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Biomedical subjects

Keith Baar

Publications and source records attributed to Keith Baar.

16 recordsLinked to original sources
PubMed2006

Denervation does not change the ratio of collagen I and collagen III mRNA in the extracellular matrix of muscle.

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type III and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 +/- 1.6%, 73 +/- 2.3%, and 83 +/- 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red confirmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity.

Animals↗
PubMed2006

Regional variation of tibialis anterior tendon mechanics is lost following denervation.

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle. The mechanical response of tendon to inactivity that has been determined experimentally differs from what is reported by patients. We investigated the hypothesis that this difference was the result of artifacts of the testing process and did not represent what occurred in vivo. To test this hypothesis, a novel approach was used to determine the mechanical properties of the tibialis anterior (TA) tendon by optically measuring the end-to-end mechanical strains as well as the local strains at specific regions of excised TA tendon units. When the end-to-end strain of normal TA tendon is determined, stress-strain response curves show considerably more extensibility than when strain is measured across only the midsection of the tendon (mid-tendon). The strain experienced by the region close to the muscle (muscle tendon) is five times greater than the strain in either the mid-tendon or near the bone (bone-tendon). Five weeks of denervation decreased muscle mass by 67%; increased tendon mass by 10%; and changed the entire shape of the nonlinear response curve, including a loss in regional variation in strain, a 3.9-fold increase in end-to-end tangent modulus, and a 70% reduction in the toe region, as a result of a drastic reduction of the extensibility in the muscle-tendon region. The stress-strain response in the mid-tendon region of a normal TA tendon is therefore not indicative of its overall ability to deform in vivo as it transmits forces from muscle to bone.

Animals↗
PubMed2006

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation.

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT(1/2)) were approximately 30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT(1/2), but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT(1/2). The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation.

Animals↗
PubMed2006

Resistance exercise, muscle loading/unloading and the control of muscle mass.

Muscle mass is determined by the difference between the rate of protein synthesis and degradation. If synthesis is greater than degradation, muscle mass will increase (hypertrophy) and when the reverse is true muscle mass will decrease (atrophy). Following resistance exercise/increased loading there is a transient increase in protein synthesis within muscle. This change in protein synthesis correlates with an increase in the activity of protein kinase B/Akt and mTOR (mammalian target of rapamycin). mTOR increases protein synthesis by increasing translation initiation and by inducing ribosomal biogenesis. By contrast, unloading or inactivity results in a decrease in protein synthesis and a significant increase in muscle protein breakdown. The decrease in synthesis is due in part to the inactivation of mTOR and therefore a decrease in translation initiation, but also to a decrease in the rate of translation elongation. The increase in degradation is the result of a co-ordinated response of the calpains, lysosomal proteases and the ATP-dependent ubiquitin-proteosome. Caspase 3 and the calpains act upstream of the ubiquitin-proteosome system to assist in the complete breakdown of the myofibrillar proteins. Two muscle specific E3 ubiquitin ligases, MuRF1 and MAFbx/atrogen-1, have been identified as key regulators of muscle atrophy. In this chapter, these pathways and how the balance between anabolism and catabolism is affected by loading and unloading will be discussed.

Animals↗
PubMed2006

Activation of Akt as a potential mediator of adaptations that reduce muscle injury.

UNLABELLED: Contraction-induced injury occurs when a muscle is stretched while activated (lengthening contraction). Exposure to a bout of lengthening contractions results in protection from subsequent lengthening contraction-induced injury as well as an elevation in phosphorylated Akt and p70S6K. Whether Akt or p70S6K is involved in the protection from contraction-induced injury is unclear. To test for a specific role of Akt and/or p70S6K to induce protective adaptations, we used a conditioning protocol of passive stretches that reduces contraction-induced injury with minimal involvement of other cellular responses that have been associated with the Akt signaling pathway, such as increased metabolism, cell growth, and cell death. PURPOSE: To determine whether activation of Akt or p70S6K is necessary to induce protective adaptations. METHODS: Extensor digitorum longus muscles of anesthetized mice were administered 75 lengthening contractions in situ with or without previous exposure to 75 passive stretches 1 h, 24 h, 3 d, or 14 d prior to lengthening contractions. RESULTS: Compared with unconditioned muscles, the deficit in isometric force and number of injured fibers 3 d following lengthening contractions were smaller by half for passive-stretch-conditioned muscles from all time points. Phosphorylation of Akt and p70S6K were analyzed by Western blot 0 or 3 h following either lengthening contractions or passive stretches. Whereas lengthening contractions increased phosphorylation of Akt at 0 h and p70S6K at 3 h, passive stretches did not at any time increase phosphorylation of Akt or p70S6K despite reducing contraction-induced injury. CONCLUSION: Activation of neither Akt nor p70S6K is necessary to induce adaptations that reduce the severity of contraction-induced injury.

