Biomedical subjects

T E Hewett

Publications and source records attributed to T E Hewett.

At least 19 recordsLinked to original sources

PubMed2001

PKA-dependent phosphorylation of cardiac myosin binding protein C in transgenic mice.

OBJECTIVE: To investigate the physiological role of cAMP-dependent protein kinase A (PKA)-mediated, cardiac myosin binding protein C (MyBP-C) phosphorylation. METHODS: A cardiac MyBP-C cDNA lacking nine amino acids, which contained a phosphorylation site, was made, and subsequently used to generate multiple lines of transgenic mice. Upon confirming that a partial replacement of endogenous protein with transgenic protein occurred, the biochemical and physiological consequences were studied. PKA-dependent phosphorylation assays were used to estimate the phosphorylation states of major cardiac PKA substrates. Myofibril Mg-ATPase activities were also measured. Isolated working heart and whole animal exercise studies were used to measure the physiological changes. RESULTS: Transgenic mice displayed a compensatory response, with PKA-mediated phosphorylation of both troponin I and phospholamban showing significant increases. The remaining endogenous cardiac MyBP-C also showed increased phosphorylation levels. Maximal Mg(2+)-ATPase activity was increased. Significant functional changes at both the whole organ and whole animal levels also occurred. Parameters reflecting cardiac contractility and relaxation increased about 22 and 25%, respectively, in the mutant relative to wild type mice (n=5, P<0.001). In young adults the capacity for stress exercise, quantitated using an exercise treadmill regimen, was substantially enhanced (n=6, P<0.01). CONCLUSIONS: Cardiac MyBP-C phosphorylation plays an important physiological role and that the protein's degree of phosphorylation is coordinated with the phosphorylation levels of other proteins within the contractile apparatus.

Animals↗

PubMed2000

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice.

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.

Actinin↗

PubMed2000

Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy.

Multiple mutations in cardiac troponin I (cTnI) have been associated with familial hypertrophic cardiomyopathy. Two mutations are located in the cTnI inhibitory domain, a highly negatively charged region that alternately binds to either actin or troponin C, depending on the intracellular concentration of calcium. This region is critical to the inhibition of actin-myosin crossbridge formation when intracellular calcium is low. We modeled one of the inhibitory domain mutations, arginine145-->glycine (TnI(146Gly) in the mouse sequence), by cardiac-specific expression of the mutated protein in transgenic mice. Multiple lines were generated with varying degrees of expression to establish a dose relationship; the severity of phenotype could be correlated directly with transgene expression levels. Transgenic mice overexpressing wild-type cTnI were generated as controls and analyzed in parallel with the TnI(146Gly) animals. The control mice showed no abnormalities, indicating that the phenotype of TnI(146Gly) was not simply an artifact of transgenesis. In contrast, TnI(146Gly) mice showed cardiomyocyte disarray and interstitial fibrosis and suffered premature death. The functional alterations that seem to be responsible for the development of cardiac disease include increased skinned fiber sensitivity to calcium and, at the whole organ level, hypercontractility with diastolic dysfunction. Severely affected lines develop a pathology similar to human familial hypertrophic cardiomyopathy but within a dramatically shortened time frame. These data establish the causality of this mutation for cardiac disease, provide an animal model for understanding the resultant pathogenic structure-function relationships, and highlight the differences in phenotype severity of the troponin mutations between human and mouse hearts.

Actins↗

PubMed2000

In vivo analysis of an essential myosin light chain mutation linked to familial hypertrophic cardiomyopathy.

Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutations in both the regulatory (Glu22Lys) and essential light chains (Met149Val) result in an unusual pattern of hypertrophy, leading to obstruction of the midventricular cavity. When a human genomic fragment containing the Met149Val essential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguously establish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, we generated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the coding regions of the gene loci. Expression of the proteins did not lead to a hypertrophic response, even in senescent animals. Changes did occur at the myofilament and cellular levels, with the myofibrils showing increased Ca(2+) sensitivity and significant deficits in relaxation in a transgene dose-dependent manner. Clearly, mice do not always recapitulate important aspects of human hypertrophy. However, because of the discordance of these data with data obtained in transgenic mice containing the human genomic fragment, we believe that the concept that these point mutations by themselves can cause hypertrophic cardiomyopathy should be revisited.

Animals↗

PubMed2000

Hypertrophic defect unmasked by calcineurin expression in asymptomatic tropomodulin overexpressing transgenic mice.

