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Biochemical Society Transactions (2014) Volume 42, part 2
The cardioprotective role of small heat-shock protein 20 Tamara P. Martin*, Susan Currie† and George S. Baillie*1 *Institute of Cardiovascular and Medical Sciences, University of Glasgow, Wolfson-Link Building, Glasgow G12 8QQ, U.K. †Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Hamnett Building, 161 Cathedral Street, Glasgow G4 0RE, U.K.
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Abstract The small HSP (heat-shock protein) HSP20 is a molecular chaperone that is transiently up-regulated in response to cellular stress/damage. Although ubiquitously expressed in various tissues, it is most highly expressed in skeletal, cardiac and smooth muscle. Phosphorylation at Ser16 by PKA (cAMP-dependent protein kinase) is essential for HSP20 to confer its protective qualities. HSP20 and its phosphorylation have been implicated in a variety of pathophysiological processes, but most prominently cardiovascular disease. A wealth of knowledge of the importance of HSP20 in contractile function and cardioprotection has been gained over the last decade. The present mini-review highlights more recent findings illustrating the cardioprotective properties of HSP20 and its potential as a therapeutic agent.
Introduction HSPs (heat-shock proteins) are a ubiquitous and diverse group of chaperone proteins that play a key role in maintenance of normal cell metabolism and function. Expression of these proteins is rapidly up-regulated to protect the cell from various kinds of damage following periods of cellular stress [1]. HSPs are classified into families determined by their molecular mass and function. The small HSPs are the most diverse family, are unrelated to the other families and are known for the protective effects they confer in neurology and cardiology [2]. There are at least ten small HSPs (HSPB1– HSPB10), with molecular masses ranging from 12 to 43 kDa. Small HSPs are characterized by their unique N-terminus and a conserved α-crystallin domain at their C-terminus, which facilitates their intrinsic chaperone activity [3]. One of these small HSPs, HSP20 (HSPB6), contains a domain inhibiting platelet aggregation and a homology sequence of troponin I [4] and thus is gaining increasing interest in the field of cardiovascular research. Although HSP20 is a ubiquitously expressed protein, it is most highly expressed in skeletal, cardiac and smooth muscle [4]. Like other proteins of the same family, the expression and activity of HSP20 is unregulated in response to cellular stress. At the basal level, HSP20 is primarily found in the cytosol, but a subpopulation may translocate into the nuclear compartment in response to stress signals [4]. In the cell, HSP20 can exist as monomers or larger oligomers and has a strong tendency to form disulfide-linked dimers [5]. Formation of larger oligomers decreases the chaperone’s activity [6]. Phosphorylation of HSP20 at Ser16 alters its ability to aggregate, thus triggering its protective effects [7]. Key words: cAMP, cardioprotection, heat-shock protein 20 (HSP20), peptide array, phosphodiesterase type 4 (PDE4). Abbreviations: AKAP, A-kinase-anchoring protein; HSP, heat-shock protein; I/R, ischaemia/ reperfusion; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase. 1 To whom correspondence should be addressed (email [email protected]).
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Initial research into the role of HSP20 in the heart showed that expression levels and phosphorylation at Ser16 were increased upon prolonged stimulation with the β-adrenergic agonist isoprenaline [8]. This was a significant finding given the importance of β-adrenergic neurohormonal stimulation in mediating augmented contractility in the heart under flight-or-fight scenarios [3]. Not surprisingly, adenoviral gene transfer of HSP20 [8] and transgenic overexpression of HSP20 [9] was associated with increases in contractility and Ca2 + transient peak, highlighting further the importance of HSP20 in cardiac function.
