Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

The impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure.

The long-predicted endocrine function of the heart has been proven by the discovery of atrial natriuretic peptide (atrial natriuretic factor, A-type natriuretic peptide; ANP) 20 years ago. This subsequently led to the description of a whole family of structurally similar but genetically distinct peptides, the natriuretic peptide family, which contributes to cardiovascular homeostasis. These looped peptides promote natriuresis and diuresis, act as vasodilators, and exert antimitogenic effects on cardiovascular tissues. Two members, ANP and brain natriuretic peptide (B-type natriuretic peptide; BNP) are secreted by the heart mainly in response to myocardial stretch induced by volume load. The natriuretic peptides are synthesized as preprohormones. The C-terminal endocrinological active peptides (ANP, BNP) and their N-terminal prohormone fragments are found in plasma. The natriuretic peptide system is activated to its highest degree in ventricular dysfunction. However, natriuretic peptides are increased in all patients with edematous disorders which lead to an increase in atrial tension or central blood volume, such as renal failure or ascitic liver cirrhosis. It could be demonstrated that in chronic heart failure patients and during the subacute phase of myocardial infarction, of all tested neurohormones, the cardiac natriuretic peptides were best markers to identify heart failure and the most powerful predictors of morbidity and mortality. Natriuretic peptides are independent markers for risk assessment. In comparative studies BNP was superior to ANP and its N-terminal prohormone fragments in myocardial infarction as well as in chronic heart failure patients. Less data on N-terminal proBNP (NT-proBNP) is available, but BNP and NT-proBNP appear to be equivalent markers. For primary care physicians natriuretic peptide measurement is useful to decide which patient with suspected heart failure warrants further investigation, particularly when assessment of left ventricular function is not readily available. Natriuretic peptides have an excellent negative predictive value, particularly in high risk patients. An increase in BNP is serious enough to warrant follow-up examinations. For the cardiologists the natriuretic peptides are helpful for guidance of therapy and monitoring disease course in heart failure patients and for risk stratification in heart failure and myocardial infarction.

Creatine kinase, myosin heavy chains and magnetic resonance imaging after eccentric exercise.

The aim of this study was to examine the relationship between myosin heavy chain (MHC) release as a specific marker of slow-twitch muscle fibre breakdown and magnetic resonance imaging (MRI) of skeletal muscle injury after eccentric exercise. The effects of a single series of 70 high-intensity eccentric contractions of the quadriceps femoris muscle group (single leg) on plasma concentrations of creatine kinase and MHC fragments were assessed in 10 young male sport education trainees before and 1 and 4 days after exercise. To visualize muscle injury, MRI of the loaded thigh was performed before and 4 days after the eccentric exercise. All participants recorded an increase (P < 0.05) in creatine kinase after exercise. In five participants, T2 signal intensity was unchanged post-exercise compared with pre-exercise and MHC plasma concentration was normal; however, they showed an increase (P < 0.05) in creatine kinase after exercise. For the remaining five participants, there was an increase in T2 signal intensity of the loaded vastus intermedius and vastus lateralis. These changes in MRI were accompanied by an increase in MHC plasma concentration (P< 0.01) as well as an increase in creatine kinase (P < 0.01). We suggest that changes in MRI T, signal intensity after muscle damage induced by eccentric exercise are closely related to damage to structurally bound contractile filaments of some muscle fibres. Additionally, MHC plasma release indicates that this damage affects not only fast-twitch fibres but also some slow-twitch fibres.

Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction.

Brain natriuretic peptide (BNP), NT-proBNP and NT-pro-atrial natriuretic peptide (NT-proANP) were measured in blood samples from 57 patients using immunoassays and immunoradiometric assays to evaluate the usefulness as diagnostic markers for the detection of heart failure. For the detection of impaired left ventricular ejection fraction (LVEF), receiver operating characteristic curves showed that BNP had the best diagnostic performance with an area under curve (AUC) of 0.75+/-0.06. However, NT-proBNP (AUC: 0.67+/-0.07) and NT-proANP (AUC: 0.69+/-0.08) showed no significant difference to BNP. In a further analysis for the detection of resting LVEF <40%, BNP again was the best marker with an AUC of 0.83+/-0.06. NT-proBNP showed only a slightly smaller AUC (0.79+/-0.07). The AUC for NT-proANP was significantly smaller (0.65+/-0.08) compared to BNP. Additionally, BNP and NT-proBNP correlated negatively with the resting LVEF (BNP: -0.472, p<0.001; NT-proBNP: -0.306, p=0.026), whereas NT-proANP showed no significant correlation. In summary, BNP was the best marker to detect patients with impaired LVEF compared to NT-proBNP and NT-proANP. However, NT-proBNP showed no significant differences to BNP and it is therefore a new promising alternative marker for the detection of left ventricular dysfunction.

