Sensitivity Of Tension Development Research Articles

Functional Significance of C-Terminal Mobile Domain of Cardiac Troponin I

C-terminal mobile domain of cTnI (cTnI-MD, residues 168-210) is a highly conserved region which plays important roles in regulating myofilament function. Due to the unresolved structure of the region, it is still unclear how cTnI-MD plays a role in modulating the sarcomere length (SL)-dependent and Ca2+-induced effects on cardiac contractility. In this study, the functional significance of the MD in contractile behavior of skinned rat papillary muscle fibers and SL dependent activation was studied by truncation of the entire MD (cTnI(1-167)) and the C-terminus of MD (cTnI(1-193)). We hypothesized that different truncations may have different impacts on SL-dependent Ca2+-regulation of tension development and cross-bridge cycling. To test this hypothesis, the tension-pCa relationships and myosin cross-bridge kinetics were measured as cTnI(1-167) and cTnI(1-193) were exchanged into skinned rat papillary muscle fibers at short (SL=1.9µm) and long SLs (SL=2.2µm). Our results indicate that the SL induced enhancement of Ca2+-sensitivity and cooperativity observed in wild-type fibers is preserved in cTnI(1-193) and is diminished in cTnI(1-167) fibers. cTnI(1-167) truncation resulted in enhanced maximum tension and cooperativity of Ca2+-activation compared to cTnI(WT) and cTnI(1-193) and enhanced Ca2+-sensitivity compared to cTnI(WT) at both short and long SLs. cTnI(1-193) truncation resulted in enhanced myofilament Ca2+-sensitivity at both SLs and a depressed maximum tension just at long SL. Both cTnI-MD truncations showed a faster cross-bridge attachment rate at both SLs. Compared to cTnI(WT), cTnI(1-167) showed a slower cross-bridge detachment rate while cTnI(1-193) showed a faster cross-bridge detachment rate at long SL. The results of our study indicate that the truncation of cTnI-MD at different regions causes destabilization of the closed-state position of tropomyosin suggested by the observed increase in the cross-bridge attachment rate to thin filament as well as Ca2+-sensitivity of tension development at both sarcomere lengths.

Chronic Treatment with Carvedilol Improves Ca2+-Dependent ATP Consumption in Triton X-Skinned Fiber Preparations of Human Myocardium

Evidence is given that beta-blocker treatment differentially influences gene expression and up-regulation of beta(1)-adrenoceptors in human myocardium. Here, we investigate whether long-term treatment with carvedilol or metoprolol may functionally alter myofibrillar function in end-stage human heart failure. Investigations were performed in Triton X (1%, 4 degrees C, 20 h)-skinned fiber preparations of explanted hearts from patients undergoing heart transplantation due to idiopathic dilative cardiomyopathy. Five patients were not on beta-adrenoceptor blocker treatment (DCM_NBB), and 5 patients received either carvedilol (DCM_CAR) or metoprolol (DCM_MET). Nonfailing (NF) donor hearts (n = 5), which could not be transplanted due to technical reasons, were investigated for comparison. Ca(2+)-dependent tension (DT) development and actomyosin-ATPase activity (MYO) were measured and tension-dependent ATP consumption was calculated by the ratio of DT and MYO ("tension cost"). In addition, we measured the phosphorylation of troponin I (TNI) by back phosphorylation. Maximal DT and TNI phosphorylation were reduced, with myofibrillar Ca(2+) sensitivity of DT and MYO as well as tension cost being increased in DCM_NBB compared with NF. Metoprolol treatment restored TNI phosphorylation, decreased Ca(2+) sensitivity of tension development and of myosin-ATPase activity, but did not alter the tension-dependent ATP consumption. Carvedilol treatment improved maximal DT and significantly decreased tension-dependent ATP consumption without altering myofibrillar Ca(2+) sensitivity. TNI dephosphorylation was increased in patients treated with carvedilol. In conclusion, chronic beta-adrenoceptor blockade functionally alters myofibrillar function. The more economic cross-bridge cycling in patients under carvedilol treatment may provide an explanation for the efficacy of carvedilol in the treatment of chronic heart failure patients.

Protein Kinase D Is a Novel Mediator of Cardiac Troponin I Phosphorylation and Regulates Myofilament Function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.

Sarcomere Thin Filament Regulatory Isoforms: EVIDENCE OF A DOMINANT EFFECT OF SLOW SKELETAL TROPONIN I ON CARDIAC CONTRACTION

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.

