Altered actin binding with myosin mutation in hypertrophic cardiomyopathy and sudden death.
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Actins↗
Biomedical subjects
J C Rüegg
Publications and source records attributed to J C Rüegg.
Ca(2+)- and GTP[gamma S]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method.
In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-toxin and incubated them for 1 h either at a pCa (i.e.--log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.
Animals↗
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↗
Myofibrillar Ca2+ sensitivity of cardiomyopathic hamster hearts.
We studied the Ca2+ responsiveness of skinned muscle fibre preparations from the right and left ventricles of normal (FIB) and genetically cardiomyopathic (Bio-To-2) Syrian hamsters. Thus, we compared the Ca2+/force relationships of preparations from myopathic hamsters to those of age-matched (11-16 months old) normal animals. The pCa (i.e. -log10 [Ca2+]) required for 50% force activation (Ca2+ sensitivity) was higher in the myopathic hamsters than in controls (pCa50 values of 5.3 +/- 0.03 and 5.17 +/- 0.04, respectively); this difference might be due to an alteration in regulatory proteins. Indeed, after extraction (with vanadate) and replacement of troponin I with bovine cardiac troponin the pCa50 values were similar (pCa 5.35) to those of bovine ventricular fibres. The Ca2+ sensitizer EMD 53998 (10 microM) increased Ca2+ sensitivity in preparations from normal and cardiomyopathic hamsters equally, by 0.4 pCa units. Incubation of fibre bundles with the catalytic subunit of cyclic-adenosine-monophosphate-dependent protein kinase decreased Ca2+ sensitivity, thereby "normalizing" the enhanced Ca2+ responsiveness of fibres from cardiomyopathic hamsters. It is not clear, however, whether the pathologically increased Ca2+ sensitivity of the hearts of aged myopathic hamsters reflects a maladaptation, or a compensatory mechanism of the failing heart.
Actin Cytoskeleton↗
Effects of troponin-I extraction with vanadate and of the calcium sensitizer EMD 53998 on the rate of force generation in skinned cardiac muscle.
The rate of tension development following release of ATP from caged-ATP in the presence of calcium was studied in skinned cardiac fibres from swine. A low-force rigor state was obtained by using butanedione monoxime (BDM) during the induction of rigor. BDM was washed out and following release of ATP in the presence of Ca2+ (pCa 4.3), the muscles contracted with an apparent rate of about 2 s-1 at 22 degrees C. After treatment with 10 mM vanadate to extract troponins I and C the fibres contracted independently of calcium. The rate of contraction upon release of ATP was slower than prior to extraction and was independent of [Ca2+]. Since treatment with vanadate has been shown to extract about 90% of troponin-I the results suggest that the muscles under these conditions are partially activated by removal of an inhibition of cross-bridge interaction by troponin I. A partial recovery of force was obtained by prolonged incubation in DTT containing solutions possibly reflecting reconstitution with troponin I still present in the fibre bundle. Treatment with a solution containing whole troponin caused almost complete recovery of calcium sensitivity and rate of force development. The calcium sensitizer EMD 53998 increased rates of contraction in a dose dependent manner, suggesting that this compound increases force and calcium sensitivity by increasing the cross-bridge attachment rates.
Adenosine Triphosphate↗
The calcium sensitizer EMD 53998 antagonizes phosphate-induced increases in energy cost of isometric tension in cardiac skinned fibres.
We have investigated whether a Ca(2+)-sensitizing substance, the thiadiazinone derivative EMD 53998, can alter the ratio of ATPase activity to force, i.e. the tension cost in skinned fibres of swine cardiac trabecula in which the tension cost was increased by inorganic phosphate. In the presence of 10 mM inorganic phosphate (Pi) and thapsigargin 20 microM, EMD 53998 reduced the energy cost of isometric tension over the entire range of activating Ca2+ concentrations, resulting in a consistent change in slope (approximately 20% decrease) of the ATPase/force relation. We confirmed that in the absence of added phosphate and at maximal Ca2+ activation EMD 53998 had little if any effect on tension cost. We had previously reported that the effects of EMD 53998 and Pi on calcium sensitivity and maximum isometric tension are mutually antagonistic and our new energy data now support the proposal that EMD 53998 functionally antagonizes the effects of Pi on crossbridges. The decrease in the slope of the relation between ATPase and force caused by EMD 53998 may be interpreted to reflect either a decrease in the rate of 'detachment' (g(app)) of crossbridges or an increase in average force per crossbridge, as predicted by classical crossbridge models. Since the Pi release step of the crossbridge cycle is associated with the rate of 'attachment' (f(app)) rather than g(app), we conclude that the decrease in tension cost with EMD 53998 most likely reflects an increased force per crossbridge.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Coordination of nuclear and mitochondrial gene expression during the development of cardiac hypertrophy in rats.
