Overcoming difficulties on synthesis of cardiac troponin-I.

Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.

Troponina I como biomarcador de lesão cardíaca em cães com sepse / Troponin I as a biomarker of cardiac injury in dogs with sepsis

Avaliou-se a troponina I como biomarcador de lesão cardíaca na sepse, além de outros parâmetros hematológicos, em cadelas com piometra. Os grupos avaliados não diferiram estatisticamente na avaliação da concentração sérica da troponina I cardíaca. A quantidade total de leucócitos (mm3) e a porcentagem de bastonetes foram significativamente maiores no grupo sepse (23.221,74±16.848,80mm3 e 5,91±10,18%) quando comparado ao grupo não sepse (14.492,86±6.828,26mm3 e 1,93±1,64%) e ao grupo controle (10.320,00±3.999,02mm3 e 1,65±2,05%). Houve diferença significativa nas concentrações séricas da proteína C reativa (mg/dL) no grupo sepse (19,57±41,69md/dL) se comparado ao grupo não sepse (10,29±12,02mg/dL) e ao grupo controle (3,60±3,53mg/dL). Na avaliação da concentração sérica do lactato, houve diferença significativa entre cães com piometra e cães saudáveis, porém não houve diferença significativa entre os grupos sepse e não sepse. Os resultados do presente estudo indicam que a troponina I cardíaca não pôde ser considerada um biomarcador precoce para injúria miocárdica nos casos de cadela com piometra, pois os resultados das mensurações foram semelhantes entre os grupos, o que indica que pode não ter ocorrido lesão dos cardiomiócitos nessa fase. Já a proteína C reativa e o lactato são possíveis marcadores para inflamação sistêmica, uma vez que demonstraram concentrações séricas significativamente maiores em cadelas com piometra.(AU)

Troponin I as a biomarker of cardiac injury in sepsis, and other hematological parameters in female dogs with pyometra were evaluated. The groups did not differ in the assessment of serum cardiac troponin I. The total amount of leukocytes (mm3) and percentage of band cells was significantly higher in the sepsis group (23,221.74±16,848.80mm3 and 5.91±10.18%) compared to the non-sepsis group (14,492.86±6,828.26mm3 and 1.93±1.64%) and the control group (10,320.00±3,999.02mm3 and 1.65±2.05% respectively). There were significant differences in serum concentrations of C-reactive protein (mg / dL) in the sepsis group (19.57±41.69md / dL) compared to the non-sepsis group (10.29±12.02mg / dL) and control group (3.60±3.53mg / dL). In the evaluation of serum lactate concentration, there was a significant difference between dogs with pyometra and healthy dogs, but there was no significant difference between the sepsis and non-sepsis groups. The results of this study indicate that troponin I could not be considered an early biomarker of myocardial injury in cases of dogs with pyometra, because the results of the measurements were similar between groups, inferring that there may not have been an cardiomyocytes injury at this stage. C-reactive protein and lactate are potential markers for systemic inflammation, as demonstrated by significantly higher serum concentrations in dogs with pyometra.(AU)

"Young at heart": Regenerative potential linked to immature cardiac phenotypes.

The adult human myocardium is incapable of regeneration; yet, the zebrafish (Danio rerio) can regenerate damaged myocardium. Similar to the zebrafish heart, hearts of neonatal, but not adult mice are capable of myocardial regeneration. We performed a proteomics analysis of adult zebrafish hearts and compared their protein expression profile to hearts from neonatal and adult mice. Using difference in-gel electrophoresis (DIGE), there was little overlap between the proteome from adult mouse (>8weeks old) and adult zebrafish (18months old) hearts. Similarly, there was a significant degree of mismatch between the protein expression in neonatal and adult mouse hearts. Enrichment analysis of the selected proteins revealed over-expression of DNA synthesis-related proteins in the cardiac proteome of the adult zebrafish heart similar to neonatal and 4days old mice, whereas in hearts of adult mice there was a mitochondria-related predominance in protein expression. Importantly, we noted pronounced differences in the myofilament composition: the adult zebrafish heart lacks many of the myofilament proteins of differentiated adult cardiomyocytes such as the ventricular isoforms of myosin light chains and nebulette. Instead, troponin I and myozenin 1 were expressed as skeletal isoforms rather than cardiac isoforms. The relative immaturity of the adult zebrafish heart was further supported by cardiac microRNA data. Our assessment of zebrafish and mammalian hearts challenges the assertions on the translational potential of cardiac regeneration in the zebrafish model. The immature myofilament composition of the fish heart may explain why adult mouse and human cardiomyocytes lack this endogenous repair mechanism.

