Mechanical restitution of the rat papillary muscle

The experiments were performed on isometrically contracting rat papillary muscles paced at 1.0 Hz and at a temperature of 37°C. The contractile response of a test contraction continuously rose when the duration of the preceding stimulus interval was gradually increased from 0.2 s. A maximum value was seen at intervals of 60–120 s. This phenomenon was called mechanical restitution of the papillary muscle. The insertion of a priming beat before the test contraction in order to increase the amount of contractile calcium elevated the mechanical restitution curve but it did not change the maximum contractile force seen after 60–120 s intervals. Lowering the extracellular calcium concentration from 2.0 mM to 1.0 mM, however, depressed this maximum contractile force to about 50%. The mechanical restitution is thought to reflect inflow of activator calcium to a cellular store, from which it is later released in response to the action potential. By using two test contractions a simple method is described to estimate the recirculating fraction of activator calcium between beats in this preparation. In 11 preparations the recirculating fraction of activator calcium was 0.72±0.03 (mean ± SE). The results are consistent with the view that the same model of metabolism of activator calcium as previously proposed for rabbit papillary muscles is also applicable to the rat heart.     

Mechanisms for hypothermia-induced increase in contractile force studied by mechanical restitution and post-rest contractions in guinea-pig papillary muscle

Lowering myocardial temperature increases contractile force, presumably by increasing intracellular calcium content. To study the mechanisms behind this, we compared the effects of some known inotropic interventions with hypothermia on mechanical restitution and post-rest contractile force in isolated guinea-pig papillary muscles. In four groups (n = 6 per group), the effects of: (1) reducing the ability for Na/Ca exchange to extrude Ca²⁺ (a) by increasing [Na⁺]₁ with ouabain or (b) by increasing [Ca²⁺]_o; and (2) activation of calcium channels with Bay-K 8644, were compared with lowering temperature from 37 to 27 °C. Normally (at 37 °C and 2 mm CaCl₂), mechanical restitution could be described by a rapid recovery phase with a time constant between 180 and 220 ms, followed by a slowly decaying phase with a time constant between 5000 and 8000 ms and post-rest contractions (1–10 min rest) were markedly depressed compared to steady-state contractions. Steady-state developed force was markedly increased at 27 °C, after 1 μm ouabain, 6 mm CaCl₂ or 0.1 μM Bay-K 8644. At 27 °C the rapid recovery phase of restitution was delayed while the slowly decaying phase was not affected. Ouabain and increased [Ca²⁺]_o caused elevation of the slowly decaying phase of restitution and markedly attenuated the post-rest depression of developed force, which may be attributed to a reduced diastolic extrusion of Ca²⁺ via the Na/Ca exchanger. Hypothermia and Bay-K 8644 on the other hand, augmented this post-rest depression. Hence, this study suggests that increased Ca²⁺ influx due to delayed inactivation of calcium channels may account for the increased developed force during hypothermia rather than reduced diastolic extrusion of Ca²⁺ via the Na/Ca exchanger.     

Effect of amiodarone on the dynamics of mechanical restitution of rat papillary muscles

We studied the effect of amiodarone (class III antiarrhythmic drug) on the dynamics of mechanical restitution of rat papillary muscle. Amiodarone produced a weak negative inotropic effect and stimulated potentiation of contractility of the muscle preparation after short-term (4-60 sec) cessation of its electrical stimulation. On the other hand, the time of attaining half-maximum amplitude of contractions after amiodarone treatment did not differ from the control. Analysis of curves presenting the drop of potentiation of muscle preparation contractility after resumption of regular electrical stimulation after 60-sec arrest until attaining a stable level showed that the amplitude returned to the initial level by the 9thcontraction-relaxation cycle both in the control and after amiodarone treatment. Coefficient of the drop of contraction amplitude potentiation was virtually the same in the two groups. Presumably, amiodarone does not modulate calcium-binding capacity of the sarcoplasmic reticulum, but improves Ca retention in the sarcoplasmic reticulum stores.     

