Secreted Protein Acidic and Cysteine Rich Matricellular Protein is Enriched in the Bioactive Fraction of the Human Vascular Pericyte Secretome.

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.

Downregulation of miR-483-5p decreases hypoxia-induced injury in human cardiomyocytes by targeting MAPK3.

BACKGROUND: MiR-483-5p was recently identified as a risk factor in the early stages of acute myocardial infarction (AMI) patients. Here, we further investigated how miR-483-5p affects cardiomyocyte apoptosis and oxidative stress under hypoxic conditions. METHODS: Plasma samples were collected from AMI patients and healthy volunteers. The expression of miR-483-5p was determined using quantitative real-time PCR. An in vitro hypoxic model was constructed to mimic AMI in AC16 cells. Cell viability, apoptosis and oxidative stress biomarker levels (MDA, SOD and CAT) were respectively determined using CCK-8, flow cytometry and commercial assay kits. RESULTS: The expression levels of miR-483-5p were significantly higher in AMI patients than in control subjects. Circulating levels of miR-483-5p positively correlated with creatine kinase MB isoform (CK-MB) and cardiac troponin I (cTnI) levels. The in vitro experiments showed that the expression levels of miR-483-5p were also upregulated in hypoxia-induced AC16 cell injury. MiR-483-5p overexpression significantly increased hypoxia-induced cardiomyocyte apoptosis and oxidative stress, while knockdown attenuated these effects. Mechanistically, miR-483-5p directly targets MAPK3 in AC16 cells. Furthermore, the protective effects of miR-483-5p knockdown against hypoxia-induced cardiomyocyte injury are partially dependent on MAPK3. CONCLUSIONS: MiR-483-5p, which targets MAPK3, might be a potential therapeutic target for the diagnosis and prevention of hypoxia-induced myocardial injury.

Extracellular ubiquitin levels are increased in coronary heart disease and associated with the severity of the disease.

Aim: This study aimed to evaluate concentration of plasma extracellular ubiquitin (UB) in coronary heart disease (CHD) patients and its correlation with the disease severity.Methods: Levels of UB and stromal cell-derived factor-1a (SDF-1a) were measured in 60 healthy controls and 67 CHD cases. Coronary atherosclerosis was assessed with Gensini scoring system. Spearman correlation was used to evaluate the correlation between UB and low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), creatine kinase-MB (CK-MB), cardiac troponin I (cTnI) or SDF-1a. The receiver-operating characteristic (ROC) curve was established to assess the predictive value of UB.Results: Plasma UB levels were significantly higher in CHD patients than in controls (p < .0001), and the levels in those with acute myocardial infarction (AMI) were higher than stable angina pectoris (SAP) and unstable angina pectoris (UAP) groups (both p < .01). UB was also positively correlated with Gensini score, CRP, CK-MB and cTnI in CHD. ROC analysis of UB showed that the area under the curve (AUC) were 0.711 (95%CI, 0.623-0.799) and 0.778 (95%CI, 0.666-0.890) for CHD and acute coronary syndrome (ACS), respectively. Plasma SDF-1a levels were elevated in CHD patients but showed no significant correlation with UB concentration or the severity of the disease.Conclusion: Plasma UB concentration was increased in CHD and the change of UB levels may reflect the progression of CHD.

miRNA-21 Expression in the Serum of Elderly Patients with Acute Myocardial Infarction.

BACKGROUND The aims of this study were to examine the expression of miRNA-21 in the serum of elderly patients (>65 years) with acute myocardial infarction (AMI) and to investigate the potential role of serum miRNA-21 as a marker of early cardiac myocyte damage. MATERIAL AND METHODS Thirty-eight elderly patients with recent AMI, 27 elderly patients with unstable angina pectoris, and 25 healthy elderly individuals were included in the study. Serum miRNA-21 expression was determined following total RNA extraction and reverse-transcribed into cDNA, followed by reverse transcription-polymerase chain reaction (RT-PCR). Serum creatine kinase MB isoenzyme (CK-MB) and cardiac troponin I (cTnI) levels were analyzed by electrochemiluminescence. Apoptosis of human cardiac myocytes (HCM) was analyzed using fluorescence-activated cell sorting (FACS), and protein expression of caspase-3 was detected using Western blot. RESULTS Expression levels of miRNA-21 in the serum of elderly patients with AMI were positively correlated with serum levels of CK-MB (r=0.3683, P=0.0229) and cTnI (r=0.5128, P=0.009). Following tumor necrosis factor (TNF)-α induction, the apoptosis rates of HCM transfected with the miRNA-21 mimic short hairpin RNA (shRNA) were downregulated by 39.1% compared with control HCM cells, and protein expression of c-Jun N-terminal kinases (JNK) and p38 were unchanged (P>0.05); protein expression of p-JNK, p-p38 and caspase-3 were downregulated by 37.1%, 35.8%, and 36.0%, respectively. CONCLUSIONS Expression of miRNA-21 was upregulated in the serum of elderly patients with AMI, which inhibited TNF-a induced apoptosis in HCM by activating the JNK/p38/caspase-3 signaling pathway.

Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.

Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Role of E-selectin for diagnosing myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia.

Backgrounds Effects of myocardial injury on E-selectin remain unclear. Thus, we investigated the diagnostic value of E-selectin for myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia. Methods In this prospective and blinded clinical study, plasma E-selectin, cardiac troponin I, creatine kinase isoenzyme MB, interleukin-6 and tumor necrosis factor alpha concentrations were measured in paediatric patients with mycoplasma pneumoniae pneumonia (MPP group, n = 138). The control group comprised 120 healthy children. The definition of cardiac injury was based on cardiac troponin I or CK-MB (with or possibly without abnormal electrocardiogram evidence). Diagnostic value of E-selectin for myocardial injury was determined by analysing receiver operating characteristic curves. Results Among the 138 mycoplasma pneumoniae pneumonia patients, 40 patients were identified with myocardial injury, while 98 patients were identified without myocardial injury. Plasma E-selectin concentrations were: 40.22 ± 4.80 ng/mL, in patients with myocardial injury; 18.55 ± 2.16 ng/mL, in patients without myocardial injury and 12.39 ± 3.27 ng/mL, in healthy children. For the 40 patients identified with myocardial injury, area under the receiver operating characteristic curve value for plasma E-selectin concentrations was 0.945 (95% CI: 0.899-0.991), and optimal diagnostic cut-off value was 29.93 ng/mL (positive likelihood ratio = 72.5). Conclusion E-selectin was shown to be an effective index for myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia, and its role in other causes of myocardial injury warrants further investigation.

Hsp70 expression induced by Co-Enzyme Q10 protected chicken myocardial cells from damage and apoptosis under in vitro heat stress.

The aim of this study was to investigate whether induction of Hsp70 expression by co-enzyme Q10 (Q10) treatment protects chicken primary myocardial cells (CPMCs) from damage and apoptosis in response to heat stress for 5 hours. Analysis of the expression and distribution of Hsp70 and the levels of the damage-related enzymes creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), as well as pathological analysis showed that co-enzyme Q10 alleviated the damage caused to CPMCs during heat stress. Further, analysis of cell apoptosis and the expression of cleaved caspase-3 indicated that co-enzyme Q10 did have an anti-apoptotic role during heat stress. Western blot analysis showed that pretreatment with co-enzyme Q10 led to a significant increase in the expression of Hsp70 during heat stress. Immunostaining assays confirmed the results of western blot analysis and also showed that co-enzyme Q10 could accelerate the translocation of Hsp70 into the nucleus during heat stress, but this was not observed in the group that was treated with only co-enzyme Q10. These findings seem to indicate that co-enzyme Q10 protected CPMCs from heat stress via the induction of Hsp70. To investigate this, 200 µM quercetin, an Hsp70 inhibitor, was used to inhibit the expression of Hsp70 2 h before heat stress. Quercetin pre-treatment was observed to suppress the expression of Hsp70 as well the protective function of co-enzyme Q10 at 5 h of heat stress. This finding confirms that Q10 brought about its effects via Hsp70 expression, but the mechanism underlying this needs further investigation.

Antioxidant effects of hydroxysafflor yellow A and acetyl-11-keto-ß-boswellic acid in combination on isoproterenol-induced myocardial injury in rats.