Nonhematopoietic Peroxisome Proliferator-Activated Receptor-α Protects Against Cardiac Injury and Enhances Survival in Experimental Polymicrobial Sepsis.

OBJECTIVES: Peroxisome proliferator-activated receptor-α is significantly down-regulated in circulating leukocytes from children with sepsis. Peroxisome proliferator-activated receptor-α null (Ppara) mice have greater mortality than wild-type mice when subjected to sepsis by cecal ligation and puncture. We sought to characterize the role of peroxisome proliferator-activated receptor-α in sepsis and to identify the mechanism whereby peroxisome proliferator-activated receptor-α confers a survival advantage. DESIGN: Prospective randomized preclinical study. SETTING: Laboratory investigation. SUBJECTS: Male C57Bl/6J and Ppara mice (B6.129S4-Ppara/J), aged 12-16 weeks. INTERVENTIONS: Bone marrow chimeric mice were generated and subjected to cecal ligation and puncture. Survival was measured for 7 days. Separate groups of nontransplanted mice underwent cecal ligation and puncture and were euthanized 24 hours later for plasma and tissue analyses. MEASUREMENTS AND MAIN RESULTS: Ppara mice had dramatically reduced survival compared with wild-type mice irrespective of the peroxisome proliferator-activated receptor-α status of the bone marrow they received (3% vs 63%; p < 0.0001). No difference in survival was observed between Ppara mice that received wild-type versus Ppara marrow or in wild-type mice receiving wild-type versus Ppara marrow. In septic, nontransplanted mice at 24 hours, Ppara mice had elevated cardiac troponin levels compared with wild-type mice. Cardiac histologic injury scores were greater in Ppara versus wild-type mice. Expression of transcription factors and enzymes related to fatty acid oxidation in the heart were profoundly down-regulated in both wild-type and Ppara mice, but more so in the Ppara mice. CONCLUSIONS: Peroxisome proliferator-activated receptor-α expression in nonhematopoietic tissues plays a critical role in determining clinical outcome in experimental polymicrobial sepsis and is more important to survival in sepsis than hematopoietic peroxisome proliferator-activated receptor-α expression. Cardiac injury due to inadequate energy production from fatty acid substrate is a probable mechanism of decreased survival in Ppara mice. These results suggest that altered peroxisome proliferator-activated receptor-α-mediated cellular metabolism may play an important role in sepsis-related end-organ injury and dysfunction, especially in the heart.

Triiodo-L-Thyronine Promotes the Maturation of Cardiomyocytes Derived From Rat Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BMMSCs) can differentiate into cardiomyocytes and be used in cardiac tissue engineering for heart regeneration. However, the effective clinical application of cardiomyocytes derived from BMMSCs is limited because of their immature phenotype. The aim of this study was to investigate the potential of triiodo-L-thyronine (T3) to drive cardiomyocytes derived from BMMSCs to a more mature state. BMMSCs were divided into 3 groups: untreated controls, differentiated, and T3 treated. The differentiation potential was evaluated by immunofluorescence microscopy and flow cytometry. Data were represented as the numbers of cells positive for the troponin I (cTnI), α-actinin, GATA4, and the connexin-43 (Cx-43). The mRNA levels of these specific markers of cardiomyocytes were determined by quantitative real-time polymerase chain reaction. The levels of cardiomyocytes markers protein and octamer-binding transcription factor 4 (Oct-4) were determined by Western blot analyses. Our data demonstrate that T3 treatment leads to a significant increase in cells positive for cTnI, GATA4, Cx-43, and α-actinin. The mRNA and protein expression levels of these specific markers of cardiomyocytes were also increased after T3 treatment. At the same time, the protein expression level of Oct-4 was substantially downregulated in T3-treated cells. These results demonstrate that T3 treatment increases the differentiation of BMMSCs induced to cardiomyocytes and promotes their maturation.

DNA methylation regulates mouse cardiac myofibril gene expression during heart development.

