Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and β-myosin heavy chain with sinusoidal analysis

Kawai, Masataka; Karam, Tarek; Michael, John Jeshurun; Wang, Li; Chandra, Murali

doi:10.1007/s10974-016-9456-2

Cited by 10 publications

(5 citation statements)

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“…The Pi study characterizes the Pi-release step 5 and the force-generation step 4, which precedes the Pi-release step (Kawai and Halvorson, 1991); 2 πb is sensitive to steps 4 and 5. The results are analyzed in terms of Scheme 1, which is based on six CB states, and which has been successfully used for characterizing rabbit fast-twitch muscle fibers (Kawai and Halvorson, 1991; Kawai and Zhao, 1993; Galler et al, 2005), rabbit slow-twitch muscle fibers (Wang and Kawai, 1996, 1997), and cardiac muscle fibers from rodents (Wang et al, 2013, 2014a; Kawai et al, 2016) and large mammals (Zhao and Kawai, 1996; Fujita et al, 2002).…”

Section: Resultsmentioning

confidence: 99%

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

Kawai

Karam

Kolb

et al. 2018

Journal of General Physiology

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Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb−) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb− fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb− fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k−4) are larger in Neb+ fibers than in Neb− fibers. The rates of the force generation step (k4) and the reversal detachment step (k−2) change in the opposite direction. These effects can be explained by Le Chatelier’s principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

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Section: Resultsmentioning

confidence: 99%

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

Kawai

Karam

Kolb

et al. 2018

Journal of General Physiology

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“…Therefore, there is a possibility that the signal of hypophosphorylation of the rod domain may be transmitted to the myosin head domain and contribute to the acceleration of CB kinetics as seen in mutant fibers. Because adult murine ventricles predominantly express the fast a-MHC isoform rather than slow b-MHC isoform (40)(41)(42)(43), it is not likely that myosin isoform switching takes place. We infer from these observations that a Ca 2þ -sensitizing mutation in TnC causes a shift in myosin rod phosphorylation profile.…”

Section: Discussionmentioning

confidence: 99%

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Kawai

Johnston

Karam

et al. 2017

Biophysical Journal

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The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca-sensitivity and a decrease (0.8×) in cooperativity (n) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k) and its equilibrium constant (K) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k) decreased (0.5×), reversal step (k) increased (2×), and the phosphate-release step (1/K) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

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“…The α-MHC isoform contracts more vigorously than the β-MHC, albeit at the cost of utilizing more energy. Furthermore, we showed that calcium sensitivity was more attenuated in β-MHC than in α-MHC containing fibers [31]. Our study revealed that the α-MHC isoform predominates in myosin heavy chain in shamcontrol rat hearts (90%), whereas, in comparison, the predominant phenotype is β-MHC in the BDL-cirrhotic heart [16].…”

Section: Abnormal Intracellular Calcium Handling Systemmentioning

confidence: 65%

Dysregulated Calcium Handling in Cirrhotic Cardiomyopathy

2023

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Cirrhotic cardiomyopathy is a syndrome of blunted cardiac systolic and diastolic function in patients with cirrhosis. However, the mechanisms remain incompletely known. Since contractility and relaxation depend on cardiomyocyte calcium transients, any factors that impact cardiac contractile and relaxation functions act eventually through calcium transients. In addition, calcium transients play an important role in cardiac arrhythmias. The present review summarizes the calcium handling system and its role in cardiac function in cirrhotic cardiomyopathy and its mechanisms. The calcium handling system includes calcium channels on the sarcolemmal plasma membrane of cardiomyocytes, the intracellular calcium-regulatory apparatus, and pertinent proteins in the cytosol. L-type calcium channels, the main calcium channel in the plasma membrane of cardiomyocytes, are decreased in the cirrhotic heart, and the calcium current is decreased during the action potential both at baseline and under stimulation of beta-adrenergic receptors, which reduces the signal to calcium-induced calcium release. The study of sarcomere length fluctuations and calcium transients demonstrated that calcium leakage exists in cirrhotic cardiomyocytes, which decreases the amount of calcium storage in the sarcoplasmic reticulum (SR). The decreased storage of calcium in the SR underlies the reduced calcium released from the SR, which results in decreased cardiac contractility. Based on studies of heart failure with non-cirrhotic cardiomyopathy, it is believed that the calcium leakage is due to the destabilization of interdomain interactions (dispersion) of ryanodine receptors (RyRs). A similar dispersion of RyRs may also play an important role in reduced contractility. Multiple defects in calcium handling thus contribute to the pathogenesis of cirrhotic cardiomyopathy.

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Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and β-myosin heavy chain with sinusoidal analysis

Cited by 10 publications

References 50 publications

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Dysregulated Calcium Handling in Cirrhotic Cardiomyopathy

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