Mechanical performance and glycolytic requirement in trout ventricular muscle

The glycolytic pathway seems to be coupled to the aerobic performance in mammalian cardiac muscle. Because many conditions are different in ectotherms, its influence on twitch force and resting force was recorded at 15 degrees C in isometric ventricular preparations from rainbow trout. To reduce glycolytic activity, preparations were exposed to 0.4 mmol l(-1) iodoacetate for 35 min or alternatively to 120 min anoxia in a glucose-free solution containing 10 &mgr;mol l(-1) adrenaline in an attempt to remove glycolytic substrates. The anoxic period was followed by recovery in an oxygenated solution containing aerobic substrates but no glucose. Control experiments indicated that this treatment, like iodoacetate, inhibits glycolysis, although glycogen was reduced by one half only. In fully oxygenated preparations with access to mitochondrial substrates, both attempts to reduce glycolytic activity tended to increase both resting force and the reductions in twitch force during high activity imposed by high stimulation rates and exposure to 10 &mgr;mol l(-1) adrenaline. Thus, the glycolytic pathway appears to be of specific importance under aerobic conditions also in the heart of ectotherms. J. Exp. Zool. 293:360-367, 2002.     

Electrical and mechanical activity in heart tissue of flounder and rainbow trout during acidosis

1. Twitch force and voltage across the sarcolemma were measured in heart tissue of flounder and rainbow trout. 2. For the trout heart, hypercapnia was followed by a loss of force and an action potential prolongation. 3. This was also observed for the flounder heart, but only initially. 4. About 5 min after the onset of hypercapnia, an increase in force and a shortening of the action potential occurred in the flounder heart. 5. After about 30 min of hypercapnia a decrease in force and a prolongation of the action potential slowly appeared. 6. These results can be interpreted in terms of a species-dependent effect of acidosis on the cellular Ca2+ handling and the influence of intracellular Ca2+ on the action potential.     

Systolic pressure gradients between the wall of the left ventricle, the left ventricular chamber, and the aorta during positive inotropic states: implications for left ventricular efficiency

To study systolic pressure gradients developed between the left ventricular wall, its chamber, and the aortic root, in one group of dogs left ventricle ventral wall intramyocardial pressure, left ventricular outflow tract pressure, and aorta pressure were compared with aortic flow as well as left ventricular dimension changes during control conditions as well as during positive intropic states induced by isoproterenol, stellate ganglion stimulation, and noradrenaline. In another group of dogs systolic pressures in the ventral wall of the left ventricle, the main portion of the left ventricular chamber, and the aorta were compared with aortic flow during similar interventions, before and after the administration of phentolamine. Pressure gradients between the wall of the left ventricle and the outflow tract of the left ventricle were minimal during control states, but during the three positive inotropic states were increased significantly. In contrast, pressure gradients between the outflow tract of the left ventricle and the aortic root were insignificant during positive inotropic states; those between the wall and main portion of the chamber were only significantly different during left stellate ganglion stimulation. The data derived from these experiments indicate that useful peak power output of the left ventricle (systolic aortic pressure X flow) is unchanged following isoproterenol infusion, but is increased by stellate ganglion stimulation and noradrenaline. The useful peak power output index (an index of left ventricular efficiency derived by dividing useful peak power output by peak intramyocardial pressure) was reduced more by isoproterenol than the other two interventions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen biosynthesis

It has been speculated that heart valve interstitial cells (VICs) maintain valvular tissue homeostasis through regulated extracellular matrix (primarily collagen) biosynthesis. VICs appear to be phenotypically plastic, inasmuch as they transdifferentiate into myofibroblasts during valve development, disease, and remodeling. Under normal physiological conditions, transvalvular pressures (TVPs) on the right and left side of the heart are vastly different. Hence, we hypothesize that higher left-side TVPs impose larger local tissue stress on VICs, which increases their stiffness through cytoskeletal composition, and that this relation affects collagen biosynthesis. To evaluate this hypothesis, isolated ovine VICs from the four heart valves were subjected to micropipette aspiration to assess cellular stiffness, and cytoskeletal composition and collagen biosynthesis were quantified by using the surrogates smooth muscle alpha-actin (SMA) and heat shock protein 47 (HSP47), respectively. VICs from the aortic and mitral valves were significantly stiffer (P < 0.001) than those from the pulmonary and tricuspid valves. Left-side isolated VICs contained significantly more (P < 0.001) SMA and HSP47 than right-side VICs. Mean VIC stiffness correlated well (r = 0.973) with TVP; SMA and HSP47 also correlated well (r = 0.996) with one another. Assays were repeated for VICs in situ, and, as with in vitro results, left-side VIC protein levels were significantly greater (P < 0.05). These findings suggest that VICs respond to local tissue stress by altering cellular stiffness (through SMA content) and collagen biosynthesis. This functional VIC stress-dependent biosynthetic relation may be crucial in maintaining valvular tissue homeostasis and also prove useful in understanding valvular pathologies.     