Cardiac natriuretic peptides: new laboratory parameters in heart failure patients.

Natriuretic peptides, atrial natriuretic peptide and brain natriuretic peptide, are key regulators in the homeostasis of salt and water excretion and in the maintenance of blood pressure. During heart failure, these peptides are highly activated because of volume overload and increased myocardial wall tension. Among all natriuretic peptides and neurohormones, brain natriuretic peptide and its N-terminal prohormone fragment have been shown to be the best markers to identify patients with heart failure. They are useful prognostic markers as well. The stability of brain natriuretic peptides and N-terminal prohormones in ethylene-diamine-tetraacetic-acid whole blood is sufficient for routine use. Sensitive and specific assays without the need for plasma extraction are commercially available. The available data indicate that natriuretic peptides are powerful diagnostic and prognostic markers in heart failure patients. First data on treatment guidance are promising.

Hypoxia induces heat shock protein expression in human coronary artery bypass grafts.

OBJECTIVE: Heat shock proteins (HSPs) are molecular chaperones which are essential for cell survival. Heat shock and hypoxia markedly increase the expression of several HSPs in various tissues, i.e. heart. In our in vitro study, we investigated whether HSPs are inducible in human vessels which are used as coronary artery bypass grafts. METHODS: We used remnants of the saphenous vein and the internal mammary artery from 34 patients undergoing coronary artery bypass surgery. Each vessel was divided into segments, one for control conditions at 37 degrees C (5% CO(2)-95% air), the remaining ones for thermal (30 min at 42 degrees C) or hypoxic treatment (6 h oxygen deprivation with nitrogen). The expression of Hsp60, Hsp72 and Hsp73 was investigated by immunohistochemistry and Western-blot analysis. RESULTS: Compared to controls, segments of the saphenous vein undergoing heat treatment showed significantly increased expression of Hsp72 in the intima (P=0.035) and Hsp73 in the media (P=0.003). In the internal mammary artery, Hsp72 and Hsp73 were expressed in the intima at significantly higher levels (P=0.042 each). A 6 h oxygen deprivation with nitrogen resulted in elevated levels of Hsp60 (media: P=0.048), of Hsp72 (intima: P<0.001 and media: P=0.004) and of Hsp73 (intima: P=0.029) in the saphenous vein. In the internal mammary artery, Hsp73 expression was significantly enhanced (intima: P=0.048 and media: P=0.017). The results were confirmed by Western-blot analysis in representative veins. CONCLUSIONS: These findings demonstrate the common cellular defense mechanism of HSP expression in response to stress in coronary artery bypass grafts. Hypoxia and heat treatment strongly induce Hsp72 and Hsp73 expression in human coronary artery bypass grafts.

Cardiac troponin and beta-type myosin heavy chain concentrations in patients with polymyositis or dermatomyositis.

Cardiac troponin T (cTnT), cardiac troponin I (cTnI), myosin heavy chains (MHC), myoglobin, creatine kinase (CK), and creatine kinase isoenzyme MB (CKMB), were measured in blood samples from 39 polymyositis (PM) or dermatomyositis (DM) patients without clinical evidence for cardiac involvement to evaluate their clinical usefulness in this patient population. MHC, myoglobin, and CKMB were frequently elevated and correlated with each other and with disease severity. Undetectable cTnI in all but one patient indicated that MHC was released from skeletal muscle, thereby providing the first laboratory evidence of frequent slow-twitch muscle fibre-necrosis in patients with PM or DM. CKMB was elevated in 51%, cTnT in 41%, and cTnI in only 2.5% of patients. cTnI did not correlate with other markers or with disease severity scores. The close correlations found between cTnT and skeletal muscle damage markers and the relationship between cTnT with disease severity without clinical evidence for myocardial damage suggest a release of cTnT from skeletal muscle. The relationship of cTnT with disease severity indicates a possible role of the marker for risk stratification. However, the prognostic values of cardiac troponins and other muscle damage markers in PM/DM patients remain to be compared in prospective outcome trials.

