N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

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Single Cardiac Ventricular Myosins Change Step-Size with Loading

10.1101/123166 ◽

2017 ◽

Author(s):

Yihua Wang ◽

Chen-Ching Yuan ◽

Katarzyna Kazmierczak ◽

Danuta Szczesna-Cordary ◽

Thomas P. Burghardt

Keyword(s):

Actin Binding ◽

Transgenic Mouse Models ◽

Cardiac Myosin ◽

Step Size ◽

Average Force ◽

Essential Light Chain ◽

C Terminus ◽

N Terminus ◽

Mutant Forms ◽

Increasing Load

ABSTRACTThe cardiac myosin motor powers the beating heart by catalyzed ATPase free energy conversion to contractile work. Transgenic mouse models for heart disease express mouse α-cardiac myosin heavy chain with human essential light chain (ELC) in wild type (WT), or hypertrophic cardiomyopathy linked mutant forms, A57G or E143K. Mutants modify the ELC actin binding N-terminus or C-terminus regions. Motility and single myosin mechanical characteristics show stark contrasts between the motors related to their average force, power, and displacement while all indicate the ability to down-shift ensemble step-size with increasing load. A57G and E143K consume more ATP than control WT in the presence of actin with A57G upregulating and E143K downregulating power compared with WT. Higher ATP consumption and downregulated power in E143K implies a lower unitary force. Effects on power are consistent with an A57G that impairs the ELC N-terminus actin binding and an E143K that reduces lever-arm rigidity.

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Deletion of 1- 43 Amino Acids from the N-terminus of Myosin Essential Light Chain in Transgenic Mice Decreases Force Production by Reducing the Number of Myosin Cross-Bridges

Biophysical Journal ◽

10.1016/j.bpj.2010.12.895 ◽

2011 ◽

Vol 100 (3) ◽

pp. 126a

Author(s):

John J. Michael ◽

Steven J. Ford ◽

Katarzyna Kazmierczak ◽

Danuta Szczesna-Cordary ◽

Chandra Murali

Keyword(s):

Amino Acids ◽

Transgenic Mice ◽

Light Chain ◽

Force Production ◽

Essential Light Chain ◽

N Terminus ◽

Cross Bridges

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Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00572.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. H253-H259 ◽

Cited By ~ 14

Author(s):

John Jeshurun Michael ◽

Sampath K. Gollapudi ◽

Steven J. Ford ◽

Katarzyna Kazmierczak ◽

Danuta Szczesna-Cordary ◽

...

Keyword(s):

Amino Acids ◽

Light Chain ◽

Sarcomere Length ◽

Cardiac Myosin ◽

Wild Type ◽

Essential Light Chain ◽

Cross Bridge ◽

Maximal Activation ◽

Cardiac Muscle Fibers

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50 (−log10 [Ca2+]free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca2+ sensitivity. Our data suggest that the NH2-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca2+ sensitivity.

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Deletion of the N-Terminus of Myosin Essential Light Chain (N-ELC) in the Background of HCM-A57G Mutation in Double Mutant Mice Rescues Hypercontractile Myosin Phenotype

Biophysical Journal ◽

10.1016/j.bpj.2019.11.1836 ◽

2020 ◽

Vol 118 (3) ◽

pp. 328a

Author(s):

Yoel H. Sitbon ◽

Katarzyna Kazmierczak ◽

Melanie Veerasammy ◽

Jingsheng Liang ◽

Danuta Szczesna-Cordary

Keyword(s):

Light Chain ◽

Double Mutant ◽

Mutant Mice ◽

Essential Light Chain ◽

N Terminus

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Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin

The Journal of General Physiology ◽

10.1085/jgp.202012801 ◽

2021 ◽

Vol 153 (7) ◽

Author(s):

Yoel H. Sitbon ◽

Francisca Diaz ◽

Katarzyna Kazmierczak ◽

Jingsheng Liang ◽

Medhi Wangpaichitr ◽

...

Keyword(s):

Light Chain ◽

Physiological Model ◽

Papillary Muscles ◽

Myosin Regulatory Light Chain ◽

Essential Light Chain ◽

Sarcomeric Protein ◽

N Terminus ◽

Ventricular Myosin ◽

Energy Conserving ◽

Cross Bridges

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K–associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.

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Abstract 314: The Interaction Of The Estrogen Receptor Alpha With The Cardiac Myosin Essential Light Chain Isoform 4 As A New Regulatory Mechanism In The Heart

Circulation Research ◽

10.1161/res.113.suppl_1.a314 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Shokoufeh Mahmoodzadeh ◽

Miriam Schanz ◽

Hang Pham ◽

Mercy M Davidson ◽

Ingo Morano ◽

...

Keyword(s):

Estrogen Receptor ◽

Light Chain ◽

Human Heart ◽

Transcriptional Activity ◽

Heart Function ◽

Luciferase Reporter ◽

Human Atrium ◽

Cardiac Myosin ◽

Human Cardiomyocytes ◽

Essential Light Chain

Estrogen (17β-estradiol, E2) is one of the key regulators of growth, differentiation and physiological function in different tissues, including the heart. The effects of E2 are mainly mediated by estrogen receptor (ER) alpha and beta, which act in concert with many cofactors to mediate estrogenic effects. So far, only a few cofactors of ER have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and characterized the novel interaction partners of ERα Yeast two hybrid screening of a human heart cDNA library revealed that ERα interacts with the cardiac myosin essential light chain isoform 4 (ALC1) in presence of E2. ALC1, as a member of contractile proteins, is expressed in the fetal heart and becomes restricted to the atria of the adult heart under physiological conditions, and is reexpressed in ventricle of adult hypertrophic hearts. This switch is accompanied by alteration of contractile performance, thus improving the heart function. Retransformation experiments showed that ALC1 interacts with full-length ERα and ERα-EF domain in presence of E2. The interaction of ERα with ALC1 was also confirmed by Co-IP in human atrium. Double irnmunofluorescence (IF) analysis of paraffin-embedded sections from human atrium tissues showed co-localization of ERα and ALC1 proteins in a striated sarcomeric pattern. Co-localization corresponds most likely to the H-zone of sarcomere. IF analysis of AC16 cells (a human cardiomyocytes cell line) showed that ERα and ALC1 were mainly localized in the cytoplasm in the absence of E2. However, E2 treatment of AC16 cells led to a translocation of ALC1 and ERα into the nuclei of AC16 cells, where they also co-localized. Expression analysis in AC16 cells showed that E2 increases the expression of ALC1 gene (MYL4). Using ERE-based luciferase reporter assays we showed that E2-induced interaction of ERα with the ALC1 represses the transcriptional activity of ERα. We characterize for the first time an E2-regulated interaction of ALC1 with ERα in cardiomyocytes that may be crucial in physiological and/or pathological processes by regulating of transcriptional activity of ERa in the heart and/or by modulating contractile properties of cardiomyocytes.

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