Kyounghwan Lee

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling

The localization of MyBP-C within cardiac muscle cells provides efficient mechanochemical excitation-contraction coupling. The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies.

Cardiac Myosin Binding Protein-C Plays No Regulatory Role in Skeletal Muscle Structure and Function

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C(t/t)). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C(t/t) mice. Slow and fast skeletal muscles in cMyBP-C(t/t) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C(t/t) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM.

Myosin binding protein C is a thick filament protein of vertebrate striated muscle. The cardiac isoform [cardiac myosin binding protein C (cMyBP-C)] is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains and is located at nine sites, 43nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament, have been proposed. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal three domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends toward neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-Cs modulation of cardiac muscle contraction.

ID: 136: N-TERMINAL REGION OF CARDIAC MYOSIN BINDING PROTEIN-C IS NECESSARY FOR CARDIAC FUNCTION

Rationale Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated. Hypothesis The N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo. Methods and Results Transgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C110 kDa), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C110 kDa hearts. Importantly, cMyBP-C110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-Cs regulation of actomyosin interaction, in cMyBP-C110 kDa heart homogenates compared to those from NTG mice. Conclusion The N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.

Micromorphology and development of the epicuticular structure on the epidermal cell of ginseng leaves

Background A leaf cuticle has different structures and functions as a barrier to water loss and as protection from various environmental stressors. Methods Leaves of Panax ginseng were examined by scanning electron microscopy and transmission electron microscopy to investigate the characteristics and development of the epicuticular structure. Results Along the epidermal wall surface, the uniformly protuberant fine structure was on the adaxial surface of the cuticle. This epicuticular structure was highly wrinkled and radially extended to the marginal region of epidermal cells. The cuticle at the protuberant positions maintained the same thickness. The density of the wall matrix under the structures was also similar to that of the other wall region. By contrast, none of this structure was distributed on the abaxial surface, except in the region of the stoma. During the early developmental phase of the epicuticular structure, small vesicles appeared on wall–cuticle interface in the peripheral wall of epidermal cells. Some electron-opaque vesicles adjacent to the cuticle were fused and formed the cuticle layer, whereas electron-translucent vesicles contacted each other and progressively increased in size within the epidermal wall. Conclusion The outwardly projected cuticle and epidermal cell wall (i.e., an epicuticular wrinkle) acts as a major barrier to block out sunlight in ginseng leaves. The small vesicles in the peripheral region of epidermal cells may suppress the cuticle and parts of epidermal wall, push it upward, and consequently contribute to the formation of the epicuticular structure.

Structural Changes in Myosin Binding Protein C in Different Biochemical Conditions

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filaments of vertebrate striated muscle. Its function in skeletal muscle is not yet understood, but it is known to modulate contractility in the heart. Defects in MyBP-C lead to both skeletal and cardiac muscle disease. MyBP-C is an elongated molecule consisting primarily of a linear array of 10 (skeletal) or 11 (cardiac) immunoglobulin- and fibronectin-like 10 kDa domains. In addition to binding to the thick filament via its C-terminus, MyBP-C can also interact with myosin subfragment-2 and with actin via its N-terminus. MyBP-C is observed in EM images of sarcomeres as a set of up to nine transverse stripes, 43 nm apart, in each half of the A-band. Electron tomography of skeletal muscle in the relaxed state suggests that these stripes are due to extension of MyBP-C from the surface of the thick filament to the thin filaments, to which it binds. To investigate whether MyBP-C might change structurally in different physiological states, we have carried out experiments on isolated myofibrils and A-segments (A-bands free of thin filaments) to determine the appearance of the stripes under different biochemical conditions. Preliminary results suggest that in myofibrils (where thin filaments overlap thick filaments), stripes are present in both relaxed and rigor states, although they are less prominent in rigor. In A-segments (lacking thin filaments), stripes are common in the relaxed and activated (ATP/high Ca2+) states, but weak or absent in rigor. The results are consistent with a model in which the N-terminus of MyBP-C binds to actin filaments when they are available, and to myosin subfragment-2 when they are not.