A treadmill exercise regimen for identifying cardiovascular phenotypes in transgenic mice

Fewell, Jason G.; Osińska, Hanna; Klevitsky, Raisa; Ng, WF; Sfyris, George; Bahrehmand, Fariborz; Robbins, Jeffrey

doi:10.1152/ajpheart.1997.273.3.h1595

Cited by 73 publications

(74 citation statements)

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“…Oligonucleotides corresponding to atrial natriuretic factor, phospholamban, sarcoplasmic reticulum ATPase 2A, skeletal ␣-actin, and glyceraldehyde-3-phosphate dehydrogenase (used as a loading control) were used to examine gene expression related to cardiac hypertrophy and sarcoplasmic reticulum calcium handling (26). Ventricular protein was loaded onto a 5% glycerol gel and electrophoresed to separate the ␣-and ␤-MHC proteins.…”

Section: Methodsmentioning

confidence: 99%

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

Miller

Palmer

Ruch

et al. 2005

Journal of Biological Chemistry

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The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1v⌬5-14, referred to as 1v⌬5-14), a region that interacts with actin (Sanbe, A., Gulick, J., Fewell, J., and Robbins, J. (2001) J. Biol. Chem. 276, 32682-32686). 1v⌬5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1v⌬5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1v⌬5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1v⌬5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

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Section: Methodsmentioning

confidence: 99%

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

Miller

Palmer

Ruch

et al. 2005

Journal of Biological Chemistry

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“…Myofibrillar sample preparation, gel preparation, electrophoretic conditions, and gel staining have been described (17). Samples used for two-dimensional gel electrophoresis were prepared as described by Kirschbaum et al (23).…”

Section: Methodsmentioning

confidence: 99%

“…Gels were stained with colloidal blue (Sigma). The phosphorylation state of fibers used in the mechanical studies was tested by Western blot analysis as described (17). Each sample consisted of two ventricular fiber bundles homogenized in sample buffer (8 M urea, 18 mM Tris (pH 8.5), 20.7 mM glycine, 9.3 mM dithiothreitol, 4.6% (v/v) saturated sucrose, and 0.004% (w/v) bromphenol blue).…”

Section: Methodsmentioning

confidence: 99%

“…This response may help maintain force production and stroke volume in the face of an increased rate of relaxation. However, the response is attenuated or absent in the mouse heart after similar exercise regimens (17). Changes in RLC phosphorylation in ischemic rabbit hearts have been noted (18), and in some patients with heart failure, decreases in light chain phosphorylation occurred (19).…”

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Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2

Sanbe¹,

Fewell²,

Gulick³

et al. 1999

Journal of Biological Chemistry

114

102

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A role for myosin phosphorylation in modulating normal cardiac function has long been suspected, and we hypothesized that changing the phosphorylation status of a cardiac myosin light chain might alter cardiac function in the whole animal. To test this directly, transgenic mice were created in which three potentially phosphorylatable serines in the ventricular isoform of the regulatory myosin light chain were mutated to alanines. Lines were obtained in which replacement of the endogenous species in the ventricle with the nonphosphorylatable, transgenically encoded protein was essentially complete. The mice show a spectrum of cardiovascular changes. As previously observed in skeletal muscle, Ca 2؉ sensitivity of force development was dependent upon the phosphorylation status of the regulatory light chain. Structural abnormalities were detected by both gross histology and transmission electron microscopic analyses. Mature animals showed both atrial hypertrophy and dilatation. Echocardiographic analysis revealed that as a result of chamber enlargement, severe tricuspid valve insufficiency resulted in a detectable regurgitation jet. We conclude that regulated phosphorylation of the regulatory myosin light chains appears to play an important role in maintaining normal cardiac function over the lifetime of the animal.The roles of the regulatory myosin light chains (RLCs) 1 and the reversible post-translational modifications they undergo in striated muscle are beginning to be defined. In skeletal muscle, a serine at the amino end of the protein can be phosphorylated by a sarcoplasmic kinase (1), and it is now clear that RLCs in the different striated muscles are phosphorylated to differing degrees, leading presumably to different physiological effects. In smooth muscle, RLC phosphorylation by myosin light chain kinase (MLCK), which is activated by a Ca 2ϩ /calmodulin-dependent pathway, is responsible for initiating muscle contraction (2, 3). However, in skeletal and cardiac muscle, in which the thin, rather than thick, filament mediates control of contraction, RLC phosphorylation does not activate contraction but appears to play a modulatory role. In skinned skeletal muscle fibers, RLC phosphorylation increases sensitivity to activating Ca 2ϩ such that there is a significant leftward shift in the force-Ca 2ϩ relationship (4 -6). Increased RLC phosphorylation in skeletal muscle is also associated with potentiation of isometric twitch tension with repeated activation and inactivation of contraction (7, 8), rate of force production (9 -11), and maximum Ca 2ϩ -stimulated MgATPase activity (12). The mechanistic basis for the effects of RLC phosphorylation in striated muscle is hypothesized to be a lessening of the weak interaction of the myosin head with the myosin backbone and is probably due to a net charge change in a critical region of the protein (13). Upon phosphorylation, the myosin heads move away from the backbone to a position closer to actin, which presumably increases the rate at which myosin-actin interaction...

