Biomedical subjects

J M Metzger

Publications and source records attributed to J M Metzger.

At least 55 records · Page 3Linked to original sources

PubMed1996

Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.

Actins↗

PubMed1995

Altered Ca2+ sensitivity of tension in single skeletal muscle fibres from MyoD gene-inactivated mice.

1. Single, fast glycolytic skeletal muscle fibres were isolated from wild-type (MyoD+/+) and MyoD mutant mice (MyoD-/-), which lack a functional copy of the MyoD gene. Fibres were chemically permeabilized to permit manipulation and control of the ionic environment of the otherwise intact myofilament apparatus. 2. Results show a fivefold greater variability in the [Ca2+] required for half-maximum tension generation among individual MyoD-/- fibres in comparison with controls (p < 0.05). 3. Consistent with this finding, Western blot analysis showed a sevenfold greater variability in the isoform expression pattern of the thin filament regulatory protein troponin T in Myod-/- compared with control fibres (p < 0.05). 4. Electrophoretic analysis of single-fibre segments indicated no apparent alteration in the isoform expression pattern of other regulatory and contractile proteins. In addition, other parameters of contractile function, including velocity of unloaded shortening, and maximum force production, were not significantly different between MyoD-/- and MyoD ø fibres. 5. These findings indicate that the thin filament structure- function relationship is altered due to the MyoD mutation and suggest that MyoD plays a role in establishing and/or maintaining the differentiated phenotype of adult fast skeletal muscle fibres.

Animals↗

PubMed1995

Synthesis and biological evaluation of NK1 antagonists derived from L-tryptophan.

The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan (3), which was derived from the screening lead N-ethyl-L-tryptophan benzyl ester, has been used as a starting point to identify high-affinity substance P receptor antagonists with improved in vivo activity. Altering the ester moiety to an amide or ether led to a substantial loss in binding affinity, but conversion to a ketone provided compounds with affinity comparable to the equivalent esters. A homochiral synthesis of the key intermediate amino ketone 15 was developed which allows its preparation on a large scale. From this intermediate a range of amine-containing acylamino derivatives were prepared with affinity optimized in the morpholinylbutyramide 161 which has an IC50 of 0.17 nM at the human NK1 receptor. In addition to improving affinity, the amino group also provided aqueous solubility for a number of these derivatives. When tested in vivo the quinuclidine derivative L-737,488 (16i) was found to be an orally active (ID50 = 1.8 mg/kg) inhibitor of substance P-induced dermal extravasation in the guinea pig.

Amines↗

PubMed1995

Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers.

Myosin binding-induced activation of the thin filament was examined in isolated cardiac myocytes and single slow and fast skeletal muscle fibers. The number of cross-bridge attachments was increased by stepwise lowering of the [MgATP] in the Ca(2+)-free solution bathing the preparations. The extent of thin filament activation was determined by monitoring steadystate isometric tension at each MgATP concentration. As pMgATP (where pMgATP is -log [MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pMgATP range of 5.0-5.4. The steepness of the tension-pMgATP relationship, between the region of the curve where tension was zero and the peak tension, is hypothesized to be due to myosin-induced cooperative activation of the thin filament. Results showed that the steepness of the tension-pMgATP relationship was markedly greater in cardiac as compared with either slow or fast skeletal muscle fibers. The steeper slope in cardiac myocytes provides evidence of greater myosin binding-induced cooperative activation of the thin filament in cardiac as compared with skeletal muscle, at least under these experimental conditions of nominal free Ca2+. Cooperative activation is also evident in the tension-pCa relation, and is dependent upon thin filament molecular interactions, which require the presence of troponin C. Thus, it was determined whether myosin-based cooperative activation of the thin filament also requires troponin C. Partial extraction of troponin C reduced the steepness of the tension-pMgATP relationship, with the effect being significantly greater in cardiac than in skeletal muscle. After partial extraction of troponin C, muscle type differences in the steepness of the tension-pMgATP relationship were no longer apparent, and reconstitution with purified troponin C restored the muscle lineage differences. These results suggest that, in the absence of Ca2+, myosin-mediated activation of the thin filament is greater in cardiac than in skeletal muscle, and this apparent cooperativity requires the presence of troponin C on thin filament regulatory strands.