OBJECTIVE: Dilation and hypertrophy often occur concurrently in cardiomyopathy, yet the interaction between these two functionally distinct conditions remains unknown. METHODS: Combinatorial effects of hypertrophy and dilation were investigated by cross-breeding of two cardiomyopathic transgenic mouse lines which develop either hypertrophy (calcineurin-mediated) or dilation (tropomodulin-mediated). RESULTS: Altering the intensity of signals driving hypertrophy and dilation in cross-bred litters resulted in novel disease phenotypes different from either parental line. Augmenting the calcineurin-dependent hypertrophic stimulus in tropomodulin overexpressing transgenics elevated heart:body weight ratios, increased ventricular wall thickness, and significantly accelerated mortality. These effects were evident in calcineurin cross-breeding to tropomodulin backgrounds of transgene homozygosity (severe dilation) or heterozygosity (mild dilation to asymptomatic). Molecular analyses indicated that tropomodulin and calcineurin signaling events in the first week after birth were critical for determination of disease outcome, substantiated by demonstration that temporary neonatal inhibition of tropomodulin expression prevents dilation. CONCLUSIONS: This study shows that postnatal timing of altered signaling in cardiomyopathic transgenic mouse models is a pivotal part of determining outcome. In addition, intensifying hypertrophic stimulation exacerbates dilated cardiomyopathy, supporting the concept of shared molecular signaling between hypertrophy and dilation.

Animals↗

PubMed2000

Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects.

Alpha- and beta-myosin heavy chain (MHC), the two MHC isoforms expressed in the mammalian heart, differ quantitatively in their enzymatic activities. The MHC composition of the heart can change dramatically in response to numerous stimuli, leading to the hypothesis that changes in cardiac function can be caused by myosin isoform shifts. However, this hypothesis has remained unproven because the stimuli used to generate these shifts are complex and accompanied by many additional physiological changes, including alterations in cardiac mass and geometry. Adult mouse ventricles normally express only alpha-MHC (the faster motor). To determine whether genetic alteration of the MHC isoform composition in the adult mouse heart would result in changes in cardiac chamber mass and contractility, we established transgenic mouse lines that express a Myc-tagged beta-MHC molecule (the slower motor) in adult ventricular tissue, one of which expresses 12% of its myosin as the transgene. There is no evidence of hypertrophy, induction of hypertrophic markers, and no histopathology. Myofibrillar Ca(2+)-activated ATPase activity is decreased by 23%, and Langendorff preparations demonstrate a significant 15% decrease in systolic function in transgenic hearts. These results suggest that even small shifts in the myosin isoform composition of the myocardium can result in physiologically significant changes in cardiac contractility and could be relevant to cardiovascular disease.

Animals↗

PubMed2000

Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1.

The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1-mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling.

Animals↗

PubMed2000

Neuromuscular and hormonal factors associated with knee injuries in female athletes. Strategies for intervention.

Female athletes who participate in jumping and cutting sports are 4 to 6 times more likely to sustain a serious knee injury than male athletes participating in the same sports. More than 30,000 serious knee injuries are projected to occur in female intercollegiate and high school athletics in the US each year. The majority of these injuries occur by non-contact mechanisms, most often during landing from a jump or making a lateral pivot while running. Knee instability, due possibly to decreased neuromuscular strength and coordination or increased ligamentous laxity, may underlie the increased incidence of knee injury in females. Neuromuscular training can significantly increase dynamic knee stability in female athletes. Female sex hormones (i.e. estrogen, progesterone and relaxin) fluctuate radically during the menstrual cycle and are reported to increase ligamentous laxity and decrease neuromuscular performance and, thus, are a possible cause of decreases in both passive and active knee stability in female athletes. Oral contraceptives stabilise hormone levels during the menstrual cycle and may function to either passively or actively stabilise the knee joint. The long term objective of clinicians and researchers should be to determine the factors that make women more susceptible than men to knee ligament injury and to develop treatment modalities to aid in the prevention of these injuries. The immediate objectives of this review are to examine how female and male athletes differ in neuromuscular and ligamentous control of the lower extremity. The review will examine the effects of neuromuscular training on knee stability. The effects of female hormone levels and oral contraceptives on neuromuscular control of the female athletes' knee will also be discussed.

Athletic Injuries↗

PubMed1999

In vivo modeling of myosin binding protein C familial hypertrophic cardiomyopathy.