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“…Treadmill exercise is one of the primary physiological methods used to increase hemodynamic stress and to detect cardiovascular abnormalities that may not be readily apparent at rest (22,23). To test the hypothesis that the abnormalities in capillary morphology observed in the mutant mice could have some consequences for blood flow and lead to pathological changes after cardiovascular stress, left ventricle samples were prepared after exercise experiment 2, and the possibility of cardiac injury was studied by assessing the extent of apoptosis (24), the activities of ␤-glucuronidase (25) and matrix metalloproteinase 2 (26), and the mRNA levels of ANP (27).…”

Section: Ultrastructural Changes In the Heart And Skeletal Muscle Capmentioning

confidence: 99%

Lack of type XV collagen causes a skeletal myopathy and cardiovascular defects in mice

Eklund¹

2001

Proceedings of the National Academy of Sciences

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Type XV collagen occurs widely in the basement membrane zones of tissues, but its function is unknown. To understand the biological role of this protein, a null mutation in the Col15a1 gene was introduced into the germ line of mice. Despite the complete lack of type XV collagen, the mutant mice developed and reproduced normally, and they were indistinguishable from their wild-type littermates. However, Col15a1-deficient mice showed progressive histological changes characteristic for muscular diseases after 3 months of age, and they were more vulnerable than controls to exercise-induced muscle injury. Despite the antiangiogenic role of type XV collagen-derived endostatin, the development of the vasculature appeared normal in the null mice. Nevertheless, ultrastructural analyses revealed collapsed capillaries and endothelial cell degeneration in the heart and skeletal muscle. Furthermore, perfused hearts showed a diminished inotropic response, and exercise resulted in cardiac injury, changes that mimic early or mild heart disease. Thus, type XV collagen appears to function as a structural component needed to stabilize skeletal muscle cells and microvessels.T ype XV collagen belongs to the heterogeneous group of non-fibril-forming collagens and is thought to be a homotrimer consisting of three ␣1(XV) collagen chains (1). It is characterized by a central highly interrupted triple helical domain and large N-and C-terminal noncollagenous domains (2-4), and it has been shown to be a chondroitin sulfate proteoglycan (5). Type XV collagen mRNAs are expressed in many tissues, but the highest mRNA levels in the mouse can be detected in the heart and skeletal muscle (4). The protein is shown by immunostaining to have a widespread tissue distribution and has been localized mainly to the basement membrane zones, although it can also be found in the fibrillar collagen matrix of some tissues (6, 7). Its function is not known, however.In terms of primary structure, type XV collagen is highly homologous with type XVIII collagen, and together they form a distinct subgroup among the collagens (1, 3). They have thrombospondin-1 sequence homology in the N terminus, seven homologous collagenous domains, and highly homologous Cterminal noncollagenous domains. Type XVIII collagen is the precursor of endostatin, which has been shown to have a potent antiangiogenic effect (8), and the highest degree of homology between collagen types XV and XVIII involves the C-terminal endostatin sequence. The corresponding fragment in type XV collagen has also been shown to have antiangiogenic activity (9, 10).To understand the biological function and significance of type XV collagen, we generated a mouse strain lacking in ␣1(XV) collagen chains by site-specific Cre-loxP-mediated deletion in embryonic stem (ES) cells (11). The data suggest a structural role for type XV collagen in providing mechanical stability between cells and the extracellular matrix in skeletal muscle fibers and microvessels. Col15a1 deficiency leads to functional rather than struct...

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A treadmill exercise regimen for identifying cardiovascular phenotypes in transgenic mice

Cited by 73 publications

References 0 publications

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2

Lack of type XV collagen causes a skeletal myopathy and cardiovascular defects in mice

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