Increase In Maximal Tension Research Articles

Functional significance of C-terminal mobile domain of cardiac troponin I

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1–167) and cTnI(1–193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 μm) and long (2.2 μm) sarcomere lengths (SLs). Our results show that the cTnI(1–167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1–167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.

Effects of Hcm-Linked Mutations in Myosin Essential Light Chain and the Role of Serine-195 Pseudo-Phosphorylation

In this study we investigated whether phosphorylation of myosin ELC at Serine 195, identified by proteomic analysis as a novel phosphorylation site, and shown to affect contractility in zebrafish cardiomyocytes, can play a role in cardiac muscle contraction. We also investigated whether S195D pseudo phosphorylation of ELC could rescue an ELC induced hypertrophic cardiomyopathy (HCM) phenotype. To test this, we exchanged human recombinant WT-ELC, WT-S195D and ELC-A57G, E143K and M173V mutants in skinned porcine papillary muscle fibers and porcine cardiac myosin, both expressing the β-myosin heavy chain (MHC). In myosin exchange experiments, we observed a stronger binding of WT-S195D to MHC (lower Kd), but the degree of ELC exchange vs. WT was significantly lower (∼48% vs. ∼69%). However, no changes in %-reconstitution were observed among all recombinant ELC mutants when compared to WT. In the ELC-reconstituted fibers, WT-S195D was seen to exchange with a significantly lower efficiency compared with WT (∼33% vs 47%), but it also caused a small increase in maximal force (by ∼3 kN/m2). An increase in maximal tension upon pseudo-phosphorylation of ELC was in accord with the MLCK-induced or pseudo (with S15D mutation)-phosphorylation observed for the myosin RLC. However, a WT-S195D-mediated increase in force was not accompanied by any increase in myofilament calcium sensitivity, as observed in RLC studies. Maximal force generation was not changed in any of ELC-reconstituted fibers when compared to WT-ELC. Two ELC mutants (A57G and E143K) slightly (but significantly) increased Ca2+-sensitivity of force while M173V produced no change. Further studies are needed to reveal whether these subtle changes seen in A57G and E143K-exchanged porcine cardiac preparations could be “rescued” by respective S195D pseudo-phosphorylation mimics. Supported by AHA-12PRE12030412 (WH) and NIH-HL108343 (DSC).

Doxorubicin-induced vasomotion and [Ca(2+)](i) elevation in vascular smooth muscle cells from C57BL/6 mice.

To explore the action of doxorubicin on vascular smooth muscle cells. Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca(2+)](i) in isolated aortic smooth muscle cells was measured by using Fluo-3. Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca(2+)](i) in single muscle cells. Treatment with 10 micromol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca(2+) or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 micromol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 micromol/L increased the frequency of the RC only. In the presence of 100 micromol/L caffeine, however, 100 micromol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 micromol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca(2+)-free solution, doxorubicin induced a transient [Ca(2+)](i) increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca(2+)](i) was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin. Doxorubicin not only releases Ca(2+) from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca(2+) into vascular smooth muscle cells.

Impaired insulin-induced attenuation of noradrenaline-mediated vasoconstriction in insulin-resistant obese Zucker rats.

1. Insulin resistance is associated with hypertension but the underlying mechanism is unclear. We tested the hypothesis that insulin-induced vasodilatation is impaired in insulin-resistant obese Zucker rats. We studied mesenteric artery (approximately 220 microns diameter) function before the development of hypertension in 3-month old obese Zucker rats and age-matched lean rats. 2. In vessels from lean rats, insulin at concentrations of 50, 500 and 5000 m-units/l attenuated the constriction in response to noradrenaline (50 m-units/l: 8 +/- 3%, P < 0.05; 500 m-units/l: 13 +/- 3%, P < 0.02; 5000 m-units/l: 13 +/- 2%, P < 0.02). 3. Vessels from obese rats failed to show any such response to insulin (2 +/- 6% increase in maximal tension with 5000 m-units/l; not significant), both in the presence and absence of L-arginine (3 mmol/l). 4. Vessels from obese rats showed slight but significant impairment in the vasodilator response to acetylcholine (5 x 10(-8)-10(-4) mol/l) (obese: 64.1 +/- 3.7% relaxation; lean: 77.3 +/- 3.7% relaxation; P < 0.05); however, relaxation in response to A23187 was not significantly different between the phenotypes (obese: 81.3 +/- 10.6% relaxation; lean: 79.1 +/- 9.7% relaxation; not significant). 5. Systolic blood pressure was not significantly different in lean (126 +/- 8 mmHg) and obese (127 +/- 7 mmHg) rats at the time of study (not significant). 6. We conclude that insulin-induced attenuation of noradrenaline-mediated vasoconstriction is impaired in the obese Zucker rat and that this defect precedes and therefore could contribute to the development of hypertension in this insulin-resistant model. The defect in insulin action could reside in the endothelial generation of nitric oxide, as endothelial function is also abnormal.

