The rates of Ca2+ dissociation and cross-bridge detachment from ventricular myofibrils as reported by a fluorescent cardiac troponin C

Abstract

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.

FIGURE 1.

Ca²⁺ dissociation from IANBD-labeled TnC in Tn and Tn-exchanged rabbit rigor myofibrils. Panel A shows the time course of IANBD fluorescence as Ca²⁺ was chelated by EGTA and removed from the regulatory binding site of TnC_IANBD^T53C reconstituted into Tn. The Tn (0.3 μm) in Buffer A + 200 μm Ca²⁺ was rapidly mixed with an equal volume of Buffer A + 10 mm EGTA at 15 °C (Ca²⁺ off Tn trace, 40.8 ± 0.5/s). The Ca²⁺-saturated Tn base line was collected by mixing the Ca²⁺-saturated Tn with Buffer A + 200 μm Ca²⁺. The Ca²⁺-free Tn base line was acquired by rapidly mixing Tn in Buffer A + 5 mm EGTA against equal volumes of Buffer A + 5 mm EGTA. Panel B shows the time course of the IANBD fluorescence as Ca²⁺ was removed by EGTA from TnC_IANBD^T53C Tn-exchanged rabbit rigor ventricular myofibrils. TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ were rapidly mixed with an equal volume of the Buffer A + 10 mm EGTA at 15 °C (Ca²⁺ off Myofibrils trace, 25.3 ± 0.7/s). The Ca²⁺-saturated and Ca²⁺-free myofibril base lines were acquired from TnC_IANBD^T53C myofibrils under same buffer conditions as described for TnC_IANBD^T53CTn in panel A.

FIGURE 2.

Effect of myofibril species on the rate of Ca²⁺ dissociation from Tn-exchanged rigor myofibrils. TnC_IANBD^T53C Tn was exchanged into rat, dog, and failing human ventricular myofibrils to acquire the apparent rates of Ca²⁺ dissociation for the respective myofibrils. The rigor Ca²⁺ dissociation rates for the different species were; rat (16.7 ± 0.5/s), dog (21.6 ± 0.7/s), and failing human ventricular myofibrils (22.9 ± 0.9/s). Buffer conditions were identical to those described for panel B of Fig. 1. All traces were acquired at 15 °C. The data traces were normalized and staggered for clarity.

FIGURE 3.

Effect of ATP on rabbit ventricular myofibrils and reconstituted thin filaments + S1. Panel A shows the time course of IANBD fluorescence as Ca²⁺ was removed from TnC_IANBD^T53C and D65A TnC_IANBD^T53C rabbit myofibrils in the presence of ATP. TnC_IANBD^T53C rabbit myofibrils (Ca²⁺ Saturated Control versus EGTA + ATP trace, no observed rate) and D65A TnC_IANBD^T53C rabbit myofibrils (D65A versus EGTA + ATP trace, 203 ± 11/s) in Buffer A + 200 μm Ca²⁺ were mixed with an equal volume of the Buffer A + 10 mm EGTA and 2 mm ATP at 15 °C. The D65A TnC_IANBD^T53C Ca²⁺-free myofibril base line was acquired by rapidly mixing the D65A myofibrils in Buffer A + 5 mm EGTA against equal volumes of Buffer A + 5 mm EGTA (D65A Rigor). Panel B shows the time course of IANBD fluorescence decay from Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils by mixing with ATP in the presence or absence of ADP. TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA ± 2 mm ADP were rapidly mixed with equal volumes of the Buffer A + 5 mm EGTA + 2 mm ATP (Ca²⁺-free Myofibrils versus ATP trace, 163 ± 8/s, and Ca²⁺-free Myofibrils + ADP versus ATP trace, 6.9 ± 0.6/s, respectively). To examine the effect of ATP on the Ca²⁺-saturated state, TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ were mixed with an equal volume of Buffer A + 200 μm Ca²⁺ + 2 mm ATP (Ca²⁺ Saturated versus Ca²⁺ + ATP trace, no observed rate). The Ca²⁺-free base line (Ca²⁺-free Control trace) from Fig. 1B was included as a reference point for the fluorescence decay induced by ATP. Panel C shows the time course of IAANS fluorescence decay from Ca²⁺-free reconstituted thin filaments containing TnC_IAANS^T53C Tn upon dissociation of myosin-S1. TnC_IAANS^T53C reconstituted thin filaments with a myosin S1 to actin subunit ratio of 1:7, 2:7 and 4:7 in Buffer A (without Tween 20) + 5 mm EGTA were rapidly mixed with equal volumes of the Buffer A (without Tween 20) + 5 mm EGTA + 2 mm ATP. The rates of S1 detachment reconstituted at the ratios of 1:7, 2:7, and 4:7 concentrations were ∼300/s, 268 ± 11/s, and 265 ± 10/s, respectively. All data were collected at 15 °C.

FIGURE 4.

