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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.

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.

Abstract —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.

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.

Objectives: The incidence of second anterior cruciate ligament (ACL) injury in the first 12 months after ACL reconstruction (ACLR) and return to sport in a young, active population has been reported to be 15 times greater than a previously uninjured cohort. It is unknown if this high relative rate of injury continues beyond the first year after return to sport following ACLR. The tested hypothesis was that the incidence rate of a subsequent ACL injury in the 2 years following ACLR and return to sports would be less than the incidence rate reported within the first 12 months after return to sport, but greater than the ACL injury incidence rate in an uninjured cohort of young athletes. Methods: Seventy-eight subjects who underwent ACLR and were ready to return to a pivoting/cutting sport (RTS) and 47 controls were prospectively enrolled. Each subject was followed for injury and athletic exposure (AE) data for a 24-month period after return to sport. Twenty-three subjects after ACLR and 4 control subjects suffered an ACL injury during this time. Incidence rate ratios (IRR) were calculated to compare the rates (per 1000 athletic exposures [AEs]) of ACL injury in athletes in the ACLR group and control group. For the ACLR group, similar comparisons were conducted for side of injury by sex. Results: The overall incidence rate of a second ACL injury within 24 months after ACLR and RTS (1.39/1000 AE) was nearly 6 times greater (IRR=5.71, 95% CI: 2.0, 22.7, p=0.0003) than healthy control subjects (0.24/1,000 AE). Female ACLR athletes demonstrated 4 times greater rate of injury within 24 months of RTS (IRR=4.51; 95% CI: 1.5-18.2, p=0.0004) than female controls. Within the ACLR group, there was a trend for female subjects to be over two (RR=2.43, 95% CI: 0.8, 8.6) times more likely to suffer a contralateral injury (1.13/1000 AEs) than an ipsilateral injury (0.47/1000 AEs). Overall, 29.5% of athletes suffered a second ACL injury within 24 months of RTS with 20.5% sustaining a contralateral injury and 9.0% incurring an ipsilateral graft re-tear injury. A higher proportion of females (23.7%) suffered a contralateral injury compared to males (10.5%). Conversely, for ipsilateral injuries, the incidence proportion between females (8.5%) and males (10.5%) were found to be similar. Conclusion: These data support the hypothesis that in the 24 months following ACLR and RTS, patients are at greater risk (nearly 6 times) to suffer a subsequent ACL injury compared to young athletes without a history of ACL injury. Additionally, the contralateral limb of female patients appears to be at greatest risk. These 24 month outcome data, analyzed in conjunction with recent 12 month outcome data which noted a 15 times greater risk of ACL injury compared to healthy control subject, indicate that the greatest risk of second ACL injury after ACLR occurs during the initial 12 months after ACLR and RTS than the second year after RTS.

Objectives: Excessive knee joint laxity may decrease overall joint stability and increase the risk of anterior cruciate ligament (ACL) injury during high risk activities. Despite the frequent clinical use of knee arthrometry, as an accurate and valid method for evaluation of knee laxity, no data exists to correlate instrumented laxity measures and ACL strain during dynamic high-risk activities. The purpose of this study was to evaluate relationships between instrumented anterior knee laxity measurements and ACL strain during simulated bi-pedal landing (as an identified high-risk injurious task). We hypothesized that a strong correlation would be observed between ACL strain and arthrometry displacement, and that specimens with greater anterior knee laxity would demonstrate increased peak ACL strain during landing. Methods: 20 cadaveric lower limbs (46±6 years, 10 female and 10 male) were tested using a CompuKT knee arthrometer to measure anterior knee joint laxity. Each specimen was tested under four continuous cycles of A-P shear force (±134 N) applied to the tibial tubercle. To quantify ACL strain, a DVRT displacement transducer was arthroscopically placed on the ACL (AM-bundle), and specimens were retested. Subsequently, bi-pedal landing from 30 cm was simulated on a subset of 14 specimens (45±5 years, 6 female and 8 male) using a novel custom-designed drop-stand. Paired sample t-tests, and a general linear model were used to evaluate changes in joint laxity and ACL strain. Results: During simulated drawer tests, 134 N of applied anterior drawer load produced an average 3.1±1.1 mm peak anterior tibial translation and 4.9±4.3% peak ACL strain. Anterior drawer load was a significant determinant of anterior tibial translation (pConclusion: This work represents the first cadaveric study to establish a strong correlation between arthrometrically measured anterior knee joint laxity and peak ACL strain during dynamic landing (as a high-risk injurious condition). Experimental findings support the tested hypothesis, as a strong correlation between arthrometry displacement collected during laxity tests and concurrent calculated ACL strain was demonstrated. Instrumented measures of anterior knee laxity were predictive of peak ACL strain during landing, while specimens with greater knee laxity demonstrated higher levels of peak ACL strain during landing. Considering ACL strain as an established quantifiable measure of ACL injury risk, current findings highlight the importance of instrumented anterior knee laxity assessments as a potential indicator of ACL injury risk in addition to its clinical utility in the evaluation of ACL integrity.