Anthracyclines enhance tension development in cardiac muscle by direct interaction with the contractile system.

Anthracyclines are highly effective anticancer agents which induce a well described but incompletely understood cardiac toxicity. In this study, a direct action of several anthracyclines on the force generating mechanism of heart muscle preparations is described. To allow discrimination between membrane related effects and a direct action of anthracyclines on the actin-myosin contractile system, both inner and outer membranes of cardiac fibres were permeabilized. All anthracyclines tested in this study [doxorubicin (Dox), epirubicin, daunorubicin and idarubicin] showed positive inotropic actions. Dox and epirubicin, which are considered the most cardiotoxic drugs of the anthracycline family, significantly increased the maximal calcium activated tension by 33% (n = 8, P < 0.01) and by 26% (n = 8, P < 0.01) respectively. Daunorubicin and idarubicin increased the maximal tension by 12% and 9% respectively (P = n.s.). Other chemotherapeutic drugs (Taxol and 5-FU) had no effect on maximal tension. To elucidate the mechanism behind this Dox-induced increase in maximal tension, calcium sensitivity curves were measured and rigor experiments were performed. A small but significant increase in pCa50 value (+0.14 +/- 0.03, P < 0.05) was observed only after incubation with 20 microM Dox. Dox acted during the transition to force generating cross-bridges as reflected by the significant increase in rigor tension (12%, P < 0.05) after preincubation of cardiac fibres with Dox. Cycling of cross-bridges is a prerequisite for Dox to increase tension because no effect on tension was seen after Dox was added to fibres in an established rigor. In summary, anthracyclines increased the maximal tension in cardiac muscle fibres by direct interaction with the actin-myosin cross-bridges. Changes in calcium sensitivity are unlikely to contribute to the observed increase in maximal tension. The rise in tension as is seen in this experimental set-up may contribute to destruction of the contractile machinery of cardiac muscle. In agreement with this hypothesis is the observation that the more cardiotoxic anthracyclines induced the largest increase in maximal tension of the cardiac fibres.

Different immunological sensitizing regimens and airway responsiveness in vitro to contractile agonists in guinea-pigs

Groups of guinea-pigs were sensitized to ovalbumin, using different regimens to induce either IgG-like antibodies or IgE-like and IgG-like antibodies and the responsiveness to histamine and carbachol in vitro of tracheal and lung parenchymal strips was determined. EC 50 values for histamine and carbachol in both tracheal and lung parenchymal strips were not significantly different for either group of sensitized guinea-pigs or their respective controls. The maximal tension developed in parenchymal strips was not significantly different for either sensitized or control groups. For tracheal strips, maximal tension was not significantly different in each group except in animals with IgG-like antibodies, which developed less maximal tension with histamine than controls. The absence of any increase in maximal tension or any change in EC 50 to histamine or to carbachol in tracheal or parenchymal strips from immunized guinea-pigs indicates that immunological sensitization does not increase the responsiveness of smooth muscle to these contractile agonists.

The Pattern and Mechanisms of Response to Oxygen by the Ductus Arteriosus and Umbilical Artery

Extract: Response of the ductus arteriosus and umbilical artery to changes in oxygen tension, to acetylcholine, and to sympathetic and parasympathetic blocking agents was studied in vitro in isolated rings obtained from 22 fetal lambs of 98− to 147-day gestation.After stabilization of tension at a baseline level (0.3—0.7 g) in a Po2, environment of 35–45 mm Hg, both increase of the Po2 to 550 mm Hg and decrease of the Po2 to 8 mm Hg of the bathing solution produced constriction. The mean maximal tension developed by the ductus arteriosus was 3.91 g at high P0, and 3.87 g at low Po2. The increase in maximal tension developed with advancing gestation was also similar at both high and low Po2.At Po2, levels of 8–550 mm Hg, acetylcholine produced a further increase in tension, whereas bradykinin only produced an increase in tension at high PO2. Alpha and beta sympathetic blockade had no effect on the constrictor response to oxygen. Atropine relaxed the ductus arteriosus and umbilical artery at both high and low Po2 levels; the degree of relaxation was related to drug concentration. Acetylcholin-esterase also relaxed the ductus arteriosus constricted by oxygen. The cholinesterase inhibitor, edrophonium, produced ductus constriction at P0, of 35–45 mm Hg with little further effect from high Po2. The constrictor effect of high or low Po2 on the ductus arteriosus and umbilical artery seems to be mediated through release of acetylcholine.