Myosin binding protein C (MyBP-C) is an integral part of the striated muscle sarcomere. As is the case for other sarcomeric genes in human populations, multiple mutations within the gene have been linked to familial hypertrophic cardiomyopathy. Although some MyBP-C lesions are the result of missense mutations, most show truncated polypeptides lacking either the myosin or myosin and titin binding sites. Previously, we generated transgenic (TG) mice with cardiac-specific expression of a MyBP-C mutant lacking the myosin and titin binding domains. Surprisingly, the mutant protein was stable and made up a majority of the MyBP-C species, with concomitant reductions in endogenous MyBP-C such that overall MyBP-C stoichiometry was conserved. In the present study, we created a second series of TG mice that express, in the heart, a mutant MyBP-C lacking only the myosin binding site. In contrast to the previous data for the MyBP-C lacking both titin and myosin binding sites, only very modest levels of protein were found, consistent with data obtained from human biopsies in which mutated MyBP-C could not be detected. Despite normal levels of wild-type MyBP-C, there were significant changes in the structure and ultrastructure of the heart. Fiber mechanics showed decreased unloading shortening velocity, maximum shortening velocity, and relative maximal power output.

Animals↗

PubMed1999

Transgenic over-expression of a motor protein at high levels results in severe cardiac pathology.

Transgenesis has become a useful tool in effecting a complete or partial remodeling of the cardiac contractile apparatus. Although gene dosage effects were initially a concern, recent data showed that the heart is able to accommodate varying levels of transgenic over-expression without detectable ill effects. The present study was designed to test the limits of the transgenic paradigm in terms of the production of a cardiac phenotype due simply to the over-expression of a contractile protein. To this end, eight lines of mice which express an isoform of the essential myosin light chain 1 that is normally found in the adult ventricle (ELC1v) were generated. Overt phenotype was correlated both with the level of expression/protein replacement and copy number of the transgene. Two of the lines showed essentially complete replacement of the atrial isoform (ELC1a) with ELC1v. However, the phenotypes of the two lines differed dramatically. The line with the lower copy number (37 copies), and moderate over-expression (16 fold) showed no overt pathology while a line with very high copy number (94 copies) and extremely high levels of over-expression (27-50 fold) developed a significant atrial hypertrophy, dilation and cardiomyopathy. These data indicate that very high expression levels of a contractile protein can cause a cardiac pathology that is unrelated to its degree of replacement in the sarcomere and the unique role(s) it may assume in motor protein function.

Actins↗

PubMed1999

Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy.

Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and beta-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.

Alleles↗

PubMed1998

A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy can be caused by mutations in genes encoding sarcomeric proteins, including the cardiac isoform of myosin binding protein C (MyBP-C), and multiple mutations which cause truncated forms of the protein to be made are linked to the disease. We have created transgenic mice in which varying amounts of a mutated MyBP-C, lacking the myosin and titin binding domains, are expressed in the heart. The transgenically encoded, truncated protein is stable but is not incorporated efficiently into the sarcomere. The transgenic muscle fibers showed a leftward shift in the pCa2+-force curve and, importantly, their power output was reduced. Additionally, expression of the mutant protein leads to decreased levels of endogenous MyBP-C, resulting in a striking pattern of sarcomere disorganization and dysgenesis.

Animals↗

PubMed1998

A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy.

Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.

Animals↗

PubMed1998

Functional significance of cardiac myosin essential light chain isoform switching in transgenic mice.

The different functions of the ventricular- and atrial-specific essential myosin light chains are unknown. Using transgenesis, cardiac-specific overexpression of proteins can be accomplished. The transgenic paradigm is more useful than originally expected, in that the mammalian heart rigorously controls sarcomeric protein stoichiometries. In a clinical subpopulation suffering from heart disease caused by congenital malformations of the outflow tract, an ELC1v-->ELC1a isoform shift correlated with increases in cross-bridge cycling kinetics as measured in skinned fibers derived from the diseased muscle. We have used transgenesis to replace the ventricular isoform of the essential myosin light chain with the atrial isoform. The ELC1v--> ELC1a shift in the ventricle resulted in similar functional alterations. Unloaded velocities as measured by the ability of the myosin to translocate actin filaments in the in vitro motility assay were significantly increased as a result of the isoform substitution. Unloaded shortening velocity was also increased in skinned muscle fibers, and at the whole organ level, both contractility and relaxation were significantly increased. This increase in cardiac function occurred in the absence of a hypertrophic response. Thus, ELC1a expression in the ventricle appears to be advantageous to the heart, resulting in increased cardiac function.

Amino Acid Sequence↗

PubMed1998

Cardiac physiology in transgenic mice.