Adenosine Triphosphate↗

PubMed1995

Effects on Ca(2+)-activated tension due to a synthetic NH2-terminal actin peptide in single skeletal muscle fibers.

Insight into the mechanism of force development in striated muscle will be provided by elucidating the specific regions of the actin molecule that interact with myosin and regulatory subunits of the thin filament during Ca(2+)-activated contraction. There is growing evidence that the acidic NH2-terminal domain of actin 1) may represent an important binding site for myosin and 2) may interact with the inhibitory region of troponin I. The purpose of this study was to determine the effects of a synthetic peptide corresponding to a specific sequence of the NH2-terminal domain of skeletal muscle actin on Ca(2+)-activated tension in chemically skinned single psoas skeletal muscle fibers. This study focused on the highly conserved Lys18-Arg28 amino acid sequence of actin, a region of native actin that is believed to interact with troponin I and myosin. The effects of synthetic actin peptide Lys18-Arg28 on tension development varied, depending on 1) the concentration of Ca2+ in the activating solutions and 2) the peptide concentration. At submaximal concentrations of Ca2+, isometric tension was reversibly potentiated in the presence of 100-500 microM synthetic actin peptide Lys18-Arg28. Importantly, scrambling the sequence of Lys18-Arg28 fully abolished the increase in Ca2+ sensitivity, providing evidence that the observed effects were specific to the sequence of peptide Lys18-Arg28. In contrast, maximum Ca(2+)-activated tension was inhibited by millimolar concentrations of Lys18-Arg28 and the scrambled peptide, indicating that this effect was nonspecific. The effect of peptide Lys18-Arg28 to increase the Ca2+ sensitivity of tension is not known but may be due to an effect of the actin peptide to alter thin filament activation, a possibility consistent with proposed interactions between this domain of actin and the inhibitory region of troponin I.

Actins↗

PubMed1995

Myosin heavy chain expression in contracting myocytes isolated during embryonic stem cell cardiogenesis.

Mouse embryonic stem (ES) cells are totipotent cells derived from the inner cell mass of the preimplantation blastocyst and are capable of differentiating in vitro into cardiac myocytes. Attached cultures of differentiating ES cells were established to document the timing of contractile development by microscopic observation and to permit the microdissection of cardiac myocytes from culture. The onset of spontaneous contraction varied markedly in differentiation culture, with contraction being maintained on average for 9 days (range, 1 to 75 days). Indirect immunofluorescence in microscopy showed that myosin expression was localized to the contracting cardiac myocytes in culture. Myosin heavy chain (MHC) isoform expression in microdissected ES cell-derived cardiac myocytes was determined by means of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The distribution of MHC isoform expression in isolated ES cell cardiac myocytes was as follows: 27% expressed the beta-MHC isoform, 33% expressed both the alpha- and beta-MHC isoforms, and 40% expressed the alpha-MHC isoform. MHC phenotype was correlated to the duration of continuous contractile activity of the myocytes. Myocytes that had just initiated spontaneous contractile activity predominantly expressed the beta-MHC (average days of contraction before isolation, 2.5 +/- 0.7). The alpha-MHC isoform was detected after mouse prolonged contractile activity in vitro (1 to 5 weeks). A strong correlation was obtained between MHC phenotype and days of contraction of the cardiac myocyte preparations isolated from ES cell cultures (r = .93). The apparent transition in MHC isoform expression during ES cell differentiation parallels the beta- to alpha-MHC isoform transition characteristic of murine cardiac development in vivo. These findings are evidence that ES cell cardiac myocyte differentiation follows the normal developmental program of murine cardiogenesis.

Animals↗

PubMed1994

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.

Animals↗

PubMed1993

Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice.

