Investigating skeletal muscle biomarkers for the early detection of Australian myotoxic snake envenoming: an animal model pilot study.

INTRODUCTION: Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. METHODS: Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. RESULTS: There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0-4 hours and 0-10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78-1.00) and 0.88 (0.73-1.00) versus 0.79 (0.63-0.95) and 0.66 (0.51-0.80). DISCUSSION: The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. CONCLUSION: Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming.

Relationship between Serum miR-106 and MYL4 Levels and the Prevalence, Risk Stratification, and Prognosis of Atrial Fibrillation.

Objective: To analyze the predictive value of serum microRNA-106 (miRNA-106), miR-106, and myosin light chain 4 (MYL4) levels on the prevalence of atrial fibrillation and to explore the relationship between serum miR-106 and MYL4 and the risk stratification and prognosis of atrial fibrillation, thereby providing basis for them to become clinical targets for the treatment of atrial fibrillation in the future. Methods: 300 patients with atrial fibrillation treated in our hospital from May 2017 to March 2019 were selected as the atrial fibrillation group, and 300 healthy people who came to our hospital for physical examination in the same period were selected as the control group. The general data of the subjects in the two groups were collected. The serum miR-106 level of the subjects in the two groups was detected by fluorescence quantitative polymerase chain reaction (PCR), and the level of MYL4 was detected by enzyme-linked immunosorbent assay (ELISA). The expression of miR-106 and MYL4 in the myocardium was observed by immunohistochemistry. The relationship between the levels of serum miR-106 and MYL4 and the prevalence of atrial fibrillation and the score of atrial fibrillation thromboembolism risk stratification scoring system (cha2ds2) was compared between the two groups. The relationship between serum level of miR-106 and prognosis of patients with atrial fibrillation was analyzed. Results: The systolic blood pressure, diastolic blood pressure, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and left anterior descending artery (LAD) in the atrial fibrillation group were significantly higher than those in the control group, while HDL-C and left ventricular ejection fraction (LVEF) were significantly lower than those in the control group (P < 0.01). The level of serum miR-106 in patients with atrial fibrillation was significantly higher than that in the control group, whereas the level of MYL4 was significantly lower than that in the control group (P < 0.01). miR-106 was mainly localized in the cytoplasm, and the positive expression rate of miR-106 was 71.43% (81/115) in patients with atrial fibrillation and 21.74% (25/115) in patients with sinus rhythm. MYL4 was mainly located in the cell membrane and the positive expression rate of MYL4 was 24.35% (28/115) in patients with atrial fibrillation and 64.35% (74/115) in patients with sinus rhythm. With the increase of the severity of atrial fibrillation, the level of serum miR-106 gradually increased and the level of MYL4 gradually decreased, which were statistically significant compared with the control group (P < 0.05). With the increase of miR-106 level and the decrease of MYL4 level, the prevalence of atrial fibrillation gradually increased. With the increase of cha2ds2 score, the level of serum miR-106 increased and the level of MYL4 decreased. The survival rate of patients with miR - 106 ≤ 1.96 was significantly higher than that of patients with miR - 106 > 1.96. The survival rate of patients with MYL4 ≥ 0.24 was significantly higher than that of patients with MYL4 < 0.24. At the same time, TC and LDL-C were included in the analysis. The results showed that the survival rate of patients with TC ≤ 4.5 mmol/L was significantly higher than that of patients with TC > 4.5 mmol/L, and that of patients with LDL-C ≤ 2.6 mmol/L was significantly higher than that of patients with LDL-C > 2.6 mmol/L. Conclusion: Serum miR-106 and MYL4 levels are closely related to the prevalence of atrial fibrillation, which can reflect the risk of thromboembolism in patients with atrial fibrillation and can be used as a biological indicator to predict the prognosis of patients with atrial fibrillation.

Elevated Myl9 reflects the Myl9-containing microthrombi in SARS-CoV-2-induced lung exudative vasculitis and predicts COVID-19 severity.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

CD247, a Potential T Cell-Derived Disease Severity and Prognostic Biomarker in Patients With Idiopathic Pulmonary Fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear. Methods: Datasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis. Results: CD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein-protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils. Conclusion: These results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF.

Local vibration induced vascular pathological structural changes and abnormal levels of vascular damage indicators.

