CAMKII REGULATION OF PHOSPHOLAMBAN AND SR Ca²⁺ LOAD

Abstract

Phospholamban (PLN) is a small phosphoprotein in the cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLN tonically inhibits the SR Ca²⁺-ATPase (SERCA2a) and phosphorylation of PLN, at either Ser¹⁶ by PKA or Thr¹⁷ by Ca²⁺-calmodulin-dependent protein kinase (CaMKII), reverses this inhibition. Consequently, there are increases in SERCA2a activity, SR Ca²⁺ uptake rate and SR Ca²⁺ load. Through this mechanism, PLN is a major regulator of basal cardiac Ca²⁺ cycling, contractility and relaxation and the main determinant of β-adrenergic mechanical responses in the heart. In this article, we briefly review the functional role of CaMKII-dependent PLN phosphorylation at Thr¹⁷ site and the new findings that link this phosphorylation to beneficial or detrimental effects in pathophysiological situations.

INTRODUCTION

During cardiac action potential, Ca²⁺ enters the cell through the L-type Ca²⁺ channels to trigger Ca²⁺ release from the SR, which activates the myofilaments to drive contraction. The decrease in cytosolic Ca²⁺ leads to relaxation. This decrease is mainly induced by SERCA2a, which mediates Ca²⁺ uptake into the SR, and to a lesser extent by the Na⁺/Ca²⁺ exchanger (NCX), which transfers Ca²⁺ to the extracellular space. By mediating SR Ca²⁺ uptake, the activity of SERCA2a also influences cardiac contractility, since it determines the size of the luminal Ca²⁺ store that is available for release in the next beat. The activity of SERCA2a, which in humans determines the rate of removal of >70% of cytosolic Ca²⁺, is under the control of the closely associated SR protein phospholamban (PLN), a small phosphoprotein of 52 amino acids. Dephosphorylated PLN inhibits the affinity of SERCA2a for Ca²⁺ and PLN-phosphorylation relieves this inhibition.

The use of gene knockout and transgenic mouse models, in which the expression levels of PLN have been altered, constituted a crucial step in the recognition of the role of PLN in the regulation of myocardial performance. Ablation of PLN produced enhanced contractility and relaxation¹. This hypercontractile function of PLN-deficient hearts (PLN⁻/⁻) was associated with increases in the affinity of SERCA2a for Ca²⁺ and in SR Ca²⁺ content. Opposite results were obtained in mice with PLN overexpression. In addition to the PLN expression levels, SERCA2a activity is also regulated by PLN phosphorylation. There are two PLN phosphorylation sites that are physiologically relevant: Ser¹⁶ residue, phosphorylated by PKA and Thr¹⁷, phosphorylated by CaMKII. Phosphorylation of these sites reverses the inhibition of SERCA2a by PLN, thus increasing the affinity of the enzyme for Ca²⁺ and the rate of SR Ca²⁺ uptake. This in turn leads to increases in SR Ca²⁺ load, SR Ca²⁺ release and myocardial contractility. The status of PLN phosphorylation also depends on the activity of the type 1 phosphatase (PP1), the major SR phosphatase, which specifically dephosphorylates PLN.

CaMKII-dependent PLN phosphorylation in physiological situations: β-adrenergic stimulation

Cardiac function is regulated on a beat-to-beat basis through the sympathetic nervous system. β1-adrenergic receptor stimulation (β-ARs) induces positive chronotropic, inotropic and relaxant effects,—the so-called “fight or flight response” —, which is considered the most effective mechanism to acutely increase cardiac output. Activation of β-AR by β1-agonists at the cell membrane, initiates a signal-transduction pathway that proceeds through Gs proteins to stimulate cyclic AMP (cAMP) formation by adenylate cyclase and PKA activation. PKA then phosphorylates and alters the function of several cardiac proteins among which PLN is predominant in determining the relaxant and inotropic effects of β-agonists¹, by increasing SR Ca²⁺ uptake and load (Figure 1).

Figure 1.

