PKA phosphorylation of the small heat-shock protein Hsp20 enhances its cardioprotective effects

Abstract

The small heat-shock protein Hsp20 (heat-shock protein 20), also known as HspB6, has been shown to protect against a number of pathophysiological cardiac processes, including hypertrophy and apoptosis. Following β-adrenergic stimulation and local increases in cAMP, Hsp20 is phosphorylated on Ser16 by PKA (protein kinase A). This covalent modification is required for many of its cardioprotective effects. Both Hsp20 expression levels and its phosphorylation on Ser16 are increased in ischaemic myocardium. Transgenic mouse models with cardiac-specific overexpression of Hsp20 that are subject to ischaemia/reperfusion show smaller myocardial infarcts, and improved recovery of contractile performance during the reperfusion phase, compared with wild-type mice. This has been attributed to Hsp20's ability to protect against cardiomyocyte necrosis and apoptosis. Phosphomimics of Hsp20 (S16D mutants) confer improved protection from β-agonist-induced apoptosis in the heart, whereas phospho-null mutants (S16A) provide no protection. Naturally occurring mutants of Hsp20 at position 20 (P20L substitution) are associated with markedly reduced Hsp20 phosphorylation at Ser16, and this lack of phosphorylation correlates with abrogation of Hsp20's cardioprotective effects. Therefore phosphorylation of Hsp20 at Ser16 by PKA is vital for the cardioprotective actions of this small heat-shock protein. Selective targeting of signalling elements that can enhance this modification represents an exciting new therapeutic avenue for the prevention and treatment of myocardial remodelling and ischaemic injury.

Figure 1

Schematic diagram of the domain structure of Hsp20 Hsp20 is a 160-amino-acid protein. A nine-amino-acid motif (WLRRAS-APL) at its N-terminus has been shown to inhibit thrombin-induced platelet aggregation [6]. This region encompasses a PKA/PKG consensus phosphorylation sequence, RRAS¹⁶, which allows the function of the protein to be regulated by β-adrenergic signalling. The C-terminal α-crystallin domain is common to all sHSPs and aids in their chaperone activities [2]. In Hsp20, this domain also encompasses a region similar to the minimal inhibitory region of the thin filament-associated protein troponin I (TnI) (GFVAREFHRRYRL), which may facilitate its interaction with actin [6]. Hsp20 has been shown to translocate to the myofilament under ischaemic conditions [27], and is able to bind actin [28], therefore it may also play a role in stabilizing the cytoskeleton during ischaemic stress [29].

Figure 2

Proposed role of Hsp20 in protecting against β-agonist-induced hypertrophy Following β-adrenergic stimulation, a local cAMP gradient is generated via the action of PDE4 isoforms associated directly with Hsp20. With sustained production of cAMP, the local PDE4 complement becomes saturated, leading to the activation of PKA, which may be scaffolded by AKAP–Lbc. PKA then phosphorylates Hsp20 on Ser¹⁶, leading to induction of its cardioprotective actions [17]. Following α-adrenergic stimulation, PKC isoforms also scaffolded by AKAP–Lbc can phosphorylate anchored PKD. Combined PKA/PKC phosphorylation leads to the activation and translocation of PKD to the nucleus, where it contributes to the depression of transcription factors involved in the fetal gene response, a key mediator of the hypertrophic phenotype [19]. AC, adenylate cyclase; α-AR, α-adrenergic receptor; β-AR, β-adrenergic receptor.