The fibrous collagens are ubiquitous in animals and form the structural basis of all mammalian connective tissues, including those of the heart, vasculature, skin, cornea, bones, and tendons. However, in comparison with what is known of their production, turnover and physiological structure, very little is understood regarding the three-dimensional arrangement of collagen molecules in naturally occurring fibrils. This knowledge may provide insight into key biological processes such as fibrillo-genesis and tissue remodeling and into diseases such as heart disease and cancer. Here we present a crystallographic determination of the collagen type I supermolecular structure, where the molecular conformation of each collagen segment found within the naturally occurring crystallographic unit cell has been defined (P1, a Ϸ 40.0 Å, b Ϸ 27.0 Å, c Ϸ 678 Å, ␣ Ϸ 89.2°, ␤ Ϸ 94.6°, ␥ Ϸ 105.6°; reflections: 414, overlapping, 232, and nonoverlapping, 182; resolution, 5.16 Å axial and 11.1 Å equatorial). This structure shows that the molecular packing topology of the collagen molecule is such that packing neighbors are arranged to form a supertwisted (discontinuous) right-handed microfibril that interdigitates with neighboring microfibrils. This interdigitation establishes the crystallographic superlattice, which is formed of quasihexagonally packed collagen molecules. In addition, the molecular packing structure of collagen shown here provides information concerning the potential modes of action of two prominent molecules involved in human health and disease: decorin and the Matrix Metallo-Proteinase (MMP) collagenase.x-ray ͉ fiber ͉ crystallography ͉ fibril ͉ extracellular matrix A lthough the general features of the structure of type I collagen have been known for a long time, the specific packing arrangement of collagen molecules in situ has remained difficult to define, despite a great deal of effort by many investigators (1-16) (the general organization of type I collagen is summarized in Fig. 5, which is published as supporting information on the PNAS web site). Recently, we approached this difficult structural problem by employing conventional crystallographic techniques in x-ray fiber diffraction experiments (13,17,18), culminating in an initial electron density map (13, 19) that allowed a crude look at some aspects of the supermolecular arrangement of collagen molecules in situ. Unfortunately, the high degree of disorder observed in the gap region of the electron density map precluded the fitting of a molecular model to the electron density; the gap region was largely uninterpretable. Without the structure of the gap region, it was impossible to determine the overall molecular arrangement of collagen molecules in situ, and therefore its potential for improving our understanding of the structural, developmental, and pathological function of the collagen fibril at the molecular level remained unrealized. We have subsequently integrated additional (nonoverlapping) intensity data into the structural determination process (b...

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.

Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

The Frank-Starling law of the heart describes the interrelationship between end-diastolic volume and cardiac ejection volume, a regulatory system that operates on a beat-to-beat basis. The main cellular mechanism that underlies this phenomenon is an increase in the responsiveness of cardiac myofilaments to activating Ca 2+ ions at a longer sarcomere length, commonly referred to as myofilament length dependent activation. This review focuses on what molecular mechanisms may underlie myofilament length dependency. Specifically, the roles of inter-filament spacing, thick and thin filament based regulation, as well as sarcomeric regulatory proteins are discussed. Although the "Frank-Starling law of the heart" constitutes a fundamental cardiac property that has been appreciated for well over a century, it is still not known in muscle how the contractile apparatus transduces the information concerning sarcomere length to modulate ventricular pressure development. KeywordsFrank-Starling Law of The Heart; Length-Tension Relationship; Sarcomere length; Regulation Frank-Starling's Law of the HeartOver a century ago, Otto Frank in Germany and Ernest Starling in England reported on the relationship between the extent of ventricular filling and pump function of the heart, a phenomenon collectively referred to as Frank-Starling's Law of the Heart. A modern view of this phenomenon [1] (illustrated in Figure 1) holds that there is a unique relationship between end-systolic volume and end-systolic pressure in the heart that is solely determined by contractile state. As a consequence, for a given contractile state, ventricular stroke volume is i) proportional to diastolic filling (i.e. preload), and ii) stroke volume can be maintained in the face of increased aortic pressures (i.e. afterload) simply by increasing preload as illustrated by the two pressure-volume loops in Figure 1. Contractile state, within this framework, can be viewed as any factor that alters end-systolic pressure independently of end-systolic volume and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Figure 1). The ESPVR-slope is a very useful index of cardiac contractility that can be measured in situ by various methods; a convenient and popular approach is the use of the pressure-volume conductance catheter [2]. The cellular mechanisms that underlie the ESPVR are discussed in the following section. NIH Public Access Relationship between whole heart property and myofilament length dependent activationPump function of the heart is intimately related to force generation, active shortening, and regulation of card...