Nebivolol, carvedilol and metoprolol do not influence cardiac Ca 2+ sensitivity

It has been argued that some β-adrenoceptor antagonists may directly influence myofibrillar cross-bridge interaction in cardiac skinned fiber preparations of animal models. The present study investigates the effects of nebivolol, metoprolol and carvedilol on tension development of Triton X-100 skinned fibers obtained from human failing myocardium as well as on force of contraction and intracellular Ca 2+ transient in isolated trabeculae. In skinned fiber preparations, none of the β-adrenoceptor antagonists (10 μM) influenced Ca 2+ sensitivity of tension development or maximal Ca 2+ activated tension (DT max): control: EC 50 for Ca 2+: 1.28±0.05 μM, DT max: 14.09±0.59 mN/mm 2; nebivolol: 1.36±0.1 μM, 14.14±0.95 mN/mm 2; carvedilol: 1.32±0.11 μM, 13.83±0.90 mN/mm 2; metoprolol: 1.34±0.14 μM, 13.72±0.36 mN/mm 2. Simultaneous measurement of force and Ca 2+ transient in the presence of the β-adrenoceptor antagonists (3 μM) showed that the decrease in force of contraction was paralleled by a similar decrease in the intracellular Ca 2+ transient. In conclusion, none of the investigated β-adrenoceptor antagonists influenced Ca 2+ sensitivity of myofibrillar tension development in human failing myocardium.

The enhanced contractility in phospholamban deficient mouse hearts is not associated with alterations in (Ca2+)-sensitivity or myosin ATPase-activity of the contractile proteins.

Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e.g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sensitivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 +/- 0.2 micromol/l; tension: 1.7 +/- 0.3 micromol/l; p < 0.001) and wild-type controls (1.1 +/- 0.01 micromol/l; 2.2 +/- 0.4 micromol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/ force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban.

Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes.

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform. We found a significant desensitization in the Ca2+ sensitivity of tension development in beta-MHC-expressing ventricular myocytes (pCa50=5. 51+/-0.03, where pCa is -log[Ca2+], and pCa50 is pCa at which tension is one-half maximal) compared with that in predominantly alpha-MHC-expressing myocytes (pCa50=5.68+/-0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca2+ activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in beta-MHC-expressing myocytes compared with the alpha-MHC-expressing cardiac myocytes (pCa50=5.47+/-0.05 versus 5.76+/-0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca2+ sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.

Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy.

Most studies indicate that failing human hearts have greater baseline myofibrillar Ca2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca2+, thus altering the Ca2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism. Using in vitro back-phosphorylation with [gamma-32P]ATP, we demonstrated a significant (P<0.05) approximately 25% reduction in baseline PKA-dependent TnI phosphorylation in human hearts with dilated cardiomyopathy (DCM) compared with nonfailing (NF) human hearts. There was no significant difference in cAMP content or maximal PKA activity between DCM and NF hearts, but expression of the regulatory subunits of PKA-I (RI) and PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by approximately 40%, P<0.05; RII by approximately 30%, P<0.01). PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the beta-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca2+ sensitivity.

Effects of constitutive overexpression of insulin-like growth factor-1 on the mechanical characteristics and molecular properties of ventricular myocytes.

Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca2+ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1 in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres.

The Ca2+ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca2+ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca2+ capacity and the rate of Ca2+ uptake decreased from the control values of 0.384 +/- 0.030 micromol (mg fibre protein)-1 and 19.8 +/- 1.9 nmol min-1 (mg fibre protein)-1, respectively, to 0.210 +/- 0.016 micromol (mg fibre protein)-1 and 13.5 +/- 0.9 nmol min-1 (mg fibre protein)-1; the calculated amount of Ca2+ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 micromol (mg fibre protein)-1. In the 7-day denervated muscle, the SR Ca2+ capacity and the rate of Ca2+ uptake increased to 0.517 +/- 0.06 micromol (mg fibre protein)-1 and 21.6 +/- 2.3 nmol min-1 (mg fibre protein)-1, respectively; the calculated amount of Ca2+ released increased to 0.217 micromol (mg fibre protein)-1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca2+ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation.