Adaptation, Physiological↗
PubMed2006

Training for endurance and strength: lessons from cell signaling.

The classic work of Hickson demonstrated that training for both strength and endurance at the same time results in less adaptation compared with training for either one alone: this has been described as the concurrent training effect. Generally, resistance exercise results in an increase in muscle mass, and endurance exercise results in an increase in muscle capillary density, mitochondrial protein, fatty acid-oxidation enzymes, and more metabolically efficient forms of contractile and regulatory proteins. In the 25 yr since Hickson's initial description, there have been a number of important advances in the understanding of the molecular regulation of muscle's adaptation to exercise that may enable explanation of this phenomenon at the molecular level. As will be described in depth in the following four papers, two serine/threonine protein kinases in particular play a particularly important role in this process. Protein kinase B/Akt can both activate protein synthesis and decrease protein breakdown, thus leading to hypertrophy, and AMP-activated protein kinase can increase mitochondrial protein, glucose transport, and a number of other factors that result in an endurance phenotype. Not only are PKB and AMPK central to the generation of the resistance and endurance phenotypes, they also block each other's downstream signaling. The consequence of these interactions is a direct molecular blockade hindering the development of the concurrent training phenotype. A better understanding of the activation of these molecular pathways after exercise and how they interact will allow development of better training programs to maximize both strength and endurance.

Adenylate Kinase↗
PubMed2005

New dimensions in tissue engineering: possible models for human physiology.

Tissue engineering is a discipline of great promise. In some areas, such as the cornea, tissues engineered in the laboratory are already in clinical use. In other areas, where the tissue architecture is more complex, there are a number of obstacles to manoeuvre before clinically relevant tissues can be produced. However, even in areas where clinically relevant tissues are decades away, the tissues being produced at the moment provide powerful new models to aid the understanding of complex physiological processes. This article provides a personal view of the role of tissue engineering in advancing our understanding of physiology, with specific attention being paid to musculoskeletal tissues.

Animals↗
PubMed2005

Development of a three-dimensional physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells.

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction.

Acetylcholine↗
PubMed2004

Self-organization of rat cardiac cells into contractile 3-D cardiac tissue.

The mammalian heart is not known to regenerate following injury. Therefore, there is great interest in developing viable tissue-based models for cardiac assist. Recent years have brought numerous advances in the development of scaffold-based models of cardiac tissue, but a self-organizing model has yet to be described. Here, we report the development of an in vitro cardiac tissue without scaffolding materials in the contractile region. Using an optimal concentration of the adhesion molecule laminin, a confluent layer of neonatal rat cardiomyogenic cells can be induced to self-organize into a cylindrical construct, resembling a papillary muscle, which we have termed a cardioid. Like endogenous heart tissue, cardioids contract spontaneously and can be electrically paced between 1 and 5 Hz indefinitely without fatigue. These engineered cardiac tissues also show an increased rate of spontaneous contraction (chronotropy), increased rate of relaxation (lusitropy), and increased force production (inotropy) in response to epinephrine. Cardioids have a developmental protein phenotype that expresses both alpha- and beta-tropomyosin, very low levels of SERCA2a, and very little of the mature isoform of cardiac troponin T.

Animals↗
PubMed2004

Rapid formation of functional muscle in vitro using fibrin gels.

