Biomedical subjects

R L Moss

Publications and source records attributed to R L Moss.

At least 127 records · Page 7Linked to original sources

PubMed1993

Altered calcium sensitivity of isometric tension in myocyte-sized preparations of porcine postischemic stunned myocardium.

Postischemic ventricular myocardial dysfunction, termed stunning, is characterized by a persistent but ultimately reversible depression of contractile function. The present study was undertaken to investigate the possibilities that reduced contractile force in stunning is due to a decrease in maximal tension-generating capability or to a decrease in the Ca2+ sensitivity of the myofilaments. The experiments combine an in vivo open-chest porcine heart model of stunning (n = 5) with in vitro measures of myocyte myofilament calcium sensitivity from these same hearts. Regional myocardial function in the left anterior descending coronary artery (LAD) perfusion bed of porcine hearts was measured with transmural ultrasonic crystals. The protocol was 45 minutes of low-flow LAD ischemia at 40% of control flow, followed by 30 minutes of postischemic reperfusion at control aerobic flow. Percent systolic wall thickening decreased to 8 +/- 5% of control during ischemia (p < 0.05) and returned to 38 +/- 8% of control in the postischemic stunned state (p < 0.05). Serial endocardial biopsies were obtained from the preischemic and postischemic myocardium in the LAD perfusion bed and from the aerobically perfused myocardium in the circumflex bed. The biopsies were mechanically disrupted, and myocyte-sized preparations of permeabilized myocardium were attached to a force transducer and a length-changing device to allow for direct measurement of steady-state tension-pCa (i.e., -log[Ca2+]) relations. The pCa for half-maximal activation of tension, i.e., pCa50, in LAD myocardium decreased from 5.88 +/- 0.05 before ischemia to 5.69 +/- 0.03 after ischemia (p < 0.05); however, maximal Ca(2+)-activated tension and the slope of the tension-pCa relation were unaffected by the ischemic episode.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1993

Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with alpha-hemolysin.

Force-velocity relations were obtained from single cardiac myocytes isolated by enzymatic digestion of rat myocardium and permeabilized with the pore-forming staphylococcal toxin alpha-hemolysin. Single cardiac myocytes were attached to a force transducer and piezoelectric translator and viewed with an inverted microscope to allow periodic monitoring of sarcomere length during experiments. Permeabilized cells were activated by immersion in a bath of known [Ca2+]. We report that the Ca2+ sensitivity of cells obtained by enzymatic digestion and permeabilized using alpha-hemolysin is similar to that reported previously for mechanically disrupted ventricular myocardium; however, the tension-pCa relation is less steep in the new preparation. During isotonic measurements, force was clamped to various loads using a rapid-response servo system. All recordings of shortening under load were distinctly curvilinear, and analysis of data involved fitting each shortening recording with a single exponential curve and calculating the value of the slope at the initial time of the load clamp. In addition, the presence of significant resting force at initial sarcomere lengths in these cells required that the possibility of alteration of velocity due to the presence of resting force be addressed. The maximum shortening velocity in fully Ca(2+)-activated single ventricular myocytes studied by this method was 2.83 muscle lengths per second on average. The basis for curvilinear shortening is postulated to be multifactorial in cardiac muscle, involving a combination of shortening inactivation and one or more passive elasticities that resist stretch or compression depending on sarcomere length. Shortening velocity shows a dependence on myosin isoform content when cells from a single heart are compared; however, this relation does not hold when cells from different hearts are compared. The behavior of single alpha-hemolysin-permeabilized myocyte shortening under loaded conditions at lower levels of Ca2+ is also described. During submaximal Ca2+ activation, initial shortening velocities are faster than those observed in maximally activated cells. This may be due to contributions of high passive force to increase shortening velocity under conditions of low active force generation, when passive force in the cell is a greater proportion of the total force and there are fewer bound crossbridges.

Animals↗

PubMed1992

Influence of a strong-binding myosin analogue on calcium-sensitive mechanical properties of skinned skeletal muscle fibers.