BACKGROUND: It is well known that epigenetic modifications play an important role in controlling the regulation of gene expression during the development. Our previous studies have demonstrated that the expression of fetal troponin I gene (also called slow skeletal troponin I, ssTnI) is predominated in the fetal stage, reduced after birth and disappeared in the adulthood. The mechanism underlying the developmentally related ssTnI gene regulation is not clear. In this study, we have explored the epigenetic role of DNA methylation in the regulation of ssTnI expression in the heart during the development. RESULTS: The DNA methylation levels of CpG island and CpG dinucleotides region were detected using methylation specific PCR (MSP) and bisulfite sequence PCR (BSP) in 2000 bp upstream and 100 bp upstream of ssTnI gene promoter. Real time RT-PCR and Western blot were used to detect ssTnI mRNA and protein expression levels. We found that DNA methylation levels of the CpG dinucleotides region in ssTnI gene promoter were increased with the development, corresponding to a decreased expression of ssTnI gene in mouse heart. However the DNA methylation levels of CpG islands in this gene were not changed during the development. Application of a methylation inhibitor, 5-Azacytidine, in cultured myocardial cells partially prevented the decline of ssTnI expression. CONCLUSION: Our results indicate that DNA methylation, as an epigenetic intervention, plays a role in the regulation of the fetal TnI gene expression in the heat during the development.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²âº Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

Metabolic efficiency promotes protection from pressure overload in hearts expressing slow skeletal troponin I.

BACKGROUND: The failing heart displays increased glycolytic flux that is not matched by a commensurate increase in glucose oxidation. This mismatch induces increased anaplerotic flux and inefficient glucose metabolism. We previously found adult transgenic mouse hearts expressing the fetal troponin I isoform, (ssTnI) to be protected from ischemia by increased glycolysis. In this study, we investigated the metabolic response of adult mouse hearts expressing ssTnI to chronic pressure overload. METHODS AND RESULTS: At 2 to 3 months of age, ssTnI mice or their nontransgenic littermates underwent aortic constriction (TAC). TAC induced a 25% increase in nontransgenic heart size but only a 7% increase in ssTnI hearts (P<0.05). Nontransgenic TAC developed diastolic dysfunction (65% increase in E/A ratio), whereas the E/A ratio actually decreased in ssTnI TAC. Isolated perfused hearts from nontransgenic TAC mice showed reduced cardiac function and reduced creatine phosphate:ATP (16% reduction), but ssTnI TAC hearts maintained cardiac function and energy charge. Contrasting nontransgenic TAC, ssTnI TAC significantly increased glucose oxidation at the expense of palmitate oxidation, preventing the increase in anaplerosis observed in nontransgenic TAC hearts. Elevated glucose oxidation was mediated by a reduction in pyruvate dehydrogenase kinase 4 expression, enabling pyruvate dehydrogenase to compete against anaplerotic enzymes for pyruvate carboxylation. CONCLUSIONS: Expression of a single fetal myofilament protein into adulthood in the ssTnI-transgenic mouse heart induced downregulation of the gene expression response for pyruvate dehydrogenase kinase to pressure overload. The consequence of elevated pyruvate oxidation in ssTnI during TAC reduced anaplerotic flux, ameliorating inefficiencies in glucose oxidation, with energetic and functional protection against cardiac decompensation.

Epigenetic regulation of cardiac myofibril gene expression during heart development.

Cardiac gene expression regulation is controlled not only by genetic factors but also by environmental, i.e., epigenetic factors. Several environmental toxic effects such as oxidative stress and ischemia can result in abnormal myofibril gene expression during heart development. Troponin, one of the regulatory myofibril proteins in the heart, is a well-known model in study of cardiac gene regulation during the development. In our previous studies, we have demonstrated that fetal form troponin I (ssTnI) expression in the heart is partially regulated by hormones, such as thyroid hormone. In the present study, we have explored the epigenetic role of histone modification in the regulation of ssTnI expression. Mouse hearts were collected at different time of heart development, i.e., embryonic day 15.5, postnatal day 1, day 7, day 14 and day 21. Levels of histone H3 acetylation (acH3) and histone H3 lysine 9 trimethylation (H3K9me(3)) were detected using chromatin immunoprecipitation assays in slow upstream regulatory element (SURE) domain (TnI slow upstream regulatory element), 300-bp proximal upstream domain and the first intron of ssTnI gene, which are recognized as critical regions for ssTnI regulation. We found that the levels of acH3 on the SURE region were gradually decreased, corresponding to a similar decrease of ssTnI expression in the heart, whereas the levels of H3K9me(3) in the first intron of ssTnI gene were gradually increased. Our results indicate that both histone acetylation and methylation are involved in the epigenetic regulation of ssTnI expression in the heart during the development, which are the targets for environmental factors.