Analysis of mechanical alternans in rabbit papillary muscle

Isometric force and action potentials were recorded in rabbit papillary muscles. It was found that the monophasic decline of the contractile potentiation that was recorded after an extrasystole (ES) was replaced by transient mechanical alternans (TMA) when temperature and calcium concentration of the perfusion medium had been lowered (from 37 degrees to 27 degrees C and from 2.0 to 0.5 mM, respectively). TMA in response to ES was also seen when the preparation was exposed to 2mM 4-aminopyridine. Furthermore, TMA could be induced by a shortening step during activity. Mechanical restitution curves were recorded by relating max. rate of force development of a test contraction to the duration of the preceding stimulus interval. It was found that the alternating contractions during TMA were associated with shifts of the mechanical restitution curve along the force axis. The duration of the action potential was inversely related to force development during TMA. It is proposed that TMA is due to a reduced damping of a regulatory feedback system between inotropic state and intake of activator calcium during the action potential. The following sequence of events are suggested: The abbreviated action potential accompanying a potentiated contraction is associated with reduced intake of activator calcium. This leads to depression of the subsequent contraction. The latter contraction is associated with increased calcium intake due to prolongation of the action potential. This will lead to potentiation of the next beat and the sequence is repeated. It is proposed that recirculation of calcium between heart beats will act as a damping factor of this system.     

Cortactin is required for integrin-mediated cell spreading

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. Here we show that expression of either the GFP-tagged N-terminal or the C-teminal halves of cortactin inhibits significantly the spreading of COS7 cells on fibronectin. Introducing inactivating point mutation into the SH3 domain of the C-terminal half of cortactin suspends the dominant negative effect of the construct. In addition, a vector-based RNA interference was used to knock-down endogenous level of cortactin in cells. We demonstrate that cortactin deficient cells were not able to spread. These results suggest that cortactin is required for integrin-mediated signalling pathways.     

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.     

Age differences in temperature dependence of repolarization of the adrenocortical cell membrane

In experiments on isolated adrenals of male rats of two age groups (5 and 28–29 months) no age differences were found in the membrane potential of cells in the zona fasciculata of the cortex. The temperature dependence of repolarization of the cell membrane in the zona fasciculata of the adrenal cortex was investigated in rats of different ages within the temperature range from 7 to 17°C after preliminary cooling of the adrenals. The temperature coefficient of repolarization, calculated in old animals (Q₁₀=2.732) was significantly higher than in the young animals (Q₁₀=1.481). With age, the contribution of reactions with high activation energy increases in total balance of processes determining repolarization of the adrenocortical cell membrane.Biophysics Group, Laboratory of Physiology, Institute of Gerontology, Academy of Medical Sciences of the USSR, Kiev. (Presented by Academician of the Academy of Medical Sciences of the USSR D. F. Chebotarev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 12, pp. 651–653, December, 1978.     

Palmitoylation of ERBIN is required for its plasma membrane localization

LAP (leucine-rich repeats (LRR) and PSD-95/Dlg/ZO-1 (PDZ)) family proteins, including Scribble, LET-413, ERBIN, Densin-180 and Lano, are involved in the regulation of cell polarity. The LRR domains of LAP proteins were reported to mediate their basolateral membrane localization and to be essential for their function. To further dissect the mechanism of the plasma membrane localization of ERBIN, we introduced various mutants of ERBIN into cultured cells and observed the intracellular localization. When an LRR domain mutant lacking amino acid residues 1–32 at the amino (N) terminal region was over-expressed in cells, the mutant did not localize at the plasma membrane, but localized in the cytoplasm. We found that cysteines 14 and 16 at the N-terminal region of ERBIN are in vivo palmitoylated. Over-expressed mutants in which cysteine 14 and/or cysteine 16 were changed to serines did not localize at the plasma membrane, indicating that the palmitoylation of ERBIN is necessary for its plasma membrane localization. The over-expressed 1–196 amino acids fragment of ERBIN, which lacked the latter half of LRR, was palmitoylated but did not localize at the plasma membrane. These results suggest that both palmitoylation and LRR are required for the plasma membrane localization of ERBIN.     

Plasma membrane cholesterol is required for efficient pseudorabies virus entry

Alphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex virus, varicella-zoster virus and pseudorabies virus (PRV). Here, using methyl-beta-cyclodextrin and fluorescently tagged PRV, we directly show that depletion of cholesterol from the plasma membrane of host cells significantly reduces PRV entry. Cholesterol depletion did not reduce PRV attachment, but stalled virus particles at the plasma membrane before penetration of the cell. Cholesterol depletion results in destabilization of lipid raft microdomains in the plasma membrane, which have been shown before to be involved in efficient entry of different viruses. A significant fraction of PRV virions appears to localize juxtaposed to GM1, a lipid raft marker, during entry. Together, these data indicate that cholesterol and possibly cholesterol-rich lipid rafts may be important during PRV entry.     

Salmonella-induced cell death is not required for enteritis in calves

Salmonella enterica serovar Typhimurium causes cell death in bovine monocyte-derived and murine macrophages in vitro by a sipB-dependent mechanism. During this process, SipB binds and activates caspase-1, which in turn activates the proinflammatory cytokine interleukin-1beta through cleavage. We used bovine ileal ligated loops to address the role of serovar Typhimurium-induced cell death in induction of fluid accumulation and inflammation in this diarrhea model. Twelve perinatal calves had 6- to 9-cm loops prepared in the terminal ileum. They were divided into three groups: one group received an intralumen injection of Luria-Bertani broth as a control in 12 loops. The other two groups (four calves each) were inoculated with 0.75 x 10(9) CFU of either wild-type serovar Typhimurium (strain IR715) or a sopB mutant per loop in 12 loops. Hematoxylin and eosin-stained sections were scored for inflammation, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were detected in situ. Fluid accumulation began at 3 h postinfection (PI). Inflammation was detected in all infected loops at 1 h PI. The area of TUNEL-labeled cells in the wild-type infected loops was significantly higher than that of the controls at 12 h PI, when a severe inflammatory response and tissue damage had already developed. The sopB mutant induced the same amount of TUNEL-positive cells as the wild type, but it was attenuated for induction of fluid secretion and inflammation. Our results indicate that serovar Typhimurium-induced cell death is not required to trigger an early inflammatory response and fluid accumulation in the ileum.     

Effect of hypoxia on mechanical properties of hyperthyroid cat papillary muscle.

It has been previously established that hyperthyroid myocardium exhibits increased performance under well-oxygenated conditions. To date, it is not known whether hyperthyroid cardiac muscle can maintain this increased performance during hypoxia. The responses of isolated right ventricular papillary muscles from hyperthyroid and euthyroid kittens to hypoxia were compared under isometric conditions at 31 degrees C. Under well-oxygenated conditions, the hyperthyroid cardiac muscle exhibited both an increased contractility and an accelerated rate of relaxation. A similar degree of acute hypoxic stress for 15 min resulted in a greater decrease in contractility in the hyperthyroid compared with the euthyroid papillary muscle as indicated by a greater fall in both peak tension development (2.2 +/- 0.25 from 4.2 +/- 0.2 vs. 0.9 +/- 0.15 from 3.2 +/- 0.4 g/mm2, P less than 0.01) and +dT/dt (12.9 +/- 2.3 from 25 +/- 3 vs. 4.0 +/- 0.6 from 14 +/- 1 g-s-1-mm-2, P less than 0.01). In addition, compared with the euthyroid data, hypoxia resulted in impaired myocardial relaxation in the hyperthyroid cardiac muscle. Thus, the hyperthyroid compared with the euthyroid papillary muscle exhibits both a greater decrease in contractility and an impairment of myocardial relaxation during hypoxia, indicating a greater susceptibility to a given hypoxic stress.     

GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.     

Intestinal smooth muscle is required for patterning the enteric nervous system

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid‐gut of the chick embryo, we find that α‐smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest‐derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS.