The role of CaMKII regulation of phospholamban activity in heart disease

1A major characteristic of human and experimental heart failure (HF) is altered Ca2+ cycling, associated with decreased contractility, which partially reflects the impaired function of the sarcoplasmic reticulum (SR) membrane. During a normal excitation-contraction-coupling cycle (ECC), Ca2+ enters the cell through the L-type Ca2+ channels leading to activation of the ryanodine receptors (RyR2) in the SR and release of Ca2+ from this membrane system. This Ca2+-induced-Ca2+-release mechanism underlies a fine-tuned synchronization of Ca2+ cycling in the heart, coordinating contraction and relaxation. Relaxation is mediated mainly by the activity of the SR Ca2+-ATPase (SERCA2a) and to a lesser extent by the Na+/Ca2+ exchanger (NCX). Thus, the SR is the major regulator of Ca2+-handling during the cardiac excitation-contraction-relaxation cycle.

2The activity of SERCA2a is under the reversible control of phospholamban (PLN), an SR associated protein. PLN is a 52 amino acid phosphoprotein, which, in the dephosphorylated state, inhibits the apparent Ca2+-affinity of SERCA2a. PLN can be phosphorylated at three distinct sites in vitro: Ser16 by cyclic AMP (cAMP)- and cGMP-dependent protein kinases, Thr17 by Ca2+-calmodulin-dependent protein kinase II (CaMKII), and Ser16 by protein kinase C. Phosphorylation of these sites in vitro relieves the inhibition of PLN on SERCA2a and increases SR Ca2+ uptake. Whereas Ser10 phosphorylation by PKC does not occur in intact hearts, cGMP phosphorylation of PLN has been described in isolated myocytes. However, the physiological significance of this pathway is still unclear. In contrast, phosphorylation of Ser16 and Thr17 by PKA and CaMKII has been shown to be a key mediator of the positive inotropic and relaxant effects of ß1-adrenergic stimulation in the heart. The increase in SERCA2a activity and Ca2+ uptake rate elicited by the phosphorylation of these sites, leads to an increase in the velocity of relaxation, SR Ca2+ load and SR Ca2+ release which, in association with L-type Ca2+ channel and RyR2 phosphorylation, mediate the enhanced contractility produced by ß1-stimulation. Dephosphorylation of PLN, occurring by a SR-associated type 1 phosphatase (PP1; ), reverses the activation of SERCA2a and the stimulatory effects of β1-agonists. This article will discuss the role of Thr17 phosphorylation of PLN and address its significance under physiological and pathological processes.

3Phospholamban was first described as a cAMP-dependent protein kinase substrate in the early 1970s. The phosphorylated amino acid was shown to be Ser16 and phosphorylation enhanced SERCA2a activity and Ca2+-uptake. Subsequently, PLN was shown to be also phosphorylated by a SR-associated Ca2+-CaM-kinase (CaMKII) at Thr17 and this phosphorylation occurred independently of its PKA-phosphorylation. Phosphorylation by CaMKII also enhances SR Ca2+-transport through an increase in the apparent affinity of the SERCA2a for Ca2+ (KCa). Thus, it was initially proposed that phosphorylated PLN acts as a stimulator of cardiac SERCA2a activity. However, in the late 1980s and early 1990s, there were two significant breakthroughs: (a) in vitro studies of reconstituted SR membrane systems; and (b) in vivo studies in mouse models with ablation or overexpression of PLN, which demonstrated that dephosphorylated PLN is actually an inhibitor of SERCA2 and phosphorylation relieves this inhibition, giving the appearance of phosphorylation-induced stimulation. These findings, together with the characterization and identification of a cardiac SR–associated protein phosphatase that can dephosphorylate PLN, has led to our current understanding of PLN as a reversible inhibitor of cardiac SR Ca2+-ATPase activity.

4Furthermore, recent studies showed that the activity of PLN can itself be regulated by the HS-1 associated protein X-1 (HAX-1), which is ubiquitously expressed in mitochondria and SR. HAX-1 physically interacts with PLN and the binding region of PLN includes amino acids 16–22 with both Ser16 and Thr17 phosphorylation sites. Interestingly, phosphorylation of PLN diminishes its binding to HAX-1, indicating that this interaction may be physiologically relevant in the heart. Indeed, HAX-1 has been found to increase PLN inhibition of SR Ca2+ cycling and cardiac contractility in vivo, whereas β1-adrenergic stimulation relieves this inhibition.

