Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Extensible behavior of titin in the miniswine left ventricle.

BACKGROUND: The sarcomeric protein titin is a molecular spring responsible for passive tension and restoring forces of cardiomyocytes. Extension of titin as a function of sarcomere length (SL) has been studied in rodents, which predominantly express the smaller, stiffer N2B titin isoform. Large mammals coexpress roughly equal proportions of N2B and N2BA titin, the larger, more compliant isoform. We hypothesized that extension of titin in relation to SL differs in large mammals and that this difference is functionally important. METHODS AND RESULTS: We characterized the filling pressure-SL relation in diastolic-arrested miniswine left ventricles. SL was 2.15 to 2.25 mum at a filling pressure of approximately 0 mm Hg and reached a maximum of approximately 2.50 mum with overfilling. In the normal filling pressure range, SL ranged from approximately 2.32 to approximately 2.40 mum. We assessed titin extension as a function of SL using immunoelectron microscopy, which allowed delineation of the behavior of specific spring segments. The major isoform difference was that the N2B-Us segment extended approximately 4-fold more as a function of SL in N2B compared with N2BA titin. Using this segment, we estimated sarcomeric force development with a worm-like chain model and found that N2B develops markedly greater force than N2BA titin. The resulting force with coexpression of N2B and N2BA titin is intermediate. CONCLUSIONS: In light of murine studies showing that operating SLs are shorter than in miniswine, our results indicate that coexpression of the 2 titin isoforms in large mammals allows longer SLs without the development of excessive diastolic tension.

Comparative biomechanics of thick filaments and thin filaments with functional consequences for muscle contraction.

The scaffold of striated muscle is predominantly comprised of myosin and actin polymers known as thick filaments and thin filaments, respectively. The roles these filaments play in muscle contraction are well known, but the extent to which variations in filament mechanical properties influence muscle function is not fully understood. Here we review information on the material properties of thick filaments, thin filaments, and their primary constituents; we also discuss ways in which mechanical properties of filaments impact muscle performance.

Cardiac myosin binding protein-C is essential for thick-filament stability and flexural rigidity.

Using atomic force microscopy, we examined the contribution of cardiac myosin binding protein-C (cMyBP-C) to thick-filament length and flexural rigidity. Native thick filaments were isolated from the hearts of transgenic mice bearing a truncation mutation of cMyBP-C (t/t) that results in no detectable cMyBP-C and from age-matched wild-type controls (+/+). Atomic force microscopy images of these filaments were evaluated with an automated analysis algorithm that identified filament position and shape. The t/t thick-filament length (1.48 +/- 0.02 microm) was significantly (P < 0.01) shorter than +/+ (1.56 +/- 0.02 microm). This 5%-shorter thick-filament length in the t/t was reflected in 4% significantly shorter sarcomere lengths of relaxed isolated cardiomyocytes of the t/t (1.97 +/- 0.01 microm) compared to +/+ (2.05 +/- 0.01 microm). To determine if cMyBP-C contributes to the mechanical properties of thick filaments, we used statistical polymer chain mechanics to calculate a per-filament-specific persistence length, an index of flexural rigidity directly proportional to Young's modulus. Thick-filament-specific persistence length in the t/t (373 +/- 62 microm) was significantly lower than in +/+ (639 +/- 101 microm). Accordingly, Young's modulus of t/t thick filaments was approximately 60% of +/+. These results provide what we consider a new understanding for the critical role of cMyBP-C in defining normal cardiac output by sustaining force and muscle stiffness.

The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments.

Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (fln(ΔC44)) are significantly shorter (2.68 ± 0.06 µm; p < 0.005) than thick filaments containing a full length flightin (fln⁺; 3.21 ± 0.05 µm) and thick filaments containing an N-terminal domain truncated flightin (fln(ΔN62); 3.21 ± 0.06 µm). Persistence length was significantly reduced in fln(ΔN62) (418 ± 72 µm; p < 0.005) compared to fln⁺ (1386 ± 196µm) and fln(ΔC44)(1128 ± 193 µm). Statistical polymer chain analysis revealed that the C-terminal domain fulfills a secondary role in thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM's dual role in flight and courtship behaviors.

Flightin is necessary for length determination, structural integrity, and large bending stiffness of insect flight muscle thick filaments.

Despite the fundamental role of thick filaments in muscle contraction, little is known about the mechanical behavior of these filaments and how myosin-associated proteins dictate differences between muscle types. In this study, we used atomic force microscopy to study the morphological and mechanical properties of fully hydrated native thick filaments isolated from indirect flight muscle (IFM) of normal and mutant Drosophila lacking flightin (fln(0)). IFM thick filaments from newly eclosed (0-1 h old) wild-type flies have a mean length of 3.04+/-0.05 microm. In contrast, IFM thick filaments from newly eclosed fln(0) flies are more variable in length and, on average, are significantly longer (3.90+/-1.33 microm) than wild-type filaments from flies of the same age. In the absence of flightin, thick filaments can attain lengths >300% of wild-type filaments, indicating that flightin is required for setting the proper filament length in vivo. Filaments lacking flightin are structurally compromised, and filament preparations from fully matured 3- to 5-day-old adult fln(0) IFM yielded fragments of variable length much shorter than 3.20+/-0.04 microm, the length obtained from wild-type flies of similar age. The persistence length, an index of bending stiffness, was calculated from measurements of filament end-to-end length and contour length. We show that the presence of flightin increases persistence length by more than 40% and that wild-type filaments increase in stiffness with age. These results indicate that flightin fulfills an essential role in defining the structural and mechanical properties of IFM thick filaments.

Nanomechanics of native thick filaments from indirect flight muscles.

During flight, the wings of Drosophila melanogaster beat nearly 200 times per second. The indirect flight muscle fibers that power this movement have evolved to resist the repetitive mechanical stress that results from the 5-ms wing beat cycle at a strain amplitude of 3.5%. In order to understand how this is achieved at the sarcomere level, we have analyzed the mechanical properties of native thick filaments isolated from indirect flight muscle. Single filaments adsorbed onto a solid support were manipulated in physiological buffer using an atomic force microscope. Images taken after the manipulation revealed that segments were stretched, on average, to 150%, with a maximum at 385% extension. The lateral-force-versus-displacement curve associated with each manipulation contained information about the bending and tensile properties of each filament. The bending process was dominated by shearing between myosin dimers and yielded a shear modulus between 3 and 13 MPa. Maximum tension along the stretched filaments was observed at approximately 200% extension and varied between 8 and 17 nN. Based on current models of thick filament structure, these variations can be attributed to cross-links between myosin dimers distributed along the filament.

Endothelin receptor blockade has an oxygen-saving effect in Dahl salt-sensitive rats with heart failure.

The effects of endothelin (ET) receptor blockade on energy utilization in heart failure (HF) are unknown. We administered ET type A (ETA), ET type B (ETB), and ETA/ETB antagonists to isolated hearts from Dahl salt-sensitive (DS) rats with HF and controls. Contractile efficiency was assessed as slope-1 of myocardial O consumption (VO2)-pressure-volume area relation. In HF, ETA and ETA/ETB but not ETB blockade decreased the contractility index (Emax)(-15 +/- 3% and -17 +/- 2%, P < 0.05), excitation-contraction (E-C) coupling VO2 (-39 +/- 4% and -37 +/- 5%, P < 0.01), and efficiency (-15 +/- 4% and -17 +/- 2%, P < 0.05). Despite decreased efficiency, ETA and ETA/ETB blockade decreased total VO2 (-24 +/- 3% and -22 +/- 2%, P < 0.05). Na+/H+ exchanger inhibition decreased Emax and E-C coupling VO2 similar to ETA and ETA/ETB blockade, but did not alter efficiency. In HF, endogenous ET-1 maintains contractility at expense of increased VO2 through ETA receptor activation, likely mediated by Na+/H+ exchange.