Chronic high-dose testosterone treatment: impact on rat cardiac contractile biology.

Androgen therapy provides cardiovascular benefits for hypogonadism. However, myocardial hypertrophy, fibrosis, and infarction have been reported in testosterone or androgenic anabolic steroid abuse. Therefore, better understanding of the factors leading to adverse results of androgen abuse is needed. The aim of the present study was to examine the impact of high dose of androgen treatment on cardiac biology, and whether exposure duration modulates this response. Male rats were treated with 10 mg/kg testosterone, three times a week, for either 4 or 12 weeks; vehicle injections served as controls. Four weeks of testosterone treatment induced an increase in ventricular wall thickness, indicative of concentric hypertrophy, as well as increased ejection fraction; in contrast, both parameters were blunted following 12 weeks of high-dose testosterone treatment. Cardiac myocyte contractile parameters were assessed in isolated electrically stimulated myocytes (sarcomere and intracellular calcium dynamics), and in chemically permeabilized isolated myocardium (myofilament force development and tension-cost). High-dose testosterone treatment for 4 weeks was associated with increased myocyte contractile parameters, while 12 weeks treatment induced significant depression of these parameters, mirroring the cardiac pump function results. In conclusion, chronic administration of high-dose testosterone initially induces increased cardiac function. However, this initial beneficial impact is followed by significant depression of cardiac pump function, myocyte contractility, and cardiac myofilament function. Our results indicate that chronic high-testosterone usage is of limited use and may, instead, induce significant cardiac dysfunction.

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers.

Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 µm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca2+ sensitivity for 2.5 vs. 2.0 µm sarcomere length fibers (pCa50 = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca2+ sensitivity of force was attenuated, though the Ca2+ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by ∼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s-1). In the presence of dextran, cross-bridge detachment was ∼20% slower at 2.5 vs. 2.0 µm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s-1). However, osmotic compression of fibers at either 2.5 or 2.0 µm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.

Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling.

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²âº transient amplitude. Effects were primarily observed at 5 and 10 µM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²âº transient amplitudes. At 10 µM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²âº handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

Non-uniform distribution of strain during stretch of relaxed skeletal muscle fibers from rat soleus muscle.

Tension and regional average sarcomere length (L(s)) behavior were examined during repeated stretches of single, permeabilized, relaxed muscle fibers isolated from the soleus muscles of rats. We tested the hypothesis that during stretches of single permeabilized fibers, the global fiber strain is distributed non-uniformly along the length of a relaxed fiber in a repeatable pattern. Each fiber was subjected to eight constant-velocity stretch and release cycles with a strain of 32% and strain rate of 54% s(-1). Stretch-release cycles were separated by a 4.5 min interval. Throughout each stretch-release cycle, sarcomere lengths were measured using a laser diffraction technique in which 20 contiguous sectors along the entire length of a fiber segment were scanned within 2 ms. The results revealed that: (1) the imposed length change was not distributed uniformly along the fiber, (2) the first stretch-release cycle differed from subsequent cycles in passive tension and in the distribution of global fiber strain, and (3) a characteristic "signature" for the L(s) response emerged after cycle 3. The findings support the conclusions that longitudinal heterogeneity exists in the passive stiffness of individual muscle fibers and that preconditioning of fibers with stretch-release cycles produces a stable pattern of sarcomere strains.

Burn-induced heart failure: lipopolysaccharide binding protein improves burn and endotoxin-induced cardiac contractility deficits.

BACKGROUND: Burn injury is frequently complicated by bacterial infection. Following burn injury, exposure to endotoxin produces a measurable decrease in cardiomyocyte sarcomere contractile function. Lipopolysaccharide-binding protein (LBP) is an acute phase protein that potentiates the recognition of lipopolysaccharide (LPS) by binding to the lipid A moiety of LPS. In this study, we sought to determine the effect of recombinant rat LBP (rLBP) on cardiomyocyte sarcomere function after burn or sham injury in the presence or absence of bacterial endotoxin. METHODS: Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 24 h post-injury, cardiomyocytes were isolated, plated at 50,000 cells/well, and incubated with 50 µg/mL LPS and rLBP or chloramphenicol acetyltransferase (BVCat, an irrelevant control protein produced using the same expression system as rLBP) at concentrations by volume of 1%, 5%, 10%, and 30%. Subsets of cardiomyocytes were incubated with 5% rat serum or 30% rLBP and blocking experiments were conducted using an LBP-like synthetic peptide (LBPK95A). In vitro sarcomere function was measured using a variable rate video camera system with length detection software. RESULTS: Co-culture of burn and sham injury derived cardiomyocytes with high-dose rLBP in the presence of LPS resulted in a significant reduction to the functional impairment observed in peak sarcomere shortening following exposure to LPS alone. LBP-like peptide LBPK95A at a concentration of 20 µg/mL, in the presence of LPS, abolished the ability of 30% rLBP and 5% rat serum to restore peak sarcomere shortening of cardiomyocytes isolated following burn injury to levels of function exhibited in the absence of endotoxin exposure. CONCLUSIONS: In the setting of LPS challenge following burn injury, rLBP at high concentrations restores cardiomyocyte sarcomere contractile function in vitro. Rather than potentiating the recognition of LPS by the cellular LPS receptor complex, rLBP at high concentrations likely results in an inhibitory binding effect that minimizes the impact of endotoxin exposure on cardiomyocyte function following thermal injury.

