Phosphorylations of Regulatory Proteins in Excitation Contraction Coupling Modify Contractility by Controlling Cellular Ca2+ Fluxes, the Response of the Myofilaments to Ca2+, and the Kinetics of the Cross-Bridge Cycle

Adrenergic and cholinergic signals in the autonomic nervous system are major controllers determining the state of contractility in the myocardium. The effects of adrenergic stimulation at the level of the cardiac myocytes are indicated in isometric twitches shown in Figure 10A and in the pressure traces in Figure 10B. The data show there is an increase in the peak amplitude of force and pressure with adrenergic stimulation and in the peak amplitude of the Ca-transient. With adrenergic stimulation There is also abbreviation of the action potential duration and an abbreviation of the over cycle time of the both tension, pressure, and the Ca transient. Linkage of signals arising from the transmitters of the autonomic nervous system as well as blood levels of neurohumors such as epinephrine and acetylcholine affect EC coupling via an elaborate signaling cascade. In Figure 2 the activity of this system was revealed by propranolol treatment of the subject performing the exercise. The binding of neurotransmitters, neurohumors, or pharmacological agonists to adrenergic or cholinergic receptors triggers the cascade or in the case of propranolol blocks the cascade. G-proteins, which are GTP binding proteins, transduce the receptor binding signal to an alteration of the enzyme activity of adenylyl cyclase, which is responsible for the generation of cyclic AMP from ATP. Stimulatory G proteins (Gs) linked to adrenergic beta-receptors promote the formation of cAMP, whereas inhibitory G proteins (Gi) inhibit adenylyl cyclase. cAMP activates protein kinase A, which phosphorylates key proteins that regulate the entry and exit of Ca²⁺ from the myofilament space. As mentioned above in the case of the SR, PLB is the major PKA substrate; which, in its dephosphorylated state, inhibits the rate of transport of SR Ca-transport through its interaction with SERCA2a. PLB is also a substrate for Ca²⁺ activated calmodulin dependent kinase (CAMK). This Ca²⁺ dependent phosphorylation appears important in a “staircase” effect in which force generated by the myocardium increases with HR. With the increased frequency associated with an increase in HR, there is a greater influx of Ca²⁺ due to increased amplitude and delayed inactivation of I_Ca. With phosphorylation of PLB by either PKA or CAMK, there is suppression of the PLB-SERCA2a interaction and a release of the Ca²⁺ pump activity from inhibition; Ca²⁺ affinity of the pump increases, without changes in the maximum velocity. This increase in Ca²⁺ uptake increases Ca²⁺ loaded into the SR and induces an accelerated relaxation of the myocytes. This increase in rate of Ca²⁺ removal from the cytoplasm and myofilaments accounts substantially for the enhanced relaxation and abbreviated contraction/relaxation cycle during adrenergic stimulation. PKA also phosphorylates a subunit in the oligomeric assembly of proteins that make up the L-type Ca²⁺ channel of the heart. The phosphorylation enhances the probability that the channel will open upon depolarization, but apparently does not affect the unitary conductance. Thus, “sleeping” Ca²⁺ channels awaken when phosphorylated. This increase in the trigger for Ca²⁺ release together with the increased Ca²⁺ loading associated with PLB phosphorylation essentially accounts for the increase in the systolic Ca²⁺ transient. Regulation of the release of Ca²⁺ through SR RyR2s by PKA also provides a mechanism to control delivery of Ca²⁺ to the myofilaments and thus to control contractility. In this case the mechanism involves PKA-mediated phosphorylation of the binding protein, FKBP12.6. In the absence of phosphorylation, FKBP12.6 stabilizes clusters of RyR and enhances coupled gating within the clusters. This coupling determines the “gain” of the Ca²⁺ release mechanism, and in the absence of phosphorylation of FKBP12.6, the gain is high for SR Ca²⁺ release. In this context, the term “gain” means the amount of Ca²⁺ released for a given change in Ca²⁺ current. Phosphorylation of FKBP12.6 removes this stabilizing effect on the clusters of RyRs, and results in reduced SR Ca²⁺ release due to decreased CICR gain, and increased Ca²⁺ leak from the SR during diastole. PKA dependent phosphorylation of an isotype of K⁺ channels is also important as a determinant of the duration of the ventricular action potential, which is reduced with adrenergic stimulation (Fig. 10A).

It is now recognized that autonomic nervous system signaling to the sarcomere is also an important factor in controlling contractility (Solaro, 2008). Two major substrates for PKA are TnI and MyBP-C. The major impact of phosphorylation of TnI, which is a unique property of the adult cardiac isoform, is to increase the off rate for Ca-exchange with TnC. Thus, not only does phosphorylation of PLB increases removal of Ca²⁺ from the cytoplasm, but phosphorylation of TnI also promotes release of Ca²⁺ from its TnC binding sites. There is also evidence that phosphorylation of MyBP-C by PKA acts to increase cross-bridge kinetics. This increase in kinetics promotes the entrance and the exit of the cross-bridges into and out of the force generating cycle. Increases in the rate of tension development are related to this increase in cross-bridge kinetics and to increases in the rate of release of Ca into the cytoplasm. In view of the kinetic changes, measurement of the maximum rate of rise of pressure (+dp/dt max) during systole provides a way of assessing contractility. Moreover, –dp/dt max provides a measure a measure of relaxation kinetics or lusitropy. The maximum rate of pressure development occurs before the opening of the aortic valve and has proved a useful index of the contractile state of the myocardium. Enhanced cross-bridge cycling rates are also well documented to promote the rate of relaxation. Figure 11 summarizes the integrated effects of modifying properties of membrane and myofilament proteins by phosphorylation, which tightly control contractility. As emphasized above, the phosphorylations are critical to the ability of the heart to tune its activity cycle to the fast heart rates during adrenergic stimulation and to accommodate the increasing VR without a significant change in EDV.