Cardioprotective actions of HSP20 Studies over the last decade have begun to unravel the cardioprotective role of HSP20. Early studies showed that transgenic overexpression of HSP20 (∼10-fold) significantly decreased the extent of infarction and reduced necrotic damage in an ex vivo and an in vivo model of I/R (ischaemia/reperfusion) injury. Additionally, these mice displayed improved functional recovery of contractile performance, as well as displaying increased levels of phospho-HSP20 [10]. Further research demonstrated that many of the cardioprotective actions of HSP20 depend on phosphorylation of a putative PKA (cAMP-dependent protein kinase)/PKG (protein kinase G) consensus site in the N-terminus of HSP20 (Ser16 ). Consequently, overexpression of non-phosphorylatable HSP20 (HSP20S16A ) resulted in hearts with larger infarcts, reduced recovery of contractile function and increased necrosis and apoptosis compared with mice overexpressing wild-type HSP20 following I/R injury [7]. The authors suggest that this could be due to suppressed autophagy, a critical catabolic pathway upregulated during cardiac stress [7]. Subsequent research has shown that HSP20 expression levels may be decreased by miR-320, a known miRNA up-regulated during I/R Biochem. Soc. Trans. (2014) 42, 270–273; doi:10.1042/BST20130272
Targeting cAMP Signalling to Combat Cardiovascular Diseases
injury [11]. Furthermore, HSP20 exerts some of its cardioprotective actions by inhibiting β-adrenergic-mediated cardiac apoptosis via direct interaction with the pro-apoptotic signalling protein Bax [12]. This association prevents Bax translocation to the mitochondria and subsequent release of cytochrome c and activation of caspase 3. Correspondingly, overexpression of a constitutively phosphorylated HSP20 mutant (S16D) conferred full protection from apoptosis, whereas a non-phosphorylatable mutant (S16A) exhibited no anti-apoptotic properties. Moreover, expression levels of the cardiac chaperone are increased in response to hypertrophic stimuli, including chronic β-adrenergic signalling and aortic constriction [1]. Chronic β-adrenergic stimulation of cardiomyocytes resulted in increased expression of both HSP20 and phospho-HSP20 (Ser16 ), with adenoviral overexpression of HSP20 improving contractility and cardiac function [8]. Interestingly, cardiac overexpression of HSP20 prevented hypertrophic remodelling in response to chronic infusion of the β-agonist isoprenaline, including reducing interstitial fibrosis and cardiomyocyte apoptosis. In this scenario, HSP20 exhibits its protective actions, at least in part, via inhibition of the ASK1 (apoptosis signal-regulating kinase 1) signalling pathway [13]. More recently, investigations from our group have also highlighted an antihypertrophic role for HSP20 and phospho-HSP20 in neonatal cardiomyocytes and using an in vivo model of aortic constriction, as discussed in the next section. In addition to the protective actions mentioned above, HSP20 also plays a role in myofilament contraction. During β-adrenergic stimulation, HSP20 translocates from the cytosol to the myofilaments [12]. The cardiac chaperone is also directly involved in sarcoplasmic reticulum Ca2 + cycling. Overexpression of HSP20 reduced the expression of protein phosphatase 1 associated with phospholamban, leading to enhanced activity of SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2 + -ATPase 2a) and enhanced cardiac function [9]. More recently, HSP20 has been described as a ‘cardiokine’ involved in regulating myocardial angiogenesis [14]. Initial experiments demonstrated that exogenously applied recombinant HSP20 to HUVECs (human umbilical vein endothelial cells) augmented proliferation, migration and tube formation. A protein-binding assay revealed an interaction between HSP20 and VEGF (vascular endothelial growth factor), indicating a role in angiogenesis. Gratifyingly, cardiac-specific overexpression of HSP20 resulted in increased circulating levels of HSP20 and enhanced capillary density in HSP20 overexpressing hearts [14], providing further confirmation of the cardioprotective nature of HSP20. Collectively, these studies support the notion that HSP20 may represent a novel therapeutic target for a myriad of cardiovascular disorders.
Therapeutic potential of HSP20 As mentioned, many of the cardioprotective actions of HSP20 are dependent on PKA phosphorylation of Ser16 located in the N-terminus [7]. Although there has been some experimental
Figure 1 Disruption of the HSP20–PDE4 complex promotes HSP20 phosphorylation on Ser16 to trigger its cardioprotective actions HSP20 interacts with AKAP-Lbc and PDE4s. (A) Under basal conditions, PDE4 bound to HSP20 hydrolyses cAMP, preventing activation of PKA, and HSP20 remains largely unphosphorylated. (B) Upon β-adrenergic stimulation, cAMP levels increase and activate AKAP-Lbc-bound PKA, which phosphorylates HSP20 on Ser16 . Mapping of the HSP20–PDE4 interaction site informed the discovery of a disruptor peptide that specifically dissociates the complex, augmenting phosphorylation of HSP20. Many of the cardioprotective actions of HSP20 are dependent on HSP20 phosphorylation.