Morphometric changes induced by amino acid supplementation in skeletal and cardiac muscles of old mice.

Aging is associated with progressive structural disorganization of muscular and cardiac fibers, decreasing functional capacity, and increased rates of disease and death. Aging is also characterized by disturbances in protein synthesis with impaired cellular organelle functions, particularly in the mitochondria. The availability of amino acids is a key factor for the overall metabolism of mammals and exogenous supplements of amino acid mixtures (AAm) could be a valid therapeutic strategy to improve quality of life, avoiding malnutrition and muscle wasting in the elderly. We investigated the morphoquantitative effects of long-term AAm supplementation on the mitochondria and sarcomeres (by electron microscope) and on collagen matrix deposition (by histologic techniques) in both skeletal and cardiac muscles of young and aged mice. Our data showed that old animals have fewer mitochondria and massive fibrosis in both muscles. Long-term AAm supplementation increased the number and volume of mitochondria and sarcomeres and decreased fibrosis in both skeletal muscle and hearts in old rats. These findings indicate that AAm restored muscular morphologic parameters and probably improved the mechanical performance of these organs.

Effects of massage on the mechanical behaviour of muscles in adolescents with spastic diplegia: a pilot study.

Calf muscles of five adolescents aged 12 to 15 years (three males, two females) with spastic diplegia were massaged for 14 minutes twice a week for 5 weeks in a controlled sequence, stretching the muscles transversely rather than longitudinally, without eliciting pain. Slow, passive test stretches were applied before and after massage. After massage, the range of movement was not consistently increased but, on average, greater force was needed to stretch the muscle than before massage. However, after massage the resting ankle angle sometimes changed so that the calf muscles were either shorter or longer. We suggest that these phenomena could be explained if massage resets sarcomere lengths which corrects for thixotropic effects (i.e. previous use modifies a muscle's mechanical behaviour). A redistribution on sarcomere lengths within muscles could also have reset proprioceptive feedback. The incidence of abnormal stretch reflexes during test stretches fell from 40 to 22%, comparing the first five sessions with the last five sessions. The amplitude of voluntary alternating ankle rotation increased in three participants. Motor skills were assessed with the Gross Motor Function Measure-66 (GMFM-66) 1 week before the test period, during the 5th week, and 12 weeks later. Our participants in Gross Motor Function Classification System (GMFCS) Levels I and II made sustained improvements in GMFM-66 scores (6.4% at 5 weeks falling to 5.5% at 17 weeks), one increase being significant. One participant in GMFCS Level III improved significantly only after massage of all leg muscles for 30 weeks.

Protein kinase D selectively targets cardiac troponin I and regulates myofilament Ca2+ sensitivity in ventricular myocytes.

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Heat, phosphorus NMR and microcalorimetry in relation to the mechanism of filament sliding.

For muscle heat measurements the methods available are sensitive and rapid, and the heat is related to the chemical changes in a manner that provides a firm outline for understanding the mechanism of contraction. For example linear dependence of the shortening heat on the sarcomere length has shown that the rate of turnover of cross-bridges increases during shortening. However, heat is bound to lack specificity. In order to cope with this problem, various methods such as rigorous chemical analyses, phosphorus NMR and microcalorimetry have been introduced. As a result of ultra-rapid freezing and chemical analysis by D. R. Wilkie (Gilbert, Kretzchmar, Wilkie and Woledge, 1971), the energy balance discrepancy between (heat + work) and the amount of phosphocreatine (PCr) split emerged, i.e. the unexplained enthalpy. Calcium ions move from the sarcoplasmic reticulum to the calcium-receptive proteins in the sarcoplasm during contraction. In an attempt to find the cause of the unexplained enthalpy, microcalorimetry of calcium binding to calcium-receptive proteins has been performed. The results have shown that calcium ions dislocated between sites within the sarcoplasm on activation may produce about 1/3 of the unexplained heat. In addition calcium pump should operate by consuming PCr to relocate the calcium after the contraction. Time-resolved phosphorus NMR has also shown that a certain amount of PCr splitting continues during early minute of recovery period after the contraction without Pi released. This delayed splitting of PCr is most likely caused by the kinetic properties of the contractile proteins and can explain another 1/3 of the unexplained enthalpy. The mechanism of how muscle is regulated is another important question. Studies of calcium binding to calcium-receptive proteins in the sarcoplasm by using titration microcalorimetry has shown that troponin C has a characteristic single calcium-binding site that is most likely to be involved in the regulation of contraction.