Oxidative stress plays an important role in the initiation and development of myocardial injury (MI). The peroxisome proliferator-activated receptor gamma coactivator-1α (PGC­1α)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is considered to be a potential target for cardioprotection in MI. Acetyl-11-keto-ß-boswellic acid (AKBA) is the major organic acid component extracted from Boswellia serrata Roxb. ex Colebr. Hydroxysafflor yellow A (HSYA) is the principal active constituent of Carthamus tinctorius L. In the present study, we aimed to investigate the cardioprotective effects of HSYA and AKBA in combination in vivo and in vitro, as well as the underlying mechanisms responsible for these effects. For this purpose, MI was produced in Sprague-Dawley rats by subcutaneous injection with isoproterenol. To model ischemic-like conditions in vitro, H9C2 cells were subjected to oxygen-glucose deprivation (OGD). The levels of creatine kinase-MB (CK­MB), lactate dehydrogenase (LDH), malondialdehyde (MDA) as well as superoxide dismutase (SOD) activity were examined as well as apoptotic cell death. Mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨm or MMP) were measured using MitoSOX Red and 5,5',6,6'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye. The expression of PGC-1α and Nrf2 was quantified by western blot analysis and immunohistochemistry. HSYA and AKBA prevented myocardial pathological changes, significantly reduced the blood levels of CK-MB and LDH, and decreased apoptotic cell death. They significantly increased the expression of PGC-1α and Nrf2, and the activity of the antioxidant enzyme SOD and also decreased the levels of MDA and ROS. Moreover, the reduction in MMP was partly prevented by HSYA and AKBA. Taken together, these findings elucidate the underlying mechanisms through which HSYA and AKBA protect against MI. Additionally, HSYA and AKBA appear to act synergistically in order to exert cardioprotective effects.

[BIOCHEMICAL MARKERS OF MYOCARDIAL DAMAGE IN CHILDREN AFTER SURGICAL CORRECTION OF CONGENITAL HEART DISEASE].

The results of observations of 184 children from the CHD after surgical correction on which explored the biochemical markers of myocardial injury in serum--myocardial fraction of creatine kinase and galectin-3. The relationship between increased serum concentrations of these markers, clinical and paraclinical characteristics of examined children.

Cardiac and oxidative stress biomarkers in Trypanosoma evansi infected camels: diagnostic and prognostic prominence.

This study was conducted to investigate the level of cardiac and oxidative stress markers in camels infected with Trypanosoma evansi and to explore the diagnostic and prognostic value of cardiac troponin I (cTnI) and creatine kinase-myocardial band (CK-MB) in response to infection. Seventy four dromedary camels with clinical and laboratory evidence of trypanosomosis and 20 healthy controls were included in this study. Serum cTnI, CK-MB, CK, malondialdehyde (MDA) and super oxide dismutase (SOD) were measured. The values of cTnI, CK-MB, CK and MDA were significantly higher, whereas SOD level was lower in T. evansi infected camel. Successfully treated camels (n = 43) had lower levels of cTnI, CK-MB, CK and MDA, but higher level of SOD compared to camels with treatment failure. Both cTnI and CK-MB showed high degree of accuracy in predicting treatment outcome (success vs failure). The area under the curve for cTnI and CK-MB was 0.98 and 0.93, respectively. However, cTnI showed better sensitivity and specificity than CK-MB (Se = 96.8% vs 83.9% and Sp = 100% vs 88.5%, respectively). These results suggest that cTnI and CK-MB could be used as diagnostic and prognostic biomarkers in camels infected with T. evansi.

Hearts of some Antarctic fishes lack mitochondrial creatine kinase.

Creatine kinase (CK; EC 2.7.3.2) functions as a spatial and temporal energy buffer, dampening fluctuations in ATP levels as ATP supply and demand change. There are four CK isoforms in mammals, two cytosolic isoforms (muscle [M-CK] and brain [B-CK]), and two mitochondrial isoforms (ubiquitous [uMtCK] and sarcomeric [sMtCK]). Mammalian oxidative muscle couples expression of sMtCK with M-CK, creating an energy shuttle between mitochondria and myofibrils. We hypothesized that the expression pattern and activity of CK would differ between hearts of red- and white-blooded Antarctic notothenioid fishes due to their striking differences in cardiac ultrastructure. Hearts of white-blooded icefishes (family Channichthyidae) have significantly higher mitochondrial densities compared to red-blooded species, decreasing the diffusion distance for ATP between mitochondria and myofibrils and potentially minimizing the need for CK. The distribution of CK isoforms was evaluated using western blotting and maximal activity of CK was measured in mitochondrial and cytosolic fractions and tissue homogenates of heart ventricles of red- and white-blooded notothenioids. Transcript abundance of sMtCK and M-CK was also quantified. Overall, CK activity is similar between hearts of red- and white-blooded notothenioids but hearts of icefishes lack MtCK and have higher activities of M-CK in the cytosol compared to red-blooded fishes. The absence of MtCK may compromise cardiac function under stressful conditions when ATP supply becomes limiting.