We studied the coordination of nuclear and mitochondrial gene expression during cardiac hypertrophy following aortic stenosis or thyroid hormone treatment in rats. We measured mRNA levels for representative subunits of cytochrome-c oxidase, two encoded by mitochondrial DNA and two encoded by the nucleus, as well as the levels of one mitochondrial rRNA. In both models of hypertrophy, an increase of total tissue RNA, reflecting mainly cytosolic ribosomes, accompanied the increase in ventricular weight. Relative levels of mitochondrial rRNA remained unchanged, indicating a net synthesis of mitochondrial ribosomes as well. In both models, cytochrome-c oxidase activity and nuclear-encoded mRNAs remained fairly constant, whereas levels of mitochondrial mRNAs were transiently decreased 24 h after the growth stimulus. We conclude that, in the initial phase of hypertrophy, the signal regulating the synthesis of mitochondrial rRNA is synchronized with nuclear gene expression, whereas the signal regulating mitochondrial mRNA synthesis is not. We postulate that differential regulation of mitochondrial transcription and premature termination of the polycistronic transcript (the latter giving rise to the mitochondrial rRNAs) account for the observed results.
Animals↗
Infusion of oxidized glutathione enhances postischemic segment-shortening in dog hearts.
Oxidized glutathione (GSSG) but not its reduced form (GSH) is taken up by intact myocardial cells, and is rapidly converted into GSH. Reduced glutathione is an important intracellular defense against oxygen-derived free radicals and has been found to enhance calcium sensitivity in skinned cardiac fibers. We have investigated the effects of intravenous GSSG on left ventricular systolic pressure, maximal rate of rise of pressure and regional segment-shortening in dogs subjected to occlusion of the left anterior descending artery for 30 minutes, followed by 45 minutes reperfusion. Starting 10 minutes before reperfusion, the dogs were randomly treated with either GSSG (100 mM, 5 ml/min, n = 5) or Ringer's solution (5 ml/min, n = 5) until 30 minutes of reperfusion. Myocardial blood flow was measured by radioactive microspheres. Infusion of GSSG increased total glutathione content in both ischemic (47 +/- 16 mumol/g protein) and nonischemic myocardium (71 +/- 17 mumol/g protein) as compared to controls (23 +/- 2 mumol/g protein, p < 0.05). In both groups paradoxical wall motion occurred in the ischemic region during occlusion. On reperfusion, regional dyskinesia persisted in controls; while, in glutathione-treated dogs, systolic segment-shortening reached half the baseline values (p < 0.05, treated vs controls, at 15, 30, 45 minutes reperfusion). During ischemia the area of pressure-length loops, obtained from simultaneous recordings of left ventricular pressure and regional segment length, decreased to 30 +/- 7% of baseline in controls and to 40 +/- 18% of baseline in GSSG-treated animals. After 45 minutes reperfusion it was restored to 78 +/- 22% baseline in treated hearts but was still 36 +/- 16 of baseline in controls (p < 0.05). We conclude that infusion of GSSG increases the intracellular stores of glutathione and improves the contractile state of postischemic myocardium.
Animals↗
Effect of myosin heavy chain peptides on contractile activation of skinned cardiac muscle fibres.
Peptides derived from the sequence of the S1 domain of the myosin heavy chain were tested for their effects on the regulation of cardiac contractility. Basal calcium responsiveness of the contractile apparatus in terms of isometric tension generation and ATPase was determined in chemically demembranated ventricular fibre bundles. Incubation with a series of peptides derived from the peptide sequence around SH thiol group (Cys 707) resulted in a measurable increase in isometric tension and ATPase activity at sub-maximal concentrations of calcium but not at saturating levels of calcium activity, thus demonstrating a "calcium-sensitizing" effect of these peptides. The effects of two of these peptides, S1 687-716 and S1 701-717, are demonstrated to mimic, but importantly were not additive with, the calcium sensitization induced by lowering ATP concentration to 10 microM from 10 mM. This suggests the possibility of a similar mechanism of action underlying both types of sensitization. Because these effects demonstrate tissue specificity, were sensitive with respect to potency to not only amino acid composition but also sequence, and could not be duplicated by a similarly charged, non-homologous peptide, we attribute the effects to be specific to the sequences of these peptides. These data provide further evidence that the sequence between residues 687 and 717 of the S1 domain of the myosin heavy chain influences the calcium responsiveness of the contractile apparatus.