success with using cell-permeant peptide analogues of phosphorylated HSP20 against diseases including vasospasm of human umbilical artery [15], airway smooth muscle relaxation [16], subarachnoid haemorrhage [17], platelet aggregation [18] and keloid scarring [19], these peptides may not be useful therapeutically due to the fact that they contain an unstable phosphate group that is easily targeted by phosphatases. A more feasible approach may be to target protein–protein interactions. A direct association of HSP20 and the cytosolic AKAP (A-kinase-anchoring protein) AKAP-Lbc promotes PKA-mediated phosphorylation [20], whereas association with cAMP-specific PDE4 (phosphodiesterase type 4) inhibits this action [21] (Figure 1). PDE4s degrade cAMP by hydrolysis [22]. Those PDEs associated with HSP20 act as local cAMP sinks to create areas of the cell devoid of the cyclic nucleotide, thus preventing phosphorylation of HSP20 by PKA [23]. The action of PDEs can be overcome by increasing the rate of cAMP production, saturating the activity of the available PDE4, or by directly inhibiting PDE4 enzymes. Chronic β-adrenergic stimulation will lead to sustained increases in levels of cAMP, thus saturating the pool of PDE4 associated with HSP20. Conversely, chronic βadrenergic stimulation will similarly augment PKA activity at Ca2 + -handling proteins (for example ryanodine receptor and troponin I), increasing the strength of contraction and C The
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rate of relaxation of the heart to increase cardiac output; a process that can become decompensatory progressing to cardiac hypertrophy and heart failure [24]. Non-specific PDE4 inhibition by pharmacological means may not be feasible due to the ubiquitous nature of PDE4 isoforms and the high expression in the brain, thus leading to off-target side effects (predominately headache, nausea and emesis) at the therapeutic dose required [25]. Targeting specific protein– protein interactions that underpin the compartmentalization of PDEs in a bid to increase phosphorylated HSP20 may be a more rational approach. Recent research from our laboratory has demonstrated that, in fact, HSP20 interacts and forms a complex with PDE4D [26].
Disruption of HSP20–PDE4D Peptide array analyses were undertaken where a library of immobilized peptides (overlapping peptides of up to 25mers, each shifted by five amino acids) of the PDE4 sequence was probed with recombinant HSP20. From these analyses, insight was gained into the sites of interaction, and it was apparent that HSP20 forms a complex with PDE4 within its conserved catalytic domain. A cell-permeant peptide on the basis of this interaction site was shown to disrupt HSP20– PDE4 complexes and induce HSP20 phosphorylation under basal conditions. The disruptor peptide, but not a scrambled control peptide, attenuated chronic β-adrenergic-induced cardiac hypertrophy in neonatal cardiomyocytes, increasing phosphorylation of HSP20 at Ser16 [26]. Intriguingly, aortic banded mice treated with the HSP20–PDE4 disruptor peptide, but not scrambled control peptide, exhibited improved left ventricular function (percentage fractional shortening), attenuated hypertrophic remodelling and reduced interstitial and perivascular fibrosis (T.P. Martin, S. Currie and G.S. Baillie, unpublished work). High-throughput drug screening of small-molecule libraries could be applied to harness the cardioprotective potential of HSP20. Identification of a small molecule that can mimic the actions of disruptor peptide represents a logical and achievable therapeutic goal. A similar approach has been used recently to discover a small-molecule modulator of HSP20 that promotes relaxation of human airway smooth muscle cells and intact tissue ex vivo [21].
Conclusions Over the last decade, a wealth of knowledge has been gathered highlighting HSP20 as a multifunctional cardioprotective agent. In this sense, HSP20 represents a novel therapeutic target for treatment of a myriad of cardiovascular disorders. Currently, a cell-permeant phosphopeptide analogue of HSP20, AZX100, is in Phase II clinical trials [19]. Nevertheless, our novel approach in targeting the interaction of HSP20 with an inhibitory protein (PDE4D) may represent a new strategy to liberate the cardioprotective properties of this cardiac chaperone. C The
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Funding T.P.M., S.C. and G.S.B. are funded by Heart Research UK [grant number RG2610/12/14] to undertake research on HSP20.
