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Biomedical subjects

T E Hewett

Publications and source records attributed to T E Hewett.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1997

Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice.

Retinoids play a critical role in cardiac morphogenesis. To examine the effects of excessive retinoid signaling on myocardial development, transgenic mice that overexpress a constitutively active retinoic acid receptor (RAR) controlled by either the alpha- or beta-myosin heavy chain (MyHC) promoter were generated. Animals carrying the alpha-MyHC-RAR transgene expressed RARs in embryonic atria and in adult atria and ventricles, but developed no signs of either malformations or disease. In contrast, beta-MyHC-RAR animals, where expression was activated in fetal ventricles, developed a dilated cardiomyopathy that varied in severity with transgene copy number. Characteristic postmortem lesions included biventricular chamber dilation and left atrial thrombosis; the incidence and severity of these lesions increased with increasing copy number. Transcript analyses showed that molecular markers of hypertrophy, alpha-skeletal actin, atrial natriuretic factor and beta-MyHC, were upregulated. Cardiac performance of transgenic hearts was evaluated using the isolated perfused working heart model as well as in vivo, by transthoracic M-mode echocardiography. Both analyses showed moderate to severe impairment of left ventricular function and reduced cardiac contractility. Thus, expression of a constitutively active RAR in developing atria and/ or in postnatal ventricles is relatively benign, while ventricular expression during gestation can lead to significant cardiac dysfunction.

Animals↗
PubMed1997

Transgenic remodeling of the regulatory myosin light chains in the mammalian heart.

The regulatory myosin light chain (MLC) regulates contraction in smooth muscle. However, its function in striated muscle remains obscure, and the different functional activities of the various isoforms that are expressed in the mammalian heart (ventricle- and atrium-specific MLC2) remain undefined. To begin to explore these issues, we used transgenesis to determine the feasibility of effecting a complete or partial replacement of the cardiac regulatory light chains with the isoform that is normally expressed in fast skeletal muscle fibers (fast muscle-specific MLC2). Multiple lines of transgenic mice were generated that expressed the transgene at varying levels in the heart in a copy number-dependent fashion. There is a major discordance in the manner in which the different cardiac compartments respond to high levels of overexpression of the transgene. In atria, isoform replacement with the skeletal protein was quite efficient, even at low copy number. The ventricle is much more refractory to replacement, and despite high levels of transgenic transcript, protein replacement was incomplete. Replacement could be further increased by breeding the transgenic lines with one another. Despite very high levels of transgenic transcript in these mice, the overall level of the regulatory light chain in both compartments remained essentially constant; only the protein isoform ratios were altered. The partial replacement of the ventricular with the skeletal isoform reduced both left ventricular contractility and relaxation, although the unloaded shortening velocity of isolated ventricular cardiomyocytes was not significantly different.

Animals↗
PubMed1997

Joint loading with valgus bracing in patients with varus gonarthrosis.

The purpose of this study was to determine whether a brace designed to unload varus degenerative knees actually alters medial compartment loads by decreasing the adduction moment. Eleven patients who had arthrosis confined to the medial compartment were fitted with a valgus brace and tested before and after brace wear with pain and function scoring instruments and by automated gait analysis. The biomechanical data from these patients were compared with those from 11 healthy control subjects. Scores from an analog pain scale decreased 48% with brace wear, and function with activities of daily living increased 79%. Mean adduction moment without the brace measured 4.0 +/- 0.8% body weight times height versus 3.6 +/- 0.8% body weight times height when wearing the brace (10% decrease). The mean adduction moment for control subjects was 3.5 +/- 0.6% body weight times height. Thus, the mean adduction moment decreased from approximately one standard deviation from the normal mean to a value that is similar to the control value. Nine of 11 patients had a decrease in the adduction moment with the brace, five of 11 patients had a reduction higher than 10%, and decreases in this moment were as high as 32%. This study shows that pain, function, and biomechanical knee loading can be altered by a brace designed to unload the medial compartment of the knee.

Arthritis↗
PubMed1996

Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Alleles↗
PubMed1996

Lower limb alignment and foot angle are related to stance phase knee adduction in normal subjects: a critical analysis of the reliability of gait analysis data.

Anatomic and mechanical factors that affect loading in the knee joint can contribute to pathologic changes seen at the knee in degenerative joint disease and should be considered in treatment planning. The objectives of this study were to quantify the relationships between the alignment of the bones of the lower extremity, foot progression angle, and knee adduction moment, and to determine the reliability of our gait measurements. Gait analysis and complete radiographic evaluation of the lower extremity were performed on 11 healthy subjects. The gait measurements were recorded with an optoelectronic digitizer and a multi-component force plate. The subjects who had radiographic measurements indicative of varus alignment of the lower extremity had statistically higher peaks in knee adduction moment in early stance. Conversely, those with valgus alignment of the lower extremity had statistically lower peaks in knee adduction moment in early stance. The subjects who had a large toe-out angle and low ankle inversion moment peaks in late stance had significantly lower peaks in knee adduction moment in late stance. These significant (low to moderate) correlations suggest that the limbs with more valgus alignment and those with a toe-out gait exhibited a reduced peak adduction moment at the knee. To verify the reproducibility of the data, gait analysis testing was performed on each lower limb on 2 separate days for each subject. Analysis of variance showed that there was no significant difference between test limbs or test days for each subject. Our results suggest that the alignment of the lower limb and the foot progression angle, which can be readily measured in a clinical setting, can serve as predictors of knee joint loading in healthy individuals. These findings may have important implications for both surgical and nonsurgical treatment of abnormalities of the knee joint.

Adult↗
PubMed1994

Alpha-skeletal actin is associated with increased contractility in the mouse heart.

BALB/c mice express abnormally high levels of alpha-skeletal actin in the heart, which may be related to a duplication in the promoter of the alpha-cardiac actin gene. To evaluate the effects of overexpression of the alpha-skeletal actin isoform on cardiac contractile function, we studied these mice using the isolated perfused work-performing murine heart model and measured actin isoform expression in the same hearts. We quantified myocardial contractility from the maximum rate of contraction (+dP/dt) and time to peak pressure and relaxation from -dP/dt and time to half relaxation of left intraventricular pressure. Dot blots of total RNA hybridized against oligonucleotide sequences specific for either alpha-skeletal or alpha-cardiac actin mRNA showed that increased levels of alpha-skeletal actin RNA correlated significantly with increased contractility of hearts from the BALB/c mice (r = .80, n = 15, P < .001). The present study demonstrates a significant functional correlation between alpha-actin isoform content and cardiac contractile function and also that alpha-skeletal actin may promote an increased contractile function in the heart compared with alpha-cardiac actin.

Actins↗