NH2-terminal truncations of cardiac troponin I and cardiac troponin T produce distinct effects on contractility and calcium homeostasis in adult cardiomyocytes.

Cardiac troponin I (TnI) has an NH2-terminal extension that is an adult heart-specific regulatory structure. Restrictive proteolytic truncation of the NH2-terminal extension of cardiac TnI occurs in normal hearts and is upregulated in cardiac adaptation to hemodynamic stress or ß-adrenergic deficiency. NH2-terminal truncated cardiac TnI (cTnI-ND) alters the conformation of the core structure of cardiac TnI similarly to that produced by PKA phosphorylation of Ser(23/24) in the NH2-terminal extension. At organ level, cTnI-ND enhances ventricular diastolic function. The NH2-terminal region of cardiac troponin T (TnT) is another regulatory structure that can be selectively cleaved via restrictive proteolysis. Structural variations in the NH2-terminal region of TnT also alter the molecular conformation and function. Transgenic mouse hearts expressing NH2-terminal truncated cardiac TnT (cTnT-ND) showed slower contractile velocity to prolong ventricular rapid-ejection time, resulting in higher stroke volume. Our present study compared the effects of cTnI-ND and cTnT-ND in cardiomyocytes isolated from transgenic mice on cellular morphology, contractility, and calcium kinetics. Resting cTnI-ND, but not cTnT-ND, cardiomyocytes had shorter length than wild-type cells with no change in sarcomere length. cTnI-ND, but not cTnT-ND, cardiomyocytes produced higher contractile amplitude and faster shortening and relengthening velocities in the absence of external load than wild-type controls. Although the baseline and peak levels of cytosolic Ca(2+) were not changed, Ca(2+) resequestration was faster in both cTnI-ND and cTnT-ND cardiomyocytes than in wild-type control. The distinct effects of cTnI-ND and cTnT-ND demonstrate their roles in selectively modulating diastolic or systolic functions of the heart.

iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes.

BACKGROUND: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. METHODS AND RESULTS: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/ß-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation. CONCLUSIONS: A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

Astragaloside IV protects heart from ischemia and reperfusion injury via energy regulation mechanisms.

OBJECTIVE: This study was designed to investigate the protective potential of AS-IV against ischemia and I/R-induced myocardial damage, with focusing on possible involvement of energy metabolism modulation in its action and the time phase in which it takes effect. METHODS: SD rats were subjected to 30 minutes LADCA occlusion, followed by reperfusion. MBF, myocardial infarct size, and cardiac function were evaluated. Myocardial structure and myocardial apoptosis were assessed by double immunofluorescence staining of F-actin and TUNEL. Content of ATP, ADP, and AMP in myocardium, cTnI level, expression of ATP5D, P-MLC2, and apoptosis-related molecules were determined. RESULTS: Pretreatment with AS-IV suppressed MBF decrease, myocardial cell apoptosis, and myocardial infarction induced by I/R. Moreover, ischemia and I/R both caused cardiac malfunction, decrease in the ratio of ATP/ADP and ATP/AMP, accompanying with reduction of ATP 5D protein and mRNA, and increase in P-MLC2 and serum cTnI, all of which were significantly alleviated by pretreatment with AS-IV, even early in ischemia phase for the insults that were implicated in energy metabolism. CONCLUSIONS: AS-IV prevents I/R-induced cardiac malfunction, maintains the integrity of myocardial structure through regulating energy metabolism. The beneficial effect of AS-IV on energy metabolism initiates during the phase of ischemia.

Recurrent low-level troponin I elevation is a worse prognostic indicator than occasional injury pattern in patients hospitalized with heart failure.

