Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Toepfer, Christopher N.; Caorsi, Valentina; Kampourakis, Thomas; Sikkel, Markus B.; West, Timothy G.; Leung, Judy; Al-Saud, Sara Abou; MacLeod, Ken; Lyon, Alexander R.; Marston, Steven B.; Sellers, James R.; Ferenczi, Michael A.

doi:10.1016/j.bpj.2012.11.1720

Cited by 13 publications

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Section: Rlc Exchangementioning

confidence: 99%

“…The methods for exchange and for measuring the extent of exchange have been described previously (Toepfer et al 2013). Briefly, recombinant cardiac RLC from Rattus norvegicus was expressed, purified and dephosphorylated or phosphorylated in vitro using shrimp alkaline phosphatase or smooth muscle myosin light chain kinase, respectively (Toepfer et al 2013). RLCs with known phosphorylation levels (0.1 mol Pi mol -1 RLC, 0.5 mol Pi mol -1 RLC and 1.1 mol Pi mol -1 RLC) were exchanged for native RLC in permeabilized trabeculae using an exchange buffer (composition in mM: 5 ATP, 5 EGTA, 5 EDTA, 10 imidazole, 150 potassium propionate, 10 KH 2 PO 4 , 5 DTT and 0.5 trifluoperazine at pH 6.5).…”

Section: Rlc Exchangementioning

confidence: 99%

“…In the presence of calcium, these sites are available and cyclical interactions between actin and myosin occur, producing force and shortening. In addition, RLC phosphorylation level plays a role in modulating the ability of cross-bridges to produce force, especially under load (Toepfer et al 2013). What has not yet been well established is the ability of RLC phosphorylation to alter the kinetics of force redevelopment (k tr ) (not created by stretch activation) (Stelzer et al 2006a;Stelzer et al 2006b) in cardiac tissue.…”

Section: Introductionmentioning

confidence: 99%

“…By carrying out experiments in the presence of saturating [Ca 2+ ], the effect of calcium-dependent activation is eliminated such that any changes in k tr depend on the RLC phosphorylation levels and not on the calcium regulatory complex (de Tombe & Stienen, 2007). The role of RLC phosphorylation warrants investigation because the observed changes in RLC phosphorylation levels in myocardium, above or below basal levels, change both power output and force production of the tissue (Toepfer et al 2013) Additionally, there is a close association between RLC phosphorylation and myocardial function, such as in disease models, both acquired and inherited (Sheikh et al 2014).…”

Section: Introductionmentioning

confidence: 99%

“…In the present study, we use an in vitro RLC exchange protocol to alter RLC phosphorylation level in rat cardiac ventricular trabeculae, without affecting phosphorylation of other sarcomeric constituents (Toepfer et al 2013). Activation of the permeabilized trabeculae is achieved by temperature-jump to ensure rapid and homogeneous activation and thereby avoiding the confounding effects of Ca-diffusion into the core of the trabeculae.…”

Section: Introductionmentioning

confidence: 99%

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Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Toepfer

West

Ferenczi

2016

The Journal of Physiology

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Key points Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (k tr) at submaximal [Ca2+].Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca2+]‐activated cardiac trabecula accelerates k tr.Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows k tr and reduces force generation. k tr is dependent on sarcomere length in the physiological range 1.85–1.94 μm and RLC phosphorylation modulates this response.We demonstrate that Frank–Starling is evident at maximal [Ca2+] activation and therefore does not necessarily require length‐dependent change in [Ca2+]‐sensitivity of thin filament activation.The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank–Starling law in the heart.These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. AbstractForce and power in cardiac muscle have a known dependence on phosphorylation of the myosin‐associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (k tr) in maximally activating [Ca2+]. Activation was achieved by rapidly increasing the temperature (temperature‐jump of 0.5–20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85–1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC‐exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates k tr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows k tr by ∼50%, relative to the k tr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of k tr following step shortening or ramp shortening. Using a two‐state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross‐bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+] and are not explained by changes in the Ca2+‐sensitivity of acto‐myosin interactions. The length‐dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank–Starling law of the heart.

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Section: Rlc Exchangementioning

confidence: 99%

Section: Rlc Exchangementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Toepfer

West

Ferenczi

2016

The Journal of Physiology

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Mn²⁺‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels

Markandran

Xuan

et al. 2021

Current Protocols

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This paper provides a guideline for optimizing and utilizing Mn2+ Phos‐tag gel technology to separate phosphorylated proteins from their unphosphorylated counterparts. It provides key insights into methods for careful sample preparation and experimental directions for determining the appropriate Phos‐tag gel compositions and electrophoresis and western blotting conditions. This protocol has been used to successfully resolve proteins extracted from cardiac and skeletal muscles. The guidelines can be extended for optimizing protocols to resolve proteins from other cells or tissue sources. With this, phosphoproteomics and the elucidation of underlying mechanisms of disease progression can be accelerated. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

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Targeted proteomics of myofilament phosphorylation and other protein posttranslational modifications

Ramírez-Correa

Martinez-Ferrando

Zhang

et al. 2014

Proteomics Clinical Apps

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Global cardiac myofilament protein phosphorylation levels, and their site-specific stoichiometry, are physiologically and clinically relevant for heart function. Unlike myofilament phosphorylation, other PTMs such as O-GlcNAcylation are just beginning to gain attention due to their potential physiological and clinical implications. This review will focus on what is currently known about cardiac troponin I phosphorylation, and on the potential physiological and clinical impact of targeted proteomics including new findings on cardiac troponin I sites and stoichiometry. We will then discuss the increasing recognition of other myofilament PTMs functional relevance and the potential of targeted MS approaches, particularly MRM, for accelerating their systematic characterization. In addition, we will broadly discuss the development and application of MRM to quantitatively assess site-specific PTMs. Finally, we will give an overview of expert's consensus on MRM methods design/validation and best practices to develop MRM assays intended to reach clinical application. The unique ability of MRM and similar methods to identify and quantify cardiac myofilament PTMs is likely to become central in answering important biological questions in the field of cardiac integrative physiology.

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Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Cited by 13 publications

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Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Mn²⁺‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels

Targeted proteomics of myofilament phosphorylation and other protein posttranslational modifications

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Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Cited by 13 publications

References 0 publications

Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr) in a length‐dependent fashion

Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr) in a length‐dependent fashion

Mn2+‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels

Targeted proteomics of myofilament phosphorylation and other protein posttranslational modifications

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Product

Resources

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Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k_tr) in a length‐dependent fashion

Mn²⁺‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels