyocytes by enhancing the expression of NF-kB and Interleukins. Moreover, in preclinical models, the association of Pembrolizumab and Trastuzumab increases the Interleukins expression of 40-50% compared to the single treatments; the expression of NF-kB and Leukotriene B4 was also increased. CONCLUSION: Pembrolizumab associated to Trastuzumab leads to strong cardiac pro-inflammatory effects mediated by overexpression of NF-kB and Leukotriene B4 related pathways.

The increased expression of the inducible Hsp70 (HSP70A1A) in serum of patients with heart failure and its protective effect against the cardiotoxic agent doxorubicin.

The aim of this study was to examine the potential association between the expression of Hsp70 protein and heart failure and to investigate the possible protective effect of Hsp70 against the doxorubicin-induced toxicity. Initially, at clinical level, the expression levels of the inducible Hsp70 were quantified in serum from patients with heart failure. Our results showed that in heart failure, Hsp70 concentration appeared to be increased in blood sera of patients compared to that of healthy individuals. The enhanced expression of Hsp70 in serum of patients with heart failure seemed to be associated with various features, such as gender, age and the type of heart failure, but not with its etiology. Next, in our study at cellular level, we used primary cell cultures isolated from embryos of Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70 and wild-type mice (F1/F1). After exposure to a wide range of doxorubicin concentrations and incubation times, the dose- and time-dependent toxicity of the drug, which appeared to be reduced in Tg/Tg cells, was demonstrated. In addition, doxorubicin administration appeared to result in a dose- and time-dependent decrease in the activity of two of the major endogenous antioxidant enzymes (SOD and GPx). The increased activity of these enzymes in Tg/Tg cells compared to the control F1/F1 cells was obvious, suggesting that the presence of Hsp70 confers enhanced tolerance against DOX-induced oxidative stress. Overall, it has been indicated that Hsp70 protein exerts a very important protective action and renders cells more resistant to the harmful effects of doxorubicin.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Injury of Adult Zebrafish Expressing Acvr1lQ204D Does Not Result in Heterotopic Ossification.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant genetic disorder in humans characterized by the gradual ossification of fibrous tissues, including skeletal muscle, tendons, and ligaments. In humans, mutations in the Type I BMP/TGFß family member receptor gene, ACVR1, are associated with FOP. Zebrafish acvr1l, previously known as alk8, is the functional ortholog of human ACVR1. We previously created and characterized the first adult zebrafish model for FOP by generating animals harboring heat shock-inducible mCherry-tagged constitutively active Acvr1l (Q204D). Since injury is a known trigger for heterotopic ossification (HO) development in human FOP patients, in this study, we investigated several injury models in Acvr1lQ204D-expressing zebrafish and the subsequent formation of HO. We performed studies of Activin A injection, cardiotoxin (CTX) injection, and caudal fin clip injury. We found that none of these methods resulted in HO formation at the site of injury. However, some of the cardiotoxin-injected and caudal fin-clipped animals did exhibit HO at distant sites, including the body cavity and along the spine. We describe these results in the context of new and exciting reports on FOP, and discuss future studies to better understand the etiology and progression of this disease.

Anthracycline and trastuzumab-induced cardiotoxicity in breast cancer.

OBJECTIVE: Breast cancer is the most common cancer among women. In the last twenty years early diagnosis, neoadjuvant and adjuvant systemic treatment that targeted to specific molecular targets have significantly reduced the mortality from breast cancer. However, the increase in survival has allowed to observe the cardiotoxic effects of anticancer therapy and increased mortality from cardiovascular causes, resulting in a large literature where experts try to identify the correct management of this critical problem. Even thought the increased attention in this field, many questions have not yet answers and new studies are needed. MATERIALS AND METHODS: We conducted a broad search of the English-language literature in Medline using the following search terms: cardiotoxicity, anthracyclines, trastuzumab, breast cancer, left ventricular dysfunction, heart failure. A manual examination of the articles found has been performed. RESULTS: We provide a comprehensive assessment of the current knowledge about cardiotoxicity induced by anthracycline plus trastuzumab in women affected by breast cancer. CONCLUSIONS: Early identification and prompt treatment of subclinical cardiotoxicity may improve cardiologic prognosis of these patients and may allow oncologists to avoid withdrawal of chemotherapy. That is why it becomes always more important the creation of multidisciplinary teams where cardiologists and oncologists work together to ensure optimal care to oncologic patients treated with cardiotoxic agents.

Cardiac Effects of Highly Active Antiretroviral Therapy in Perinatally HIV-Infected Children: The CHAART-2 Study.

BACKGROUND: Before the introduction of highly active antiretroviral therapy (HAART), cardiac mortality and morbidity were common in HIV-infected children. OBJECTIVES: This study sought to identify long-term cardiovascular effects of HAART in HIV-infected children. METHODS: The CHAART-2 (HAART-Associated Cardiotoxicity in HIV-Infected Children) study prospectively compared 148 echocardiograms from 74 HAART-exposed children to 860 echocardiograms from 140 HAART-unexposed but HIV-infected children from the Pulmonary and Cardiac Complications of Vertically Transmitted HIV Infection (P2C2 HIV) study. Both studies used similar protocol, centralized echocardiographic interpretation, and measures expressed as z-scores referenced to healthy controls. Associations between HAART exposure and echocardiographic measures were evaluated using generalized estimating equations. RESULTS: Comparing the HAART-exposed and HAART-unexposed groups, any HAART exposure was positively associated with left ventricular (LV) fractional shortening (z-score for difference = 1.07; p = 0.02) and HAART exposure duration (z-score difference per year = 0.17; p = 0.003. LV mass was negatively associated with any HAART exposure (z-score difference = -0.64; p = 0.01) as was septal thickness (z-score difference = -0.93; p = 0.001). Duration of HAART exposure was negatively associated with LV end-systolic dimension and heart rate (z-score difference per year= -0.11; p = 0.05; and z-score difference per year = -0.10; p = 0.002, respectively). During 11 years of follow-up, in the HAART-exposed group, LV mass and LV end-diastolic septal thickness were lower whereas LV contractility and LV fractional shortening were higher when compared to the HAART-unexposed group. CONCLUSIONS: Cardiac structure and function were better in perinatally HIV-infected children exposed to HAART than in those of similar children from the pre-HAART era but did decline over time. Evidence-based strategies for cardiovascular monitoring are needed to inform treatment decisions to improve long-term cardiovascular health.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells.

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2 -Sema3Afl °x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. Stem Cells 2017;35:1815-1834.

Pattern of cardiotoxin-induced muscle remodeling in distinct TLR-4 deficient mouse strains.

Tissue damage triggers innate immune response mediated by Toll-like receptor 4 (TLR) that recognizes endogenous host danger molecules associated with cell death and tissue inflammation, although the precise role of TLR-4 signaling in muscle tissue repair is still uncertain. Previously, we observed that TLR-4 exerted a protective effect preventing excessive muscular damage induced by Bothrops jararacussu crude venom. This study aimed to evaluate the involvement of TLR-4 at early stages of muscular tissue remodeling in distinct mouse strains after injection of purified snake venom. Muscular injury was induced by injection of 25 µl (0.05 mg/ml) of cardiotoxin (CTX) from Naja mossambica in the gastrocnemius muscle of C3H/HeN (wild-type); C3H/HeJ mice that express a non-functional TLR-4 receptor, C57BL/6 and Tlr4 -/- (B6 background) mice. Comparing to control, Tlr4 -/- mice presented at early stages (3 DPI) of muscle injury mild inflammation with low MMP-9 activity, scarce macrophage infiltration and premature change to anti-inflammatory phenotype, low TNF-α mRNA levels and reduced myogenin expression, with low regeneration and tissue remodeling. The presence of more Ly6Cneg macrophages in Tlr4 -/- mice at 3 DPI indicates that TLR-4 may influence the differentiation into Ly6Cneg or likely affect proliferation of such cells in the muscle. The present study shows that TLR-4 deficiency and genetic background influence the outcome of muscular tissue repair in aseptic lesions and yet still maintaining some level of signaling in the TLR4-mutant mice.

