cAMP/ PKA signalling mediates the sympathetic control of excitation-contraction
coupling (ECC) in cardiomyocytes. ECC is the process in which electrical excitation of the cardiomyocytes is coupled to contraction and relaxation of the heart (Bers 2008).
Calcium (Ca2+) is a key mediator of the ECC process. During the cardiac action potential (AP) and membrane depolarisation, Ca2+ entry through L-type Ca2+ channels (L-TCC) triggers the Ca2+induced Ca2+release (CICR) from the sarcoplasmic reticulum (SR), a specialised intracellular Ca2+ store. Increasing ICa via L-TCC leads to a local increase in
Ca2+ in a region where the SR is in close proximity to the sarcolemma and where
ryanodine receptors (RyR) are localised. The local rise in Ca2+ concentration triggers the release of large concentrations of stored Ca2+ from the SR into the cytosol via RyR. Ca2+influx and release lead to an increase of the intracellular Ca2+ concentration, allowing Ca2+ to bind to the myofilament protein troponin C (TnC), resulting in cardiac contraction (Bers 2008). In order for the bound Ca2+ to dissociate from TnC and the cardiac muscle to relax (lusitropy) the cytosolic concentration of Ca2+ must decline. This is mainly achieved by the SR Ca ATPase (SERCA), which pumps Ca2+ back into SR, and the sarcolemmal Na+/ Ca2+ exchange (NCX), which removes Ca2+ from the myocyte (Bers and Guo 2005).
Regulation of cAMP/ PKA signalling is crucial for the appropriate
catecholamine‐mediated modulation of cardiac contractility (Bers 2008). As shown in Figure 1-7, β-ARs stimulation activates ACs to generate cAMP; cAMP in turn activates PKA. PKA then phosphorylates several proteins involved in the ECC process such as L-
TCC, RyR, troponin I (TnI), myosin binding protein C (MBP-C) and phospholamban (PLB).
PKA mediates the phosphorylation of L-TCCs and RyRs, increasing the open probability of the channels and thus increasing Ca2+influx into the cell. PKA phosphorylation of these targets increases the amount of Ca2+ releasedfrom the SR, and as a result, a larger amount of Ca2+ions are available for sarcomere contraction at systole (Bers and Guo 2005). The lusitropic effect occurs upon PKA phosphorylation of TnI, PLB and MBP-C.
Phosphorylation of TnI reduces the sensitivity of the sarcomeric myofilaments to Ca2+ and encourages the rapid dissociation of Ca2+ from the myofilaments (Zhang et al. 1995). Both TnI and MCB-P play an imperative role in modulating the crossbridge cycling in heart muscle (Winegrad 1999). PLB exhibits an inhibitory effect on the transport of Ca2+ by the SERCA pump. When PLB is closely associated with SERCA, the rate of Ca2+ transport is reduced. PKA phosphorylation of PLB dissociates it from SERCA, blocking the inhibitory effect of PLB (Verboomen et al. 1992), resulting in a more efficient Ca2+re-uptake in the SR and results in relaxation.
Figure 1-7. Activation of β-adrenergic receptors triggers the PKA-mediated phosphorylation of several targets involved in the excitation-contraction coupling (ECC) process.
(AC, adenylyl cyclase; ACh, acetylcholine; AKAP, A kinase anchoring protein; β-AR, β - adrenergic receptor; M2-Rec, M2-muscarinic receptor; PLB, phospholamban; Reg, PKA regulatory subunit; SR, sarcoplasmic reticulum). Adapted from (Bers 2002).
It has also been demonstrated that cAMP-activated PKA controls the frequency of contraction by phosphorylation of ryanodine receptors and other Ca2+-cycling proteins in the sino-atrial node (SAN), leading to altered calcium cycles and increased diastolic currents through Na+/Ca2+exchangers (Lakatta et al. 2010).
Pacemaker activity in the heart originates in the SAN. Myocytes in the SAN produce spontaneous action potentials; these propagate, through specialised conduction systems, first to the atria and then to the ventricles, and thus drive cardiac rhythmic contraction. It has been shown that spontaneous diastolic depolarisation of SAN cells initiate action potentials to set the rhythm of the heart. This mechanism of spontaneous depolarisation has traditionally been attributed to a "voltage clock" mechanism, mediated by voltage-sensitive membrane currents, such as the hyperpolarisation-activated pacemaker current (If)
regulated by cyclic adenosine monophosphate (cAMP) (Brown et al. 1979). This current is also referred to as a "funny" current because, unlike the majority of voltage-sensitive
currents, it is activated by hyperpolarisation rather than depolarisation. The funny channel becomes activated at the end of the action potential (at voltages from -40/-50 mV to -100/- 110 mV), which corresponds to the range in which diastolic depolarisation occurs. The
funny channel then depolarises the membrane to a level at which L-type Ca2+channels open to
initiate the upstroke in the SA node action potential. If is a mixed Na+-K+ inward current
activated by hyperpolarisation and modulated by the autonomic nervous system (DiFrancesco 1985; DiFrancesco et al. 1986).
The f-channels are encoded by the hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channel gene family. These are intermembrane proteins that serve as non-selective ligand-gated cation channels in the plasma membranes of heart cells. Of the four known HCN subunits, HCN4 is the most highly expressed in the mammalian SAN (Ishii et al. 1999; Liu et al. 2007). cAMP exerts its modulatory effects on the If current by directly
binding to HCN channels (DiFrancesco and Tortora 1991). This is more likely to occur when channels are in the open state and binding of cAMP to the channel stabilises this open configuration. This mechanism results in a shift of the If activation curve to more
positive voltages, accelerates activation and slows deactivation kinetics. Point mutations of HCN4 are associated with baseline sinus bradycardia, though the maximum heart rate achieved during exercise is normal (Nof et al. 2007). This implies that If is not the only
mechanism of SAN automaticity, especially during sympathetic activation.