Inhibition of JNK mitochondrial localization and signaling is protective against ischemia/reperfusion injury in rats.

To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.

Prognostic implications of creatine kinase-MB elevation after percutaneous coronary intervention: results from the Evaluation of Drug-Eluting Stents and Ischemic Events (EVENT) registry.

BACKGROUND: Creatine kinase-MB (CK-MB) elevation after percutaneous coronary intervention (PCI) has been associated with increased risk for mortality. Although most studies have defined periprocedural myocardial infarction (pMI) as an elevation in CK-MB >3× upper limit of normal (ULN), use of different CK-MB assays and variation in site-specific definitions of the ULN may limit the value of such relative thresholds. METHODS AND RESULTS: We used data from the multicenter Evaluation of Drug-Eluting Stents and Ischemic Events (EVENT) registry to examine the impact of variations in site-specific thresholds for CK-MB elevation on the incidence of pMI as well as the relationship between absolute peak levels of CK-MB after PCI and 1-year mortality. The study cohort consisted of 6347 patients who underwent nonemergent PCI and had normal CK-MB at baseline. Across the 59 study centers, the ULN for CK-MB ranged from 2.6 to 10.4 ng/mL (median, 5.0 ng/mL), and there was an inverse relationship between the site-specific ULN and the incidence of pMI (defined as CK-MB elevation >3× ULN). Although any postprocedure elevation of CK-MB was associated with an adverse prognosis, in categorical analyses, only CK-MB ≥50 ng/mL was independently associated with increased 1-year mortality (hazard ratio, 4.71; 95% confidence interval, 2.42 to 9.13; P<0.001). Spline analysis using peak CK-MB as a continuous variable suggested a graded, nonlinear relationship with 1-year mortality, with an inflection point at ≈30 ng/mL. CONCLUSIONS: Among unselected patients undergoing PCI, there is a graded relationship between CK-MB elevation after PCI and 1-year mortality that is particularly strong for large CK-MB elevations (>30 to 50 ng/mL). Future studies that include pMI as a clinical end point should consider using a core laboratory to assess CK-MB (to ensure consistency) and raising the threshold for defining pMI above current levels (to enhance clinical relevance).

Rearrangement of energetic and substrate utilization networks compensate for chronic myocardial creatine kinase deficiency.

Plasticity of the cellular bioenergetic system is fundamental to every organ function, stress adaptation and disease tolerance. Here, remodelling of phosphotransfer and substrate utilization networks in response to chronic creatine kinase (CK) deficiency, a hallmark of cardiovascular disease, has been revealed in transgenic mouse models lacking either cytosolic M-CK (M-CK(-/-)) or both M-CK and sarcomeric mitochondrial CK (M-CK/ScCKmit(-/-)) isoforms. The dynamic metabolomic signatures of these adaptations have also been defined. Tracking perturbations in metabolic dynamics with (18)O and (13)C isotopes and (31)P NMR and mass spectrometry demonstrate that hearts lacking M-CK have lower phosphocreatine (PCr) turnover but increased glucose-6-phosphate (G-6-P) turnover, glucose utilization and inorganic phosphate compartmentation with normal ATP γ-phosphoryl dynamics. Hearts lacking both M-CK and sarcomeric mitochondrial CK have diminished PCr turnover, total phosphotransfer capacity and intracellular energetic communication but increased dynamics of ß-phosphoryls of ADP/ATP, G-6-P and γ-/ß-phosphoryls of GTP, indicating redistribution of flux through adenylate kinase (AK), glycolytic and guanine nucleotide phosphotransfer circuits. Higher glycolytic and mitochondrial capacities and increased glucose tolerance contributed to metabolic resilience of M-CK/ScCKmit(-/-) mice. Multivariate analysis revealed unique metabolomic signatures for M-CK(-/-) and M-CK/ScCKmit(-/-) hearts suggesting that rearrangements in phosphotransfer and substrate utilization networks provide compensation for genetic CK deficiency. This new information highlights the significance of integrated CK-, AK-, guanine nucleotide- and glycolytic enzyme-catalysed phosphotransfer networks in supporting the adaptivity and robustness of the cellular energetic system.