The ability of strong-binding myosin heads to activate the thin filament was investigated by incubating skinned single muscle fibers with N-ethylmaleimide-(NEM) modified myosin subfragment-1 (S1). Isometric force was influenced in a complex manner: during maximal calcium activation, NEM-S1 inhibited force with half-maximal inhibition at 20 microM while at submaximal calcium, NEM-S1 potentiated force with greatest effect at 6 microM. When fibers were treated with NEM-S1 (4-8 microM), the tension-pCa (-log [Ca2+]) relationship became less steep (i.e. the Hill coefficient decreased from 5.4 to 3.0 upon treatment with NEM-S1), but the midpoint was unchanged. These results support the idea that strong binding of intrinsic heads contributes to the cooperativity observed in Ca2+ activation of force. The NEM-S1-induced increase in force at low Ca2+ was associated with an acceleration of a kinetic transition, and this transition was activated to near maximum while force was not. The rate of force redevelopment following restretch (ktr) at submaximal calcium was increased by NEM-S1 in a concentration-dependent manner, yielding a maximum rate at low [Ca2+] which was similar to that observed during full activation. The effects of NEM-S1 on force and ktr indicate that strong-binding myosin cross-bridges are involved in activation of the thin filament.

Animals↗

PubMed1992

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.

Animals↗

PubMed1992

Modulation of single-unit activity in the rat medial amygdala by neurotransmitters, estrogen priming, and synaptic inputs from the hypothalamus and midbrain.

The medial amygdala (m-AMG) appears to act as an integrative center for sensory, synaptic, and endocrine signals important in the regulation of reproductive function. Extracellular single-unit recordings from anesthetized, ovariectomized female rats were used to investigate neuropharmacological, hormonal, and synaptic modulation of neurons in the m-AMG. Electrical stimulation of the ventromedial hypothalamus (VMH) elicited excitatory or inhibitory orthodromic responses in 72% and antidromic responses in 7% of m-AMG neurons, whereas stimulation of the midbrain central gray (MCG) induced orthodromic responses in 43% of m-AMG neurons. Interestingly, most cells that were influenced by MCG stimulation were also orthodromically driven by the VMH, as 40% of all m-AMG cells responded orthodromically to both the VMH and MCG. Furthermore, the majority of these cells tended to be modulated by both areas in the same direction. Iontophoretic application of glutamate, GABA, ACh, and LHRH could modulate the spontaneous firing rate of m-AMG neurons. In particular, ACh had a predominantly excitatory action, which was more effective on m-AMG neurons that were orthodromically driven by the VMH and that were from estrogen-primed animals. In addition to increasing chemical responsiveness to ACh, estrogen priming of ovariectomized animals also increased the spontaneous firing rate of m-AMG neurons and decreased the number of silent cells. These modulatory actions on m-AMG neurons may be important in the medial amygdala's regulation of the behavioral and endocrine aspects of reproductive function in the female rat.

Amygdala↗

PubMed1992

LHRH neurons in the medial septal-diagonal band-preoptic area do not project directly to the hippocampus: a double-labeling immunohistochemical study.

While neurons containing immunoreactive luteinizing hormone-releasing hormone (LHRH) are scattered primarily in the medial septal-diagonal band of Broca-medial preoptic area (mS-dbB-PO) complex, autoradiographic studies have demonstrated dense concentrations of LHRH receptors in the hippocampus. The route by which LHRH is transported to its hippocampal receptors is unknown. The present study was designed to test the hypothesis that LHRH-containing neurons in the mS-dbB-PO complex project to hippocampal sites containing LHRH receptors, thereby serving as a source of innervation to these receptors. Large (0.10 microliters) or small (0.02 microliters) volumes of the retrograde tracer wheat germ agglutinin (WGA) were injected unilaterally into four separate hippocampal locations in six ovariectomized female rats. In an additional five females, a 0.15 microliter volume of the retrograde tracer fluorogold (FG) was similarly injected. After a five day survival period, the animals were sacrificed. Vibratome sections of the brain were stained for both WGA and LHRH with a dual immunohistochemical technique. Since FG is a fluorescent chromagen, brains of animals injected with FG only required processing for LHRH immunofluorescence. As a positive control, some sections containing retrogradely labeled cells filled with either WGA or FG were processed for choline acetyltransferase (CHAT) immunoreactivity. The WGA and FG injections covered targeted hippocampal sites and neurons containing retrogradely transported WGA or FG were found in abundance in the mS-dbB-PO complex. In accord with previous reports, many CHAT-positive and fewer LHRH-positive neurons were found in this complex. Approximately 5-10% of the CHAT-positive neurons also contained WGA or FG; however, no neurons were found to co-localize LHRH and either of the retrograde tracers. The results indicate that LHRH neurons in the mS-dbB-PO complex do not project directly to hippocampal sites containing LHRH receptors.