BACKGROUND: The vascular component of the hand-arm-vibration syndrome (HAVS) is often characterized by vibration-induced white fingers (VWF). Active substances secreted by the vascular endothelial cells (VEC) maintain a dynamic balance but damage to the blood vessels may occur when the equilibrium is altered, thus forming an important pathological basis for VWF. This study was aimed at investigating vascular damage indicators as a basis for an early detection of disorders caused by vibration, using the rat tail model. METHODS AND RESULTS: Experiments were conducted using a control group of rats not exposed to vibration while two exposed groups having different exposure durations of 7 and 14 days were randomly formed. Following exposure, the structural changes of tail tissue samples in anesthetized rats were observed. Enzyme-linked immunosorbent assay (ELISA) was used for analyzing four vascular damage indicators myosin regulatory light chain (MLC2), endothelin-1 (ET-1), vinculin (VCL) and 5-hydroxytryptamine (5-HT) in tail blood samples. We found that both vascular smooth muscle and endothelial cells displayed changes in morphology characterized by vacuolization and swelling in the vibration-exposed group. The levels of vascular damage indicators were altered under the vibration. CONCLUSION: The degree of vascular pathology increased with the longer duration exposure. Furthermore, the levels of MLC2, ET-1 and 5-HT in rat plasma were associated with vascular injury caused by local vibration.

Statin treatment before stroke reduces pro-inflammatory cytokine levels after stroke.

OBJECTIVE: In this clinical case-control study, we investigated statin treatment in stroke patients on a range of inflammatory effectors in peripheral blood. We focus on RhoA GTPase and its downstream effectors as a future inflammatory target in stroke treatment. METHODS: Data from 10 patients already on statins at stroke onset (Pre-S group) was compared with data from both 29 patients starting statin treatment right after stroke onset (Post-S group) and with 8 healthy controls. In T-cells isolated from stroke patients, we analyzed the activity of the main cytoskeletal regulator RhoA GTPase and its downstream effectors: rho-associated protein kinase (ROCK), myosin phosphatase targeting protein subunit 1 (pMYPT1), myosin light chain kinase (pMLC) and cofilin. In the blood samples, we further determined levels of 12 key plasma cytokines as well as C-reactive protein (CRP) and kallikrein. RESULTS: Compared to healthy controls, the Post-S group achieved significantly higher RhoA and ROCK activities, while the Pre-S did not differ from controls. Levels of pMYPT1, pMLC and cofilin did not differ from controls in the Pre-S and Post-S groups. At day 90 after stroke, interferon γ and IL-18 were significantly increased in the Post-S group compared to the Pre-S group. We found a positive correlation between CRP and NIHSS, whereas kallikrein levels showed no correlation with NIHSS at any of the days. CONCLUSION: Stroke induces changes in the RhoA-ROCK pathway in T-cells. CRP and NIHSS score correlated positively in the study. Statins may have an anti-inflammatory effect as statin treatment before stroke reduces post-stroke pro-inflammatory levels. RhoA GTPase and its downstream effectors are possibly the key to improve statin treatment in stroke.

Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation.

INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

Changes in serum cardiac myosin light chain 1 levels in children with fulminant myocarditis during continuous blood purification.

OBJECTIVE: To investigate the changes in serum cardiac myosin light chain 1 (CMLC-1) levels in children with fulminant myocarditis (FM) during continuous blood purification (CBP), as well as to analyze its correlation with other laboratory indexes. METHOD: Twenty-four (24) children with FM who underwent CBP were enrolled. Before and during treatment (48 and 72 hours after treatment, or death), the optical density value of serum CMLC-1 was measured using enzyme-linked immunosorbent assay, and then the serum CMLC-1 concentration was calculated. The correlations between CMLC-1 OD value change and laboratory indexes including creatine kinase-MB (CK-MB), troponin, myohemoglobin and N-terminal pro-brain natriuretic peptide (NT-proBNP) were analyzed. RESULTS: The serum CMLC-1 concentration significantly increased in the children with FM and decreased obviously during CBP therapy. In the same period, the change of CMLC-1 concentration were positively correlated with creatine kinase-MB (r=0.528), troponin (r=0.726), myohemoglobin (r=0.702), and NT-proBNP levels (r=0.589). CONCLUSION: The serum CMLC-1 concentration increases significantly in children with FM, but CBP therapy can effectively control this increase.

