Immunohistochemical analysis of cardiac troponin inhibitor in an experimental model of acute myocardial infarction experimental model and in human tissues.

Acute obstruction of coronary arteries leads to acute myocardial infarction (AMI), which causes unexpected death in humans. However, AMI cannot be easily detected in forensic examinations with traditional hematoxylin and eosin (H&E) staining. We analyzed whether cardiac troponin inhibitor (CTnI) could serve as a sensitive and specific early marker for diagnosing AMI in forensic medicine. We established an AMI model in rabbits by ligating the left ventricular branch and observed CTnI expression with immunohistochemistry after different ligation times. We found increased CTnI staining at the 0.5-h time point and depletion of CTnI staining with a 1-h ligation. The areas in which CTnI staining was depleted as seen with immunohistochemical analysis were consistent with the results of H&E staining. Next, human myocardium tissues from 30 persons who died from AMI and were subsequently examined in our forensic center were studied using immunohistochemistry with an antibody to human CTnI. Areas of infarction also showed depletion of CTnI staining. These findings suggested that immunohistochemical detection of CTnI is earlier, more sensitive, and myocardial tissue - specific as compared with H&E staining. CTnI may serve as an ideal marker for diagnosing AMI in forensic investigations.

The stress-responsive MAP kinase p38 is activated by low-flow ischemia in the in situ porcine heart.

Stress-responsive p38 MAP kinase is activated by phosphorylation during global and severe regional myocardial ischemia. However, it is unknown whether or not moderate, low-flow ischemia also activates p38 MAP kinase. Therefore, we investigated p38 MAP kinase activation in an established model of short-term hibernation and stunning. In anesthetized swine, coronary blood flow into the left anterior descending coronary artery was decreased in order to reduce regional contractile function by identical with 50%. Transmural myocardial biopsies were taken before (controls) and during ischemia as well as after reperfusion. Creatine phosphate content, after an early ischemic reduction, recovered to control values at 90 min ischemia. The expression of phospholamban, SERCA2a, calsequestrin, and troponin inhibitor was unchanged under these conditions (Northern and Western blotting). At 8 min of ischemia, however, p38 MAP kinase was activated to 221% of the pre-ischemic value as judged by its elevated phosphorylation state. Then, it returned to control values by 85 min ischemia. We conclude that low-flow ischemia transiently activates the stress-responsive p38 MAP kinase which might act to trigger cardioprotective events.

Evidence for two-site binding of troponin I inhibitory peptides to the N and C domains of troponin C.

The interactions of two troponin I peptides, Ip1 (residues 96-116) and Ip2 (residues 104-116), with spectral probe mutants F29W and F105W of intact troponin C (TnC) and of isolated N (residues 1-90) and C (residues 88-162) domains of TnC have been examined. Ip-induced fluorescence emission spectral changes were observed with all four proteins in the presence of Ca2+. Different dependencies of these spectral changes on Ip concentration for intact F29W and F105W are interpreted in terms of two binding sites on TnC. The binding of Ip1 to the C domain (KD1 = 0.50 microM) is 20-40-fold stronger than to the N domain. The binding affinity of Ip1 to both the N and C domains is greater than that of Ip2. The binding strengths of Ip1 to the N domain of intact F29W and isolated F29W/ND are the same within experimental error; that to isolated F105W/CD is weakened by 5-6-fold relative to the C domain of intact F105W. Ip-induced fluorescence changes are dependent on the presence of Ca2+ and are not seen in the presence of Mg2+ alone nor in the absence of divalent cations. This is true even though Ip2 binds to TnC under all three conditions, as demonstrated by affinity chromatography. The accumulated evidence indicates that the F-->W mutations have not significantly affected the binding of Ip peptides to TnC.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-peptide monoclonal antibody imaging of a common binding domain involved in muscle regulation.