Depressed contractile function plays a primary role in the pathophysiology of acute myocardial ischemia. Intracellular acidification is an important factor underlying the inhibition of force production in the ischemic myocardium. The effect of acidosis to depress contractility is markedly greater in cardiac as compared to skeletal muscle; however, the molecular basis of this difference in sensitivity to acidosis is not clearly understood. In this report, we describe transgenic mice that express the fast skeletal isoform of troponin C (sTnC) in cardiac muscle. In permeabilized single cardiac myocytes the shift in the midpoint of the tension-pCa relationship (i.e., pCa50, where pCa is -log[Ca2+]) due to lowering pH from 7.00 to 6.20 was 1.27 +/- 0.03 (n = 7) pCa units in control cardiac TnC (cTnC) expressing myocytes and 0.96 +/- 0.04 (n = 11) pCa unit in transgenic cardiac myocytes (P < 0.001). The effect of pH to alter maximum Ca(2+)-activated tension was unchanged by TnC isoforms in these cardiac myocytes. In a reciprocal experiment, contractile sensitivity to acidosis was increased in fast skeletal muscle fibers following extraction of endogenous sTnC and reconstitution with purified cTnC in vitro. Our findings demonstrate that TnC plays an important role in determining the profound sensitivity of cardiac muscle to acidosis and identify cTnC as a target for therapeutic interventions designed to modify ischemia-induced myocardial contractile dysfunction.

Acidosis↗

PubMed1992

Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle.

We investigated the mechanism of the Ca2+ sensitivity of cross-bridge transitions that limit the rate of force development in vertebrate skeletal muscle. The rate of force development increases with Ca2+ concentration in the physiological range. We show here that at low concentrations of Ca2+ the rate of force development increases after partial extraction of the 20-kD light chain 2 subunit of myosin, whereas reconstitution with light chain 2 fully restores native sensitivity to Ca2+ in skinned single skeletal fibers. Furthermore, elevated free Mg2+ concentration reduces Ca2+ sensitivity, an effect that is reversed by extraction of the light chain but not by disruption of thin-filament activation by partial removal of troponin C, the Ca2+ binding protein of the thin filament. Our findings indicate that the Ca2+ sensitivity of the rate of force development in vertebrate skeletal muscle is mediated in part by the light chain 2 subunit of the myosin cross-bridge.

Animals↗

PubMed1991

Kinetics of a Ca(2+)-sensitive cross-bridge state transition in skeletal muscle fibers. Effects due to variations in thin filament activation by extraction of troponin C.

The rate constant of tension redevelopment (ktr; 1986. Proc. Natl. Acad. Sci. USA. 83:3542-3546) was determined at various levels of thin filament activation in skinned single fibers from mammalian fast twitch muscles. Activation was altered by (a) varying the concentration of free Ca2+ in the activating solution, or (b) extracting various amounts of troponin C (TnC) from whole troponin complexes while keeping the concentration of Ca2+ constant. TnC was extracted by bathing the fiber in a solution containing 5 mM EDTA, 10 mM HEPES, and 0.5 mM trifluoperazine dihydrochloride. Partial extraction of TnC resulted in a decrease in the Ca2+ sensitivity of isometric tension, presumably due to disruption of near-neighbor molecular cooperativity between functional groups (i.e., seven actin monomers plus associated troponin and tropomyosin) within the thin filament. Altering the level of thin filament activation by partial extraction of TnC while keeping Ca2+ concentration constant tested whether the Ca2+ sensitivity of ktr results from a direct effect of Ca2+ on cross-bridge state transitions or, alternatively, an indirect effect of Ca2+ on these transitions due to varying extents of thin filament activation. Results showed that the ktr-pCa relation was unaffected by partial extraction of TnC, while steady-state isometric tension exhibited the expected reduction in Ca2+ sensitivity. This finding provides evidence for a direct effect of Ca2+ on an apparent rate constant that limits the formation of force-bearing cross-bridge states in muscle fibers. Further, the kinetics of this transition are unaffected by disruption of near-neighbor thin filament cooperativity subsequent to extraction of TnC. Finally, the results support the idea that the steepness of the steady-state isometric tension-calcium relationship is at least in part due to mechanisms involving molecular cooperativity among thin filament regulatory proteins.

Actin Cytoskeleton↗

PubMed1991

Financial and operational impact of a transfer center.

A tertiary care hospital established a transfer center to manage the large number of transfer requests from other hospitals in its catchment region. Motivating factors included occurrence of economic transfers, medically inappropriate transfers, poor interfacility communication, and a bed shortage. An interdisciplinary task force was convened to design and implement the transfer center, requiring two months from conception to implementation. Non-recurring direct costs were $3000. At the time of implementation, written transfer agreements were promoted and signed with many transferring hospitals. A retrospective audit covering 28 weeks of operation indicates that 1141 inpatient days were avoided for an average of 21.5 days per back-transferred patient. (Back-transfer refers to the return of a patient to the original hospital.) An additional 140 admissions were possible as a result of these avoided days. Approximately $550,000 in variable costs were saved on an annualized basis. Moreover, substantial improvements in communications and interfacility cooperation were realized. No cases of inappropriate denials or delays in transfer were identified. We conclude that a transfer center can be a highly cost-effective mechanism to manage the large number of patients transferred to a tertiary care center.

Admitting Department, Hospital↗

PubMed1990

Calcium-sensitive cross-bridge transitions in mammalian fast and slow skeletal muscle fibers.

The fundamental mechanism underlying the differing rates of tension development in fast and slow mammalian skeletal muscle is still unknown. Now, in skinned (membrane-permeabilized) single fibers it has been shown that the rate of formation of the strongly bound, force-producing cross-bridge between actin and myosin is calcium-sensitive in both fast and slow fibers and that the rate is markedly greater in fast fibers. The transition rates obtained at high calcium concentrations correlated with myosin isoform content, whereas at low calcium concentrations the thin filament regulatory proteins appeared to modulate the rate of tension development, especially in fast fibers. Fiber type-dependent differences in rates of cross-bridge transitions may account for the characteristic rates of tension development in mammalian fast and slow skeletal muscles.

Actins↗

PubMed1990

Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers.

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO4(2-) as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC2 resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca2+] by as much as 0.2 pCa units after LC2 extraction. At low concentrations of Ca2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC2 to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC2. LC2 extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.

Animals↗

PubMed1990

Effects of tension and stiffness due to reduced pH in mammalian fast- and slow-twitch skinned skeletal muscle fibres.

1. The pH dependence of the Ca2+ sensitivities of isometric tension and stiffness was investigated at 10 and 15 degrees C in skinned single fibres from rat and rabbit fast- and slow-twitch skeletal muscles. Stiffness was determined by recording the tension responses to sinusoidal length changes (3.3 kHz); peak-to-peak amplitude of the length change was monitored by laser diffraction and averaged approximately 1.3 nm (half-sarcomere)-1. We have assumed that stiffness provides a measure of the number of cross-bridge attachments to actin. 2. At maximal Ca2+ activation, stiffness was depressed by 22 +/- 2% (mean +/- S.E.M.) in fast-twitch fibres but was unchanged in slow-twitch fibres when pH was lowered from 7.00 to 6.20. As reported previously, maximum tension was depressed by 20-45% at low pH, with the effect being greater in fast-twitch compared to slow-twitch fibres. 3. In fast-twitch fibres at 10 and 15 degrees C the Ca2+ concentrations for half-maximal activation of tension and stiffness were increased at low pH. In slow-twitch fibres, similar effects were observed at 15 degrees C, but at 10 degrees C there were no changes in the Ca2+ sensitivities of tension and stiffness when pH was lowered. 4. Linear relationships between relative tension and relative stiffness were obtained at all temperatures, with slopes of 1.04 +/- 0.01 at pH 7.00 and 0.76 +/- 0.01 at pH 6.20 in fast- and slow-twitch fibres, indicating that force per cross-bridge attachment is similarly reduced at low pH in both types of fibres. 5. In both fast- and slow-twitch fibres, rigor tension (no added ATP or creatine phosphate; pCa 9.0) was depressed by 35 +/- 7% and stiffness by 12 +/- 4% when pH was reduced from 7.00 to 6.20. Since cross-bridge cycling is inhibited in rigor the effect of low pH to depress rigor tension suggests that pH directly modulates the strength of the bond formed between actin and myosin. 6. The effect of low pH to reduce the apparent number of cross-bridge attachments during maximal Ca2+ activation in fast- but not slow-twitch fibres could account for the greater H(+)-induced depression of maximum force in fast-twitch fibres. In both fibre types, the decrease in the number of cross-bridge attachments at submaximal concentrations of Ca2+ may in part account for the loss in Ca2+ sensitivity of tension at low pH, due perhaps to a reduction in co-operative activation of the thin filament by bound cross-bridges.

Animals↗

PubMed1990

pH modulation of the kinetics of a Ca2(+)-sensitive cross-bridge state transition in mammalian single skeletal muscle fibres.