Changes in serum cardiac myosin light chain 1 levels in children with fulminant myocarditis during continuous blood purification

Summary Objective: To investigate the changes in serum cardiac myosin light chain 1 (CMLC-1) levels in children with fulminant myocarditis (FM) during continuous blood purification (CBP), as well as to analyze its correlation with other laboratory indexes. Method: Twenty-four (24) children with FM who underwent CBP were enrolled. Before and during treatment (48 and 72 hours after treatment, or death), the optical density value of serum CMLC-1 was measured using enzyme-linked immunosorbent assay, and then the serum CMLC-1 concentration was calculated. The correlations between CMLC-1 OD value change and laboratory indexes including creatine kinase-MB (CK-MB), troponin, myohemoglobin and N-terminal pro-brain natriuretic peptide (NT-proBNP) were analyzed. Results: The serum CMLC-1 concentration significantly increased in the children with FM and decreased obviously during CBP therapy. In the same period, the change of CMLC-1 concentration were positively correlated with creatine kinase-MB (r=0.528), troponin (r=0.726), myohemoglobin (r=0.702), and NT-proBNP levels (r=0.589). Conclusion: The serum CMLC-1 concentration increases significantly in children with FM, but CBP therapy can effectively control this increase.

Assessment of Preclinical Liver and Skeletal Muscle Biomarkers Following Clofibrate Administration in Wistar Rats.

Clofibrate is a known rodent hepatotoxicant classically associated with hepatocellular hypertrophy and increased serum activities of cellular alanine aminotransferase/aspartate aminotransferase (ALT/AST) in the absence of microscopic hepatocellular degeneration. At toxic dose, clofibrate induces liver and skeletal muscle injury. The objective of this study was to assess novel liver and skeletal muscle biomarkers following clofibrate administration in Wistar rats at different dose levels for 7 days. In addition to classical biomarkers, liver injury was assessed by cytokeratin 18 (CK18) cleaved form, high-mobility group box 1, arginase 1 (ARG1), microRNA 122 (miR-122), and glutamate dehydrogenase. Skeletal muscle injury was evaluated with fatty acid binding protein 3 (Fabp3) and myosin light chain 3 (Myl3). Clofibrate-induced hepatocellular hypertrophy and skeletal muscle degeneration (type I rich muscles) were noted microscopically. CK, Fabp3, and Myl3 elevations correlated to myofiber degeneration. Fabp3 and Myl3 outperformed CK for detection of myofiber degeneration of minimal severity. miR-122 and ARG1 results were significantly correlated and indicated the absence of liver toxicity at low doses of clofibrate, despite increased ALT/AST activities. Moreover, combining classical and novel biomarkers (Fabp3, Myl3, ARG1, and miR-122) can be considered a valuable strategy for differentiating increased transaminases due to liver toxicity from skeletal muscle toxicity.

Response of Novel Skeletal Muscle Biomarkers in Dogs to Drug-Induced Skeletal Muscle Injury or Sustained Endurance Exercise.

The skeletal muscle (SKM) injury biomarkers, skeletal troponin I (sTnI), myosin light chain 3 (Myl3), and creatine kinase muscle isoform (Ckm) have been shown recently to be more sensitive and specific for monitoring drug-induced SKM injury than the conventional biomarkers, aspartate transaminase (AST) and creatine kinase (CK) enzymatic assays in rat toxicology studies. To evaluate the utility of these SKM biomarkers across species, they were assessed in 2 dog models: a drug-induced injury study in Beagle dogs and a 160 km endurance exercise run completed by Alaskan sled dogs. In the drug-induced injury model, mean sTnI and Myl3 plasma levels were 6- and 18-fold, respectively, compared with baseline as early as Study Day (SD) 15, while mean plasma AST and CK levels did not increase, and biopsy samples were non-remarkable for histopathology prior to SD 29 when degeneration was first noted. Peak group mean plasma responses over baseline for sTnI, Myl3, and Ckm biomarkers were 96-, 103-, and 11-fold, respectively, compared with 2.5-fold for AST and 3.8-fold for CK-enzymatic (CK-enz) assay. In the sled dog sustained exercise model, the peak response for all biomarkers was observed at the first sampling (2 h) after the completion of the run. The sTnI, Myl3, and Ckm mean fold peak values compared with baseline were 170-, 120-, and 150-fold, respectively, while AST increased 7-fold and CK-enz increased 29-fold. These findings support the conclusion that sTnI, Myl3, and Ckm are sensitive early tissue leakage biomarkers for monitoring SKM injury and effects of exercise in dog, extending their utility across preclinical species beyond the rat, and provide further support to investigate their translational utility to clinical trial settings to monitor for drug-induced SKM injury and ensure patient safety.

