Troponin I phosphorylation enhances crossbridge kinetics during beta-adrenergic stimulation in rat cardiac tissue

Abstract

Inotropic agents that increase the intracellular levels of cAMP have been shown to increase crossbridge turnover kinetics in intact rat ventricular muscle, as measured by the parameter f(min) (the frequency at which dynamic stiffness is minimum). These agents are also known to increase the level of phosphorylation of two candidate myofibrillar proteins: myosin binding protein C (MyBPC) and Troponin I (TnI), but have no effect on myosin light chain 2 phosphorylation (MyLC2). The aim of this study was to investigate whether the phosphorylation of TnI and/or MyBPC was responsible for the increase in crossbridge cycling kinetics (as captured by f(min)) seen with the elevation of cAMP within cardiac tissue. Using barium-activated intact rat papillary muscle, we investigated the actions of isobutylmethylxanthine (IBMX), an inhibitor of cAMP-dependent phosphatase, which simulates the action of beta-adrenergic agents, and the chemical phosphatase 2,3-butanedione monoxime (BDM), which has been shown to dephosphorylate a number of contractile proteins. The presence of 0.6 mM IBMX approximately doubled the f(min) value of intact rat papillary muscle. This action was unaffected by the addition of BDM. In the presence of IBMX and BDM, the level of phosphorylation of MyBPC was unchanged, that of MyLC2 was reduced to 60 % of control, yet that of TnI was markedly increased (to 30 % above control levels). We conclude that TnI phosphorylation, mediated by cAMP-dependent protein kinase A, is the molecular basis for the enhanced crossbridge cycling seen during beta-adrenergic stimulation of the heart.

Figure 1. The effect of phosphatase 2,3-butanedione monoxime (BDM) and isobutylmethylxanthine (IBMX) on isometric twitch parameters of intact rat papillary muscle

Twitch parameters (peak force, time to peak (TTP) and half-relaxation time (RT₅₀))are normalised to the control value (C, ▪) and presented as mean ± s.e.m. All muscles act as their own control. BDM (B, , n = 10) sampled at 5 min after addition to muscle bath, IBMX (I, □, n = 8) sampled at 3.5 min, BDM + IBMX (B + I, , n = 20) sampled at plateau, typically 5 min after addition of the second agent. All timing parameters are significantly different from control and between treatments. For peak isometric tension: ^a significantly different when compared to control value; ^b significantly different when compared to BDM value; ^c significantly different when compared to IBMX value.

Figure 2. The effect of BDM and IBMX on the relationship between tension (T) and stiffness (S) in intact rat papillary muscle

Simultaneous measurements of T and S and the effect of the addition of BDM then IBMX (□, n = 7) or IBMX then BDM (•, n = 5) were recorded. The arrows indicated the addition of the first and then second agent to the muscle bath. The T/S ratio is normalised to the control ratio (Time = 0) for each papillary muscle and is presented as mean ± s.e.m.

Figure 3. Dynamic stiffness and phase plots from intact rat papillary muscle

Representative dynamic stiffness (dashed lines) and phase plots (continuous lines) from rat papillary muscle under steady-state 0.5 mm Ba²⁺ contracture at 25 ° C are shown in A, B and C. The pseudorandom binary noise length signal and corresponding dynamic force response gave rise to dynamic stiffness and phase data at 340 equally spaced frequencies in the range 0.1-19 Hz. A stiffness value of 1 is equivalent to 11.1 N m⁻¹. f_min corresponds to the frequency at which the dynamic stiffness minimum occurs. This value corresponds to the point of inflexion of the phase plot. A, stiffness and phase plots before (thin line) and 3.5 min after (thick line) the addition of 0.6 mm IBMX to the muscle bath. The effect of IBMX was to shift the stiffness and phase plots to the right, indicating an increase in the crossbridge cycling kinetics. The value of f_min for this experiment increased from 2.0 to 3.5 Hz with the addition of IBMX. B, stiffness and phase plots before (thin line) and 5 min after (thick line) the addition of 5 mm BDM to the muscle bath. BDM did not alter f_min, indicating that the crossbridge cycling kinetics were unchanged. C, stiffness and phase plots before (thin line) and 8 min after (thick line) the addition of 0.6 mm IBMX and 5 mm BDM to the muscle bath. The effect of the combination of IBMX and BDM was to shift the stiffness and phase plots to the right, indicating an increase in the crossbridge cycling kinetics. The value of f_min for this experiment increased from 2.1 to 3.5 Hz. D, pooled results for all experiments. Steady-state tension and f_min values are given for rat papillary muscle in response to BDM (B, , n = 12), IBMX (I, □, n = 8) and a combination of BDM and IBMX (B + I, , n = 15). All values are normalised to the control (C, ▪). The control f_min value was 2.1 ± 0.7 Hz (n = 20). ^aSignificantly different when compared to the control value; ^bsignificantly different from BDM value; ^csignificantly different from IBMX value.

Figure 4. The effect of BDM and IBMX on the phospholabelling of contractile proteins in rat cardiac tissue

A, B and C, representative experiments of the phospholabelling of contractile proteins. Papillary muscles were phospholabelled with ³²P for 4 h and then incubated without (control) or with 0.6 mm IBMX for 5 or 10 min (A), 5 mm BDM for 5 min (B), or a combination of BDM and IBMX for 10 min (C). Samples were freeze-clamped, homogenised and separated on 10 % SDS-PAGE gels. The gels were dried and exposed to autoradiographic film. Proteins were identified by Coomassie blue staining. D, pooled data from all of the experiments. Autoradiographs were scanned and analysed by laser scanning densitometry. Values were normalised to their relative control and are presented as mean ± s.e.m. The bars represent control (C, ▪), BDM (B, , n = 4), IBMX (I, □, n = 4) and BDM + IBMX (B + I, , n = 5). MyBPC, Myosin binding protein C; TnI, troponin I; MLC2, myosin light chain 2. ^a Significantly different when compared to control value; ^b significantly different from BDM value; ^c significantly different from IBMX value; ^d significantly different from BDM + IBMX value.