Force, sarcomere shortening velocity and ATPase activity.

UNLABELLED: We have tested the hypothesis that the transition rate (G) of the cardiac XB from the strong force generating state to the weak state is a linear function V of the sarcomere (VSL); furthermore, we tested whether the ATPase rate of the two isoforms of myosin can be held responsible for the difference between V0 of rat cardiac trabeculae containing V1 isomyosin versus those containing V3 isomyosin. METHODS: V1 isomyosin was induced by thyroid hormone treatment of the rats for 2 weeks, V3 isomyosin by PTU treatment for 1 month. Force was measured with a strain gauge in trabeculae from the rat right ventricle in K-H solution ([Ca]o=1.5 mM, 25 degrees C). Sarcomere length (SL) was measured with laser diffraction techniques. Twitch force at constant SL, and the force response to shortening at constant VSL (0-8 microm/s; deltaSL 50-100 nm) were measured at varied time during the twitch. RESULTS: The force response to shortening consisted of a fast initial exponential decline (tau = 2 ms) followed by a slow decrease of F. The instantaneous difference (deltaF) between isometric force (FM) and the declining force depended on shortening duration (deltat), VSL and instantaneous FM: deltaF = G1 x FM x deltat x VSL x (1-VSL/VMAX), where VMAX is the unloaded VSL and G1 was 6.15 +/- 2.12 microm(-1) (mean +/- s.d.; n=6). deltaF/FM was independent of the time onset of shortening. G1 of V1 and V3 trabeculae did not differ. V0 of V1 and V3 trabeculae differed 2-2.5 fold, as did both the ATPase rate and the velocity of actin sliding in a motility assay of the myosin purified from V1 or V3 hearts. The temperature dependence of the ATPase rate (Q10: 4.03 and 4.33, respectively; n.s.) was similar to that of V0 that has previously been reported for predominantly V1 trabeculae. Cross-linking of actin to myosin with the short chain cross linker EDC increased the ATPase rate of the two isomyosins (200-fold and 600-fold respectively) to exactly the same final level and reduced their Q10 by 50%. CONCLUSION: The linear interrelation between deltaF and VSL is consistent with feedback, whereby XB kinetics depends on VSL. This feedback provides an integrated description of cardiac muscle mechanics and energetics. The results, also, suggests that it is unlikely that the hydrolytic domain of the cross bridge determines V0 and warrant ongoing experiments to investigate the role of the actin binding domain of the XB in cardiac sarcomere kinetics. In order to further investigate the role of the actin binding domain, we have expressed chimeric cardiac myosin, co-assembled with MLC, by mutual substitution of actin binding loop on alpha MHC and beta MHC.

Shortening properties of two biochemically defined muscle fibre types of the Norway lobster Nephrops norvegicus L.

Mechanical properties of myofibrillar bundles from single chemically skinned fibres from the superficial abdominal flexor muscle of the Norway lobster Nephrops norvegicus were measured, and the protein content of these fibres was analysed by SDS-PAGE. Two slow fibre phenotypes (S1, S2) were distinguished on the basis of their myofibrillar protein assemblages. Data from 9 S1 and 8 S2 fibres obtained at similar sarcomere length demonstrate significant differences between the fibre types in maximal tension (N cm-2, S1: 10.5 +/- 3.9; S2: 3.1 +/- 0.8), in the delay of the peak of stretch activation (ms, S1: 122 +/- 18; S2: 412 +/- 202), in fibre stiffness (N cm-2 per nm half sarcomere, S1: 0.36 +/- 0.19; S2: 0.09 +/- 0.03) and in maximal shortening velocity (fibre length s-1, S1: 0.53 +/- 0.10; S2: 0.27 +/- 0.06). Furthermore, the maximal power output of the type S1 fibres was about five times larger than that of S2 fibres. The power output was maximal at lower loads in S1 fibres (relative load = 0.37 +/- 0.04) than in S2 fibres (relative load = 0.44 +/- 0.05). This study represents a comprehensive investigation of two slow muscle fibre types which are thought to be specialized for slow movements (S1 fibres) and for the postural control of the abdomen (S2 fibres).

Actin mRNA levels and myofibrillar growth in leg muscles of the European lobster (Homarus gammarus) in response to passive stretch.

An actin cDNA clone from the European lobster, Homarus gamarus, has been generated by RT-PCR and used as a probe to quantify the relative abundance of actin mRNA in lobster leg muscles following imposition of passive stretch by leg flexion. The sarcomere lengths of a population of fibers in the same muscles were measured to provide an indirect marker of myofibrillar growth. Stretch resulted in a 70% increase in actin mRNA levels compared with unstretched controls animals between weeks 1 and 2 after flexion of the legs. Sarcomere lengths increased by 23% immediately after imposition of the stretch. During the same period of observed increase in actin mRNA, the sarcomere lengths returned to their initial values, indicating that longitudinal growth of the myofibrils had occurred. Results are discussed in relation to the role of stretch in crustacean muscle growth during the moult.