The transition of a muscle cell from a differentiated myotube into an adult myofiber is largely unstudied. This is primarily due to the difficulty of isolating specific developmental stimuli in vivo and the inability to maintain viable myotubes in culture for sufficient lengths of time. To address these limitations, a novel method for rapidly generating three-dimensional engineered muscles using fibrin gel casting has been developed. Myoblasts were seeded and differentiated on top of a fibrin gel. Cell-mediated contraction of the gel around artificial anchors placed 12 mm apart culminates 10 days after plating in a tubular structure of small myotubes (10-microm diameter) surrounded by a fibrin gel matrix. These tissues can be connected to a force transducer and electrically stimulated between parallel platinum electrodes to monitor physiological function. Three weeks after plating, the three-dimensional engineered muscle generated a maximum twitch force of 329 +/- 26.3 microN and a maximal tetanic force of 805.8 +/- 55 microN. The engineered muscles demonstrated normal physiological function including length-tension and force-frequency relationships. Treatment with IGF-I resulted in a 50% increase in force production, demonstrating that these muscles responded to hormonal interventions. Although the force production was maximal at 3 wk, constructs can be maintained in culture for up to 6 wk with no intervention. We conclude that fibrin-based gels provide a novel method to engineer three-dimensional functional muscle tissue and that these tissues may be used to model the development of skeletal muscle in vitro.

Animals↗
PubMed2004

Involvement of PPAR gamma co-activator-1, nuclear respiratory factors 1 and 2, and PPAR alpha in the adaptive response to endurance exercise.

Endurance exercise training induces an increase in the respiratory capacity of muscle, resulting in an increased capacity to generate ATP as well as improved efficiency of muscle contraction. Such adaptations are largely the result of a coordinated genetic response that increases mitochondrial proteins, fatty acid oxidation enzymes and the exercise- and insulin-stimulated glucose transporter GLUT4, and shifts the contractile and regulatory proteins to their more efficient isoforms. In recent years a number of the transcriptional regulators involved in this genetic response have been identified and these factors can be classified into two different groups. The first group comprises transcription factors such as nuclear respiratory factors (NRF) 1 and 2 and PPAR alpha that bind DNA in a sequence-specific manner. The second group, referred to as transcriptional co-activators, alter transcription without directly binding to DNA. The PPAR gamma co-activator (PGC) family of proteins have been identified as the central family of transcriptional co-activators for induction of mitochondrial biogenesis. PGC-1 alpha is activated by exercise, and is sufficient to produce the endurance phenotype through direct interactions with NRF-1 and PPAR alpha, and potentially NRF-2. Furthering the understanding of the activation of PGC proteins following exercise has implications beyond improving athletic performance, including the possibility of providing targets for the treatment of frailty in the elderly, obesity and diseases such as mitochondrial myopathies and diabetes.

Adaptation, Physiological↗
PubMed2003

Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity.

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.

Animals↗
PubMed2002

Exercise induces an increase in muscle UCP3 as a component of the increase in mitochondrial biogenesis.

Previous studies have indicated that exercise acutely induces large increases in uncoupling protein-3 (UCP3) in skeletal muscle, whereas endurance training results in marked decreases in muscle UCP3. Because UCP3 expression appears to be regulated by the same mechanism as other mitochondrial constituents, it seemed unlikely that exercise would result in such large and divergent changes in mitochondrial composition. The purpose of this study was to test the hypothesis that major changes in UCP3 protein concentration do not occur independently of mitochondrial biogenesis and that UCP3 increases as a component of the exercise-induced increase in mitochondria. We found a large increase in UCP3 mRNA immediately and 3 h after a bout of swimming. UCP3 protein concentration was increased approximately 35% 18 h after a single exercise bout, approximately 63% after 3 days, and approximately 84% after 10 days of exercise. These increases in UCP3 roughly paralleled those of other mitochondrial marker proteins. Our results are consistent with the interpretation that endurance exercise induces an adaptive increase in mitochondria that have a normal content of UCP3.

Animals↗
PubMed2002

Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1.

Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

Adaptation, Physiological↗
PubMedDate not supplied

Engineering of functional tendon.

Surgical tendon repair is limited by the availability of viable tissue for transplantation. Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. To address this problem, cells isolated from rat Achilles tendon were grown to confluence in culture and allowed to self-assemble into a cylinder between two anchor points. The resulting scaffold-free tissue was composed of aligned, small-diameter collagen fibrils, a large number of cells, and an excess of noncollagenous extracellular matrix; all characteristics of embryonic tendon. The stress-strain response of the constructs also resembles the nonlinear behavior of immature tendons, and the ultimate tensile strength is approximately equal to that of embryonic chick tendon, roughly 2 MPa. These physical and mechanical properties indicate that these constructs are the first viable tendons engineered in vitro, without the aid of artificial scaffolding.

Animals↗