Phospholamban interactome in cardiac contractility and survival: A new vision of an old friend

Abstract

Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR Ca-transport and Ca-release, is a key and universal characteristic of human and experimental heart failure. These SR processes are regulated by multimeric protein complexes, including protein kinases and phosphatases as well as their anchoring and regulatory subunits that fine-tune Ca-handling in specific SR sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory effects. Recent studies identified additional regulatory proteins, associated with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal histidine-rich calcium binding protein (HRC) has been shown to interact with both SERCA2a and triadin. Notably, there is physical and direct interaction between these protein players, mediating a fine-cross talk between SR Ca-uptake, storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA interactome does not only impact cardiomyocyte contractility, but also survival and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes modulate their activity and interactions with other protein partners, resulting in depressed contractility and accelerated remodeling. These genetic variants may serve as potential prognostic or diagnostic markers in cardiac pathophysiology.

Keywords: Calcium; Contractility; Heart failure; Human variants; Phospholamban; Sarcoplasmic reticulum.

Figure 1. Schematic Representation of the PLN/SERCA Interactome

In the classical view of EC-coupling, membrane depolarization activates sarcolemmal DHPR, causing an influx of calcium that activates the RyR complex, releasing calcium from the SR store. This calcium triggers myofilament contraction and is then transported back into the SR by SERCA2a, which is regulated by its binding partner phospholamban (PLN). However, this model is expanding with the identification of novel interacting and regulatory partners generating a much larger “interactome.” At the center of this scheme is the classical PLN/SERCA-transport complex. HAX-1 binds to PLN and stabilizes its inhibitory interaction with SERCA2a. PLN also interacts with the RGL, the regulatory subunit of protein phosphatase 1 (PP1c), which anchors this enzyme along with its regulators inhibitor-1 (I-1) and Hsp20 to PLN/SERCA, thereby modulating SERCA2a function through PLN phosphorylation. SERCA2a is also regulated post transcriptionally by the small ubiquitin-related modifier (SUMO-1), improving SERCA2a protein stability and activity. The histidine rich calcium binding protein (HRC) is a multi-functional protein that binds and inhibits SERCA2a as well as modulates RyR function through its interaction with triadin (TRI). The SR calcium release complex is comprised of RyR, TRI, junctin (JNC) and calsequestrin (CSQ).

Figure 2. Amplification of PKA Effects at the Level of PLN

PLN phosphorylation increases the apparent calcium affinity of SERCA2a. This is achieved by: a) PKA direct phosphorylation of PLN; and b) PKA phosphorylation of inhibitor-1 (I-1) and Hsp20, which inhibit PP1 and sustain PLN phosphorylation. Dephosphorylation of PLN occurs by protein phosphatase 1 (PP1c), which decreases the calcium affinity of SERCA2a. The balance of PKA and PP1 activities controls the phosphorylation status of PLN, leading to the leftward (PKA) or rightward (PP1) shifts in the calcium response curve of SERCA2a.

Figure 3. Regulation of SERCA2a Activity by PLN

PLN exists in equilibrium between its monomeric and pentameric forms. Monomeric PLN is mostly the active inhibitory unit that binds to SERCA2a when dephosphorylated by PP1, while the pentamer acts as the monomeric reservoir. Some studies indicate that pentameric PLN may still interact with SERCA2a but not inhibit it. SERCA2a bound by monomeric PLN has reduced affinity for calcium in comparison to SERCA2a not bound by PLN. The PLN inhibition of SERCA2a results in decreased SR calcium load, which is available for release and decreased force of contraction. PLN phosphorylation favors pentamer formation, relieving SERCA2a inhibition and resulting in increased SR calcium load and enhanced contractility. Therefore, the balance of activity between PKA and PP1 modulate SERCA2a function and force generation. In addition, HAX-1 binds to PLN and promotes monomer formation, stabilizing the PLN interaction with SERCA2a and reducing calcium uptake and force.

Figure 4. Effects of HRC on Calcium Handling

HRC is a multifunctional protein that can bind and inhibit SERCA2a as well as modulate RyR function through its interaction with triadin. (A): Overexpression of HRC inhibits SERCA2a activity, reducing SR calcium uptake as well as controlling calcium release through RyR. The resultant effect is decreased contractility. (B): Ablation of HRC increases SERCA2a activity but also enhances RyR opening, leading to increased calcium leak and arrhythmia occurrence.