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bß3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

Biomarker evaluation of skeletal muscle toxicity following clofibrate administration in rats.

The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Identification of phosphorylated MYL12B as a potential plasma biomarker for septic acute kidney injury using a quantitative proteomic approach.

Acute kidney injury (AKI) is a common and increasingly encountered complication in hospitalized patients with critical illness in intensive care units (ICU). According to the etiology, Sepsis-induced AKI (SAKI) is a leading contributor to AKI and significantly has very poor prognosis, which might be related to the late detection when the elevation of BUN and serum creatinine (SCr) is used. Many genes are up-regulated in the damaged kidney with the corresponding protein products appearing in plasma and urine. Some of these are candidate biomarkers for more timely diagnosis of SAKI. Therefore, extensive research efforts over this past decade have been directed at the discovery and validation of novel SAKI biomarkers to detect injury prior to changes in kidney function, a number of serum and urinary proteins, including NGAL, KIM-1, cystatin-C, IL-18, and L-FABP, have been identified for predicting SAKI before a rise in BUN and serum creatinine in several experimental and clinical trainings. Unfortunately, an ideal biomarker of SAKI with highly sensitivity and specificity has not been identified yet. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for SAKI. In the present study, kidney tissue samples from SAKI mice were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and 4 up-regulated proteins, which were actin (ACTB), myosin regulatory light chain 12B (MYL12B), myosin regulatory light polypeptide 9 (MYL9), and myosin regulatory light chain 12A (MYL12A) were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Among all the varied proteins, MYL12B was validated by western blot. Interestingly, there was no change between the SAKI and control kidney tissues, however, phosphorylated MYL12B was detected to be consistent with the proteomics data. Furthermore, phosphorylated MYL12B was found similarly to be increased in SAKI plasma, while MYL12B was changeless in plasma of control group. Taking together, phosphorylated MYL12B may be employed as a potential plasma biomarker for the early diagnosis of SAKI.

Free light chains in patients with acute heart failure secondary to atherosclerotic coronary artery disease.

Increased combined free light chains (cFLCs) are strongly prognostic of death in general populations and in patients with chronic kidney disease, but scarce data are available on cFLC in heart failure (HF). The aim of this study was to assess the dynamics and prognostic significance of cFLC levels in patients after admission with acute HF (AHF). cFLC measurements were compared in 49 patients with AHF, 37 patients with stable HF, 43 patients with stable coronary artery disease and without HF ("disease controls"), and 37 healthy controls. The association of cFLC with death and/or rehospitalization was assessed. Patients with AHF had significantly elevated cFLC levels, compared with other groups (p <0.001). Patients with stable HF showed higher levels of cFLCs than healthy controls. In patients with AHF, cFLC levels were correlated with cystatin C (Spearman's r = 0.63, p <0.001) and creatinine (Spearman's r = 0.47, p = 0.002). During 3-month follow-up, brain natriuretic peptide was reduced significantly (p = 0.017), but cFLCs did not change significantly. In a multivariate Cox regression analysis, the higher quartiles of cFLCs were significantly associated with death or readmission (hazard ratio 8.34, 95% confidence interval 2.38 to 29.22, p = 0.0009) after adjustment for age, gender, brain natriuretic peptide and cystatin C levels. Higher quartiles of cFLCs were prognostic for death alone (hazard ratio 14.0, 95% confidence interval 1.72 to 113.8, p = 0.014). In conclusion, increased serum cFLC concentrations in patients with AHF were independently associated with prognosis. In patients with AHF, elevated cFLC levels persist long after clinical stabilization, which may reflect immune disturbances and/or the reduced capacity of (perhaps functionally impaired) kidneys and the endothelium to eliminate them.

Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

Proteome expression and carbonylation changes during Trypanosoma cruzi infection and Chagas disease in rats.