Animals↗

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↗

PubMed1992

Developmental changes in troponin T isoform expression and tension production in chicken single skeletal muscle fibres.

1. The Ca2+ sensitivity of tension development was characterized in single skinned fibres from the slow anterior latissimus dorsi (ALD), fast posterior latissimus dorsi (PLD), and fast pectoralis major (PM) muscles of the chicken at adult and neonatal (2 weeks post-hatch) stages of development. In the adult, the PM was most sensitive, the ALD intermediate, and the PLD least sensitive to Ca2+. 2. PM and PLD fibres were less sensitive to Ca2+ at the neonatal stage of development than in the adult. However, ALD fibres exhibited no age-dependent changes in Ca2+ sensitivity. 3. Characterization of regulatory protein composition indicated that the PM and PLD fibres had identical fast isoforms of troponin C and troponin I at each developmental stage examined, but there were muscle-specific and age-dependent expressions of troponin T isoforms in these fibres. 4. In the ALD fibres, identical slow isoforms of troponin C, troponin I and tropomyosin were found at each stage. In addition, the troponin T isoform that was present did not change with age. 5. The results suggest a relationship between the specific troponin T isoform composition of individual muscle fibres and their calcium sensitivities of tension development.

Aging↗

PubMed1992

Ca2+ regulation of mechanical properties of striated muscle. Mechanistic studies using extraction and replacement of regulatory proteins.

Extraction of regulatory proteins from thick and thin filaments of vertebrate striated muscle has proven to be an important approach in elucidating roles of these proteins in regulating contraction and in probing specific mechanisms of activation. For some proteins, such as LC2 and C protein, extraction has been fundamental in demonstrating the importance of these proteins in modulating contraction and the kinetics of cross-bridge interaction. For other proteins, such as TnC and troponin, extraction has provided significant insight into the importance of thin-filament intermolecular cooperativity in modulating Ca2+ sensitivity of the contractile process. A combination of extraction and readdition has provided a means of introducing mutated or derivatized proteins into fibers to accomplish a variety of experimental objectives. The use of this approach is likely to grow with the need to test the functional consequences of site-specific mutations as part of studies directed to mechanisms of regulation or altered regulation in heart and skeletal muscles under normal and pathophysiological conditions. Such studies are likely to include extraction in combination with other probes of function such as flash photolysis of reaction substrates or products within the cross-bridge interaction cycle. Although extraction is a powerful approach and is likely to be extended to proteins not discussed in this review, an essential element of experimental design in studies such as these is that appropriate control experiments be done to verify that observed effects of the extraction protocol are specifically attributable to the protein that is removed.

Animals↗

PubMed1992

Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat.

Effects of [Ca2+] on isometric tension and unloaded shortening velocity were characterized in single chemically skinned myocytes from frog atrium and in mechanically disrupted myocardium from rat ventricle. The preparations were attached to a force transducer and piezoelectric translator and were viewed with an inverted microscope to allow continuous monitoring of sarcomere length during mechanical measurements. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of each preparation. Ca2+ sensitivity of isometric tension was assessed as pCa50, i.e., the Ca2+ concentration at which tension was 50% maximal, and was greater for frog atrial myocytes (pCa50 6.17) than for rat ventricular myocytes (pCa50 6.06). This difference in Ca2+ sensitivity may be due to variations in myofibrillar protein isoform composition in the two preparations. Inclusion of caffeine in the activating solutions substantially increased the Ca2+ sensitivity of tension, which may be a manifestation of a direct effect of caffeine on the myofibrillar proteins. Unloaded shortening velocity during maximal activation averaged 4.32 muscle lengths per second in frog atrial myocytes and 4.46 muscle lengths per second in rat ventricular myocytes. When [Ca2+] was reduced, unloaded shortening velocity decreased substantially in both preparations. Possible mechanisms for the effect of Ca2+ on shortening velocity in myocardium include Ca2+ dependence of the rate of ADP dissociation from actomyosin complexes or a shortening-dependent internal load involving structures such as C protein or long-lived myosin cross-bridges.