Schematic representation of cAMP/PKA/CaMKII cascades triggered by βAR-stimulation. β-ARs leads to increases in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA). PKA-dependent phosphorylation of different proteins involved in Ca²⁺ handling increases intracellular Ca²⁺. The increase in intracellular Ca²⁺ would favor CaMKII activation and CaMKII-dependent phosphorylation of various targets like the Thr¹⁷ site of PLN. PKA activation also inhibits PP1, the major phosphatase that dephosphorylates PLN. This inhibition would contribute to maintain both PKA and CaMKII-dependent phosphorylations.

Although β-AR-stimulation results in PLN phosphorylation at Ser¹⁶ (PKA site) and Thr¹⁷ (CaMKII site), the relevance of Thr¹⁷ phosphorylation in the relaxant and inotropic effects of β1-agonists has remained largely equivocal. Experiments in transgenic mice, expressing either wild type-PLN or the Ser16→Ala mutant PLN, demonstrated that the phosphorylation of Ser¹⁶ of PLN is a prerequisite for the phosphorylation of Thr¹⁷. As will be further discussed below, phosphorylation of Ser¹⁶ may be required to enhance cytosolic Ca²⁺ to the necessary level for CaMKII activation and Thr¹⁷ phosphorylation. Experiments in Thr17→Ala mutant PLN hearts further showed that phosphorylation of Ser¹⁶ was sufficient for mediating the maximal cardiac responses to β-ARs. More recent studies demonstrated that transgenic mice expressing a CaMKII inhibitory peptide targeted to the longitudinal SR (AIP4-LSR TG) exhibit reduced PLN Thr¹⁷ phosphorylation, decreased SR Ca²⁺ uptake, prolonged twitch Ca²⁺ transient decline and a decrease in basal contraction and relaxation rates. However, the response to isoproterenol remained unaltered. Similarly, although SR Ca²⁺ content was significantly reduced in cardiomyocytes from another genetic model of cardiac CaMKII inhibition (AC3-I mice), these cells exhibited normal physiological responses to acute isoproterenol application². These findings suggested either a predominant role of the phosphorylation of Ser¹⁶ over that of Thr¹⁷ in the mechanical effect produced by β-ARs or that cardiomyocytes can successfully compensate for Thr¹⁷ mutation and/or CaMKII inhibition. Supporting the first possibility, kinetic experiments comparing phosphorylation of Ser¹⁶ and Thr¹⁷ sites of PLN, showed a correlation between contractility and cAMP elevation as well as phosphorylation of the PKA site of PLN but not of the CaMKII site of PLN, during acute β-ARs. However, experiments which combined phosphorylation site-specific antibodies with quantification of ³²P incorporation into PLN in intact hearts indicated that phosphorylation of Thr¹⁷ accounted for approximately 50% of total PLN phosphorylation and enhancement of the relaxation rate at high isoproterenol concentrations (≥10 nM). In these experiments, no contribution of CaMKII to PLN phosphorylation could be detected at the lower isoproterenol doses³. In line with these findings, other experiments demonstrated that the dose-response curve of Thr¹⁷ phosphorylation to isoproterenol was shifted to the right, compared to that of Ser¹⁶ phosphorylation, clearly indicating that Ser¹⁶ was the only phosphorylated site at the lowest isoproterenol concentrations. These results might explain the failure to find significant PLN phosphorylation in the Ser16→Ala mutant PLN mice, since the lack of phosphorylation of Ser¹⁶ would preclude the increase in intracellular Ca²⁺ necessary to phosphorylate Thr¹⁷ (Figure 1). Similarly, they might also provide a clue in interpreting results of experiments performed with relatively low extracellular Ca²⁺, in which the contribution of Thr¹⁷ to total PLN phosphorylation was much lower than that observed in isolated rat hearts labeled with ³²P³. Experiments using the PKA inhibitor H-89 further confirmed that activation of PKA is required for β-AR mediated phosphorylation of the Thr¹⁷ site. Taken together, these findings would support the idea that CaMKII is a β-AR mediator, with PKA as its upstream activator through the increase in intracellular Ca²⁺. Interestingly, sustained β-ARs enhanced cell contraction and Ca²⁺ transients by a mechanism which is largely PKA-independent but sensitive to CaMKII-inhibitors, underscoring the role of CaMKII during β-ARs under these conditions.