Actins↗
The positive inotropic calcium sensitizer EMD 53998 antagonizes phosphate action on cross-bridges in cardiac skinned fibers.
The diazinone derivative EMD 53998 sensitizes skinned myocardial fibers to Ca2+ and enhances maximal calcium-activated force (pCa = 4.5) by approximately 100%; the EC50 is 10 microM in the absence and about 30 microM in the presence of added inorganic phosphate (10 mM). Although concentrations of added phosphate as low as 0.5 mM inhibit force, at high concentrations of EMD 53998 (> or = 50 microM), phosphate only inhibits at concentrations exceeding 20 mM. These data suggest that the effects of EMD 53998 and phosphate are mutually antagonistic. Importantly, both EMD 53998 and phosphate had similar effects on force generation in troponin I-depleted (Ca(2+)-independent) skinned fibers, thus demonstrating that these compounds are likely to affect cross-bridges directly and not via the Ca(2+)-regulatory system.
Animals↗
Troponin replacement in permeabilized cardiac muscle. Reversible extraction of troponin I by incubation with vanadate.
Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca2+ solution (pCa greater than 8), the permeabilized muscle produced 84.8% +/- 20.1% (+/- S.D., n = 98) of the isometric force elicited by high Ca2+ (pCa approximately 4.5) prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% +/- 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (greater than 90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.
Animals↗
Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues.
In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.
Animals↗
Modulation of myosin phosphorylation-contraction coupling in skinned smooth muscle.
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Animals↗
Peptide competition of actin activation of myosin-subfragment 1 ATPase by an amino terminal actin fragment.
The amino-terminal region of actin participates in the binding of myosin subfragment 1 (S1) during cross-bridge cycling, thereby assisting in the activation of the magnesium-dependent myosin ATPase. Effects of three actin fragments on the magnesium-dependent S1 and acto-S1 ATPase activities in solution were studied. One of the peptides, containing residues actin 1-44, mimicked the S1 ATPase-activating properties of actin and in turn inhibited acto-S1 ATPase both in a concentration-dependent manner. This suggests peptide competition for the actin binding site on myosin. The other fragments, residues actin 1-18 and 82-119, respectively, had no detectable effect on S1- and acto-S1 ATPase activity.
Actins↗
What does TnCDANZ fluorescence reveal about the thin filament state?
Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnCDANZ) increases upon activation with Ca2+. This fluorescence enhancement is due to Ca2+ binding to the Ca(2+)-specific binding sites of TnCDANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca2+ binding to TnCDANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnCDANZ fluorescence. "Weakly" bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnCDANZ fluorescence. "Strongly" attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.
Actin Cytoskeleton↗
Contractile protein interactions in smooth muscle.
Smooth muscle tone and 'holding economy' depend on the rate constants governing the cross-bridge cycle. Thus, calcium activation via calmodulin-dependent myosin light chain phosphorylation may determine the apparent rate constant ('f') at which cross-bridges enter the force-generating state, forming actin-attached, strongly bound cross-bridges. This phosphorylation of the light chain may be inhibited in skinned fibers by a peptide mimic of the calmodulin recognition site of the myosin light chain kinase (RS 20) that relaxes smooth muscle. In smooth muscle, the apparent cross-bridge detachment rate constant ('g') also seems to be variable, a low constant allowing for a high holding economy and low shortening velocity in the 'latch state'. It may also account for force maintenance at low levels of myosin phosphorylation. Additionally, cross-bridge attachment may, however, be also controlled by other regulatory proteins such as calponin and caldesmon.
Actins↗
Modulation of crossbridge kinetics by myosin isoenzymes in skinned human heart fibers.
Skinned fibers from the normal human heart with the beta-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2+ sensitivity than skinned fibers with the alpha-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (g(app)), myosin heavy chain isoenzymes seem to have different crossbridge turnover kinetics. Modulation of g(app) by myosin heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. g(app) was independent of Ca2+.
Adenosine Triphosphatases↗
Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.
Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)
3',5'-Cyclic-AMP Phosphodiesterases↗