Animals↗

PubMed1992

Long-term and short-term electrophysiological effects of estrogen on the synaptic properties of hippocampal CA1 neurons.

The ovarian steroids exert both long-term and short-term actions on neurons involving different cellular mechanisms. We have investigated the long-term and short-term effects of estrogen on the electrophysiological properties of CA1 neurons utilizing intracellular recordings in hippocampal slices prepared from ovariectomized female rats. An in vivo estrogen-priming paradigm was used to examine long-term genomic actions of estrogen. Subcutaneous estrogen injections 2 d prior to recording had no effect on the intrinsic membrane properties of CA1 neurons, but increased synaptic excitability by prolonging the EPSP and inducing repetitive firing in response to Schaffer collateral stimulation. Short-term effects of estrogen that presumedly involve direct membrane interactions were tested by application of steroids directly to the slice. Superfusion of 17 beta-estradiol, but not 17 alpha-estradiol, caused a rapid and reversible increase in the amplitude of the Schaffer collateral-activated EPSP. This potentiation of the EPSP by 17 beta-estradiol still occurred in the presence of the NMDA antagonist 2-amino-5-phosphonovalerate, but was blocked by the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Depolarizing responses to iontophoretic pulses of exogenous glutamate were also potentiated by 17 beta-estradiol, suggesting a post-synaptic site of action. In addition, 17 beta-estradiol potentiated the responses to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate, and quisqualate, but not NMDA, further implicating non-NMDA receptors in the short-term action of estrogen. In contrast, 17 beta-estradiol had no effect on responses to exogenous GABA or on the Schaffer collateral-induced late IPSP.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1991

Electrophysiological evidence for a rapid membrane action of the gonadal steroid, 17 beta-estradiol, on CA1 pyramidal neurons of the rat hippocampus.

The rapid electrophysiological effects of 17 beta-estradiol on CA1 pyramidal neurons (n = 86) were investigated utilizing intracellular recording from the rat hippocampal slice preparation. Bath application of 17 beta-estradiol, but not 17 alpha-estradiol, caused a reversible depolarization and increased input resistance with a latency of less than 1 min in 19.8% of CA1 neurons tested. There was no significant difference in the percentage of estradiol-responsive cells between male and female rats. Estradiol-responsive cells were identified from prepubertal female rats, as well as females in all stages of the estrous cycle. 17 beta-estradiol had no effect on the slow afterhyperpolarization or accommodative properties of CA1 neurons. In 2 out of 4 cells tested, the specific antiestrogen, tamoxifen, blocked the excitatory response to 17 beta-estradiol.

Animals↗

PubMed1991

Inguinal hernia repair in early infancy.

Controversy regarding the management of inguinal hernias in young infants stimulated this study. It was our hypothesis that inguinal hernia in young infants can and should be repaired at the time of presentation. In order to address this issue, the following questions were examined: How frequently are these hernias incarcerated and how should this be managed? What is the best timing of repair? Should the asymptomatic contralateral groin be explored? How do the recurrence and complication rates compare with those in older children? Have advances in anesthesia affected the treatment of these infants? The records of 384 infants less than 2 months of age who underwent inguinal herniorrhaphy from January 1985 to January 1990 at Children's Hospital and Medical Center in Seattle were reviewed. Nearly one fourth (24%) of the patients had incarcerated hernias. Preoperative reduction was successful 96% of the time. The hernia was then repaired within 48 hours. Six patients required urgent operation, but bowel resection was not necessary in any. Contralateral groin exploration was performed 96% of the time and was positive in 85%. The recurrence rate was 1.0%. Complications occurred in 2.3% of patients. There were no hernia-related deaths. Spinal anesthesia was used in 63 patients with no postoperative apnea. An aggressive approach toward prompt repair of hernias in this age group can result in a very low incidence of hernia-related complications. Repair can be carried out safely in the first 2 months of life with recurrence and complication rates comparable with those in older children. Spinal anesthesia may lower the rate of postoperative apnea.

Age Factors↗

PubMed1991

Role of the ventromedial nucleus of hypothalamus in the male-induced enhancement of lordosis in female rats.