Overproduction and rapid purification of human fast skeletal β troponin T using Escherichia coli expression vectors: functional differences between the α and β isoforms

Troponin T (TpnT), an essential component of the Ca 2+-regulatory troponin complex, is involved in protein-protein interactions with other thin-filament proteins during muscle contraction in vertebrate striated muscle (VSM). The isoforms of TpnT are encoded by members of a multigene family which, by alternate splicing, produces a complex pattern of isoproteins in VSM. The functional domains of TpnT are only tentatively identified and structure-function analysis on this protein is limited due to the heterogeneity of the multiple isoforms. We reasoned that the overproduction and purification of a single TpnT species in Escherichia coli would provide an insight into these studies, besides being useful in crystallizing the protein. We cloned the human fast skeletal αTpnT-encoding cDNA (βTpnT f) in three expression vectors. Overexpression was achieved in an E. coli BL21(DE3) lysogen using a T7 RNA polymerase promoter-based vector, pET17b. The unfused recombinant protein was purified by a simple and rapid procedure in a biologically active and immunoreactive form. This is the first successful synthesis of a complete βTpnT f polypeptide from any species using an in vitro expression system. Purified human βTpnT f, a predominant fetal form, was less Ca 2+-sensitive and exhibited considerably reduced affinity for troponin C and tropomyosin, as compared to the rabbit fast skeletal αTpnT, a predominant adult isoform. These results provide a biochemical correlate to the age-related differences in Ca 2+ sensitivity of tension development in vertebrate fast skeletal muscles

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.

Variations in contractile properties of rabbit single muscle fibres in relation to troponin T isoforms and myosin light chains.

1. The maximal velocity of shortening (Vmax), tension-pCa relationships and the contractile and regulatory protein composition were determined in single, chemically skinned fibres from adult rabbit plantaris muscles. 2. Three groups of fibres were identified based on their protein compositions. One group had exclusively the slow-type myosin heavy chain (MHC) and myosin light chains (LC) and had low velocities. Another group of fibres had mixtures of fast-type and slow-type MHCs and LCs and had intermediate shortening velocities. The third group of fibres had fast-type myosin heavy and light chains and high velocities. 3. The low-velocity fibres had a mean velocity (+/- S.E.M.) of 0.86 +/- 0.03 muscle lengths/s (ML/s) at 15 degrees C. The remaining fibres formed a continuum with respect to Vmax from 1.37 to 3.94 ML/s. These results indicate that a much greater diversity exists among single fibres from adult mammalian skeletal muscle than previously recognized. The intermediate- and high-velocity fibres formed a continuum (from slow to fast) with respect to the amount of myosin light chain 3 (LC3). That is, Vmax increased with the relative LC3 content in single fibres in the intermediate- and high-velocity groups in a quantitative, statistically significant manner. 4. Three isoforms of fast-type troponin T were identified among the intermediate- and high-velocity fibres. These fibres also contained fast-type troponin C and troponin I. As was the case with the relative LC3 content, these fibres also formed a continuum with respect to the relative proportions of the three isoforms of fast-type troponin T. It appears that different isoforms of troponin T are responsible for a slightly higher Ca2+ sensitivity of tension development in the high-velocity fibres compared to the intermediate fibres. The continuum in troponin T isoform composition paralleled an increase in Vmax among these fibres. 5. The low-velocity fibres had the highest Ca2+ sensitivity of the three groups and had exclusively the slow-type isoforms of the regulatory proteins in the troponin complex. 6. The co-ordinated variations in troponin T and LC3 compositions among the intermediate- and high-velocity fibres are discussed as a possible means for the further differentiation of the contractile properties of the fibres in these two groups, beyond that provided by myosin heavy chain isoforms alone.

Greater hydrogen ion-induced depression of tension and velocity in skinned single fibres of rat fast than slow muscles.