Clinical and experimental results on cardiac troponin expression in Duchenne muscular dystrophy.

BACKGROUND: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). METHODS: Plasma from 14 patients (mean age, 7.5 years; range, 5.7-19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). RESULTS: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 microg/L; range, 0.06-0.16 microg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763-5030 U/L) with age (median, 7.5 years; range, 6.8-10.9 years; r = -0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. CONCLUSIONS: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

Release of muscle proteins after downhill running in male and female subjects.

The release of muscle proteins after downhill running, which mainly includes eccentric muscle action, was compared in females (F; n=9) and males (M; n=9). They performed 20 min of downhill treadmill running with 16% decline with a target heart rate of 70% of the individual VO2peak, which was determined two weeks before. Blood samples were drawn before, 6 and 24 h after exercise to measure plasma levels of skeletal troponin I (sTnI), myosin heavy chain fragments (MHC), creatine kinase (CK), and myoglobin (Mb). Baseline levels before exercise were significantly higher in males compared to females for the cytoplasmic proteins CK and Mb, but the difference for MHC and sTnI was not significant. Both groups displayed marked and significant early (6 h) increases (P<0.05) for sTnI (median: F: 8.2 microg/L; M: 22.0 microg/L), Mb (median: F: 86.8 microg/L; M: 407 microg/L), and CK (median: F: 162 U/L; M: 339 U/L). A significant (P<0.05) but delayed (24 h) increase was found for MHC (median: F: 482 microU/L; M: 651 microU/L). The absolute values for all four parameters were significantly (P<0.05) higher in males compared to females; however, no difference was found for the relative increases and the time course of all parameters between females and males. We conclude 1) that there were no significant differences in the basal concentrations of predominantly bound proteins, and 2) that there were no differences in the relative muscle protein release between females and males before and after one bout of high-intensive eccentric exercise. The higher plasma concentrations of all measured muscle proteins in males are probably caused by the higher muscle mass compared to females.

Natriuretic peptides stimulate cyclic guanosine monophosphate production in human saphenous vein and internal mammary artery.

OBJECTIVE: It has been shown previously that the internal mammary artery releases more cyclic guanosine monophosphate after stimulation with atrial natriuretic peptide than the saphenous vein, and that C-type natriuretic peptide possesses a cyclic guanosine monophosphate stimulating potential on saphenous vein bypass grafts. The present study was undertaken to investigate intracellular content and extracellular release of cyclic guanosine monophosphate, by the internal mammary artery and saphenous vein, after challenge with further members of the natriuretic peptide family. METHODS: Specimens of human internal mammary artery and saphenous vein from 29 patients were cut into segments and stimulated with 10(-6) M concentrations of atrial natriuretic peptide (internal mammary artery n=8, saphenous vein n=10), brain natriuretic peptide (internal mammary artery n=9, saphenous vein n=13), C-type natriuretic peptide (internal mammary artery n=12, saphenous vein n=15), and urodilatin (internal mammary artery n=8, saphenous vein n=12). Intracellular content and extracellular release of cyclic guanosine monophosphate were determined using an (125)I radioimmunoassay. RESULTS: The following median stimulated intracellular cyclic guanosine monophosphate concentrations were measured in the internal mammary artery and saphenous vein: 35358 and 8672 fmol/cm(2) (P<0.001) after atrial natriuretic peptide, 45632 and 7830 fmol/cm(2) (P=0.003) after brain natriuretic peptide, 10144 and 13216 fmol/cm(2) (P=NS for intergraft comparison) after C-type natriuretic peptide, and 20949 and 6690 fmol/cm(2) (P=0.001) after urodilatin. Stimulation with atrial natriuretic peptide, brain natriuretic peptide and urodilation also led to a significant increase of extracellular cyclic guanosine monophosphate release by the internal mammary artery. CONCLUSIONS We conclude that brain natriuretic peptide and urodilatin exhibit a similarly effective cyclic guanosine monophosphate-stimulating potential on the internal mammary artery as atrial natriuretic peptide. In contrast, C-type natriuretic peptide shows comparable effects on the internal mammary artery and saphenous vein.