Mechanical interactions between collagen and proteoglycans: implications for the stability of lung tissue.

Collagen and elastin are thought to dominate the elasticity of the connective tissue including lung parenchyma. The glycosaminoglycans on the proteoglycans may also play a role because osmolarity of interstitial fluid can alter the repulsive forces on the negatively charged glycosaminoglycans, allowing them to collapse or inflate, which can affect the stretching and folding pattern of the fibers. Hence, we hypothesized that the elasticity of lung tissue arises primarily from 1) the topology of the collagen-elastin network and 2) the mechanical interaction between proteoglycans and fibers. We measured the quasi-static, uniaxial stress-strain curves of lung tissue sheets in hypotonic, normal, and hypertonic solutions. We found that the stress-strain curve was sensitive to osmolarity, but this sensitivity decreased after proteoglycan digestion. Images of immunofluorescently labeled collagen networks showed that the fibers follow the alveolar walls that form a hexagonal-like structure. Despite the large heterogeneity, the aspect ratio of the hexagons at 30% uniaxial strain increased linearly with osmolarity. We developed a two-dimensional hexagonal network model of the alveolar structure incorporating the mechanical properties of the collagen-elastin fibers and their interaction with proteoglycans. The model accounted for the stress-strain curves observed under all experimental conditions. The model also predicted how aspect ratio changed with osmolarity and strain, which allowed us to estimate the Young's modulus of a single alveolar wall and a collagen fiber. We therefore identify a novel and important role for the proteoglycans: they stabilize the collagen-elastin network of connective tissues and contribute to lung elasticity and alveolar stability at low to medium lung volumes.     

Respirometry increases cortisol levels in rainbow trout Oncorhynchus mykiss: implications for measurements of metabolic rate

This study aimed to assess the extent to which chasing, handling and confining Oncorhynchus mykiss to a small respirometer chamber during respirometric experiments is stressful and affects metabolic measurements. The study observed increased cortisol levels in animals tested using a chase protocol and subsequent intermittent‐flow respirometry, suggesting that this procedural treatment may stress animals.     

Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout

We investigated whether there are compensatory changes in the coronary microvasculature, cardiac lipid metabolism, and myocyte ultrastructure associated with ventricular enlargement in male rainbow trout. Epicardial tissue was sampled at different stages of sexual maturation, and we estimated arterial capillary density, intercapillary diffusion distance, and applied a diffusion model to predict PO₂ at different workloads. We also measured biochemical indices of lipid metabolism and estimated fractional volumes of mitochondria and myofibrils in myocytes. Immature fish with nonenlarged ventricles had the highest capillary length densities (1620±158 mm mm⁻³). Maturing trout with moderate ventricular hypertrophy had lower capillary length densities (1103±58 mm mm⁻³) and similar diffusion distances (13.9±0.7 μm) compared with immature fish (11.7±0.9 μm). The largest ventricles had intermediate capillary length densities (1457±288 mm mm⁻³) and diffusion distances (12.8±0.8 μm). Modelling predicted that enlarged ventricles would not become anoxic even at maximal workloads. Biochemical markers of fatty acid metabolism and aerobic capacity were unchanged with hypertrophy. Volume densities of mitochondria and myofibrils were also not influenced by cardiac growth. In summary, ventricle hypertrophy results in expansion of the coronary capillary bed and the maintenance of the epicardial capacities for fat and oxidative metabolism.     

Na+-K+ pump and metabolic activities of trout erythrocytes during anoxia

Metabolic activity in the red blood cells of brown trout wasmonitored under conditions of oxygen depletion and chemically inducedanoxia. Although metabolic activity was reduced during anoxia toone-third of the normoxic value, these cells maintained their ATPcontents stable and were viable for hours in the absence of oxygen. Inaddition,Na⁺-K⁺pump activity was not down-regulated when metabolic activity wasreduced during anoxia. The compatibility of this finding with energyequilibrium and ion homeostasis was investigated.