5Besides HAX-1, other regulatory proteins such as PKA, CAMKII and PP1 are also associated with PLN, achieving an efficient and compartmentalized complex that regulates SR Ca2+-cycling and cardiac function. PP1 is a negative regulator of PLN activity through its dephosphorylation and increased inhibition of SERCA2a. Interestingly, the type 1 enzyme is modulated by its endogenous inhibitors, Inhibitor-1 (I-1) and Inhibitor-2 (I-2). Inhibitor-1 gets activated upon its PKA phosphorylation at Thr-35 resulting in potent inhibition of PP1 activity and amplification of the β1-adrenergic receptor stimulatory effects. More recently, the small heat shock protein 20 (Hsp20) was also identified as a novel interacting partner of PP1 and inhibitor of its enzymatic activity, resulting in diminished PLN inhibition and enhanced cardiac function. Thus, there is a multimeric functionally coupled signaling complex, which reversibly regulates SR Ca2+ cycling in the cell, composed of SERCA, PLN, HAX-1, PKA, CAMKII, PP1, I-1, and Hsp20 (Figure 1).

6It is well established that stimulation by β1-agonists at the cell membrane, initiates a signal-transduction pathway that involves the Gs proteins to stimulate cAMP formation by adenylate cyclase, followed by PKA activation (Figure 2). PKA then phosphorylates several proteins in the cardiac myocytes to induce 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. The underlying phosphoproteins include PLN and RyR2 at the SR level, the L-type Ca2+ channel and phospholemman, at the sarcolemma level, and troponin I (TnI), C protein, and myosin light chain, at the level of the myofibrils.

7The role of PLN phosphorylation vs. the phosphorylation of other proteins, which are also involved in ECC, was demonstrated by the generation and characterization of gene knockout and transgenic models with ablation, reduction or overexpression of PLN in the heart. Ablation of PLN was associated with enhanced affinity of SERCA2a for Ca2+, contractility and relaxation. In contrast, overexpression of PLN decreased Ca2+affinity of SERCA2a and diminished SR Ca2+-load and cardiac contractility. Importantly, studies at the cardiomyocyte, organ and intact animal levels from PLN-deficient mice, indicated a significant attenuation of the inotropic and lusitropic effects of isoproterenol, compared with wild types, and revealed that PLN is a major mediator of the β1-adrenergic response in the mammalian heart. However, as noted above, PLN is not only phosphorylated by PKA, at Ser16, but also by CaMKII at Thr17, during β1-adrenergic stimulation. These phosphorylations appear to occur independently of each other in vitro. However, in vivo attempts to phosphorylate PLN by CaMKII indicated that an increase in cAMP levels was a requirement for CaMKII activation. This may be due to required PKA-phosphorylation of PLN, as well as L-type Ca2+ channels and RyR2, to enhance cytosolic Ca2+, necessary to activate CaMKII. Indeed the availability of transgenic models, expressing either wild type PLN (PLN-WT), Ser16 → Ala mutant PLN (PLN-S16A) or Thr17 → Ala mutant PLN (PLN-T17A) in the cardiac compartment of PLN knockout mice, indicated that the phosphorylation of Ser16 of PLN is a prerequisite for the phosphorylation of Thr17. In addition, these studies showed that Ser16 can be phosphorylated independently of Thr17 in vivo and that phosphorylation of Ser16 was sufficient for mediating the maximal cardiac responses to β1-adrenergic stimulation.

8The role of CaMKII-phosphorylation of PLN was also addressed in a model with expression of a CaMKII inhibitory peptide targeted to the longitudinal SR (AIP4-LSR TG; ). The results indicated that Thr17 PLN-phosphorylation as well as SR Ca2+-uptake and contractile parameters were decreased. However, the response to isoproterenol remained unaltered. Similarly, transgenic mice with CaMKII inhibition (AC3-I mice), decreased SR Ca2+-content without changes in the myocyte response to isoproterenol. These findings suggested a predominant role of Ser16 phosphorylation over that of Thr17 in the β1-adrenergic response. Furthermore, experiments in perfused rat hearts using the PKA inhibitor H-89, confirmed that PKA activation is required for β1-adreniceptor mediated phosphorylation of the Thr17 site in PLN. It was further demonstrated that, when both PLN phosphorylation sites are present, the CaMKII site contributes to PLN phosphorylation and enhanced mechanical effects only at relatively high levels of β1-adrenergic stimulation, i.e., isoproterenol concentrations ≥10 nM. The lack of contribution of Thr17 site to PLN phosphorylation at lower isoproterenol concentrations was attributed to a moderate increase in PKA activity, which would raise intracellular Ca2+ to a level not sufficient to activate CaMKII and phosphorylate Thr17 site. Taken together, these findings support the notion that CaMKII is a contributor in the stimulatory effects of β1-adrenergic receptor in the heart. However, PKA activation is required to create the necessary conditions for CaMKII activation and Thr17 phosphorylation (Figure 2). A similar conclusion should hold true for the different Ca2+ handling proteins which are phosphorylated by both kinases, like L-type Ca2+ channels or RyR2. Interestingly, sustained β1-adrenergic receptor stimulation enhanced cell contraction and Ca2+ transients by a mechanism which is largely PKA-independent but sensitive to CaMKII-inhibitors. In these studies, a shift from Ser16 to Thr17 phosphorylation pathway was observed, underscoring the role of CaMKII during prolonged β1-adrenergic stimulation. In addition, β1-adrenoceptors activate the guanine nucleotide exchange protein that is directly activated by cAMP (Epac), independently of, and in parallel with, PKA. Indeed, identified RyR2 and PLN as two effector targets of a pathway mediated by Epac-PLCє-PKCє-CaMKII. These authors described an increase in Ca2+ transient mainly attributed to an increase in RyR2 sensitivity by Ca2+ influx activation. Although the specific role of Thr17 phosphorylation of PLN in these effects was not directly tested, they showed that β-adrenergic stimulation-mediated enhancement of SR Ca2+ load and myoplasmic Ca2+ clearance were not significantly altered by PLCє ablation, suggesting a poor role of the pathway described, on SR Ca2+ uptake. Moreover, other results showed that Epac activation decreases the amplitude of evoked Ca2+ transient due to Epac-induced SR Ca2+ leak by CaMKII-phosphorylation of RyR2 and SR depletion. The different outcomes of the effects of Epac on Ca2+ transient amplitude may be due to different experimental protocols, since Epac activation produces an initial increase in Ca2+ transients before reaching a steady state, in which Ca2+ transients are decreased. Yet, both results are consistent with an increase in RyR2 activation produced by Epac. The more recent study by further showed that inhibition of PKA-dependent effects of isoproterenol by H-89 pretreatment blocked the isoproterenol-induced increase of Ca2+ transient amplitude, speed of relaxation and SR Ca2+ load. In contrast, isoproterenol still greatly increased SR Ca2+ spark frequency and decreased Ca2+ transient amplitude. Both of these effects were similar to the steady state responses produced by Epac activation. These results would imply that: (1) most of the isoproterenol-induced PKA-independent Ca2+ leak enhancement is mediated by Epac; and (2) the contribution of Epac to isoproterenol–induced SR Ca2+ reuptake through PLN phosphorylation, is very modest, if any, since no relaxant effects of isoproterenol could be detected after PKA inhibition.

9As indicated above, several studies showed that CaMKII-dependent PLN phosphorylation can only occur in the intact beating heart in the presence of ß1-adrenergic stimulation, while it occurs independently of cAMP-PKA activation in vitro. To address this apparent discrepancy, the phosphatase inhibitor okadaic acid was used in the presence of high extracellular Ca2+. Under phosphatase inhibition, increasing Ca2+, increased contractility, relaxation and phosphorylation of Thr17 of PLN, without significantly changing either cAMP or Ser16 phosphorylation. These findings indicated that Thr17 can be phosphorylated independently of Ser16 of PLN in the intact heart, in accordance with the in vitro studies. Thus, the relative balance of protein kinase (PKA and CaMKII) and phosphatase activities appears to regulate phosphorylation of Thr17 and Ser16 in PLN.

10Stimulation frequency (SF), a fundamental physiological modulator of myocardial performance, is another example in which Thr17 phosphorylation of PLN can occur in the absence of prior Ser16 phosphorylation. These findings are in concert with the fact that CaMKII can decode the frequency of Ca2+ spikes into distinct amounts of kinase activity, and indicate that SF can produce a sustained increase in CaMKII, which leads to the phosphorylation of Thr17 in PLN, without the requirement of phosphatase inhibition. Moreover, these results prompted the link between the observed Thr17 phosphorylation and the relaxant effect of increasing SF (frequency-dependent acceleration of relaxation or FDAR,; ). Indeed, FDAR was inhibited in the presence of CaMKII-inhibitors and in cardiomyocytes expressing the mutant T17A-PLN. However, although the involvement of SR and CaMKII in FDAR was supported by several studies, a recent report challenged these previous findings by showing that FDAR was still present in CaMKIIδ-KO mice. These results would suggest that either a CaMKIIδ-independent mechanism or another CaMKII isoform, like CaMKIIγ, is playing a role in FDAR.

11The role of Thr17 phosphorylation of PLN on FDAR was also questioned on the basis of three main findings: (1) FDAR precedes the phosphorylation of Thr17 site of PLN; (2) Most studies concur that the main regulatory effect of PLN phosphorylation is to increase the apparent Ca2+ affinity of SERCA2a, while FDAR is associated with an increase in the maximal velocity of SR Ca2+ uptake; and (3) FDAR has been also detected in PLNKO mice in one study, although this finding was not observed in other studies.

12Taken together, the underlying molecular steps that encompass the FDAR process are currently unclear. Although most of the experimental evidence indicates that CaMKII is involved in FDAR, some studies have challenged this possibility and the participation of PLN in FDAR. Thus, it is likely that several rather than a single mechanism, are associated with this phenomenon.

13An understanding of how pH changes alter cardiac function is important for a better comprehension of some cardiac pathological situations, which are important in the clinical setting. Myocardial ischemia is particularly relevant along these lines: in human, acidosis can be detected 15 s after the occlusion of the coronary artery and is a major mechanism for the loss of contractility during ischemia. Substantial changes in intracellular pH may also occur in disorders of different origins which affect cardiac function, like sleep apnea/hypopnea syndrome, diabetic ketoacidosis or in patients on dialysis.

14Acidosis produces a rapid decrease in the strength of contraction, which is largely due to a decrease in myofilament Ca2+ responsiveness. This decrease displaces Ca2+ from troponin C, and would be the main mechanism responsible for the early increase in diastolic Ca2+ during acidosis. The initial fall in contractility is followed by an increase in the amplitude of intracellular Ca2+ transients and contractile force (,;, the mechanism of which is not intuitively obvious, because acidosis inhibits most of the steps of excitation-contraction-coupling. Earlier experiments suggested that acidosis-induced activation of Na+-H+ exchanger (NHE), by increasing cytosolic Na+ and then Ca2+ through the NCX, was sufficient to overcome the inhibitory effect of acidosis on SERCA2a, increasing SR Ca2+ and intracellular Ca2+ transients. However, inhibition of NHE does not always prevent intracellular Ca2+ and mechanical recovery, indicating that additional mechanisms may play a role (Figure 3A). A major clue supporting this possibility was given by experiments showing that Ca2+ and contractile recovery during acidosis require an intact SR and CaMKII activity, suggesting that CaMKII-dependent phosphorylation at the SR level is involved in the recovery mechanism. Indeed, it was shown that phosphorylation of the Thr17 site of PLN transiently increased at the onset of acidosis, possibly favored by the increase in intracellular (diastolic) Ca2+ and the inhibition of phosphatase induced by acidosis. This phosphorylation was associated with the initial and most significant portion of the contractile/relaxation recovery, and both were blunted by CaMKII-inhibition. Thus, CaMKII-dependent PLN phosphorylation provides a mechanism to overcome the depressant effect of acidosis on SERCA2a. These increases in SR Ca2+ content and release also counteract the effect of acidosis on contractile proteins, thereby helping to maintain contractile force. More recent experiments showed that CaMKII also activates NHE which may add to the direct activation of the exchanger induced by acidosis (; Figure 3A). Interestingly, experiments by showed absence of mechanical recovery in myocytes lacking PLN (PLNKO). This finding may be taken to indicate that PLN is essential for SR Ca2+ and mechanical recovery during acidosis. However, the effects of PLN ablation mimic maximal PLN phosphorylation. Therefore the results of actually raise the question of whether accelerating SR Ca2+ reuptake during acidosis is beneficial, favoring mechanical recovery as discussed above, or harmful, hindering it. An explanation to these apparent contradictory results may lie on the fact that intracellular Ca2+ and mechanical recovery during acidosis require an increase in SR Ca2+ uptake above steady state, a condition that cannot be accomplished in PLNKO mice in which basal SR Ca2+ uptake is already at maximal levels. In line with DeSantiago’s results, showed that the mechanical recovery from acidosis did not occur in highly phosphorylated myocytes treated with isoproterenol and a phosphatase inhibitor.