Recently, spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) has been suggested to modulate sinus rhythm (Vinogradova et al. 2005). When the SR is full, the probability of spontaneous Ca2+ release increases. Because the SR Ca2+ content is
controlled in part by the membrane voltage, it is important to recognise that the activation of the Ca2+ clock and the membrane ionic clock are interdependent. Lakatta et al. proposed that the Ca2+ clock is manifested by spontaneous, but precisely timed, rhythmic, local Ca2+ releases from sarcoplasmic reticulum (SR) that appear shortly before firing of the next AP (Bogdanov et al. 2001; Vinogradova et al. 2002; Vinogradova et al. 2006). The elevated Cai activates NCX inward current causing diastolic depolarisation, which co-ordinately
regulates the sinus rate along with the voltage clock. PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, PLB and L- TCC, controls the phase and size of SR Ca2+ release, NCX current and thus is crucial for pacemaker function (Vinogradova et al. 2006).
Therefore cAMP regulation of ECC results in an increase in contractile force and
frequency of the heart in response to acute catecholamine exposure. However, prolonged catecholamine stimulation of the cAMP pathway results in detrimental effects such as cardiac remodelling, cardiac hypertrophy and the development of heart failure which is accompanied by a marked down-regulation of β1-AR expression and thus a significant
desensitisation of the heart to inotropic β-adrenergic stimulation (Fowler et al. 1986; Engelhardt et al. 1999; Lohse et al. 2003; Barry et al. 2008).
Both β1- and β2-ARs are expressed in cardiac myocytes and mediate an increase in
contractility via Gs-dependent coupling to adenylyl cyclase to generate cAMP (Xiao and
Lakatta 1993). However, β2-ARs can also couple to signalling pathways independent of
cAMP or Gs and, in particular, to a pertussis toxin (PTX)–sensitive pathway mediated by
Gi. Overexpression of β2-AR in transgenic mice resulted in a limited contractile response
due to Gi signalling (see 1.10.1.3), which inhibits adenylyl cyclase (Milano et al. 1994).
Only when Gi proteins were inactivated by PTX treatment did these animals with
overexpressed β2-ARs fully stimulate contractility (Xiao and Lakatta 1993). In addition to
this, activation of Gi has the potential to couple β2-ARs to other important signalling
pathways, such as the MAP kinases (Daaka et al. 1997).
It has been reported that selective stimulation or transgenic overexpression of β2-AR
coupled to cAMP, does not lead to hypertrophy, cardiomyocyte apoptosis and heart failure (Milano et al. 1994; Communal et al. 1999). Communal and colleagues measured
apoptosis using flow cytometry and terminal deoxynucleotidyl transferase (TdT)–mediated nick end-labelling (TUNEL) staining in isolated adult rat ventricular myocytes (ARVM) and found that β-AR–stimulated apoptosis was abolished by the β1-AR–selective
antagonist but was potentiated by the β2-AR–selective antagonist. The effect of β2-AR was
found to be mediated by Gi and the authors suggest that β2-AR stimulation may even be
protective (Communal et al. 1999). A number of other studies have confirmed protective and anti-apoptotic effect of β2-AR stimulation via Gi (Ahmet et al. 2004; Ahmet et al.
2005). Chesley et al. demonstrate that β2-ARs activate a PI3K–dependent, pertussis toxin–
sensitive signalling pathway in neonatal rat cardiac myocytes that is required for protection from apoptosis-inducing stimuli (Chesley et al. 2000).
cAMP signalling via Epac seems to be one of the key factors downstream of chronic catecholamine stimulation of β1-ARs, which mediates the development of cardiac
hypertrophy via the activation of the pro-hypertrophic gene transcription and via the activation of CaMKII (see 1.10.1) (Metrich et al. 2008; Breckler et al. 2011). Metrich and co-workers demonstrated that Epac1 expression is increased in the hearts of thoracic aortic constriction (TAC) treated rats. ARVM isolated from these animals displayed exaggerated cellular growth in response to Epac activation. This response involved the small GTPase Ras, the phosphatase calcineurin, and Ca2+/calmodulin-dependent protein kinase II rather than its classic effector, Rap1 (1.5.2) (Metrich et al. 2008). Although these effects were shown to be PKA-independent, PKA does play a major role in a number of hypertrophy models associated with chronic catecholamine stimulation and activation of the cAMP pathway (discussed in detail in 1.10.1.4).
In cardiac myocytes, a variety of proteins other than those involved in the ECC process, are also affected by cAMP and PKA phosphorylation. These proteins include metabolic enzymes and transcription factors (Muller et al. 2001; Sheridan et al. 2002). Moreover, β- ARs are not the only receptors that signal via cAMP and activation of PKA; many other GPCRs whose activation leads to cAMP production are expressed in cardiomyocytes. The result is that an overabundance of chemical signals will propagate responses in the heart, some of which will lead to the generation of cAMP and activation of PKA. This raises the question of how myocytes can translate the multiple signals generated in response to individual extracellular stimuli appropriately, so to achieve the required functional outcome, while avoiding off-target phosphorylation and unwanted effects.