By use of gene targeting and/or transgenesis, it is now possible to make defined changes in genes whose functions underlie mammalian cardiovascular function. Because of technical and economic considerations, these experiments are largely confined to the mouse. Genetic modification of the loci responsible for aspects of cardiac development, differentiation, and function via gene targeting, as well as modulation of the cardiac protein complement using transgenesis, has begun to provide mouse models of cardiac hypertrophy, dilated cardiomyopathy, and hypertrophic cardiomyopathies. In order to use these animal models fully and explore their phenotypes at the whole organ and whole animal levels, the extension of cardiovascular physiological methodologies to the mouse is imperative. Techniques for exploring aspects of cardiovascular function are well developed for larger animal models, but their modification for the small size of the mouse heart and for the animal's rapid cardiac cycle has proven to be a formidable challenge, requiring the combined efforts of the molecular biology, physiology, and cardiology communities. We review here the ability of present-day technology to obtain reproducible data on murine cardiac function at the whole organ and animal levels.

Animals↗

PubMed1998

Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice.

Loss of myofibril organization is a common feature of chronic dilated and progressive cardiomyopathy. To study how the heart compensates for myofibril degeneration, transgenic mice were created that undergo progressive loss of myofibrils after birth. Myofibril degeneration was induced by overexpression of tropomodulin, a component of the thin filament complex which determines and maintains sarcomeric actin filament length. The tropomodulin cDNA was placed under control of the alpha-myosin heavy chain gene promoter to overexpress tropomodulin specifically in the myocardium. Offspring with the most severe phenotype showed cardiomyopathic changes between 2 and 4 wk after birth. Hearts from these mice present characteristics consistent with dilated cardiomyopathy and a failed hypertrophic response. Histological analysis showed widespread loss of myofibril organization. Confocal microscopy of isolated cardiomyocytes revealed intense tropomodulin immunoreactivity in transgenic mice together with abnormal coincidence of tropomodulin and alpha-actinin reactivity at Z discs. Contractile function was compromised severely as determined by echocardiographic analyses and isolated Langendorff heart preparations. This novel experimentally induced cardiomyopathy will be useful for understanding dilated cardiomyopathy and the effect of thin filament-based myofibril degeneration upon cardiac structure and function.

Animals↗

PubMed1998

Physical and arthroscopic examination techniques of the patellofemoral joint.

A systematic approach to the clinical history, physical, and arthroscopic examination of patellofemoral disorders will lead to improved diagnostic accuracy and clinical treatment success. We review important aspects of physical and arthroscopic examination of patellofemoral disorders Basic and advanced physical examination techniques are presented, and their clinical significance is reviewed. Arthroscopic examination of the patellofemoral joint is used as an adjunct to physical examination to evaluate chondral lesions of the patella and femoral sulcus and to visualize patella tracking. Techniques to assess patellar tracking and the integrity of patellar restraints and to grade chondral lesions are outlined. Utilization of these techniques will improve clinical studies on the treatment of patellofemoral disorders.

Arthroscopy↗

PubMed1998

Decrease in knee joint pain and increase in function in patients with medial compartment arthrosis: a prospective analysis of valgus bracing.

We studied a brace designed to decrease loads on the medial tibiofemoral compartment in knees with chronic pain and arthrosis to determine if pain symptoms decreased, function improved, and dynamic gait characteristics altered during walking. Eighteen patients with symptomatic medial compartment arthrosis were fitted with a commercially available brace. All were evaluated after an average of 9 weeks of brace wear, and 13 patients were evaluated after 1 year of brace wear. The Cincinnati Knee Rating System and additional pain scales were used to analyze symptoms and functional limitations. Nine subjects underwent a dynamic gait analysis and were compared with a control group of 11 normal subjects matched for age and walking speed. The brace was worn an average of 7 hours a day, 5 days a week. Following 9 weeks of brace wear, statistically significant improvements were found for all pain parameters, and these improvements continued at the 1 year evaluation. Before brace wear, 78% had pain with activities of daily living, but after the first evaluation, only 39% continued to have such pain, and at the second evaluation, only 31% were so affected. Before brace wear, patients had a walking tolerance of 51 minutes prior to the onset of pain symptoms. At the first evaluation, patients could walk 138 minutes without pain, and after 1 year, they could walk 107 minutes without pain. Before brace wear, 78% rated their overall knee condition as fair or poor whereas at the first evaluation, only 33% continued to provide this rating. No differences were found in the dynamic gait parameters measured with and without the brace. While this brace did not provide the dramatic improvements in symptoms, function, and patient satisfaction obtainable after high tibial osteotomy, it did help the majority of patients. If the goal of brace use is to buy a short amount of time for patients who cannot undergo or wish to avoid osteotomy or knee arthroplasty, then bracing appears to offer a reasonable alternative for short-term pain relief and improved function.

Adult↗