Multiple-component regulatory protein systems function through a generalized mechanism where a single regulatory protein or ligand binds to a variety of receptors to modulate specific functions in a physiologically sensitive context. Muscle contraction is regulated by the interaction of actin with troponin I (TnI) or myosin in a Ca(2+)-sensitive manner. Actin utilizes a single binding domain (residues 1-28) to bind to residues 104-115 of TnI (Van Eyk JE, Sönnichsen FD, Sykes BD, Hodges RS, 1991, In: Rüegg JC, ed, Peptides as probes in muscle research, Heidelberg, Germany: Springer-Verlag, pp 15-31) and to myosin subfragment 1 (S1, an enzymatic fragment of myosin containing both the actin and ATP binding sites) (Van Eyk JE, Hodges RS, 1991, Biochemistry 30:11676-11682) in a Ca(2+)-sensitive manner. We have utilized an anti-TnI peptide (104-115) monoclonal antibody, Mab B4, that binds specifically to TnI, to image the common binding domain of actin and thus mimic the activity of actin including activation of the S1 ATPase activity and TnI-mediated regulation of the S1 ATPase. Mab B4 has also been utilized to identify a receptor binding domain on myosin (residues 633-644) that is recognized by actin. Interestingly, Mab B4 binds to the native protein receptors TnI and S1 with relative affinities of 100- and 25,000-fold higher than the binding affinity to the 12-residue peptide immunogen. Thus, anti-peptide monoclonal antibodies prepared against a receptor binding domain can mimic the ligand binding domain and be utilized as a powerful tool for the detailed analysis of complex multiple-component regulatory systems.

The inhibitory effect of troponin I on the ATPase activity of myofibrils treated with troponin T and troponin T1.

Troponin in bullfrog skeletal myofibrils was substituted by troponin T and troponin T1 from rabbit skeletal muscle. The inhibitory effect of troponin I was examined on the ATPase activity of these troponin-substituted myofibrillar preparations. The ATPase of myofibrils substituted by troponin T was more inhibited by troponin I than the ATPase of myofibrils substituted by troponin T1.

[Effect of neurotropic drugs on calmodulin and troponin C-dependent processes]. / Vliianie neirotropnykh soedinenii na kal'modulin- i troponin C-zavisimye protsessy.

The effects of neurotropic compounds on Ca-binding proteins (calmodulin, troponin C) were investigated. It was shown that the majority of neuroleptics of the phenothiazine group effectively interact with the both proteins and inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+-activated actomyosin. ATPase. Neuroleptics of the butyrophenone group as well as imipramine and diphenehydramine having a low efficiency interact only with calmodulin. Methophenazine, a phenothiazine neuroleptic, being an effective inhibitor of calmodulin and of calmodulin-dependent phosphodiesterase, does not influence troponin C or Ca-dependent actomyosin ATPase. Therefore, this compound may be used as a convenient tool in the study of processes controlled by these Ca-binding proteins. It is concluded that troponin C possesses Ca-dependent sites which bind pharmacological agents structurally similar to that of calmodulin. However, these sites bind pharmacological agents with a low efficiency and exhibit selectivity towards certain drugs. Despite the obvious homology of the both Ca-binding proteins, i.e., calmodulin, troponin C, their effects on the processes under their control appear to be selective.

[Effect of calmodulin inhibitors on the contraction and relaxation of the heart muscle]. / Vliianie ingibitorov kal'modulina na sokrashchenie i rasslablenie serdechnoi myshtsy.

The calmodulin inhibitor trifluoroperazine (5 microM) diminished the velocity of contraction and relaxation of the guinea-pig papillary muscle by 31 and 53%, respectively. Methophenazine showed approximately the same affinity to both calmodulin and trifluoroperazine and nearly 40-fold less affinity to troponin. Methophenazine (5 microM) did not change the contraction velocity and diminished the relaxation velocity ty 21%. It is suggested that calmodulin participates mainly in the control of myocardial relaxation.

Erythrocyte troponin inhibitor-like protein: isolation and characterization.

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000--24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca2+ and K+-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg2+-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.