Inflammation and oxidative stress, elicited by Trypanosoma cruzi infection, are important pathologic events during progressive Chagasic cardiomyopathy. In this study, we infected Sprague-Dawley rats with T. cruzi, and treated with phenyl-α-tert-butylnitrone (PBN-antioxidant) and/or benznidazole (BZ-anti-parasite). We employed two-dimensional gel electrophoresis/mass spectrometry to investigate (a) the plasma proteomic changes associated with infection and disease development, and (b) the beneficial effects of PBN and BZ in controlling the disease-associated plasma profile. Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) tandem MS (MS/MS) analysis of differentially expressed (total 146) and oxidized (total 48) protein spots yielded 92 unique proteins. Our data showed that treatment with PBN and BZ restored the differential expression of 65% and 30% of the disease-associated proteins to normal level, respectively, and PBN prevented development of oxidative adducts on plasma proteins. Western blotting to detect dinitrophenyl-derivatized carbonyl-proteins revealed plasma proteins were maximally oxidized during acute infection. Functional and disease/disorder analyses allocated a majority of the differentially expressed and oxidized proteins into inflammation/immunity and lipid metabolism categories and to molecular pathways associated with heart disease (e.g. cardiac infarction, contractile dysfunction, hypertrophy, and hypertension) in chagasic rats, and to curative pathways (e.g. ROS scavenging capacity, immune regulation) in infected rats treated with PBN and/or BZ. We validated the two-dimensional gel electrophoresis results by Western blotting, and demonstrated that the disease-associated increased expression of gelsolin and vimentin and release of cardiac MYL2 in the plasma of chagasic rats was returned to control level by PBN/BZ treatment. Increased plasma levels of gelsolin, MYL2 and vimentin were directly correlated with the severity of cardiac disease in human chagasic patients. Together, these results demonstrate the plasma oxidative and inflammatory response profile, and plasma detection of cardiac proteins parallels the pathologic events contributing to Chagas disease development, and is of potential utility in diagnosing disease severity and designing suitable therapy for management of human chagasic patients.

A comparison of mortality and cardiac biomarker response between three outbred stocks of Sprague Dawley rats treated with isoproterenol.

The authors compared the mortality and cardiac biomarker responses in three outbred stocks of Sprague Dawley rats (CD/IGS, Sasco, Harlan) treated with isoproterenol hydrochloride. Cardiac injury was confirmed by histologic evaluation, and increases in cardiac troponin I concentration in serum were measured by two methods. CD/IGS rats had a higher incidence and earlier mortality compared with Sasco or Harlan rats. Harlan rats had lower severity scores for cardiomyocyte degeneration/necrosis compared with the other stocks. Post-isoproterenol treatment cardiac troponin I concentrations were greater in CD/IGS and Sasco rats compared with Harlan rats. Concentrations of cardiac troponin T followed a similar pattern to that of cardiac troponin I in rats treated with isoproterenol. Myosin, light chain 3 concentrations increased in all rats treated with isoproterenol, but there was no difference between the three stocks in the magnitude or pattern of the dose response. Increases in fatty acid binding protein 3 concentrations were detected in only the highest dose group at the earliest timepoint postdose for all three stocks of rats. Results of these studies illustrate the need for investigators to recognize the potential differences in response between stocks of Sprague Dawley rats treated with cardiotoxicants or novel chemical entities.

Evaluation of the usefulness of biomarkers for cardiac and skeletal myotoxicity in rats.

Since cardiac and skeletal myotoxicity affect the development of drug candidates, it is important to detect their toxicity at an early stage of drug development. For that purpose, in this study, the usefulness of several cardiac and skeletal myotoxic biomarkers in blood were evaluated using two rat models treated intraperitoneally with an acetylcholinesterase inhibitor carbofuran (CAF) or a synthetic catecholamine isoproterenol (ISO). The biomarkers assayed were fatty acid binding protein 3 (Fabp3), myosin light chain 1 (MLC1), cardiac troponin I (cTnI), cardiac troponin T (cTnT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK). CAF and ISO treatment of rats induced greater increases in the levels of Fabp3, MLC1, cTnI and cTnT than in the levels of AST, LDH and CK. A kinetic analysis indicated that the levels of all of the biomarkers had returned to the basal level by 24h after drug administration. Pathological examination revealed lesions in the heart, mainly at the left ventricle and septum, in both CAF- and ISO-treated rats. CAF-treated rats showed widespread lesions of skeletal muscle that were independent of muscle fiber type, while in ISO-treated rats locoregional lesions were observed only in slow twitch muscle. Receiver operating characteristic curve analysis of the sensitivity of the tested biomarkers indicated that MLC1 and cTnT were the most effective biomarkers of cardiotoxicity. For skeletal myotoxicity, Fabp3 and MLC1 were the most effective biomarkers based on the specific tissue distribution of these proteins. Conversely, the rapid blood clearance of these markers should be taken into account when considering the use of these biomarkers.