Dose dependence of ATP and ADP on the rate of cross-bridge detachment. Panel A shows the effect of increasing ATP on the apparent rate (fast phase) of cross-bridge detachment from Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. TnC_IANBD^T53C rabbit myofibrils in Buffer A + 5 mm EGTA were mixed against equal volumes of Buffer A + 5 (0.51 ± 0.02/s), 10 (1.1 ± 0.2/s), 50 (5.5 ± 0.6/s), or 100 (16 ± 2/s) μm ATP (traces shown top to bottom, respectively). Panel B shows the triphasic fluorescence change that occurs upon ATP addition to the Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils with increasing ATP from 5 to 100 μm over an extended period of time. The Ca²⁺-free base line over these long times displayed a linear decrease in fluorescence that was due to either the bleaching of the signal or myofibril settling, which was subtracted from the experimental traces. Buffer conditions were identical to those described for panel A. Panel C shows the dose-dependent effect that increasing ATP had on the apparent fast rate of cross-bridge detachment from Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. The inset shows the transmitted light microscopy image of the myofibrils after being mixed with 50 μm ATP in the stopped flow. Panel D shows the effect that increasing ADP had on the apparent fast rate of cross-bridge detachment of Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils. Increasing concentrations of ADP (0–2000 μm) were added to the Ca²⁺-free TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA and rapidly mixed against Buffer A + 5 mm EGTA + 2 mm ATP at 15 °C. Both the concentrations of ATP and ADP shown in panels C and D were the final concentration after mixing in the stopped-flow. The top inset shows the transmitted light microscopy image of the myofibrils after being mixed with 2 mm ATP in the stopped flow. The bottom inset shows an enlarged myofibril from this population.

FIGURE 5.

Cross-bridge detachment rates from varying species of myofibrils. Panel A displays the rates of cross-bridge detachment in the absence of ADP at 15 °C from failing human (41 ± 4/s), dog (125 ± 17/s), rabbit (163 ± 8/s), and rat ventricular myofibrils (159 ± 6/s) that were exchanged with TnC_IANBD^T53C Tn. The TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA were rapidly mixed with equal volumes of the Buffer A + 5 mm EGTA + 2 mm ATP. Panel B displays the rates of cross-bridge detachment in the presence of 2 mm ADP from failing human (2.3 ± 0.3/s), dog (6.3 ± 0.6/s), rabbit (6.9 ± 0.6/s), and rat ventricular myofibrils (42 ± 3/s) that were exchanged with TnC_IANBD^T53C Tn at 15 °C. TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA + 2 mm ADP were rapidly mixed with equal volumes of Buffer A + 5 mm EGTA + 2 mm ATP.

FIGURE 6.

Effect of slowed cross-bridge detachment on the rate of Ca²⁺ dissociation from rabbit myofibrils. Panel A represents the biphasic kinetic trace for TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ + 2 mm ADP when mixed with an equal amount of Buffer A + 10 mm EGTA and 2 mm ATP at 15 °C (Myofibrils + ADP versus EGTA + ATP trace, biphasic). The Ca²⁺-free myofibrils + ADP versus ATP trace from Fig. 3, panel B (6.9 ± 0.6/s), was shown for a graphic comparison. Panel B shows the effect that ADP and cross-linking had on the rate of Ca²⁺ dissociation at 15 °C. The Ca²⁺-free myofibrils + ADP versus ATP trace (panel A) was subtracted from the biphasic myofibrils + ADP versus EGTA + ATP trace (panel A) to recover the Ca²⁺ dissociation signal (subtraction, ∼ 25/s). TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ + 2 mm ADP were rapidly mixed with an equal volume of the Buffer A + 10 mm EGTA at 15 °C (Myofibril + ADP versus EGTA trace, 24 ± 1/s). Cross-linked TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ were rapidly mixed with an equal volume of the Buffer A + 10 mm EGTA + 2 mm ATP (Cross-linked Myofibrils versus EGTA + ATP trace, 22 ± 1/s).

FIGURE 7.

Effect of temperature on the rates of Ca²⁺ dissociation and cross-bridge detachment as reported by TnC in rabbit myofibrils. Panel A shows the comparison between the rates of rigor Ca²⁺ dissociation (△), cross-bridge detachment + 2 mm ADP (○), and maximal proposed rate of ADP dissociation (□) at increasing temperatures (* denotes calculated value). Rigor Ca²⁺ dissociation (△) was determined by mixing TnC_IANBD^T53C myofibrils in Buffer A + 200 μm Ca²⁺ with an equal volume of the Buffer A + 10 mm EGTA. The Cross-Bridge Detachment (No ADP) plot (inset, □) represents cross-bridge detachment from ADP free myofibrils. Cross-bridge detachment in presence of ADP was determined by mixing TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA + 2 mm ADP (○) with an equal volume of the Buffer A + 5 mm EGTA + 2 mm ATP. The shaded area is used to highlight the difference between the maximal proposed rate of ADP dissociation to that with 2 mm ADP. Panel B shows the effect that increasing ADP had on the apparent fast rate of cross-bridge detachment of Ca²⁺-free TnC_IANBD^T53C rabbit myofibrils at 35 °C. Increasing concentrations of ADP (0–2000 μm) were added to the Ca²⁺-free TnC_IANBD^T53C myofibrils in Buffer A + 5 mm EGTA and rapidly mixed against Buffer A + 2 mm ATP at 35 °C. The concentrations of ADP shown were the final concentration after mixing in the stopped-flow.