BACKGROUND: Elevated troponin at baseline is associated with higher mortality in heart failure (HF) patients, but the prognostic role of recurrently elevated troponin is not well described. METHODS AND RESULTS: We performed chart reviews of 196 HF patients without acute coronary syndrome, with at least three Troponin I (TnI) measurements on different admissions. For the analyses, three sets of TnI values closest to baseline, one year and two years were selected for each patient. Based on the three sets of TnI, the lowest value of TnI (minimum), the highest value of TnI (maximum), median value of TnI and delta TnI (3rd TnI-baseline TnI) were derived for each patient. The study population of 196 patients had 632 person-year follow-up, consisted predominantly of elderly (68 ± 10 years) male patients (99%) with mean ejection fraction of 26 ± 13%. Using multivariate Cox proportional hazards model only minimum TnI, but not the maximum, median or delta of TnI values, was significantly associated with mortality (HR: 13.7, 95% CI: 3.7 to 50.8, p<0.001). As a categorical variable, minimum TnI value of >0.04ng/ml was also independently associated with mortality (p=0.01, HR=1.6, 95% CI: 1.1 to 2.3). CONCLUSIONS: In HF patients without acute coronary syndrome, the persistence of TnI elevation, even at low levels, is associated with a worse survival than sporadic TnI elevations of higher magnitude or any single elevation in TnI; and a recurrent elevation of TnI >0.04ng/ml portends a poor prognosis.

Mobilization of CD34+CXCR4+ stem/progenitor cells and the parameters of left ventricular function and remodeling in 1-year follow-up of patients with acute myocardial infarction.

Mobilization of stem cells in acute MI might signify the reparatory response. Aim of the Study. Prospective evaluation of correlation between CD34+CXCR4+ cell mobilization and improvement of LVEF and remodeling in patients with acute MI in 1-year followup. Methods. 50 patients with MI, 28 with stable angina (SAP), and 20 individuals with no CAD (CTRL). CD34+CXCR4+ cells, SDF-1, G-CSF, troponin I (TnI) and NT-proBNP were measured on admission and 1 year after MI. Echocardiography and ergospirometry were carried out after 1 year. Results. Number of CD34+CXCR4+ cells in acute MI was significantly higher in comparison with SAP and CTRL, but lower in patients with decreased LVEF ≤40%. In patients who had significant LVEF increase ≥5% in 1 year FU the number of cells in acute MI was significantly higher versus patients with no LVEF improvement. Number of cells was positively correlated (r = 0,41, P = 0,031) with absolute LVEF change and inversely with absolute change of ESD and EDD in 1-year FU. Mobilization of CD34+CXCR4+ cells in acute MI was negatively correlated with maximum TnI and NT-proBNP levels. Conclusion. Mobilization of CD34+CXCR4+ cells in acute MI shows significant positive correlation with improvement of LVEF after 1 year.

Usefulness of heart-type fatty acid-binding protein in patients with severe sepsis.

PURPOSE: The purpose of the study was to evaluate the value of heart-type fatty acid-binding protein (hFABP) as a novel clinical biomarker in patients with severe sepsis. METHODS: Serum concentrations of hFABP and traditional cardiac biomarkers including cardiac troponin I, creatine kinase-MB, and B-type natriuretic peptides levels were measured within 6 hours after admission in 93 severe septic patients. The value of hFABP for the diagnosis of sepsis-related myocardial dysfunction (SRMD) and for the prediction of 28-day mortality was evaluated by receiver operating characteristics curve analysis. The prognostic value of elevated hFABP was subsequently confirmed by multivariate Cox proportional hazards analysis and Kaplan-Meier survival analysis. RESULTS: Heart-type fatty acid-binding protein was elevated (≥ 4.5 ng/mL) in 58 (62.4%) patients; patients with elevated hFABP appeared more likely to have SRMD (84.5% vs 31.4%, P < .001) and have higher prevalence of 28-day death (37.9% vs 8.6%, P = .002). Heart-type fatty acid-binding protein offered superior value over conventional biomarkers in both diagnosis of SRMD (area under the curve, 0.767; P < .001) and prediction of 28-day death (area under the curve, 0.805; P < .001). CONCLUSIONS: Serum hFABP is frequently elevated among patients with severe sepsis and appears to be associated with SRMD. Elevated hFABP independently predicts 28-day mortality in severe sepsis.

Delayed preconditioning by sevoflurane elicits changes in the mitochondrial proteome in ischemia-reperfused rat hearts.

BACKGROUND: Delayed myocardial preconditioning by volatile anesthetics involves changes in DNA transcription and translation. Mitochondria play a central role in myocardial ischemia/reperfusion (I/R) injury and in ischemic or pharmacologic preconditioning. In this study, we investigated whether there are alterations in myocardial mitochondrial protein expression after volatile anesthetic preconditioning (APC) to examine the underlying mechanisms of delayed cardioprotection. METHODS: Thirty-six Sprague-Dawley rats were randomly assigned to 1 of 3 groups (n = 12 for each group). Rats in the delayed APC group were exposed to sevoflurane (2.5% for 60 minutes) 24 hours before myocardial ischemia was induced. Myocardial ischemia in the I/R and APC groups was induced by left coronary artery occlusion for 30 minutes, followed by 120 minutes of reperfusion. The control group received no treatment. The mitochondria fractions were prepared by differential centrifugation with density gradient isolation for proteomic analysis. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry was used to identify differences in the protein expression from mitochondria of the rat hearts. RESULTS: Fifteen differentially expressed mitochondrial proteins between the APC group and I/R group were identified and the expression patterns of 2 of the proteins were confirmed by Western blot analysis. These proteins were associated with mitochondrial substrate metabolism, respiration, and adenosine triphosphate (ATP)/adenosine diphosphate transport. The modifications of the mitochondrial proteome suggest an enhanced capacity of mitochondria to maintain myocardial ATP levels after I/R injury. CONCLUSION: Delayed sevoflurane myocardial preconditioning induces mitochondrial proteome remodeling, which mainly involves proteins that are related to ATP generation and transport. Therefore, proteomic changes related to bioenergetic balance may be the mechanistic basis of delayed anesthetic myocardial preconditioning.

Real-time reverse transcription-PCR expression profiling of porcine troponin I family in three different types of muscles during development.

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.

Inhibition of mitochondrial permeability transition to prevent the post-cardiac arrest syndrome: a pre-clinical study.

AIMS: Resuscitated cardiac arrest (CA), leading to harmful cardiovascular dysfunction and multiple organ failure, includes a whole-body hypoxia-reoxygenation phenomenon. Opening of the mitochondrial permeability transition pore (mPTP) appears to be a pivotal event in ischaemia-reperfusion injury. We hypothesized that pharmacological inhibition of mPTP opening may prevent the post-CA syndrome. METHODS AND RESULTS: Anaesthetized New Zealand White rabbits underwent a 15 min primary asphyxial CA and 120 min of reperfusion following resuscitation. At reflow, animals received an intravenous bolus of either cyclosporine A (CsA, 5 mg/kg) or NIM 811 (2.5 mg/kg), two potent inhibitors of mPTP opening, or the CsA vehicle (control). Short-term survival, haemodynamics, regional (sonomicrometry), and global cardiac function (dP/dt and aortic flow) were assessed. We measured markers of cellular injuries and/or organ failure, including troponin Ic release, lacticodehydrogenase, lactate, creatinine, and alanine aminotransferase. Cyclosporine A and NIM 811 significantly improved short-term survival, post-resuscitation cardiac function, as well as liver and kidney failure (P < 0.05). CsA and NIM 811 both attenuated in vitro mPTP opening (calcium retention capacity by spectrofluorimetry) and restored oxidative phosphorylation when compared with controls (P < 0.05). CONCLUSION: These data suggest that pharmacological inhibition of mPTP opening, added to basic life support, attenuates the post-CA syndrome and improves short-term outcomes in the rabbit model.

Atrial fibrillation associated with exogenous subclinical hyperthyroidism, changing axis deviation, troponin-I positive and without acute coronary syndrome.

Changing axis deviation has been rarely reported also during atrial fibrillation or atrial flutter. Changing axis deviation has been also rarely reported during acute myocardial infarction associated with atrial fibrillation or at the end of atrial fibrillation during acute myocardial infarction. Subclinical hyperthyroidism is an increasingly recognized entity that is defined as a normal serum free thyroxine and free triiodothyronine levels with a thyroid-stimulating hormone level suppressed below the normal range and usually undetectable. It has been reported that subclinical hyperthyroidism is not associated with coronary heart disease or mortality from cardiovascular causes but it is sufficient to induce arrhythmias including atrial fibrillation and atrial flutter. It has also been reported that increased factor X activity in patients with subclinical hyperthyroidism represents a potential hypercoagulable state. Serum troponin-I is a sensitive indicator of myocardial damage but abnormal troponin-I levels have been also reported without acute coronary syndrome and without cardiac damage. Abnormal troponin-I levels after supraventricular tachycardia have been also reported. We present a case of changing axis deviation in a 49-year-old Italian man with atrial fibrillation, exogenous subclinical hyperthyroidism and troponin-I positive without acute coronary syndrome. Also this case focuses attention on changing axis deviation, on subclinical hyperthyroidism and on the importance of a correct evaluation of abnormal troponin-I levels.