     

Acidosis,glycolysis and energy state in anaerobic heart tissue from rainbow trout

With focus on metabolism not depending on contractility in myocardial tissue from rainbow trout, Oncorhynchus mykiss, the effects of high CO₂ on lactate production, phosphocreatine, creatine, ATP, ADP, AMP and intracellular pH were examined under a blockage of cell respiration either alone or in combination with a glycolytic inhibition. Irrespective of metabolic interventions, a change in CO₂ from 1 to either 11 or 5% of the gas mixture perfusing the muscle bath with 15 mmol·l^-1 HCO _- ³ caused a drop of intracellular pH from 7.4 to either 6.5 or 7.0, respectively. An elevation of CO₂ to 11% diminished the rate of anaerobic lactate formation and slightly lowered anaerobic energy degradation. The further addition of 1 mmol·l^-1 iodoacetate to inhibit glycolysis strongly enhanced the tendency of acidosis to lower energy degradation. Moreover, iodoacetate induced a parallel decrease in ATP and total concentration of phosphorylated adenylates and an increase in resting tension. These effects were all substantially dampened by acidosis and could not immediately be related to tissue content of energy-rich phosphates. Tentatively, the depression of resting tension was the prime effect and a cause of the other effects acidosis. However, these were not affected by an inhibition of resting tension development with 2,3-butadione monoxime. The results suggest that glycolysis protects the anaerobic myocardium also by means not immediately related to tissue energy state. Acidosis exerts a similar protection, which is marginal as long as glycolysis is fully active, but substantial with an inhibited glycolysis.Abbreviations Cr _t total tissue concentration of creatine - G _PCr energy liberated per mol PCr hydrolyzed - IAA iodoacetate - PCr phosphocreatine - PE total tissue concentration of energy-rich phosphate bonds - pH _i intracellular pH - P _i inorganic phosphate - TAN total tissue concentration of phosphorylated adenylates - 2,3-BDM 2,3-butadione monoxime - SE standard error of the mean     

Mechanical and metabolic alterations in rat diaphragm during electrical stimulation

To investigate the hypothesis that the rate of fatigue development is not influenced by the absolute duration of contraction (train duration) and relaxation (off-phase of duty cycle) at constant duty cycle, strips of the diaphragm from 36 male adult rats (mean +/- SD wt 152 +/- 21 g) were stimulated directly for periods of 180, 250, and 320 ms at a constant duty cycle of 50%. The frequency of stimulation was adjusted to produce 40% of maximal tetanic tension at supramaximal voltages. After 30 min of stimulation, analysis of twitch characteristics between control and experimental groups indicated a prolongation of contraction time of 9% (P less than 0.05), an increase in relaxation time of 75% (P less than 0.05), and a decrease in twitch tension by 78% (P less than 0.05). Similarly, reductions (P less than 0.05) in isometric force output at high stimulation frequency (100 Hz) of 58% and at low frequency (20 Hz) of 67% were also noted. These changes were accompanied by an approximately 60% reduction in the maximal velocity of shortening. No difference was observed for any of the mechanical measures between experimental conditions. After 30-min stimulation, decreases of between 43 and 46% were noted for ATP (P less than 0.05) and increases of between three- and fourfold noted for IMP (P less than 0.05). No changes were found for either ADP or AMP. Total adenine nucleotide concentrations declined (P less than 0.05) an average of 24%. As with the mechanical data, no differences were found between the different stimulation conditions. It is concluded that for the conditions studied, fatigue mechanisms become manifest early in the stimulation period and are only minimally altered by the duration of specific contractions provided the relaxation period is of equal duration.     

Vitis vinifera root and leaf metabolic composition during fruit maturation: implications of defoliation

Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen (N) sources, either as recent assimilates, or mobilized from labile or storage pools. This study examined the response of root and leaf primary metabolism following defoliation treatments applied to fruiting vines during ripening. The objective was to link alterations in root and leaf metabolism to carbohydrate and N source functioning under conditions of increased fruit sink demand. Potted grapevine leaf area was adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves for the control. An additional group of vines were completely defoliated. Fruit sugar and N content development was assessed, and root and leaf starch and N concentrations determined. An untargeted GC/MS approach was undertaken to evaluate root and leaf primary metabolite concentrations. Partial and full defoliation increased root carbohydrate source contribution towards berry sugar accumulation, evident through starch remobilization. Furthermore, root myo‐inositol metabolism played a distinct role during carbohydrate remobilization. Full defoliation induced shikimate pathway derived aromatic amino acid accumulation in roots, while arginine accumulated after full and partial defoliation. Likewise, various leaf amino acids accumulated after partial defoliation. These results suggest elevated root and leaf amino N source activity when leaf N availability is restricted during fruit ripening. Overall, this study provides novel information regarding the impact of leaf source restriction, on metabolic compositions of major carbohydrate and N sources during berry maturation. These results enhance the understanding of source organ carbon and N metabolism during fruit maturation.     

Mechanical stimulation of tendon tissue engineered constructs: effects on construct stiffness, repair biomechanics, and their correlation

The objective of this study was to determine how in vitro mechanical stimulation of tissue engineered constructs affects their stiffness and modulus in culture and tendon repair biomechanics 12 weeks after surgical implantation. Using six female adult New Zealand White rabbits, autogenous tissue engineered constructs were created by seeding mesenchymal stem cells (0.1 x 10(6) cells/ml) in collagen gel (2.6 mg/ml) and combining both with a collagen sponge. Employing a novel experimental design strategy, four constructs from each animal were mechanically stimulated (one 1 Hz cycle every 5 min to 2.4% peak strain for 8 h/day for 2 weeks) while the other four remained unstretched during the 2 week culture period. At the end of incubation, three of the mechanically stimulated (S) and three of the nonstimulated (NS) constructs from each animal were assigned for in vitro mechanical testing while the other two autogenous constructs were implanted into bilateral full-thickness, full-length defects created in the central third of rabbit patellar tendons (PTs). No significant differences were found in the in vitro linear stiffnesses between the S (0.15+/-0.1 N/mm) and NS constructs (0.08+/-0.02 N/mm; mean+/-SD). However, in vitro mechanical stimulation significantly increased the structural and material properties of the repair tissue, including a 14% increase in maximum force (p=0.01), a 50% increase in linear stiffness (p=0.001), and 23-41% increases in maximum stress and modulus (p=0.01). The S repairs achieved 65%, 80%, 60%, and 40% of normal central PT maximum force, linear stiffness, maximum stress, and linear modulus, respectively. The results for the S constructs exceed values obtained previously by our group using the same animal and defect model, and to our knowledge, this is the first study to show the benefits of in vitro mechanical stimulation on tendon repair biomechanics. In addition, the linear stiffnesses for the construct and repair were positively correlated (r=0.56) as were their linear moduli (r=0.68). Such in vitro predictors of in vivo outcome hold the potential to speed the development of tissue engineered products by reducing the time and costs of in vivo studies.     

Mechanical control of tissue morphogenesis during embryological development

Twenty years ago, we proposed a model of developmental control based on tensegrity architecture, in which tissue pattern formation in the embryo is controlled through mechanical interactions between cells and extracellular matrix (ECM) which place the tissue in a state of isometric tension (prestress). The model proposed that local changes in the mechanical compliance of the ECM, for example, due to regional variations in basement membrane degradation beneath growing epithelium, may result in local stretching of the ECM and associated adherent cells, much like a "run-in-a-stocking". Cell growth and function would be controlled locally though physical distortion of the associated cells, or changes in cytoskeletal tension. Importantly, experimental studies have demonstrated that cultured cells can be switched between different fates, including growth, differentiation, apoptosis, directional motility and different stem cell lineages, by modulating cell shape. Experiments in whole embryonic organ rudiments also have confirmed the tight correlation between basement membrane thinning, cell tension generation and new bud and branch formation during tissue morphogenesis and that this process can be inhibited or accelerated by dissipating or enhancing cytoskeletal tension, respectively. Taken together, this work confirms that mechanical forces generated in the cytoskeleton of individual cells and exerted on ECM scaffolds, play a critical role in the sculpting of the embryo.     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Renal tissue metabolism in the rat during chronic metabolic alkalosis: importance of glycolysis