15The increase in SR Ca2+ load during acidosis, responsible for the mechanical recovery, may also increase spontaneous SR Ca2+ release and produce extra-systoles. Moreover, returning to normal pH after acidosis is also arrhythmogenic: recovery of pH induces an increase in SR Ca2+ leak. This effect was attributed to the increase in the opening probability of RyR2 due to the pH increase after acidosis and the acidosis-induced increase in SR Ca2+ content, still present at the beginning of post-acidosis. The return to normal pH also leads to recovery of the previous acidosis-induced inhibition of NCX, favoring Ca2+ extrusion and Na+ gain into the cell, membrane depolarization and eventually triggered arrhythmias (; Figure 3B). Together, these results indicate that post-acidosis CaMKII-dependent DADs are triggered by two concurrent factors: (1) acidosis-induced increase in SR Ca2+ content; and (2) relief of RyR2 and NCX, previously inhibited by acidosis.

16The role of CaMKII in I/R will be addressed in detail elsewhere in this issue. We will briefly refer here to the role of CaMKII-dependent PLN phosphorylation in this pathological situation. 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 usually refers to the intracellular Ca2+ and contractile recovery that occurs during stunning, a fully reversible post-ischemic dysfunction. Initially, this beneficial effect was associated with an increase in the phosphorylation of Thr17 site in PLN at the onset of reperfusion. Further experiments in transgenic mice in which Thr17 and/or Ser16 sites of PLN were mutated to Ala and direct measurements of intracellular Ca2+, demonstrated that Thr17 phosphorylation was essential for the recovery of Ca2+ transients and contractility in the stunned heart. These findings confirmed that the increase in Thr17 phosphorylation of PLN upon reperfusion, although transient, offers a mechanism that helps to limit cytosolic Ca2+ overload, by accelerating SR Ca2+ reuptake and thereby ameliorating intracellular Ca2+ handling (Figure 4). In contrast, when SR Ca2+ reuptake is highly enhanced by ablation of PLN, post-ischemic recovery of contractile function was negligible. A possible explanation for this apparent paradox is the higher ATP consumption of PLNKO hyperactive hearts relative to WTs, which may greatly influence contractile recovery. A second possibility that does not exclude the first one, is that under conditions in which RyR2 are altered, a persistent and exacerbated SR Ca2+ uptake, would greatly elevate SR Ca2+ content and enhance the propensity for SR Ca2+ leak, which may conspire against contractile recovery and favor reperfusion arrhythmias. Indeed, a rise in CaMKII phosphorylation of Ser2814 in RyR2 and an abrupt increase in SR Ca2+ release at the onset of reflow were recently associated with early reperfusion arrhythmias. This occurs in spite of the fact that Thr17 site of PLN was also phosphorylated. These results strongly suggest that the beneficial effects of increasing SR Ca2+ uptake in I/R, may turn to be deleterious under conditions in which the balance between SR Ca2+ uptake and leak is lost (Figure 4).

17As will be discussed below, this unbalance may constitute a major cause of the detrimental effect of CaMKII activation in the irreversible I/R.

