Ventricular myosin modifies in vitro step-size when phosphorylated

Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

doi:10.1016/j.yjmcc.2014.03.022

Cited by 38 publications

(61 citation statements)

References 32 publications

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“…Step-frequency expectation values for Δ0βmys are statistically equivalent to previous results where the 5 nm step predominates (2, 3). Probability for the 5 nm step increased while probabilities for the 3 and 8 nm steps declined in Δ17βmys compared to Δ0βmys as the fractional contribution from the truncated vELC increased (Figure 3 middle column).…”

Section: Resultssupporting

confidence: 82%

“…Step-size values for Δ0βmys are statistically equivalent to previous results (2, 3). We compared 3 step-sizes for the pooled βmys data from Δ0 and Δ17βmys @ 60% (60% truncated) using one-way ANOVA.…”

Section: Resultssupporting

confidence: 79%

“…For cardiac myosin, step-frequency varies among the myosin step-sizes and the continuity of actin motility with multiple actomyosin interactions implies that each step-size has a unique duty-ratio and characteristic time strongly actin bound (27). Step-frequency is a second fundamental motor characteristic likely involved in adapting myosin work production to the changing power demands in a normal heart (3). …”

Section: Discussionmentioning

confidence: 99%

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

Wang¹,

Ajtai²,

Kazmierczak

et al. 2015

Biochemistry

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Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ~19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method.

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Section: Resultssupporting

confidence: 82%

Section: Resultssupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Wang¹,

Ajtai²,

Kazmierczak

et al. 2015

Biochemistry

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“…Fractional phosphorylation levels of 0, 40%, and 85% (maximally phosphorylated) were investigated. We found that phosphorylation causes a dramatic re-distribution of step-frequencies (Wang et al 2014b). These data are summarized in Table 1 for the 0 (native) and 85% phosphorylated βmys (pβmys).…”

Section: Natural Myosin Activationmentioning

confidence: 88%

“…Muscle myosins produce multiple unitary steps with differing step-sizes necessitating introduction of the relative step-frequency, ω j , for unitary step j (Wang et al 2013b; Wang et al 2014b). It is proportional to the rate of cross-bridge cycling with the higher rate producing a more frequent j th step.…”

Section: In Vitro Step-size and Step-frequency Of Single Myosins In Amentioning

confidence: 99%

In vitro and in vivo single myosin step-sizes in striated muscle

Burghardt

Sun

Wang

et al. 2015

J Muscle Res Cell Motil

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Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a “second characterization” is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover “bottom-up” and “top-down” assaying of myosin characteristics.

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Mechanistic basis for rescuing hypertrophic cardiomyopathy with myosin regulatory light chain phosphorylation

Liang,

Kazmierczak,

Veerasammy

et al. 2024

Cytoskeleton

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We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D‐RLC) on the Super‐Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D‐D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg‐D166V model and wild‐type (WT) RLC mice. In the Tg‐D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V‐mutated myosin motors. The presence of the S15D moiety in Tg‐S15D‐D166V mice restored the SRX/DRX balance to levels comparable to Tg‐WT, thus mitigating the hypercontractile behavior associated with the HCM‐D166V mutation. Additionally, we investigated the impact of delivering the S15D‐RLC molecule to the hearts of Tg‐D166V mice via adeno‐associated virus (AAV9) and compared their condition to AAV9‐empty vector‐injected or non‐injected Tg‐D166V animals. Tg‐D166V mice injected with AAV9 S15D‐RLC exhibited a significantly higher proportion of myosin heads in the SRX state compared to those injected with AAV9 empty vector or left non‐injected. No significant effect was observed in Tg‐WT hearts treated similarly. These findings suggest that AAV9‐delivered phosphomimetic S15D‐RLC modality mitigates the abnormal Tg‐D166V phenotype without impacting the normal function of Tg‐WT hearts. Global longitudinal strain analysis supported these observations, indicating that the S15D moiety can alleviate the HCM‐D166V phenotype by restoring SRX stability and the SRX ↔ DRX equilibrium.

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Ventricular myosin modifies in vitro step-size when phosphorylated

Cited by 38 publications

References 32 publications

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

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

In vitro and in vivo single myosin step-sizes in striated muscle

Mechanistic basis for rescuing hypertrophic cardiomyopathy with myosin regulatory light chain phosphorylation

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