Biomedical subjects

J Van Eyk

Publications and source records attributed to J Van Eyk.

6 recordsLinked to original sources

PubMed1997

Reversal of phosphate induced decreases in force by the benzimidazole pyridazinone, UD-CG 212 CL, in myofilaments from human ventricle.

UD-CG 212 Cl, (Fig. 1: 4,5-dihydro-6-[2-(4-hydroxyphenyl)-1 H-benzimidazole-5-yl]-5-methyl-3(2H)-pyridazinone), is the primary metabolite of the positive inotropic agent pimobendan (UDCG 115 BS, Acardi). Our previous studies showed in detergent extracted preparations of canine ventricular muscle that sub-nanomolar concentrations of UD-CG 212 Cl increased submaximal myofilament force, but only when the activation state had been altered by relatively high (5-10 mM) concentrations of inorganic phosphate (Pi) or relatively low (20 microM) concentrations of MgATP. In the present study, we investigated the effects of UD-CG 212 Cl on the pCa-force relationship of detergent extracted bundles of human cardiac fibers before and after addition of Pi. As expected, treatment with 5 mM Pi depressed maximal force at pCa 4.5 by 27.0 +/- 0.4% (mean +/- SEM). Force generated at the half-maximally activating Ca2+ concentration (pCa50) of control fibers (5.98 +/- 0.2) was significantly (p < .05) reduced following the addition of 5 mM Pi (pCa50 = 5.69 +/- 0.3). The addition of UD-CG 212 Cl over a range of concentrations (10(-11)-10(-6) M) had no effect on Ca(2+)-sensitivity under control conditions, but in the presence of 5 mM Pi, there was a 23.1 +/- 0.1% increase in the percent maximal force at pCa5.9. Ca(2+)-sensitivity was also significantly increased in the presence of Pi and 10(-8) M UD-CG 212 Cl (pCa50 = 5.74 +/- 0.3, p < .05). We conclude that UD-CG 212 Cl potentially increases sub-maximal force of human ventricular myofilaments with an inotropic action depending on a state of myofilament activation associated with ischemic conditions.

Actin Cytoskeleton↗

PubMed1996

Altered interactions among thin filament proteins modulate cardiac function.

The transition of cardiac muscle myofilament activity from the diastolic to the systolic state is a complex allosteric/co-operative process, rich in potential control mechanisms. The rate and intensity of the transition is modulated by the mechanical state of the myofilaments, by covalent and non-covalent mechanisms, and by the isoform population of myofilament proteins. Moreover, the process is altered in pathological states and subject to modification by pharmacological agents with potential use as inotropic drugs. We present here a current perception of the process, with focus on molecular interactions of the thin filament components, especially troponin I. Our discussion is couched in terms of what we believe to be pressing questions in the current state of knowledge of this system. These questions are as follows: what is the topology of the thin filament and how do thin filament proteins regulate the activation of cross-bridge cycling? What is the relative role of protein phosphorylation of thin filament proteins in the regulation of the cardiac activity and dynamics? What is the relative role of feedback effects of cross-bridge binding on thin filament activity? Answers to these questions have taken on new significance, with the identification of familial hypertrophic cardiomyopathy as a "sarcomeric" disease related to missense mutations in myosin, troponin T. and tropomyosin. As discussed, new and exciting developments in this area are bringing us closer to the answers to these questions.

Actin Cytoskeleton↗

PubMed1995

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles.

Skinned fibres from porcine ventricles exhibited a higher Ca2+ sensitivity (pCa50, i.e. -log10 Ca2+ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca2+ sensitivity and Ca2+ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca2+ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa50 = 5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca2+ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca2+ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca(2+)-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 microM). The Ca(2+)-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137-148) and, in addition, this effect was antagonized by EMD 53998.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI [104-115].

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104-115] at various Ca2+ concentrations. The increase in negative ellipticity and pCa2+ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca2+ binding constant and the pCa50 value required to induce 50% of Ca2+ -dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa50 and the apparent Ca2+ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa50 values required for 50% Ca2+ activation. The magnitude of the inhibition of tension development and the shift in the Ca2+ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1986

Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115].

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.

Actins↗

PubMed1985

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits.

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

Animals↗