In addition, β-ARs activates the cAMP-binding protein Epac, independently of PKA. Activation of Epac has been shown to increase CaMKII activity and phosphoryltion of Thr¹⁷ of PLN. However, the consequences of Epac-dependent Thr¹⁷ phosphorylation remain unclear since Epac has been shown to either increase or decrease Ca²⁺ transients. These apparently disparate results may arise from Epac-dependent effects on other proteins involved in Ca²⁺ handling, since Epac activation also produces SR Ca²⁺ leak. Unfortunately, a detailed analysis of the effects of Epac on SR Ca²⁺ uptake and load is still lacking.

CaMKII-dependent phosphorylation of PLN in pathological situations

Recent studies showed the involvement of CaMKII-dependent PLN phosphorylation in different pathophysiological situations. Interestingly, CaMKII-dependent phosphorylation of PLN and the consequent increase in SR Ca²⁺ uptake, have beneficial effects for heart performance in some cases, whereas they are detrimental in others. Obviously, in all situations the effects of CaMKII-dependent PLN phosphorylation is the same, i.e. to increase SR Ca²⁺ refilling. As will be discussed below, the extent of SR Ca²⁺ load produced by PLN phosphorylation as well as the status/characteristics/activity of other proteins [ryanodine receptors (RyR2) and NCX], which share with PLN the task of regulating intracellular Ca²⁺ handling, may be key determinants of whether CaMKII-dependent PLN phosphorylation and the resultant increase in SR- Ca²⁺ load, evolves towards a final beneficial or detrimental outcome.

Acidosis

Intracellular acidosis is associated with a decrease in the ability of the heart to generate tension, which is largely due to a decrease in myofilament Ca²⁺ responsiveness. This initial impairment in cardiac performance is followed by a spontaneous recovery, which requires an intact SR and has been shown to be dependent on the activity of CaMKII. It was further demonstrated that phosphorylation of the Thr¹⁷ site of PLN transiently increased at the onset of acidosis and is responsible for the increase in SR Ca²⁺ load and most of the mechanical recovery that follows the acidotic insult. This CaMKII phosphorylation of PLN would thus provide a mechanism to overcome the direct depressant effect of acidosis on the Ca²⁺ pump. Interestingly, upon returning to normal pH, the CaMKII-dependent increase in SR Ca²⁺ load during acidosis produces SR Ca²⁺ leak, (promoted by the relief of the acidosis-induced inhibition of RyR2 when the pH is restored to normal), followed by Ca²⁺ efflux and Na⁺ influx through the NCX. This provides the molecular bases for the CaMKII-dependent ventricular arrhythmias, observed when pH is normalized after acidosis. Thus, the increase in SR Ca²⁺ load produced by CaMKII-dependent PLN phosphorylation during acidosis, is responsible for apparently opposite outcomes: beneficial, since it favors Ca²⁺ handling and mechanical recovery, and detrimental, because it puts the heart at risk of arrhythmias.

Ischemia/reperfusion (I/R)

