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Humans heart consists of an atrium and a ventricle which are separated by an atrioventricular valve at each right and left side of the heart. During each heart beat, the heart undergoes a complete cardiac cycle which comprises of a diastole and a systole phase. During diastole phase, the atria and the ventricles are at a relaxation state. This allows the heart refilled with blood with the right atrium gets the deoxygenated blood from the venae cavae while the left atrium receives oxygenated blood from the pulmonary vein. This is followed by atria contraction to push the blood into the ventricles through the opened AV valves. Then, during the systole phase, left ventricle will contract and force the oxygenated blood into the aorta subsequently to the rest of the body whereas contraction of the right ventricle will force the deoxygenated blood into the pulmonary artery subsequently to the lung.
From the complete cardiac cycle, it is obvious that atria and ventricles have different physiological function. Atria are acting as a relay chamber as they collect the blood from sources outside the heart and subsequently pump it into the ventricles. As the distance travelled by the blood is short, atria generate weak contraction force. On the other hand, ventricles are playing the pumping role of the heart to pump the blood out of the heart to other parts of the body. In order to do this, ventricles generate strong contraction force. In between ventricles, left ventricle generates greater contraction force than right ventricle in order to deliver the blood to further distance from the heart.
Contraction of the heart is initiated by depolarization of the pacemaker, sinoatrial (SA) node. The action potential is then spread through the atria and causes atria contraction. After delaying for 0.1 seconds, the action potential is then relayed to the ventricles by atrioventricular (AV) node through bundle of His and Purkinje fibers. Lastly, synchronized contraction from both sides of ventricles is formed.
Cardiac Excitation-Contraction Coupling
From above, it is known that following electrical excitation, cardiac myocytes contract which leads to blood excretion from the heart. These two processes are linked by excitation-contraction coupling (ECC), a process which is highly regulated by Ca2+ ion. Signal from the SA node causes myocytes depolarization. This in turn causes voltage-dependent Ca2+ channels to open and leads to an influx of Ca2+ current (Ica) which subsequently triggers the release of Ca2+ from sarcoplasmic reticulum (SR) via SR Ca2+ release channels (ryanodine receptors,RyR), a mechanism termed calcium-induced calcium release (CICR). The increase of intracellular Ca2+ concentration ( [Ca2+]i) following Ca2+ influx and Ca2+ release makes Ca2+ available to bind to the myofilament protein troponin C (TnC) and results in muscle contraction.(1)
There are two classes of voltage-dependent Ca2+ channel : L-type (LTCC) and T-type (TTCC) which serve different functional role. LTCCs play a major role in cardiac ECC compared to TTCCs. In ventricular myocytes, LTCCs are located at sarcolemma either in invaginated t-tubule or on surface in close proximity to RyR on SR. 10-25 LTCC and 100-200 RyR form a couplon at the sarcolemmal-SR junction. This local Ca2+ release unit acting independently from each other. During depolarization, all of the couplons are activated instantaneously, hence produces a homogeneous Ca2+ transient throughout the cell result in synchronous contraction.(2) However, in atrial myocytes, a spatiotemporal Ca2+ transient gradient is found. This is due to the lack of t-tubule and the presence of RyR at both junctional SR (j-SR) at cell periphery near to sarcolemma and non-junctional SR (nj-SR) at cell centre. During depolarization, Ica,L will first trigger the release of Ca2+ from j-SR forming a subsarcolemmal Ca2+ transient ring. However, whether this Ca2+ transient will then diffuse to the cell centre and trigger nj-SR Ca2+ release under basal condition is inconclusive and varies between species.(3, 4)
To limit the influx of Ca2+ , Ica,L is inactivated by a calcium-dependent mechanism through the negative feedback effect exerted by Ica,L and SR Ca2+ release. When local calcium concentration is high, Ca2+ bind to the calmodulin which is pre-attached to the L-type Ca2+ channels, hence accelerates their inactivation.(2) The release of SR Ca2+ upon Ica,L induction is also depends on SR Ca2+ load where a high load will increase the open probability of RyR, and thus the fraction of Ca2+ release. To avoid SR Ca2+ depletion and a key step to initiate diastole phase, SR Ca2+ release need to be terminated. There are three mechanisms proposed include RyR being inactivated when [Ca2+] is high in the sarcolemmal-SR junction. It is also regulated by SR luminal [Ca2+] where low [Ca2+] brings to the closure of RyR. Lastly, stochastic attrition where instantaneous closure of LTCC and RYR in a couplon will prevent the further release of SR Ca2+ by CICR. All these three mechanisms may contribute to the stop of Ca2+ release. (1)
After contraction, myocyte relaxation is vital for blood refilling of the heart during diastole phase. To do this, [Ca2+]I need to be reduced. This is achieved through several routes with SR Ca2+-ATPase (SERCA) reuptakes Ca2+ into SR lumen and sarcolemmal Na+/Ca2+ exchanger (NCX) removes Ca2+ into the extracellular fluid being the main routes. Sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter also contributes minorly to the removal of Ca2+ out of cytosol.(1)
Modulation of Cardiac Excitation-Contraction Coupling by Protein Kinases
Apart from the key proteins involved in calcium handling, there are other proteins involved to refine the control of ECC. One of the example are protein kinases which have a big and diverse family. The main role of protein kinases is to modulate cellular function by converting extracellular signals into cellular response through a second messenger system. In ECC modulation, they act by phosphorylating calcium handling proteins once activated by a second messenger, modulating their function, hence regulating Ca2+ indirectly and this results in alteration in myocyte contractility. Phosphatases are then dephosphorylate the targeted proteins to restore their function. Ca2+- Calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) are some of the protein kinases known to be involved in modulating ECC by phosphorylating serine or threonine monomer of the targeted proteins.(5)In this study, only PKA will be studied in detail.