Chronic metabolic alkalosis was induced in rats drinking 0.3 M NaHCO3 and receiving 1 mg furosemide/100 g body weight per day intraperitoneally. Another group of animals received a potassium supplement in the form of 0.3 M KHCO3. In this group, hypokalemia did not develop and muscle potassium fell by only 18% versus 50% in those not receiving potassium. In vitro renal production of ammonia and uptake of glutamine fell by 40% with a decrease in the activity of glutaminase I and glutamate dehydrogenase. Activity of phosphofructokinase, a major enzyme of glycolysis, rose only in the kidney of animals receiving a potassium supplement. Fructose-1,6-diphosphatase fell as well as phosphoenolpyruvate carboxykinase. Malate dehydrogenase also fell. The activity of phosphofructokinase also rose in the liver, heart, and leg muscle. The major biochemical changes in the renal cortex were the following: glutamate, alpha-ketoglutarate, malate, lactate, pyruvate, alanine, aspartate, and citrate rose as well as calculated oxaloacetate. The concentration of intermediates like 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate fell. The cytosolic redox potential (NAD+/NADH) decreased. In addition to the fall in ammoniagenesis, it could be demonstrated in vitro that the renal tubules incubated with glutamine showed decreased glucose production and increased production of lactate and pyruvate. The concentration of lactate was elevated in all tissues examined including liver, heart, and leg muscle. This study confirms in the rat that decreased renal ammoniagenesis takes place following decreased uptake of glutamine in metabolic alkalosis. All other changes are accounted for by the process of increased glycolysis, which appears to take place in all tissues in metabolic alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noninvasive reconstruction of the three-dimensional ventricular activation sequence during pacing and ventricular tachycardia in the canine heart

Single-beat imaging of myocardial activation promises to aid in both cardiovascular research and clinical medicine. In the present study we validate a three-dimensional (3D) cardiac electrical imaging (3DCEI) technique with the aid of simultaneous 3D intracardiac mapping to assess its capability to localize endocardial and epicardial initiation sites and image global activation sequences during pacing and ventricular tachycardia (VT) in the canine heart. Body surface potentials were measured simultaneously with bipolar electrical recordings in a closed-chest condition in healthy canines. Computed tomography images were obtained after the mapping study to construct realistic geometry models. Data analysis was performed on paced rhythms and VTs induced by norepinephrine (NE). The noninvasively reconstructed activation sequence was in good agreement with the simultaneous measurements from 3D cardiac mapping with a correlation coefficient of 0.74 ± 0.06, a relative error of 0.29 ± 0.05, and a root mean square error of 9 ± 3 ms averaged over 460 paced beats and 96 ectopic beats including premature ventricular complexes, couplets, and nonsustained monomorphic VTs and polymorphic VTs. Endocardial and epicardial origins of paced beats were successfully predicted in 72% and 86% of cases, respectively, during left ventricular pacing. The NE-induced ectopic beats initiated in the subendocardium by a focal mechanism. Sites of initial activation were estimated to be ～7 mm from the measured initiation sites for both the paced beats and ectopic beats. For the polymorphic VTs, beat-to-beat dynamic shifts of initiation site and activation pattern were characterized by the reconstruction. The present results suggest that 3DCEI can noninvasively image the 3D activation sequence and localize the origin of activation of paced beats and NE-induced VTs in the canine heart with good accuracy. This 3DCEI technique offers the potential to aid interventional therapeutic procedures for treating ventricular arrhythmias arising from epicardial or endocardial sites and to noninvasively assess the mechanisms of these arrhythmias.     

Strain-rate dependence of cartilage stiffness in unconfined compression: the role of fibril reinforcement versus tissue volume change in fluid pressurization

The strain and strain-rate-dependent response of articular cartilage in unconfined compression was studied theoretically. The transient stress and stiffness of cartilage were determined for strain rates ranging from zero to infinity. It is shown, for a given compressive strain, that the axial stress initially increases quickly as a function of strain rate, and then increases progressively more slowly towards the stress corresponding to the instantaneous response. The volume change of the tissue does not give its transient stiffness uniquely, because of the strong strain-rate dependence. The variation of tissue stiffness is primarily determined by the transient stiffness of the radial fibrils. Load sharing between the solid matrix and fluid pressurization also depends on the strain rate. At 15% axial compression, the matrix bears more than 80% of the applied load at a strain rate of 0.005%/s, while the fluid pressurization contributes more than 80% of the load at a strain rate of 0.15%/s. These results show the interplay between fibril reinforcement and fluid pressurization in articular cartilage: the fluid drives fibril stiffening which in turn produces high pore pressure at high strain rates.As a secondary objective of the present work, a fibrillar continuum element was formulated to replace the fibrillar spring element used previously in fibril-reinforced modeling, in order to eliminate the deformation incompatibility between the spring system and the nonfibrillar matrix. The results obtained using the two fibrillar elements were compared with the closed-form solutions for the static and instantaneous responses for the case of large deformation. It was found for unconfined compression that using the spring elements did not generally result in greater numerical errors than using the fibrillar continuum elements.