Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres

Güth, K.; Wojciechowski, Reinhold

doi:10.1007/bf00657515

Cited by 114 publications

(62 citation statements)

References 6 publications

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“…The NADH-linked fluorometric technique used to measure ATP iso in single permeabilized fibers has been previously described (13,14,20,27). Permeabilized fibers were mounted at optimal length (2.5 m) between force and displacement transducers in a quartz cuvette that was perfused with solutions containing a free ionized Ca 2ϩ concentration of either 1 nM (pCa 9.0) or 100 M (pCa 4.0) maintained at 15°C.…”

Section: Methodsmentioning

confidence: 99%

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

Sieck¹,

Prakash²,

Han³

et al. 2003

Journal of Applied Physiology

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Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development. J Appl Physiol 94: 1896-1902, 2003. First published January 31, 2003 10.1152/ japplphysiol.00617.2002-Early postnatal development of rat diaphragm muscle (Diam) is marked by dramatic transitions in myosin heavy chain (MHC) isoform expression. We hypothesized that the transition from the neonatal isoform of MHC (MHCNeo) to adult fast MHC isoform expression in Diam fibers is accompanied by an increase in both the maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) and the ATP consumption rate during maximum isometric activation (ATPiso). Rat Diam fibers were evaluated at postnatal days 0, 14, and 28 and in adults (day 84). Across all ages, Vmax ATPase of fibers was significantly higher than ATPiso. The reserve capacity for ATP consumption [1 Ϫ (ratio of ATPiso to Vmax ATPase)] was remarkably constant (ϳ55-60%) across age groups, although at day 28 and in adults the reserve capacity for ATP consumption was slightly higher for fibers expressing MHCSlow compared with fast MHC isoforms. At day 28 and in adults, both Vmax ATPase and ATPiso were lower in fibers expressing MHCSlow followed in rank order by fibers expressing MHC2A, MHC2X, and MHC2B. For fibers expressing MHCNeo, Vmax ATPase, and ATPiso were comparable to values for adult fibers expressing MHCSlow but significantly lower than values for fibers expressing fast MHC isoforms. We conclude that postnatal transitions from MHCNeo to adult fast MHC isoform expression in Diam fibers are associated with corresponding but disproportionate changes in Vmax ATPase and ATPiso. myosin heavy chain; fiber types; skeletal muscle; immunohistochemistry IN SKELETAL MUSCLES SUCH AS the diaphragm muscle (Dia m ), myosin heavy chain (MHC) functions as a molecular motor, converting chemical (ATP) into mechanical energy, thus driving muscle contraction (5). In adult skeletal muscle, several MHC isoforms exist, and the singular expression of these MHC isoforms in muscle fibers forms the basis for histochemical classification of different fiber types (18,24,25,31). In the neonatal rat Dia m , a neonatal isoform of MHC (MHC Neo ) is predominantly expressed, most often in combination with MHC Slow or MHC 2A isoforms (10,16,18,30,33). During the first 3-4 postnatal weeks, a dramatic transition in MHC isoform expression occurs in the rat Dia m , with the complete disappearance of MHC Neo expression by ϳ28 days of age (D-28). Concurrently, expression of MHC Slow and MHC 2A increases, and expression of MHC 2X and MHC 2B emerges (16,18,33).The expression of different MHC isoforms is associated with varying contractile properties of muscle fibers (3, 10-12, 24, 27, 28, 32). For example, muscle fibers expressing adult fast MHC isoforms (MHC 2A , MHC 2X , and MHC 2B ) have faster maximum shortening velocities than fibers expressing MHC Slow . In developing muscle fibers coexpressing MHC isoforms, Reiser and colleagues (22, 23) reported that maximum shortening velocity correlated...

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Section: Methodsmentioning

confidence: 99%

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

Sieck¹,

Prakash²,

Han³

et al. 2003

Journal of Applied Physiology

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“…The skinned fibers test solutions contain 85 mM K ϩ , 2 mM MgATP 2Ϫ , 1 mM Mg 2ϩ , 7 mM EGTA, 10 Ϫ6 Ϫ 10 Ϫ5 M Ca 2ϩ , 5 mM phospho(enol)pyruvate (PEP), 100 units/ml pyruvate kinase (PK), and propionate as the major anion. Subsequently, the fibers were mounted in the Guth Muscle Research System (27), which allows for simultaneous force and ATPase measurements. The quartz cuvette surrounding the preparation has a square cross-section of 1.0 mm 2 .…”

Section: Cardiac Fiber Experimentsmentioning

confidence: 99%

“…The solution in the cuvette was changed every 20 s using a peristaltic pump triggered by a computer. The hydrolysis of ATP was measured by the NADH fluorescence method, in which ATP is regenerated from ADP and PEP by the enzyme PK (27,28). This reaction is coupled to the oxidation of NADH (fluorescent) to NAD (nonfluorescent), and the reduction of pyruvate to lactate by L-lactic dehydrogenase (LDH) (27,28).…”

Section: Cardiac Fiber Experimentsmentioning

confidence: 99%

F110I and R278C Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy Affect Muscle Contraction in Transgenic Mice and Reconstituted Human Cardiac Fibers

Hernandez

Szczesna‐Cordary

Knollmann

et al. 2005

Journal of Biological Chemistry

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We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca 2؉ sensitivity of force and ATPase activity, and likewise an increased Ca 2؉ sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca 2؉ concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca 2؉ sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation.

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“…The solution in the cuvette was changed every 20 s using a peristaltic pump triggered by a computer. The hydrolysis of ATP was measured by the NADH fluorescence method, in which ATP was regenerated from ADP and phospho(enol)pyruvate by the enzyme pyruvate kinase (Reaction 1) (44). The reaction scheme is as follows.…”

Section: Simultaneous Force and Atpase Measurements In Fresh (Not Glymentioning

confidence: 99%

Abnormal Contractile Function in Transgenic Mice Expressing a Familial Hypertrophic Cardiomyopathy-linked Troponin T (I79N) Mutation

Miller

Szczêsna

Housmans

et al. 2001

Journal of Biological Chemistry

114

106

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This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine ␣-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca 2؉ sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pHinduced shifts in the Ca 2؉ dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca 2؉ from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

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Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres

Cited by 114 publications

References 6 publications

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

F110I and R278C Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy Affect Muscle Contraction in Transgenic Mice and Reconstituted Human Cardiac Fibers

Abnormal Contractile Function in Transgenic Mice Expressing a Familial Hypertrophic Cardiomyopathy-linked Troponin T (I79N) Mutation

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