PKA has a wide substrate target range. It has a tetrameric holoenzyme complex which is made up of 2 catalytic (C) subunits and 2 regulatory (R) subunits which form a dimer. Each C subunit contains a catalytic core of about 250 amino acids sequence responsible for substrate binding, ATP binding and phosphoryltransfer reaction. Each R subunit contains subdomains for dimerization, 2 cAMP binding sites, A-kinase anchoring protein (AKAP) binding sites for subcellular localization and inhibition of catalytic reaction of C subunit. Before activation, each C subunit is held in the inactive state by association with the inhibitory sequence of the R subunit. Once cAMP binds to the R subunit, C subunits dissociate from the inhibitory sequences of the R subunits and catalyse the phosphorylation of the protein substrate by transferring a phosphate group from the bound ATP to the deprotonated hydroxyl side chain of the targeted serine or threonine monomer. Three C subunit isoforms, CÎ±, CÎ² and CÏ’ have been found in mammalian cells but they all have similar catalytic reaction and substrate specificity. However, for R subunit, two isoforms for each class of R subunit (R1 and R2) have been identified. They have different affinity towards cAMP analogs and subcellular localization, hence contributes to intracellular PKA pathway organization which will be discussed further below.(6)
R 2 2
Figure 1: Topology of an anchored (R2)PKA holoenzyme. (1) Dimerization site. (2)Two cAMP binding sites. (3) AKAP binding site. (4) Catalytic activity inhibition site.
The main role of PKA is to transduce extracellular signals into cellular biological reactions by phosphorylating protein substrates in response to the increase of intracellular cAMP level. This occurs when an agonist binds to a receptor which leads to the activation of GTP-binding protein (Gs) and subsequently stimulates adenynl cyclase (AC) to produce cAMP. Activation of PKA pathway in the heart brings to the modulation of cardiac function in the aspects of contraction, metabolism, ion channels activity and gene expression. PKA pathway can be activated by various hormones which operate through the same second messenger cascade but produce different cardiac responses. This shows the existence of subcellular localization of PKA pathway in the heart. (7) In fact, many experiments have been carried out and proved the existence of spatial segregation of the receptor, G-protein, AC enzyme, phosphateses enzyme and PKA.(8) PKA can be divided into particulate (R2) and soluble (R1) form where the former is attached to the subcellular organelles through AKAP and the latter is resides in the cytosol.(6) Subcellular organization of the components involved in PKA pathway ensures the integrity of subcellular PKA signaling and produces distinct cellular responses corresponding to different stimulants.