References 1 Willis, M.S. and Patterson, C. (2010) Hold me tight: role of the heat shock protein family of chaperones in cardiac disease. Circulation 122, 1740–1751 2 Edwards, H.V., Cameron, R.T. and Baillie, G.S. (2011) The emerging role of HSP20 as a multifunctional protective agent. Cell. Signal. 23, 1447–1454 3 Fan, G.-C. and Kranias, E.G. (2011) Small heat shock protein 20 (HspB6) in cardiac hypertrophy and failure. J. Mol. Cell. Cardiol. 51, 574–577 4 Fan, G.-C., Chu, G. and Kranias, E.G. (2005) Hsp20 and its cardioprotection. Trends Cardiovasc. Med. 15, 138–141 5 van de Klundert, F.A.J.M., Smulders, R.H.P.H., Gijsen, M.L.J., Lindner, R.A., Jaenicke, R., Carver, J.A. and de Jong, W.W. (1998) The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone. Eur. J. Biochem. 258, 1014–1021 6 Lee, S., Carson, K., Rice-Ficht, A. and Good, T. (2005) Hsp20, a novel α-crystallin, prevents Aβ fibril formation and toxicity. Protein Sci. 14, 593–601 7 Qian, J., Ren, X., Wang, X., Zhang, P., Jones, W.K., Molkentin, J.D., Fan, G.-C. and Kranias, E.G. (2009) Blockade of Hsp20 phosphorylation exacerbates cardiac ischemia/reperfusion injury by suppressed autophagy and increased cell death. Circ. Res. 105, 1223–1231 8 Chu, G., Egnaczyk, G.F., Zhao, W., Jo, S.-H., Fan, G.-C., Maggio, J.E., Xiao, R.-P. and Kranias, E.G. (2004) Phosphoproteome analysis of cardiomyocytes subjected to β-adrenergic stimulation: identification and characterization of a cardiac heat shock protein p20. Circ. Res. 94, 184–193 9 Qian, J., Vafiadaki, E., Florea, S.M., Singh, V.P., Song, W., Lam, C.K., Wang, Y., Yuan, Q., Pritchard, T.J., Cai, W. et al. (2011) Small heat shock protein 20 interacts with protein phosphatase-1 and enhances sarcoplasmic reticulum calcium cycling. Circ. Res. 108, 1429–1438 10 Fan, G.-C., Ren, X., Qian, J., Yuan, Q., Nicolaou, P., Wang, Y., Jones, W.K., Chu, G. and Kranias, E.G. (2005) Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury. Circulation 111, 1792–1799 11 Ren, X.-P., Wu, J., Wang, X., Sartor, M.A., Qian, J., Jones, K., Nicolaou, P., Pritchard, T.J. and Fan, G.-C. (2009) MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation 119, 2357–2366 12 Fan, G.-C., Chu, G., Mitton, B., Song, Q., Yuan, Q. and Kranias, E.G. (2004) Small heat-shock protein Hsp20 phosphorylation inhibits β-agonist-induced cardiac apoptosis. Circ. Res. 94, 1474–1482 13 Fan, G.-C., Yuan, Q., Song, G., Wang, Y., Chen, G., Qian, J., Zhou, X., Lee, Y.J., Ashraf, M. and Kranias, E.G. (2006) Small heat-shock protein Hsp20 attenuates β-agonist mediated cardiac remodeling through apoptosis signal regulating kinase 1. Circ. Res. 99, 1233–1242 14 Zhang, X., Wang, X., Zhu, H., Kranias, E.G., Tang, Y., Peng, T., Chang, J. and Fan, G.-C. (2012) Hsp20 functions as a novel cardiokine in promoting angiogenesis via activation of VEGFR2. PLoS ONE 7, e32765 15 Flynn, C.R., Brophy, C.M., Furnish, E.J., Komalavilas, P., Tessier, D., Thresher, J. and Joshi, L. (2005) Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle. J. Appl. Physiol. 98, 1836–1845 16 Komalavilas, P., Penn, R.B., Flynn, C.R., Thresher, J., Lopes, L.B., Furnish, E.J., Guo, M., Pallero, M.A., Murphy-Ullrich, J.E. and Brophy, C.M. (2008) The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L69–L78 17 Dreiza, C., Komalavilas, P., Furnish, E., Flynn, C., Sheller, M., Smoke, C., Lopes, L. and Brophy, C. (2010) The small heat shock protein, HSPB6, in muscle function and disease. Cell Stress Chaperones 15, 1–11