18After a prolonged ischemic period, reperfusion evokes irreversible cardiac injury. Under these conditions, myocytes die by apoptosis, autophagy and necrosis. The rise in Ca2+ during ischemia and reperfusion leads to mitochondrial Ca2+ accumulation, which is greatly favored by the close association between mitochondria and SR and constitutes a main event in the initiation of cell death.

19Experimental evidence consistently indicates that CaMKII-inhibition is protective in the irreversible I/R injury. Although the mechanisms for myocardial protection by CaMKII inhibition are still unclear, the CaMKII deleterious pathway in I/R certainly involves the SR and the mitochondria. Phosphorylation of Thr17, the CaMKII site of PLN, has been shown to increase at the onset of reperfusion. This finding may suggest either that the effect of PLN phosphorylation is part of the deleterious cascade of CaMKII activation, or that this phosphorylation is beneficial, although insufficient to counteract the effect of simultaneous detrimental mechanisms. The experimental outcome of testing these possibilities has remained controversial. Referent to the first one, demonstrated that the protective effect of chronic CaMKII inhibition in AC3-I mice was lost, when they were interbred with PLNKO mice and submitted to myocardial infarction, supporting a detrimental effect of enhancing of SR Ca2+ uptake. Referent to the second possibility, several studies demonstrated that accelerating SR Ca2+ uptake by different means (i.e., overexpressing SERCA1a, with higher kinetics than SERCA2a, or expressing a repressor of PLN activity, PP1 inhibitor-1), alleviated post-ischemic cardiac injury, supporting a beneficial effect of accelerating SR Ca2+ uptake. A possible clue to explain these controversial findings is given by results showing that proteins, different from PLN, may be involved in the cascade by which CaMKII activity is deleterious in I/R. A decrease in the expression of RyR2 has been described in I/R, compatible with a degradation/damage of these channels by the concerted action of calpains and proteasomes, that would lead to an increase in the opening probability of RyR2. Moreover, redox alterations or CaMKII-dependent phosphorylation might also influence the activity of RyR2 and SR Ca2+ leak in I/R. These alterations would favor the unbalance between SR Ca2+uptake and leak, promoting mitochondrial Ca2+ overload and cell death. This cascade would be further stimulated by the recently described CaMKII-dependent phosphorylation of mitochondrial Ca2+ uniporter. Taken together, these findings suggest that the progression toward a beneficial or detrimental effect of CaMKII activation and PLN phosphorylation in I/R would critically depend on the balance between the extent of SR Ca2+ reuptake and SR Ca2+ leak, largely given by the status/characteristics of other proteins, also involved in SR Ca2+ handling, like RyR2 (Figure 4).

20Heart failure develops when the heart is unable to provide an adequate cardiac output to meet the metabolic needs of the organism. Mechanical dysfunction and arrhythmias are hallmark features of HF, being aberrant Ca2+ handling a main cause of these two characteristic alterations. Indeed, there is evidence supporting a decrease in intracellular Ca2+-transient and diminished SR Ca2+ content, an outcome that constitutes the major origin of the altered contractility in HF, and that can be attributed to alterations in the expression/activity of different Ca2+ regulatory proteins. In particular, a decrease in SERCA2a and an increase in NCX expressions have been described in different HF models and species, including human. An enhanced SR Ca2+ leak, through hyperphosphorylated RyR2, would also contribute to the decrease in SR Ca2+ content and Ca2+ release, typical of HF.

21The decrease in SERCA2a expression is not associated with a parallel decrease in PLN, which would produce an increase in the functional stoichiometry PLN/SERCA, with a decrease in SERCA2a Ca2+ affinity and SR Ca2+ uptake rate and a prolongation of relaxation times. Moreover, phosphorylation of PLN has been found to be decreased, either at Ser16, Thr17, or both, accounting for increased inhibition of SERCA2a. These findings may be due to the attenuation of β1-adrenergic cascade, due to receptor desensitization, down-regulation and uncoupling, typical of the disease progression, and/or the increase in PP1 activity, described in HF. Indeed, in human failing myocardium, phosphorylation of Ser16 in PLN decreased because of increases in PP1 activity, whereas phosphorylation of Thr17 decreased due to increased activity of PP2B (calcineurin; ). Interestingly, this decrease occurred despite an increase in CaMKII activity characteristic of HF. Taken together, these results indicate that the increase in SERCA2a/PLN ratio and the diminished phosphorylation of PLN, are key determinants of the depressed SR Ca2+ uptake in HF, leading to an increase in diastolic Ca2+, a decrease in SR Ca2+ stores and therefore in Ca2+ available for contraction. This results in reduced contractile force, impaired relaxation and altered force-frequency relationship.