The purpose of this study was to test the effect of a jump-training program on landing mechanics and lower extremity strength in female athletes involved in jump ing sports. These parameters were compared before and after training with those of male athletes. The program was designed to decrease landing forces by teaching neuromuscular control of the lower limb dur ing landing and to increase vertical jump height. After training, peak landing forces from a volleyball block jump decreased 22%, and knee adduction and abduc tion moments (medially and laterally directed torques) decreased approximately 50%. Multiple regression analysis revealed that these moments were significant predictors of peak landing forces. Female athletes demonstrated lower landing forces than male athletes and lower adduction and abduction moments after training. External knee extension moments (hamstring muscle-dominant) of male athletes were threefold higher than those of female athletes. Hamstring-to- quadriceps muscle peak torque ratios increased 26% on the nondominant side and 13% on the dominant side, correcting side-to-side imbalances. Hamstring muscle power increased 44% with training on the dom inant side and 21 % on the nondominant. Peak torque ratios of male athletes were significantly greater than those of untrained female athletes, but similar to those of trained females. Mean vertical jump height in creased approximately 10%. This training may have a significant effect on knee stabilization and prevention of serious knee injury among female athletes.

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.

Five patients with symptomatic knee hyperextension thrusting patterns due to posterolateral ligament com plex injury underwent gait analysis before and after a gait retraining program. Patients were trained to avoid knee hyperextension by 1) walking with their knees slightly flexed throughout stance, 2) maintaining ankle dorsiflexion in early stance, and 3) maintaining an erect trunk-hip attitude during stance. Kinematic and kinetic measurements were obtained using automated gait analysis. Four of the five patients significantly reduced hyperextension at the knee and abnormal motion pat terns at the hip and ankle. Patients showed increases in knee flexion throughout stance conversions of knee flexion-extension moments to more normal biphasic patterns with a 79% decrease in extension moments at terminal extension, and a 22% decrease in knee ad duction moments. Posttraining values also showed a 30% decrease in the calculated medial tibiofemoral loads ( P < 0.05). At the hip, there were significant decreases in abduction and adduction moments (36% and 18%, respectively, P < 0.01). Ankle plantar flexion motion decreased significantly by 42% ( P < 0.01). Gait retraining can alter the biomechanics of hip, knee, and ankle function to approximately normal levels, and therefore is recommended before ligament reconstruc tion because abnormal knee motions, if resumed post operatively, can stretch soft tissue reconstructions.

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.

1. The relations between mechanical parameters and myosin heavy chain isoforms were studied in myometrial smooth muscle from ovariectomized rats (O) and oestrogen-treated, ovariectomized rats (E). 2. Treatment of the rats for three days with beta-oestradiol (2 micrograms kg-1 day-1) 2-4 weeks postsurgery, produced maximal changes in uterine mass and myosin content of approximately threefold. 3. Myosin heavy chain isoform SM1 (204 kDa) was increased from 65.5 +/- 0.8% to 72.9 +/- 0.6% of the total isoform species (P < 0.001, n = 24, O and E respectively) after oestrogen treatment. 4. To avoid complications associated with activation processes, mechanical parameters were measured in permeabilized myometrial fibre bundles activated at a calcium concentration of 12.6 microM. After oestrogen treatment the maximum velocity of shortening (Vmax) measured by the slack test increased from 0.044 +/- 0.006 of the reference length (Lo) s-1 to 0.101 +/- 0.006 Lo s-1, and maximal isometric force (Pmax) increased from 23.3 +/- 4.4 mN mm-2 to 74.1 +/- 13.9 mN mm-2 (P < 0.001, n = 24, respectively). Series elasticity and the half-time to peak force were not significantly altered. 5. Both Vmax and Pmax correlated significantly with percentage SM1 in O and E fibre bundles (r = 0.61 and 0.56, n = 48 fibres; or r = 0.87 and 0.89, n = 8 grouped data per rat). Vmax, however, was only weakly correlated with Pmax (r = 0.39, n = 48). 6. To assess the relative significance of the correlation between Vmax and the percentage of SM1 and that between Vmax and Pmax, we used a multiple regression analysis with the model Vmax = intercept + beta 1 x % SM1 + beta 2 x Pmax, where intercept, beta 1 and beta 2 are regression parameters. This analysis (n = 48) indicated that Vmax was significantly dependent on the percentage of SM1 (P < 0.0002) but not on Pmax (P < 0.61). 7. There were no significant differences in the levels of myosin light chain phosphorylation between O and E fibre bundles, indicating that light chain phosphorylation is unlikely to be the basis for the differences in mechanical parameters demonstrated by these fibres.

Forty-one endomyocardial biopsies of the right interventricular septum have been investigated in 24 immunosuppressed patients after orthotopic heart transplantation. Monoclonal antibodies 27E10, 25F9, and RM3/1, which react with different macrophage phenotypes, and antisera MRP-8 and MRP-14, specific for proteins expressed on endothelial and monocyte cell surfaces in inflammation as well as markers for CD4+ and CD8+ T-lymphocytes, were employed in an indirect immunoperoxidase staining technique. This methodology permits more physiological recognition of the inflammatory process within the myocardium. It was possible to verify and to distinguish acute early, late and down-regulatory stages of inflammation in 33 biopsies (80%). No evidence of inflammation was found in seven biopsies (17%). Conventional histopathology with haematoxylin-eosin and Masson's trichrome was performed simultaneously, and demonstrated inflammation to be present in 23 of 41 biopsies (56%). An important findings is that CD4+ and CD8+ lymphocytes were absent in 15 of 41 specimens (37%) although there was inflammation proven by the presence of different macrophage phenotypes. The results indicate the necessity of long-term serial investigations of the physiological role of specific inflammatory macrophage phenotypes during the rejection process. It is concluded that the phenotyping of macrophage and endothelial cell differentiation antigens offers a sensitive approach to assess diagnosis of myocardial inflammation as a consequence of ongoing rejection in cardiac allografts.

Isoforms of striated muscle myosin have a long history. They have been extensively studied on biochemical and functional levels and more recently in terms of molecular biology. Under various environmental stresses the architecture of the contractile apparatus of striated muscle can be remodelled to meet the imposed demands.