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18 Kanno, Y. and Matsuno, H. (2006) The possibility of novel antiplatelet peptides: the physiological effects of low molecular weight HSPs on platelets. Curr. Pharm. Des. 12, 887–892 19 Lopes, L.B., Furnish, E.J., Komalavilas, P., Flynn, C.R., Ashby, P., Hansen, A., Ly, D.P., Yang, G.P., Longaker, M.T., Panitch, A. and Brophy, C.M. (2009) Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-β1-induced CTGF expression in keloid fibroblasts. J. Invest. Dermatol. 129, 590–598 20 Edwards, H.V., Scott, J.D. and Baillie, G.S. (2012) The A-kinase-anchoring protein AKAP-Lbc facilitates cardioprotective PKA phosphorylation of Hsp20 on Ser16 . Biochem. J. 446, 437–443 21 An, S.S., Askovich, P.S., Zarembinski, T.I., Ahn, K., Peltier, J.M., von Rechenberg, M., Sahasrabudhe, S. and Fredberg, J.J. (2011) A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening. Respir. Res. 12, 8 22 Conti, M. and Beavo, J. (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76, 481–511
23 Lee, L.C.Y., Maurice, D.H. and Baillie, G.S. (2013) Targeting protein–protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease. Future Med. Chem. 5, 451–464 24 Vidal, M., Wieland, T., Lohse, M.J. and Lorenz, K. (2012) β-Adrenergic receptor stimulation causes cardiac hypertrophy via a Gβγ /Erk-dependent pathway. Cardiovasc. Res. 96, 255–264 25 Zhang, K.Y., Ibrahim, P.N., Gillette, S. and Bollag, G. (2005) Phosphodiesterase-4 as a potential drug target. Expert Opin. Ther. Targets 9, 1283–1305 26 Sin, Y.Y., Edwards, H.V., Li, X., Day, J.P., Christian, F., Dunlop, A.J., Adams, D.R., Zaccolo, M., Houslay, M.D. and Baillie, G.S. (2011) Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)–HSP20 complex attenuates the β-agonist induced hypertrophic response in cardiac myocytes. J. Mol. Cell. Cardiol. 50, 872–883
Received 10 December 2013 doi:10.1042/BST20130272
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Biochemical Society Transactions (2014) Volume 42, part 2
The cardioprotective role of small heat-shock protein 20 Tamara P. Martin*, Susan Currie† and George S. Baillie*1 *Institute of Cardiovascular and Medical Sciences, University of Glasgow, Wolfson-Link Building, Glasgow G12 8QQ, U.K. †Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Hamnett Building, 161 Cathedral Street, Glasgow G4 0RE, U.K.
Biochemical Society Transactions
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Abstract The small HSP (heat-shock protein) HSP20 is a molecular chaperone that is transiently up-regulated in response to cellular stress/damage. Although ubiquitously expressed in various tissues, it is most highly expressed in skeletal, cardiac and smooth muscle. Phosphorylation at Ser16 by PKA (cAMP-dependent protein kinase) is essential for HSP20 to confer its protective qualities. HSP20 and its phosphorylation have been implicated in a variety of pathophysiological processes, but most prominently cardiovascular disease. A wealth of knowledge of the importance of HSP20 in contractile function and cardioprotection has been gained over the last decade. The present mini-review highlights more recent findings illustrating the cardioprotective properties of HSP20 and its potential as a therapeutic agent.