In the last few years, a dual effect of CaMKII-dependent protein phosphorylation (beneficial and detrimental) has been described in the scenario of I/R in the intact heart. The beneficial effect of CaMKII occurs in the stunned heart, a fully reversible post-ischemic dysfunction and is due to an increase in the phosphorylation of the Thr¹⁷ site of PLN that takes place at the onset of reperfusion. Experiments in transgenic mice in which Thr¹⁷ and/or Ser¹⁶ sites of PLN were mutated to Ala and direct measurements of intracellular Ca²⁺ were performed, demonstrated that this phosphorylation was essential for the recovery of Ca²⁺ transients and contractility in the stunned heart during reperfusion. The detrimental action of CaMKII takes place during infarction, a post-ischemic irreversible cardiac injury in which cardiac cells die by necrosis and apoptosis. It has been shown that CaMKII-dependent phosphorylation of PLN at the onset of reperfusion is not only unsuccessful in rescuing cardiac myocytes from death, but appears as part of the deleterious cascade mediated by CaMKII responsible for apoptosis and necrosis⁴. Interestingly, these deleterious effects appear to be associated with a degradation of RyR2 and an increase in SR Ca²⁺ release. Similarly opposite results regarding the final outcome of PLN phosphorylation and increase in SR Ca²⁺ uptake on myocardial performance, were described in a variety of experimental conditions. For example, the protective effect (decreased number of apoptotic cells) of chronic CaMKII inhibition in transgenic hearts expressing a CaMKII inhibitory peptide (AC3-I) and submitted to myocardial infarction, was absent in the cross mice of AC3-I overexpression and PLN ablation. In these animals, the absence of the inhibitory effect of PLN greatly enhanced SR Ca²⁺ uptake and load⁵. In contrast, other results suggest that increased PLN phosphorylation observed after inducible expression of inhibitor-1 has beneficial effects in I/R injury.

CONCLUSIONS

The results summarized above indicate that increases in SR Ca²⁺ uptake by Thr¹⁷ phosphorylation of PLN may contribute to the mechanical effects of acute β-ARs and are responsible for the sustained actions of β-1 agonists in the intact heart. Moreover, CaMKII-dependent PLN phosphorylation may paradoxically produce either favorable or harmful cardiac effects. Since the sole effect of PLN phosphorylation is to increase SR Ca²⁺ uptake and since SR Ca²⁺ content is the net result of SR Ca²⁺ uptake and release processes, one can hypothesize that the progression towards a beneficial or detrimental effect of CaMKII activation and PLN phosphorylation depends on two main factors: 1) The extent of SR Ca²⁺ reuptake; and 2) The status/characteristics of other proteins that are also involved in SR Ca²⁺ handling, among which the RyR2 is the main candidate (Figure 2). A moderate or even high increase in SR Ca²⁺ uptake (and content) due to PLN phosphorylation, would enhance RyR2 opening due to the regulatory effect of intra-SR Ca²⁺. However, in the absence of additional RyR2 modifications, the increase in SERCa2a activity produced by PLN phosphorylation may still cope with the enhanced diastolic SR Ca²⁺ release and improve SR Ca²⁺ handling. In contrast, even moderate increases in SR Ca²⁺ content may increase diastolic SR Ca²⁺ release under conditions where the RyR2 activity is altered independently of intra-SR Ca²⁺-induced modifications, enhancing the propensity to arrhythmias and leading to mitochondrial Ca²⁺ overload. This would favor apoptosis and eventually necrosis. Of interest, a similar situation to the one depicted in Figure 2B has been recently suggested to take place when the dilated cardiomyopathy that occurred in mice with CaMKIIδC overexpression could not be rescued by crossing with PLN−/− mice, in an attempt to attenuate PLN inhibitory effects on SERCA2a and ameliorate Ca²⁺ handling. Thus, increasing SERCA2a activity by PLN phosphorylation has indeed the potential of producing salutary effects in a number of diseases. However, these effects may be achieved only under certain conditions in which diastolic Ca²⁺ release could be satisfactory controlled.

Figure 2.

Hypothetical explanation of the dual effects of CaMKII in ischemia/reperfusion (See text for details).

Abbreviations

PLN

Phospholamban

SR

Sarcoplasmic Reticulum

SERCA2a

Sarco(endo)plasmic Reticulum Ca²⁺-ATPase, isoform 2a

CaMKII

Ca²⁺-calmodulin-dependent protein kinase

PKA

Protein Kinase A

NCX

Na⁺/Ca²⁺ exchanger

PP1

type 1 phosphatase

β-ARs

β1-adrenergic receptor stimulation

cAMP

cyclic adenosine monophosphate

Epac

exchange protein activated by cAMP

RyR2

Ryanodine Receptors type 2

I/R

Ischemia/reperfusion.