1. The effects of variations in pH between 7.00 and 6.20 on Ca2+ -activated tension development and maximum velocity of shortening (Vmax) were examined in skinned single skeletal fibres from rat slow-twitch soleus and fast-twitch superficial (s.v.l.) and deep (d.v.l.) regions of the vastus lateralis muscle. 2. At pH 6.50, Vmax was depressed to a similar degree in each of the soleus, d.v.l., and s.v.l. fibres. Lowering pH to 6.20 resulted in a further decline in Vmax in all fibres; however, differences between the slow fibres, identified by SDS-polyacrylamide gel electrophoresis, and fast fibres were apparent, with soleus retaining a significantly greater proportion of its control Vmax (0.83 +/- 0.03 in soleus vs. 0.69 +/- 0.03 in s.v.l.; mean +/- S.E.M.). 3. Maximum force production decreased significantly as pH was reduced. Peak force at pH 6.50, relative to that at pH 7.00, was significantly greater in soleus (0.80 +/- 0.01) than in the s.v.l. (0.75 +/- 0.01) fibres. At pH 6.20 these differences between slow and fast fibres were still greater, in that soleus fibres generated significantly greater relative forces (0.73 +/- 0.01) than did d.v.l. (0.67 +/- 0.02) or s.v.l. (0.63 +/- 0.02) fibres. 4. As pH was lowered the tension-pCa relationship shifted to the right (i.e. to higher [Ca2+]), indicating a reduction in the Ca2+ sensitivity of tension development. The [Ca2+] necessary to achieve half-maximal tension in both the slow- and fast-twitch fibres increased approximately 5-fold when pH was lowered from 7.00 to 6.20. Furthermore, in the case of the soleus, the Ca2+ threshold for tension development was 45 times greater at pH 6.20 than at pH 7.00, while in the fast-twitch fibres, this increase was 4-fold. 5. Increased [H+] differentially affected the steepness of the tension-pCa relationship between slow and fast fibres. As pH was lowered, the steepness of the lower portion of the tension-pCa curve increased in the soleus and decreased in d.v.l. and s.v.l., suggesting that apparent positive co-operativity of tension development had increased in soleus and decreased in d.v.l. and s.v.l. fibres. 6. These results (1) demonstrate an increased resistance to H+ ion-mediated contractile dysfunction in slow- compared to fast-twitch single fibres, and (2) support the hypothesis that muscular fatigue resulting from short-term, intense muscular contraction may in part be related to elevated H+ ion concentration.

Factors influencing the ascending limb of the sarcomere length‐tension relationship in rabbit skinned muscle fibres.

1. The length dependence of Ca2+-activated tension within the ascending limb of the length-tension relationship, corresponding to sarcomere lengths below about 2.25 micron, was investigated in skinned fibres from rabbit psoas muscle. At high [Ca2+] a shallow phase and then a steep phase of tension decline were observed as sarcomere length was reduced, while at low [Ca2+] tension decreased monotonically with decreases in sarcomere length. The sarcomere length at which the ascending limb intersected zero tension was greater for lower concentrations of Ca2+. 2. The length tension relationship from maximally activated fibres changed when filament lattice spacing was reduced by osmotic compression. Relationships obtained in the presence of 5% (w/v) dextran T500 more distinctly demonstrated both the shallow and steep portions of the ascending limb than did relationships from untreated fibres. 3. As striation spacing was decreased a progressive decline in the Ca2+ sensitivity of tension development was observed. Tension-pCa relationships from both control and dextran-treated fibres underwent a rightward shift (i.e. to a higher [Ca2+]) by 0.23 pCa units as sarcomere length was reduced between 2.46 and 1.54 microns. 4. Fibre stiffness was studied by applying a 3.3 kHz sinusoidal length change at one end of the fibre and measuring the resultant tension change. At submaximal activation (pCa 5.8), stiffness increased relative to tension as sarcomere length was decreased below approximately 2.4 microns, suggesting that there is an activation-related internal load at low [Ca2+]. At maximal activation, a significant increase in this ratio occurred only at sarcomere lengths less than approximately 1.8 microns, and presumably involved collision of the thick filaments with the Z-lines. 5. Length-dependent changes in the Ca2+ sensitivity of tension development do not appear to be the result of shortening-induced dissociation of Ca2+ from troponin-C, the Ca2+ binding subunit of troponin. Fibres activated in the absence of Ca2+, by the partial removal of whole troponin complexes, produced length-tension relationships similar to those observed in the same fibres before troponin removal at a submaximal [Ca2+] yielding similar active tensions.

Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament.

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.

ATP-induced tension development in glycerinated fibers of scallop adductor striated muscle. Role of regulatory light chain of myosin in calcium regulation of muscle contraction.

Removal of the regulatory light chain subunit (EDTA light chain) of myosin from glycerinated fibers of scallop adductor striated muscle resulted in an immediate loss of calcium sensitivity of tension development and in a subsequent decrease in tension developed in the presence of calcium. It is suggested that removal of EDTA light chain results in a change in the myosin heavy-chain conformation which is probably responsible for the decrease in tension development.