Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

Residual force enhancement in myofibrils and sarcomeres

Residual force enhancement has been observed following active stretch of skeletal muscles and single fibres. However, there has been intense debate whether force enhancement is a sarcomeric property, or is associated with sarcomere length instability and the associated development of non-uniformities. Here, we studied force enhancement for the first time in isolated myofibrils (n=18) that, owing to the strict in series arrangement, allowed for evaluation of this property in individual sarcomeres (n=79). We found consistent force enhancement following stretch in all myofibrils and each sarcomere, and forces in the enhanced state typically exceeded the isometric forces on the plateau of the force-length relationship. Measurements were made on the plateau and the descending limb of the force-length relationship and revealed gross sarcomere length non-uniformities prior to and following active myofibril stretching, but in contrast to previous accounts, revealed that sarcomere lengths were perfectly stable under these experimental conditions. We conclude that force enhancement is a sarcomeric property that does not depend on sarcomere length instability, that force enhancement varies greatly for different sarcomeres within the same myofibril and that sarcomeres with vastly different amounts of actin-myosin overlap produce the same isometric steady-state forces. This last finding was not explained by differences in the amount of contractile proteins within sarcomeres, vastly different passive properties of individual sarcomeres or (half-) sarcomere length instabilities, suggesting that the basic mechanical properties of muscles, such as force enhancement, force depression and creep, which have traditionally been associated with sarcomere instabilities and the corresponding dynamic redistribution of sarcomere lengths, are not caused by such instabilities, but rather seem to be inherent properties of the mechanisms of contraction.     

Synchronous behavior of spontaneous oscillations of sarcomeres in skeletal myofibrils under isotonic conditions.

An isotonic control system for studying dynamic properties of single myofibrils was developed to evaluate the change of sarcomere lengths in glycerinated skeletal myofibrils under conditions of spontaneous oscillatory contraction (SPOC) in the presence of inorganic phosphate and a high ADP-to-ATP ratio. Sarcomere length oscillated spontaneously with a peak-to-peak amplitude of about 0.5 microns under isotonic conditions in which the external loads were maintained constant at values between 1.5 x 10(4) and 3.5 x 10(4) N/m2. The shortening and yielding of sarcomeres occurred in concert, in contrast to the previously reported conditions (isomeric or auxotonic) under which the myofibrillar tension is allowed to oscillate. This synchronous SPOC appears to be at a higher level of synchrony than in the organized state of SPOC previously observed under auxotonic conditions. The period of sarcomere length oscillation did not largely depend on external load. The active tension under SPOC conditions increased as the sarcomere length increased from 2.1 to 3.2 microns, although it was still smaller than the tension under normal Ca2+ contraction (which is on the order of 10(5) N/m2). The synchronous SPOC implies that there is a mechanism for transmitting information between sarcomeres such that the state of activation of sarcomeres is affected by the state of adjacent sarcomeres. We conclude that the change of myofibrillar tension is not responsible for the SPOC of each sarcomere but that it affects the level of synchrony of sarcomere oscillations.     

Sarcomere Length Nonuniformity and Force Regulation in Myofibrils and Sarcomeres

The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.     

Force–length relation of skeletal muscles: from sarcomeres to myofibril

Sarcomeres are building blocks of skeletal muscles. Given force–length relations of sarcomeres serially connected in a myofibril, the myofibril force–length relation can be uniquely determined. Necessary and sufficient conditions are derived for capability of fully lengthening or completely shortening a myofibril under isometric, eccentric or concentric contraction, and for the myofibril force–length relation to be a continuous single-valued function. Intriguing phenomena such as sarcomere force–length hysteresis and myofibril regularity are investigated and their important roles in determining myofibril force–length relations are explored. The theoretical analysis leads to experimentally verifiable predictions on myofibril force–length relations. For illustration, simulated force–length relations of a myofibril portion consisting of a sarcomere pair are presented.     

Passive and active tension in single cardiac myofibrils.

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Sarcomere dynamics in skeletal muscle myofibrils during isometric contractions

The main goal of this study was to evaluate the dynamics of sarcomeres during isometric activation of skeletal muscle myofibrils. Rabbit psoas myofibrils (n=14) were attached between a pair of cantilevers for force measurements at one side and a rigid glass needle at the other side, and their images were used for measurements of individual sarcomere lengths (SL) during contractions. Myofibrils were set at average SL between 2.13 and 3.06 μm, and were activated and held isometric for 20–35 s during which SL and force were continuously measured. SL dispersion increased from the rest state to activation, but it remained mostly constant during the activation period. Even with the length non-uniformity developed during myofibril activation, most sarcomeres stabilized their length changes during the isometric contraction. As a result, sarcomeres contracted at different degrees of filament overlap while producing similar forces. When the myofibrils were separated in two groups that produced force at averaged short (≤2.5 μm) or long (≥2.5 μm) SL, the initial non-uniformity was greater in long lengths, but changes observed in sarcomeres during the activation period were similar, suggesting that sarcomere stability is not length-dependent.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (~ 10.0 µ) and intermediate (~ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–~6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.     

Resting Sarcomere Length-Tension Relation in Living Frog Heart

The sarcomere pattern and tension of isolated resting frog atrial trabeculae were continuously monitored. In the absence of any resting tension the sarcomere lengths varied with the diameter of the trabeculae. In over 75 % of the trabeculae the value exceeded 2.05 µm, the estimated in vivo length of the thin filaments, and it was never less than 1.89 µm. When the trabeculae were stretched the increase in length of the central undamaged portion could be completely accounted for by an increase in sarcomere length. The width of the A band was constant only at sarcomere lengths between 2.3 and 2.6 µm it decreased at smaller and increased at larger sarcomere lengths. A group of spontaneously active cells stretched the sarcomeres in cells in series to longer lengths than could be produced by passive tension applied to the ends of the trabeculae, but they did not influence the sarcomeres of adjacent cells. It is proposed that the connective tissue is a major factor in determining sarcomere length and that there are interactions between thick and thin filaments in resting muscles.     

Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres

Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod''s localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.     

Is Treatment with Trimetazidine Beneficial in Patients with Chronic Heart Failure?

Background

Whether additional benefit can be achieved with the use of trimetazidine (TMZ) in patients with chronic heart failure (CHF) remains controversial. We therefore performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the effects of TMZ treatment in CHF patients.

Methods

We searched PubMed, EMBASE, and Cochrane databases through October 2013 and included 19 RCTs involving 994 CHF patients who underwent TMZ or placebo treatment. Risk ratio (RR) and weighted mean differences (WMD) were calculated using fixed or random effects models.

Results

TMZ therapy was associated with considerable improvement in left ventricular ejection fraction (WMD: 7.29%, 95% CI: 6.49 to 8.09, p<0.01) and New York Heart Association classification (WMD: −0.55, 95% CI: −0.81 to −0.28, p<0.01). Moreover, treatment with TMZ also resulted in significant decrease in left ventricular end-systolic volume (WMD: −17.09 ml, 95% CI: −20.15 to −14.04, p<0.01), left ventricular end-diastolic volume (WMD: −11.24 ml, 95% CI: −14.06 to −8.42, p<0.01), hospitalization for cardiac causes (RR: 0.43, 95% CI: 0.21 to 0.91, p = 0.03), B-type natriuretic peptide (BNP; WMD: −157.08 pg/ml, 95% CI: −176.55 to −137.62, p<0.01) and C-reactive protein (CRP; WMD: −1.86 mg/l, 95% CI: −2.81 to −0.90, p<0.01). However, there were no significant differences in exercise duration and all-cause mortality between patients treated with TMZ and placebo.

Conclusions

TMZ treatment in CHF patients may improve clinical symptoms and cardiac function, reduce hospitalization for cardiac causes, and decrease serum levels of BNP and CRP.     

Associations of parental depression during adolescence with cognitive development in later life in China: A population-based cohort study

BackgroundPrior research has underscored negative impacts of perinatal parental depression on offspring cognitive performance in early childhood. However, little is known about the effects of parental depression during adolescence on offspring cognitive development.Methods and findingsThis study used longitudinal data from the nationally representative China Family Panel Studies (CFPS). The sample included 2,281 adolescents aged 10–15 years (the median age was 13 years with an interquartile range between 11 and 14 years) in 2012 when their parents were surveyed for depression symptoms with the 20-item Center for Epidemiologic Studies Depression Scale (CES-D). The sample was approximately balanced by sex, with 1,088 females (47.7%). We examined the associations of parental depression in 2012 with offspring cognitive performance (measured by mathematics, vocabulary, immediate word recall, delayed word recall, and number series tests) in subsequent years (i.e., 2014, 2016, and 2018) using linear regression models, adjusting for various offspring (i.e., age, sex, and birth order), parent (i.e., parents’ education level, age, whether living with the offspring, and employment status), and household characteristics (i.e., place of residence, household income, and the number of offspring). We found parental depression during adolescence to be significantly associated with worse cognitive performance in subsequent years, in both crude and adjusted models. For example, in the crude models, adolescents whose mothers had depression symptoms in 2012 scored 1.0 point lower (95% confidence interval [CI]: −1.2 to −0.8, p < 0.001) in mathematics in 2014 compared to those whose mothers did not have depression symptoms; after covariate adjustment, this difference marginally reduced to 0.8 points (95% CI: −1.0 to −0.5, p < 0.001); the associations remained robust after further adjusting for offspring earlier cognitive ability in toddlerhood (−1.2, 95% CI: −1.6, −0.9, p < 0.001), offspring cognitive ability in 2012 (−0.6, 95% CI: −0.8, −0.3, p < 0.001), offspring depression status (−0.7, 95% CI: −1.0, −0.5, p < 0.001), and parents’ cognitive ability (−0.8, 95% CI: −1.2, −0.3, p < 0.001). In line with the neuroplasticity theory, we observed stronger associations between maternal depression and mathematical/vocabulary scores among the younger adolescents (i.e., 10–11 years) than the older ones (i.e., 12–15 years). For example, the association between maternal depression and 2014 vocabulary scores was estimated to be −2.1 (95% CI: −2.6, −1.6, p < 0.001) in those aged 10–11 years, compared to −1.2 (95% CI: −1.6, −0.8, p < 0.001) in those aged 12–15 years with a difference of 0.9 (95% CI: 0.2, 1.6, p = 0.010). We also observed a stronger association of greater depression severity with worse mathematical scores. The primary limitations of this study were the relatively high attrition rate and residual confounding.ConclusionsIn this study, we observed that parental depression during adolescence was associated with adverse offspring cognitive development assessed up to 6 years later. These findings highlight the intergenerational association between depression in parents and cognitive development across the early life course into adolescence.

In this cohort study, Zhihui Li and colleagues explore associations between parental depression and offspring cognitive development up to six years later.     

Genetic dissection of novel myopathy models reveals a role of CapZα and Leiomodin 3 during myofibril elongation

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

Aliskiren and Amlodipine in the Management of Essential Hypertension: Meta-Analysis of Randomized Controlled Trials

Background

Aliskiren is a novel renin-angiotensin aldosterone system (RAAS) inhibitor, the combination therapy of aliskiren and amlodipine for blood pressure control have been reported recently. The primary objective of this analysis is to review recently reported randomized controlled trials (RCTs) to compare antihypertensive effects and adverse events between mono (amlodipine or aliskiren alone) and combination therapy of both medicines.

Methods

Databases for the search included Pubmed, Embase and the Cochrane Central Register of Controlled Trials. Revman v5.0 statistical program was used to analyze the data. Weighted mean differences (WMD) with a 95% confidence interval (CI) were used for the calculation of continuous data, and relative risk (RR) with a 95% CI was used for dichotomous data.

Results

We analyzed the data from 7 RCTs for a total of 6074 participants in this meta-analysis. We found that the aliskiren/amlodipine combination therapy had a stronger effect in lowering blood pressure as compared with the monotherapy using aliskiren (SBP: WMD = −10.42, 95% CI −13.03∼−7.82, P<0.00001; DBP: WMD = −6.60, 95% CI −7.22∼−5.97, P<0.00001) or amlodipine (SBP: WMD = −4.85, 95% CI −6.88∼−2.81, P<0.00001; DBP: WMD = −2.91, 95% CI −3.85∼−1.97, P<0.00001). No differences were found in terms of adverse events between combination therapy and monotherapy, except for the rates of peripheral edema and hypokalaemia which were significantly lower in the combination therapy than in the amlodipine monotherapy (RR = 0.78, 0.66∼0.92, P = 0.004; RR = 0.51, 0.27∼0.97, P = 0.04). Similar antihypertensive effects were found in both obese (body mass index > = 30 kg/m²) hypertensive and non-obese (body mass index <30 kg/m²) hypertensive patients. Moreover, there was no difference with the blood pressure lowering or adverse effects with regards to the combination therapy in both subgroups.

Conclusion

We found that aliskiren/amlodipine combination therapy provided a more effective blood pressure reduction than monotherapy with either drug without increase in the occurrence of adverse events.     

Assessing Interactions between Common Genetic Variant on 2q35 and Hormone Receptor Status with Breast Cancer Risk: Evidence Based on 26 Studies

Genome-wide association studies have identified 2q35-rs13387042 as a new breast cancer (BC) susceptibility locus in populations of European descent. Since then, the relationship between 2q35-rs13387042 and breast cancer has been reported in various ethnic groups; however, these studies have yielded inconsistent results. To investigate this inconsistency, we performed a meta-analysis of 26 studies involving a total of 101,529 cases and 167,363 controls for 2q35-rs13387042 polymorphism to evaluate its effect on genetic susceptibility for breast cancer. An overall random effects odds ratio of 1.14 (95% CI: 1.11–1.16, P<10⁻⁵) was found for rs13387042-A variant. Significant results were also observed using dominant (OR = 1.14, 95% CI: 1.12–1.17, P<10⁻⁵), recessive (OR = 1.17, 95% CI: 1.13–1.21, P<10⁻⁵) and co-dominant genetic model (heterozygous: OR = 1.15, 95% CI: 1.12–1.19, P<10⁻⁵; homozygous: OR = 1.20, 95% CI: 1.15–1.24, P<10⁻⁵). There was strong evidence of heterogeneity, which largely disappeared after stratification by ethnicity. Significant associations were found in East Asians, and White populations when stratified by ethnicity; while no significant associations were observed in Africans and other ethnic populations. An association was observed for both ER-positive (OR = 1.17, 95% 1.15–1.19; P<10⁻⁵) and ER-negative disease (OR = 1.08, 95% CI: 1.04–1.13; P<10⁻⁴) and both progesterone receptor (PR)-positive (OR = 1.18, 95% CI: 1.15–1.21; P<10⁻⁵) and PR-negative disease (OR = 1.10, 95% CI: 1.05–1.15; P<10⁻⁴). In conclusion, this meta-analysis demonstrated that the A allele of 2q35-rs13387042 is a risk factor associated with increased breast cancer susceptibility.     

Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293–296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (~2.0 μm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ~2.0 to 2.7 μm, the “contracted” tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ~2.7 μm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112–1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force–SL relation that is not achievable with a single elastic segment.     

Chicken breast muscle connectin: passive tension and I-band region primary structure

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A-I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles.     

Structural fluctuations in the steady state of muscular contraction.

Recent studies of the intensity fluctuation spectra of coherent light scattered from striated muscle have demonstrated the existence of large scale fluctuations in position and polarizability at the level of the myofibrillar sarcomere and its major structural subunits during the steady state of contraction. The existence of these fluctuations implies a fluctuating driving force. Various possible fluctuating motions of the thick and thin filaments, A and I bands, and entire sarcomeres are described. The magnitude of the fluctuating forces associated with the making and breaking of cross bridges is estimated. A mechanical model is proposed for coupling structural elements of a single sarcomere to one another and for coupling myofibrillar sarcomeres to one another. It is shown that the fluctuating force generated by the spontaneous making and breaking of cross bridges in conjunction with the model accounts for some of the features of the observed intensity fluctuation spectra.