Activation Kinetics of Skinned Cardiac Muscle by Laser Photolysis of Nitrophenyl-EGTA

Martin, Hunter; Bell, Marcus G.; Ellis‐Davies, Graham C. R.; Barsotti, Robert

doi:10.1016/s0006-3495(04)74173-9

Cited by 15 publications

(16 citation statements)

References 68 publications

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“…In fibres containing wild-type troponin, after the photolysis of the caged Ca 2+ chelator, the halftime of activation of a muscle fibre decreased with increasing relative force produced, as expected from previous reports [19,20]. With the mutant protein incorporated, if anything, the halftime increased as the relative force increased, despite the level of post-photolysis force being on average slightly greater in the TnC Gly159Asp replaced fibres (Fig.…”

Section: Replacement Of Whole Troponin In Rabbit Psoas Fibressupporting

confidence: 87%

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

Preston

Lipscomb

Robinson

et al. 2006

Pflugers Arch - Eur J Physiol

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We recently reported a dilated cardiomyopathy (DCM) causing mutation in a novel disease gene, TNNC1, which encodes cardiac troponin C (TnC). We have determined how this mutation, Gly159Asp, affects contractile regulation when incorporated into muscle fibres. Endogenous troponin in rabbit skinned psoas fibres was partially replaced by recombinant human cardiac troponin containing either wild-type or Gly159Asp TnC. We measured both the force-pCa relationship of these fibres and the activation rate using the caged-Ca(2+) compound nitrophenyl-EGTA. Gly159Asp TnC had no significant effect on either the Ca(2+) sensitivity or cooperativity of force generation when compared to wild type. However, the mutation caused a highly significant (ca. 50%) decrease in the rate of activation. This study shows that whilst not affecting the force-pCa relationship, the mutation Gly159Asp causes a significant decrease in the rate of force production and a change in the relationship between the rate of force production and generated force. In vivo, this mutation may cause both a slowing of force generation and reduction in total systolic force. This represents a novel mechanism by which a cardiomyopathy-causing mutation can affect contractility.

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Section: Replacement Of Whole Troponin In Rabbit Psoas Fibressupporting

confidence: 87%

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

Preston

Lipscomb

Robinson

et al. 2006

Pflugers Arch - Eur J Physiol

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“…Consistent with this assumption, force was found to change in proportion with stiffness which is thought to measure cross-bridge attachment [16]. However, recent investigations refining the force-stiffness relation reveal significant non-linearities [84]and non-proportional changes of force and stiffness accompanying force activation and force relaxation in fibres [6,100]. Whether or not these findings imply a significant non-proportional behaviour of force states depends in turn on the proportionality and nature of the stiffness.…”

Section: Cross-bridge Kinetics and Thin-filament Inactivation During mentioning

confidence: 86%

“…This problem can be overcome by loading fibres with photo-labile caged compounds that can rapidly release products, e.g. Ca 2+ or Ca 2+ chelators, by light flashes [4,9,30,69,93,96,100,111,149,156]. However, the use of caged compounds has other drawbacks (discussed in [46,117]).…”

mentioning

confidence: 99%

“…For these reasons, skinned fibres had been mostly used to investigate the mechanisms of Ca 2+ -regulated contraction under steady [Ca 2+ ] [16,17,21,28,34,78,120] and only few studies investigated the force kinetics induced by rapid changes in [Ca 2+ ] (e.g. [9,100,111,149]). Several studies that investigate force kinetics in intact and skinned fibres induce force transients by small sinusoidal or step-shaped length changes (e.g.…”

mentioning

confidence: 99%

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Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Stehle

Solzin

Iorga

et al. 2009

Pflugers Arch - Eur J Physiol

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Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca(2+) via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca(2+)-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca(2+) regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca(2+)]. Among the processes studied on myofibrils are: (1) the Ca(2+)-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.

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“…It therefore seems unlikely that the troponin isoforms directly contribute to the observed effects of the mutation on the activation kinetics. Instead, we favour a model in which force development kinetics are primarily rate‐limited by turnover kinetics of the crossbridge cycle (Stehle et al 2002 a ; Martin et al 2004). The cTn–Tm complex rapidly fluctuates between off (Ca 2+ bound) and on (no Ca 2+ bound) states (Brenner & Chalovich, 1999).…”

Section: Discussionmentioning

confidence: 99%

Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction–relaxation cycle in isolated cardiac myofibrils

Zittrich¹,

Redwood²,

Blaudeck³

et al. 2005

The Journal of Physiology

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Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnIR145G on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI WT or hcTnI R145G . Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI R146G mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mM BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca 2+ . Maximum force development (F max ) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI R145G in vitro. Ca 2+ sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k ACT is reduced at maximal [Ca 2+ ] but not at low [Ca 2+ ] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k REL . We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.

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Activation Kinetics of Skinned Cardiac Muscle by Laser Photolysis of Nitrophenyl-EGTA

Cited by 15 publications

References 68 publications

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction–relaxation cycle in isolated cardiac myofibrils

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