Strong Binding of Myosin Modulates Length-Dependent Ca ²⁺ Activation of Rat Ventricular Myocytes

Abstract

Abstract-Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca 2ϩ sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca 2ϩ sensitivity of tension (ie, pCa 50 ) were measured in the absence and presence of N-ethylmaleimide-modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non-tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of Ϸ1.90 m, application of NEM-S1 markedly increased submaximal Ca 2ϩ -activated tensions and thereby increased Ca 2ϩ sensitivity; ie, pCa 50 increased from 5.40Ϯ0.02 to 5.52Ϯ0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca 2ϩ sensitivity of tension, in that the ⌬pCa 50 between short and long lengths was 0.02Ϯ0.01 pCa units in the presence of NEM-S1 compared with a ⌬pCa 50 of 0.10Ϯ0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca 2ϩ sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges. (Circ Res. 1998;83:602-607.) Key Words: Ca 2ϩ sensitivity Ⅲ muscle length Ⅲ ventricular myocyte W ithin normal physiological limits, alterations in ventricular end-diastolic volume result in marked changes in cardiac output. Since the heart normally operates on the positive slope of the pressure-volume relationship, increases in end-diastolic volume induce the heart to increase either stroke volume, ejection pressure, or both in a way that closely matches myocardial work to the load on the heart. This intrinsic ability of the heart to alter ventricular peak systolic pressure due to beat-to-beat variations in end-diastolic volume constitutes the basis for the well-known Frank-Starling relationship. The present study was performed to investigate molecular mechanisms underlying this relationship.Twitch tension and Ca 2ϩ sensitivity of tension (ie, pCa 50 ) in cardiac muscle preparations are known to decrease as sarcomere length (SL) is shortened within its working range (ie, from Ϸ2.30 m to Ϸ1.80 m).1,2 The regulation of myocardial contraction requires the binding of Ca 2ϩ to a low-affinity site on troponin C, which permits strong interactions of myosin crossbridges with actin.3 However, experimental evidence has shown that Ca 2ϩ alone is unable to fully activate the thin filament: complete activation, in terms of force and the kinetics of for...

The Frank–Starling mechanism in vertebrate cardiac myocytes

“…This is demonstrated for frog myocardium in Fig.·2C by the leftward shift in the tension-pCa curve (δpCa 50 ) of about 0.55·pCa units over the SL range 2.2-3.1·μm at 15°C (Fabiato and Fabiato, 1978b). For comparison, in rat myocytes at 22°C, a δpCa 50 of 0.1·pCa unit from 5.41 was seen when SL increased from 1.9 to 2.25·μm (Fitzsimons and Moss, 1998), and a δpCa 50 of 0.13 to 0.19 units from 5.7 when SL increased from 1.9 to 2.3·μm (Cazorla et al, 2005). Ca 2+ sensitivity falls with falling temperature (Harrison and Bers, 1990) but if this is ignored, these figures roughly equate to a threefold fall in the Ca 2+ required for half maximal activation ([Ca 2+ ] 50 ) per 1·μm increase in SL in both amphibians and mammals (with frog having a lower [Ca 2+ ] 50 at resting length; 1·μmol·l -1 frog versus 2-4·μmol·l -1 rat).…”

“…( 2001). When a strong binding cross-bridge forms it generates tension but also shifts the tropomyosin molecule further into the actin groove, which increases the probability of a neighbouring myosin head forming a cross-bridge (Fitzsimons and Moss, 1998;Konhilas et al, 2002a). Strong binding cross-bridges also induce cooperative activation of actin by increasing the apparent affinity of TnC for Ca 2+ (Gordon and Ridgway, 1993) indicating coupling between the Ca 2+ regulatory sites on TnC and cross-bridge interactions in the thin filament (Fukuda and Granzier, 2005).…”

“…Strong binding cross-bridges also induce cooperative activation of actin by increasing the apparent affinity of TnC for Ca 2+ (Gordon and Ridgway, 1993) indicating coupling between the Ca 2+ regulatory sites on TnC and cross-bridge interactions in the thin filament (Fukuda and Granzier, 2005). This mechanism of propagated activation may be particularly important in cardiac muscle (compared with skeletal muscle) where relatively small changes in the proportion of strongly bound cross-bridges have significant effects on the Ca 2+ sensitivity of force (Fitzsimons and Moss, 1998). Indeed, recent work has shown cardiac muscle activation is controlled locally, within a regulatory unit (made up of seven actin monomers, 1 tropomyosin and 1 troponin complex) and has greater reliance on cross-bridge attachment for promoting cooperative activation than skeletal muscle (Gillis et al, 2007).…”

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension

“…Mechanical measurements were performed on an experimental apparatus described previously [31][32]. In brief, approximately 200 µl of a suspension of skinned ventricular myocytes was placed on a glass cover-slip.…”

Increasing mammalian cardiomyocyte contractility with residues identified in trout troponin C

“…An experimental chamber was constructed for attaching single cardiac myocytes to a force transducer and a servomotor for the activation and relaxation of the attached cell. Cells were attached using the method described by Fitzsimons and Moss (8). The experimental chamber was positioned on the stage of a Nikon (Mississauga, ON, Canada) Diaphot inverted microscope.…”