1. The rate constant of tension redevelopment (ktr) following a rapid release and subsequent re-extension of muscle length has been demonstrated to be Ca2+ sensitive and is thought to reflect the rate-limiting step in the cross-bridge cycle leading to the formation of the strongly bound, force-bearing state. The kinetics of this cross-bridge state transition were investigated at 15 degrees C over a wide range of Ca2+ concentrations while varying pH from 7.00 to 6.20 in rat slow-twitch soleus, rat fast-twitch superficial vastus lateralis (SVL) and rabbit fast-twitch psoas skinned single fibres. 2. At maximal levels of Ca2+ activation, ktr was unaffected by changes in pH from 7.00 to 6.20 while isometric tension was depressed to 0.60 +/- 0.02 P0 (mean +/- S.E.M.) at the low pH in fast-twitch fibres and to 0.78 +/- 0.01 P0 in slow-twitch fibres (P0 is the maximum isometric tension obtained at pH 7.00). 3. At reduced levels of Ca2+ activation, corresponding to pCa (-log [Ca2+]) greater than 5.0, ktr was markedly depressed in all fibre types when pH was lowered. The Ca2+ sensitivity of steady-state isometric tension was also reduced in all fibres at pH 6.20 compared to pH 7.00. 4. The results suggest that pH has a modulatory effect upon an apparent rate constant which is rate limiting in terms of the formation of the strongly bound, force-bearing cross-bridge state. This effect of altered pH may in part account for the reduction in the Ca2+ sensitivity of isometric force at low pH as well as the depression of the rate of rise of tension in living fibres during fatiguing stimulation.

Animals↗

PubMed1989

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle.

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Depression of Ca2+ insensitive tension due to reduced pH in partially troponin-extracted skinned skeletal muscle fibers.

Previous studies on skinned muscle fibers have demonstrated a direct effect of elevated levels of H+ ion to depress force production; however, the molecular basis for this effect is presently unknown. Here, whole troponin complexes were removed from skinned single fiber preparations of rat slow-twitch and fast-twitch muscles, and the effect of H+ ions on the resultant Ca2+-insensitive force was examined. The effect of H+ ions to depress force was found to be virtually identical in untreated control fibers activated in the presence of Ca2+ and in fibers activated in the absence of Ca2+ by troponin removal. Thus, the effect of H+ ions to depress force occurs at a step in activation beyond the disinhibition of the thin filament by Ca2+, probably involving reductions in the number of attached cross-bridges or in the force per attachment.

Animals↗

PubMed1988

Thin filament regulation of shortening velocity in rat skinned skeletal muscle: effects of osmotic compression.

1. Maximum shortening velocity (Vmax) was examined in skinned single fibres from rat slow-twitch soleus muscles at various degrees of Ca2+ activation of the thin filament, in the presence and absence of osmotic compression induced by 5% dextran. 2. At maximal levels of Ca2+ activation, Vmax remained constant as the extent of shortening was varied, with values averaging 1.43 +/- 0.05 muscle lengths/s (mean + S.E.M., n = 13). When thin filament activation was reduced by lowering the concentration of Ca2+, unloaded shortening consisted of an initial high-velocity phase for extents of shortening in the range 20-80 nm/half-sarcomere, and a subsequent low-velocity phase for greater extents of shortening. 3. In the absence of dextran, Vmax in the high-velocity phase of shortening was relatively invariant over a wide range of activation; however, at very low levels of activation, yielding tensions less than 5% of the peak value. Vmax declined precipitously. In contrast, fibres compressed radially with dextran to diameters comparable to those of intact fibres demonstrated a marked decrease in Vmax in the high-velocity phase when thin filament activation was varied over a wide range. These findings are consistent with the idea that cross-bridges in the expanded filament lattice of skinned fibres do not bear as great an axial compressive force as in intact fibres (Goldman & Simmons, 1986; Goldman, 1987) but instead buckle as the fibre shortens. 4. The value of Vmax in the low-velocity phase of shortening decreased as thin filament activation was reduced in both control and osmotically compressed fibres. The low-velocity phase, which occurred only at reduced levels of thin filament activation, may be a manifestation of a population of slowly dissociating crossbridges which with shortening become negatively strained and oppose contraction.

Animals↗