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin & Eosin staining that is widely used for subsequent morphological and morphometric analysis.

Can Bias Evaluation Provide Protection Against False-Negative Results in QT Studies Without a Positive Control Using Exposure-Response Analysis?

The revised ICH E14 document allows the use of exposure-response analysis to exclude a small QT effect of a drug. If plasma concentrations exceeding clinically relevant levels is achieved, a positive control is not required. In cases when this cannot be achieved, there may be a need for metrics to protect against false-negative results. The objectives of this study were to create bias in electrocardiogram laboratory QT-interval measurements and define a metric that can be used to detect bias severe enough to cause false-negative results using exposure-response analysis. Data from the IQ-CSRC study, which evaluated the QT effect of 5 QT-prolonging drugs, were used. Negative bias using 3 deterministic and 2 random methods was introduced into the reported QTc values and compared with fully automated data from the underlying electrocardiogram algorithm (COMPAS). The slope estimate of the Bland-Altman plot was used as a bias metric. With the deterministic bias methods, negative bias, measured between electrocardiogram laboratory values and COMPAS, had to be larger than approximately -20 milliseconds over a QTcF range of 100 milliseconds to cause failures to predict the QT effect of ondansetron, quinine, dolasetron, moxifloxacin, and dofetilide. With the random methods, the rate of false-negatives was ≤5% with bias severity < -10 milliseconds for all 5 drugs when plasma levels exceeded those of interest. Severe and therefore detectable bias has to be introduced into reported QTc values to cause false-negative predictions with exposure-response analysis.

Body Surface Area and Baseline Blood Pressure Predict Subclinical Anthracycline Cardiotoxicity in Women Treated for Early Breast Cancer.

BACKGROUND AND AIMS: Anthracyclines are highly effective chemotherapeutic agents which may cause long-term cardiac damage (chronic anthracycline cardiotoxicity) and heart failure. The pathogenesis of anthracycline cardiotoxicity remains incompletely understood and individual susceptibility difficult to predict. We sought clinical features which might contribute to improved risk assessment. METHODS: Subjects were women with early breast cancer, free of pre-existing cardiac disease. Left ventricular ejection fraction was measured using cardiovascular magnetic resonance before and >12 months after anthracycline-based chemotherapy (>3 months post-Trastuzumab). Variables associated with subclinical cardiotoxicity (defined as a fall in left ventricular ejection fraction of ≥5%) were identified by logistic regression. RESULTS: One hundred and sixty-five women (mean age 48.3 years at enrollment) completed the study 21.7 months [IQR 18.0-26.8] after starting chemotherapy. All received anthracyclines (98.8% epirubicin, cumulative dose 400 [300-450] mg/m2); 18% Trastuzumab. Baseline blood pressure was elevated (≥140/90mmHg, mean 147.3/86.1mmHg) in 18 subjects. Thirty-four subjects (20.7%) were identified with subclinical cardiotoxicity, independent predictors of which were the number of anthracycline cycles (odds ratio, OR 1.64 [1.17-2.30] per cycle), blood pressure ≥140/90mmHg (OR 5.36 [1.73-17.61]), body surface area (OR 2.08 [1.36-3.20] per standard deviation (0.16m2) increase), and Trastuzumab therapy (OR 3.35 [1.18-9.51]). The resultant predictive-model had an area under the receiver operating characteristics curve of 0.78 [0.70-0.86]. CONCLUSIONS: We found subclinical cardiotoxicity to be common even within this low risk cohort. Risk of cardiotoxicity was associated with modestly elevated baseline blood pressure-indicating that close attention should be paid to blood pressure in patients considered for anthracycline based chemotherapy. The association with higher body surface area suggests that indexing of anthracycline doses to surface area may not be appropriate for all, and points to the need for additional research in this area.

Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice.

Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

Suppression of Myostatin Stimulates Regenerative Potential of Injured Antigravitational Soleus Muscle in Mice under Unloading Condition.

Effects of myostatin (MSTN)-suppression on the regeneration of injured skeletal muscle under unloading condition were investigated by using transgenic mice expressing a dominant-negative form of MSTN (MSTN-DN). Both MSTN-DN and wild-type (WT) mice were subjected to continuous hindlimb suspension (HS) for 6 weeks. Cardiotoxin (CTX) was injected into left soleus muscle under anesthesia 2 weeks after the initiation of HS. Then, the soleus muscles were excised following 6-week HS (4 weeks after CTX-injection). CTX-injection caused to reduce the soleus fiber cross-sectional area (CSA) in WT mice under both unloading and weight-bearing conditions, but not in MSTN-DN mice. Under unloading condition, CTX-injected muscle weight and fiber CSA in MSTN-DN mice were significantly higher than those in WT mice. CTX-injected muscle had many damaged and regenerating fibers having central nuclei in both WT and MSTN-DN mice. Significant increase in the population of Pax7-positive nuclei in CTX-injected muscle was observed in MSTN-DN mice, but not in WT mice. Evidences indicate that the suppression of MSTN cause to increase the regenerative potential of injured soleus muscle via the increase in the population of muscle satellite cells regardless of unloading conditions.

Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom.

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 µg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 µM) and the ß-adrenergic antagonist propranolol (10 µM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

Subacute Cardiotoxicity of Yessotoxin: In Vitro and in Vivo Studies.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTX content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study, we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recordings of electrocardiograms, arterial blood pressure, plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the ß isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIß.

Trastuzumab Induces an Immediate, Transient Volume Increase in Humans: A Randomised Placebo-Controlled Trial.

BACKGROUND: The exact extent of and the mechanism by which trastuzumab causes cardiac side effects are not completely unravelled. We investigated the (cardiotoxic) side effects of trastuzumab in a relatively large homogeneous population. METHODS: Healthy male volunteers (n = 54) with a left ventricle ejection fraction (LVEF) > 55% were administered 6 mg/kg trastuzumab (n = 46) IV in 90 min in a placebo-controlled, parallel study. Placebo consisted of 0 · 9% NaCl (n = 8). Assessments included body weight, routine and cardiac laboratory markers and serial echocardiographic examinations (8 placebo and 9 trastuzumab treated participants) up to 63 days after dosing. Statistical analysis was done using repeated measurements of variance. FINDINGS: Following trastuzumab infusion, fluid retention was observed: mean body weight increased over the first 4 days post-administration with 0 · 4 kg (95%-confidence interval: - 0 · 2, 0 · 9, p = 0 · 2261) compared to placebo, mean haemoglobin concentration decreased with 0 · 3 mM (- 0 · 6, - 0 · 1; p = 0 · 0043), as did haematocrit (- 0 · 013 L/L [- 0 · 024, - 0 · 002], p = 0 · 0216), and protein (- 2 g/L [- 4, - 0], p = 0 · 0443) and albumin (- 2 g/L [- 3, - 1], p < 0 · 0001) concentrations. Elevations in NT-proBNP levels, parallel to the weight increase, were observed in individual cases, but not on a group level. Troponin-T concentrations did not increase. The only echocardiographic parameter that changed significantly at all studied dose levels was E/A-ratio, a load-dependent parameter: from 1 · 81 (SD 0 · 42) to 1 · 98 (0 · 31) 3-5 days after administration, contrast to placebo of 0 · 57 (90%-CI: 0 · 21-0 · 93, p = 0 · 0034). Ejection fraction and pulsed-wave Doppler recorded parameters remained unchanged. INTERPRETATION: Single dose administration of trastuzumab in humans is associated with an immediate, transient extracellular volume increase, either as a primary or secondary (compensatory) response, which can be detected easily using routine clinical assessments. Echocardiographic changes, both short and long term, could not be found after single dose administration to drug-naive patients.