22Given this central role of SERCA2a and PLN in the defective Ca2+ handling typical of HF, the strategy of altering SERCA2a and/or PLN levels or activity to restore perturbed Ca2+ uptake into the SR are potential therapeutic strategies for HF treatment. Indeed, overexpression of SERCA2a can restore Ca2+ handling and contractile function in animal models and in human HF, suggesting that repairing SERCA2a expression may be a viable therapy. Moreover PLN ablation prevented HF in a mouse model of dilated cardiomyopathy caused by deficiency of the muscle-specific LIM protein. In isolated human HF myocytes, gene therapy with antisense against PLN improved contractile and diastolic function. In contrast, PLN ablation increased SR Ca2+ filling and contractility in mice with cardiomyopathy attributable to overexpression of CaMKII. This led to premature death and mitochondrial Ca2+ overload, suggesting that accelerating SR Ca2+ uptake and increasing SR Ca2+ load, is disadvantageous at least in the presence of excessive CaMKII activity. These findings are consistent with the idea already discussed for I/R: in the face of phosphorylated RyR2 channels, as is the case of CaMKII overexpressing mice, repletion of Ca2+ stores through PLN ablation could further worsen overall heart function, via mitochondrial Ca2+ loading, cell death, and arrhythmias.

23We have described that PLN and its CaMKII-dependent phosphorylation are part of a multimeric functionally coupled signaling complex, composed of SERCA, PLN, HAX-1, PKA, CaMKII, PP1, I-1, and Hsp20, which reversibly regulates SR Ca2+ cycling. Although CaMKII-dependent PLN phosphorylation contributes to β1-adrenergic mechanical response, its role is subordinate to the PKA-dependent increase in cytosolic Ca2+ and inhibition of phosphatase, necessary to activate CaMKII and phosphorylate Thr17 of PLN. These requirements are also achieved under different pathological situations, like acidosis and I/R, independent of PKA activation. Under these conditions, CaMKII-dependent PLN phosphorylation may paradoxically produce either favorable or harmful cardiac effects. The findings summarized in this review also suggest that the beneficial or detrimental effects associated with CaMKII activation and PLN phosphorylation depend on a tight balance between SR Ca2+ reuptake and leak, determined by the status/characteristics of other SR proteins, among which the RyR2 is a main candidate. 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 increased SERCA2a activity, produced by PLN phosphorylation, may cope with the enhanced diastolic SR Ca2+ release/leak. In contrast, even moderate increases in SR Ca2+ may increase diastolic SR Ca2+ release under conditions where RyR2 activity is altered independently of intra-SR Ca2+-induced modifications, enhancing the propensity to arrhythmias and leading to mitochondrial Ca2+ overload, which favors apoptosis and necrosis. Thus, increasing SERCA2a activity by PLN phosphorylation seems to have the potential of producing salutary effects in a number of diseases, as long as these effects are achieved under conditions in which diastolic Ca2+ release is satisfactorily controlled. Future research in this area is needed to parse the contribution of different players involved in the balance/interaction between SR Ca2+ reuptake and leak, including the RyR2 regulators or the L-type Ca2+ channels. More specifically to the subject of this review, the recently described multimeric SERCA/PLN-ensemble may represent a nodal point in the interaction of several protein partners, regulating and modifying the fine-tuned control of Ca2+ cycling achieved by the duo SERCA-PLN. An intensive scrutiny of the various proteins of this new pathway will give new insights into their role in SR Ca2+ uptake control and may provide novel therapeutic avenues which can contribute to solve the abnormalities in Ca2+ handling underlying different pathological process.

24The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.