Although the PKA pathway can be activated by various stimulants, It is specifically important in the sympathetic regulation of the heart. B-adrenergic stimulation enhances the cardiac contractility (inotropy) and heart rate (chronotropy) to increase blood supply to the major organs during the "fight" or "flight" stages and increases heart relaxation rate (lusitropy)to allow proper filling of the heart at high pumping rate. This is manifested through the activation of B-adrenoceptor which leads to the downstream cAMP pathway activation and followed by phosphorylation of the calcium handling proteins involved in ECC by PKA. Phosphorylation of LTCC has been accounted for the positive inotropic effect.(1) PKA is targeted to LTCC through AKAP 18Î± which then phosphorylates the channel at serine 1928 of Î±1C subunit and a few sites on Î² subunit. (9, 10)Phosphorylation increases the mean channel open time and channel open probability as well as number of functional channel varies according to species which overall enhances the inward Ica,L.(10) Greater inward calcium current increases [Ca2+]I and SR Ca2+ release by CICR, hence enhances myocyte contractility corresponds to the increase in Ca2+ transient amplitude. (1)
The effect of increase in heart relaxation rate is mediated by the PKA phosphorylation of phospholamban ( PLB) and cardiac troponin I (cTnI).(1) PKA is anchored to PLB through AKAP 18Î´ and causes phosphorylation at N-terminal serine-16 upon B-adrenergic stimulation.(9)Phosphorylation of PLB removes its inhibitory effect on SERCA protein hence increases the rate of SR Ca2+ reuptake by SERCA. This accelerates myocyte relaxation.(11) Besides, faster SR Ca2+ reuptake raises the SR Ca2+ load for subsequent heart beat, thus generates stronger contraction. (1) PLB is the dominant protein in producing lusitropic effect as it has a faster rate of phosphorylation than cTnI.(11, 12)PKA phosphorylation is the main mechanistic pathway for its role in enhancing heart relaxation rate although CaMKII also involves in this process.(11)
Phosphorylation of human cTnI at N-terminal serine residues 23 and 24 reduces myofilament Ca2+ sensitivity and increases the rate of dissociation of Ca2+ from the TnC, hence speeds up myocyte relaxation.(13) Some data suggest increase in crossbridge cycling rate following cTnI phosphorylation may also contribute to the lusitropic effect.(14) However, this result remains controversial . (13)
The role of RyR in regulating heart performance upon B-adrenergic stimulation however remain unknown. While there is finding shows that phophorylation of RyR increases its open probability by dissociating its inhibitory protein, FKBP12.6(15), some data suggest it does not have effect on sympathetic regulation of cardiac function.(16, 17)There is also data suggests the channel activity is varied according to the extent of phosphorylation.(18) Besides, the site of phosphorylation on RyR which is link to PKA via mAKAP is also remain unresolved with several suggested sites including serine-2808, serine-2809 or serine 2030.(15, 18, 19)
Figure 2: Activation of PKA pathway by Î²-adrenergic stimulant leads to the phosphorylation of the PKA-targeted proteins involved in excitation-contraction coupling. Î² -AR, beta-adrenoceptor; AC, adenynl cyclase; Reg, PKA regulatory subunit; AKAP, A-kinase anchoring protein; RyR, ryanodine receptor; PLB, phospholamban; SR, sarcoplasmic reticulum. (1)
Role of PKA in Heart Diseases
In view of the important role of PKA in regulating cardiac calcium handling, alteration of PKA function can lead to calcium handling defect and subsequently result in heart dysfunction. Heart failure and cardiac arrhythmia are two of the examples in which PKA has a main role in them. Heart failure is a condition in which cardiac output is insufficient to meet the body demand whereas cardiac arrhythmia is an irregular heartbeat condition. The pathology of both conditions can be explained in terms of impaired calcium balance in the myocyte.
Hyperphosphorylation of RyR has been found in failing heart. As hyperphosphorylated RyR has higher channel open probability and undergoes conformational change due to dissociation of FKBP12.6 , this causes diastolic Ca2+ leak and the channel becomes hypersensitized to Ica,L stimulation. This in turn reduces SR Ca2+ reservoir. Hence, smaller Ca2+ transient amplitude is produced following each depolarization and this generates weaker contraction at systole phase which is one of the phenotype of cardiac failure.(15, 20, 21) Diastolic Ca2+ leak is also a predispose factor to cardiac arrhythmia. Ca2+ leakage at rest can cause membrane depolarization just after repolarization of the previous action potential. This delayed afterdepolarization (DAD) causes early ventricular contraction. Small Ca2+ transient amplitude following CICR delays the inactivation of Ica,L. This is turn prolong the duration of action potential and causes early afterdepolarization (EAD) arrhythmia. (20, 21) As heart failure is associated with hyperadrenergic activity, RyR hyperphosphorylation is believed attributed to the downstream increase in PKA activity. However, a reduction in the amount of phosphatases (PP1 and PP2A) in the RyR macromolecular complex have also been found. (21)