The involvement of the ventromedial nucleus of the hypothalamus (VMH) in the mating-induced enhancement of lordosis in ovariectomized estrogen-primed rats was investigated. In the first experiment, females with bilateral VMH or sham lesions were primed with 2 micrograms estradiol benzoate, and 48 h later they were subjected to repeated-mating tests. The VMH-lesioned rats failed to exhibit lordosis during the tests; however, the sham-operated females exhibited a gradual increase in lordosis quotient (LQ) with repetitive matings. In the second experiment, ovariectomized females were bilaterally implanted with estradiol (E2) or cholesterol (C) in the VMH, 48 h prior to behavioral testing. Repeated-mating-induced elevation in LQ was observed in the females when they were bilaterally implanted with E2 in the VMH; C was ineffective. To exclude the possibility of the spread of E2 to areas adjacent to VMH, plasma-luteinizing hormone (LH) was measured. Elevation in the circulating LH levels following ovariectomy was not suppressed in the females following bilateral E2 implants in the VMH, suggesting that the effect of estrogen is localized within or immediately around the VMH. The results suggest that the integrity of the VMH is critical for the potentiation of lordosis behavior in ovariectomized estrogen-primed females by male-originating sensory cues, and that selective priming of the VMH with estrogen is sufficient for the male-induced enhancement of lordosis.

Animals↗

PubMed1991

Facilitation of sexual receptivity in the female rat by C-terminal fragments of LHRH.

Ovariectomized female rats implanted with cannulae directed bilaterally towards the ventromedial nucleus of the hypothalamus (VMH) were primed with estrogen (2 micrograms) and tested for sexual receptivity before and after infusion of the C-terminal LHRH fragment, Ac-LHRH5-10. Animals responsive to the Ac-LHRH5-10 fragment were tested at two-week intervals for responsiveness to other LHRH fragments modified in the C-terminal or saline in the following order: LHRH5-10, des-Gly6LHRH, LHRH5-9OH, saline, and LHRH7-9OH. The lordosis-to-mount (L/M) ratio was used as an index of sexual receptivity. The quality of lordotic posturing and the number of proceptive and resistive behaviors were also recorded for each test. A one-way ANOVA performed on the difference in L/M between pre- and postinfusion tests revealed a significant overall treatment effect. The nonacetylated LHRH5-10 fragment and the LHRH5-9OH fragment were effective in enhancing the L/M ratio, whereas des-Gly6LHRH and LHRH7-9OH were not. The enhancement of sexual behavior by C-terminal fragments of LHRH was associated with a lack of proceptive behavior and moderate levels of resistive behavior. The results indicate that several C-terminal fragments of LHRH are capable of elevating the L/M ratio and suggest that amino acids in positions 6 through 9 may be important for this effect.

Amino Acid Sequence↗

PubMed1991

Alterations in Ca2+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers.

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.

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

C-protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation.

1. Effects on maximum shortening velocity (Vmax) due to partial extraction of C-protein were investigated in skinned fibres from rabbit psoas muscles. Up to 80% of endogenous C-protein was extracted, as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of fibre segments obtained before and after the extraction protocol. Vmax was obtained at 15 degrees C by measuring the times required to take up various amounts of slack imposed at one end of the fibre. 2. During maximal activation with Ca2+, Vmax in control fibres was 4.26 +/- 0.16 (mean +/- S.E.M., n = 7) muscle lengths per second (ML/s). Following extraction of approximately 40% of endogenous C-protein, Vmax in these same fibres was 4.41 +/- 0.24 ML/s. 3. At sufficiently low levels of submaximal activation, high- and low-velocity phases of unloaded shortening were observed. Partial extraction of C-protein significantly increased Vmax in the low-velocity phase but had no effect on the high-velocity phase. The effect on low-velocity Vmax was fully reversed by re-addition of purified C-protein. 4. At low levels of activation, the amount of shortening to the break-point between the high- and low-velocity phases was not significantly affected by C-protein extraction. Under control conditions the average break-point was 85.6 +/- 3.1 nm/half-sarcomere, while 84.1 +/- 3.1 nm/half-sarcomere was obtained following partial extraction of C-protein. 5. These results are considered in terms of a model in which an internal load slows Vmax at low levels of activation once a given amount of active shortening has occurred. C-protein may contribute to this internal load either by binding to actin and myosin or by influencing mechanical properties of myosin cross-bridges.

Animals↗