In contrast to what describe above, there is finding suggests that RyR activity does not change in heart failure. Instead of the abnormal SR Ca2+ release , it is the change in Ca2+ reuptake that contributes to the decrease SR Ca2+ load, smaller Ca2+ transient amplitude and slower decrease of [Ca2+]I during diastole phase. (22) Hypophosphorylation of PLB found in failing heart which decreases the affinity of SERCA for Ca2+ reuptake can be one of the factor accounted for these changes which leads to decrease in heart contractility and heart rate . (23)
Besides, alteration in Ca2+ extruding activity will also diminish Ca2+ balance in the myocyte. Hyperphosphorylation of NCX found in failing heart increases the transporter activity and more Ca2+ is extruded. This in combination with the decrease activity of SERCA leads to SR Ca2+ depletion and subsequently smaller Ca2+ transient amplitude is formed at each depolarization. This in turn causes weaker contraction. Similarly to RyR, hyperphosphorylation of NCX is associated with a decrease in phosphatases activity. Since the mode of action of NCX is producing an electrophysiological effect with 1 Ca2+ out coupled to 3 Na+ in, increase in NCX activity will result in depolarization and causes afterdepolarization arrhythmia. (24)
Besides PLB, hypophosphorylation of cTnI is also contributes to the slow decrease of [Ca2+]I after systole phase as under-phosphorylated cTnI will cause myofilaments more sensitive to Ca2+ and this slows down the dissociation of Ca2+ from TnC. This would debilitate myocardial relaxation and causes diastolic dysfunction. However, this might also be an useful adaptive change to allow myocytes contracts at a lower Ca2+ transient in the failing myocyte.(25)
Lastly, increase level of LTCC phosphorylation also found in failing heart. This increases the channel activity with higher channel open probability. This might probably explain why the Ica,L is maintained although lower LTCC density is found in failing heart.(26, 27)
After exploring the phosphorylation state of each PKA targeted proteins involved in ECC in failing myocytes, it presents a puzzled situation due to the unorganized phosphorylation state of the proteins. It is well-established that heart failure patient has high plasma catecholamine level. This sequentially triggers the adaptive process of the B-adrenoceptor and the downstream components in the heart leading to desensitization of the receptor, dissociation of Gs protein and subsensitization of AC. (21)Theoretically, this would lead to hypophosphorylation of the PKA targeted proteins as shown by PLB and cTnI. However, certain proteins have been identified to be present in the hyperphosphorylated state. This probably can be explained in terms of the local activity of the subcellular PKA and phosphatases which determine the response of the proteins to B-adrenergic stimulation.(25) Besides this, basal hyperphosphorylated proteins have also been shown to have less response to B-adrenergic stimulation.(24, 25)
In viewing PKA-mediated phosphorylation is playing a central role in the heart failure phenotypes and cardiac arrhythmia , it can be targeted as an approach to treat both conditions. Hence, in this study, the specific role of PKA in affecting atrial and ventricular myocytes contractility will be studied by using pharmacological PKA inhibitors.
In order to study the effect of PKA on myocyte contractility in an intact cell condition, selective PKA inhibitor is needed to achieve this by excluding the effect of other protein kinases. In this study, two PKA inhibitors have been employed to strengthen the evidence for the involvement of PKA in producing the result.
H-89 or N-[2-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide is the most potent and most selective PKA inhibitor among the H-series members with an IC50 value of 135nM. It binds reversibly to the ATP-binding site on the catalytic core by competitively inhibit the binding of ATP , hence inhibits the action of the PKA. As the catalytic core has a well conserved amino acid sequence among the kinases, this postulates a problem of selectivity among the kinases. In fact, H-89 has been found to inhibit eight other kinases at the concentration of 10uM. However, even with the high similarity in the ATP-binding site among the kinases, there is still some variability in the binding site for the adenosine component of the ATP which is also where H-89 bind to. Thus, this may confer H-89 its limited selectivity and its distinct affinity towards different kinases.(28)
Protein kinase inhibitor peptide (PKI) is a peptide found in a variety of tissues which acts to inhibit the action of PKA. PKI binds reversibly to the free C subunit and competitively inhibit PKA substrate phosphorylation. This happens because PKI has an amino acid sequence which is similar to the subdomain on R subunit which is responsible to bind to and inhibit catalytic reaction of C subunit. PKI can only acts in the present of intracellular cAMP as it only inhibits the action of free C subunit which has been pre-dissociated from R subunit by the binding of cAMP to R subunit. Besides inhibiting PKA, PKI also has a role in PKA subcellular localization as it is able to transport the nuclear free C subunit back into the cytoplasm and reform the PKA enzyme. PKI has a high affinity towards free C subunit and this makes it very selective towards PKA with an IC50 value of 2nM.(29)
By recognizing the fact that atrium and ventricle have different contractile activities and PKA plays a vital role in modulating cardiac function as well as causing cardiac diseases, the aim of this study is to examine the importance of PKA pathway in regulating rat's atrial and ventricular contractile activities in the presence and absence of a beta-agonist, isoprenaline.