Introduction HSPs (heat-shock proteins) are a ubiquitous and diverse group of chaperone proteins that play a key role in maintenance of normal cell metabolism and function. Expression of these proteins is rapidly up-regulated to protect the cell from various kinds of damage following periods of cellular stress [1]. HSPs are classified into families determined by their molecular mass and function. The small HSPs are the most diverse family, are unrelated to the other families and are known for the protective effects they confer in neurology and cardiology [2]. There are at least ten small HSPs (HSPB1– HSPB10), with molecular masses ranging from 12 to 43 kDa. Small HSPs are characterized by their unique N-terminus and a conserved α-crystallin domain at their C-terminus, which facilitates their intrinsic chaperone activity [3]. One of these small HSPs, HSP20 (HSPB6), contains a domain inhibiting platelet aggregation and a homology sequence of troponin I [4] and thus is gaining increasing interest in the field of cardiovascular research. Although HSP20 is a ubiquitously expressed protein, it is most highly expressed in skeletal, cardiac and smooth muscle [4]. Like other proteins of the same family, the expression and activity of HSP20 is unregulated in response to cellular stress. At the basal level, HSP20 is primarily found in the cytosol, but a subpopulation may translocate into the nuclear compartment in response to stress signals [4]. In the cell, HSP20 can exist as monomers or larger oligomers and has a strong tendency to form disulfide-linked dimers [5]. Formation of larger oligomers decreases the chaperone’s activity [6]. Phosphorylation of HSP20 at Ser16 alters its ability to aggregate, thus triggering its protective effects [7]. Key words: cAMP, cardioprotection, heat-shock protein 20 (HSP20), peptide array, phosphodiesterase type 4 (PDE4). Abbreviations: AKAP, A-kinase-anchoring protein; HSP, heat-shock protein; I/R, ischaemia/ reperfusion; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase. 1 To whom correspondence should be addressed (email [email protected]).
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Initial research into the role of HSP20 in the heart showed that expression levels and phosphorylation at Ser16 were increased upon prolonged stimulation with the β-adrenergic agonist isoprenaline [8]. This was a significant finding given the importance of β-adrenergic neurohormonal stimulation in mediating augmented contractility in the heart under flight-or-fight scenarios [3]. Not surprisingly, adenoviral gene transfer of HSP20 [8] and transgenic overexpression of HSP20 [9] was associated with increases in contractility and Ca2 + transient peak, highlighting further the importance of HSP20 in cardiac function.
Cardioprotective actions of HSP20 Studies over the last decade have begun to unravel the cardioprotective role of HSP20. Early studies showed that transgenic overexpression of HSP20 (∼10-fold) significantly decreased the extent of infarction and reduced necrotic damage in an ex vivo and an in vivo model of I/R (ischaemia/reperfusion) injury. Additionally, these mice displayed improved functional recovery of contractile performance, as well as displaying increased levels of phospho-HSP20 [10]. Further research demonstrated that many of the cardioprotective actions of HSP20 depend on phosphorylation of a putative PKA (cAMP-dependent protein kinase)/PKG (protein kinase G) consensus site in the N-terminus of HSP20 (Ser16 ). Consequently, overexpression of non-phosphorylatable HSP20 (HSP20S16A ) resulted in hearts with larger infarcts, reduced recovery of contractile function and increased necrosis and apoptosis compared with mice overexpressing wild-type HSP20 following I/R injury [7]. The authors suggest that this could be due to suppressed autophagy, a critical catabolic pathway upregulated during cardiac stress [7]. Subsequent research has shown that HSP20 expression levels may be decreased by miR-320, a known miRNA up-regulated during I/R Biochem. Soc. Trans. (2014) 42, 270–273; doi:10.1042/BST20130272
Targeting cAMP Signalling to Combat Cardiovascular Diseases
injury [11]. Furthermore, HSP20 exerts some of its cardioprotective actions by inhibiting β-adrenergic-mediated cardiac apoptosis via direct interaction with the pro-apoptotic signalling protein Bax [12]. This association prevents Bax translocation to the mitochondria and subsequent release of cytochrome c and activation of caspase 3. Correspondingly, overexpression of a constitutively phosphorylated HSP20 mutant (S16D) conferred full protection from apoptosis, whereas a non-phosphorylatable mutant (S16A) exhibited no anti-apoptotic properties. Moreover, expression levels of the cardiac chaperone are increased in response to hypertrophic stimuli, including chronic β-adrenergic signalling and aortic constriction [1]. Chronic β-adrenergic stimulation of cardiomyocytes resulted in increased expression of both HSP20 and phospho-HSP20 (Ser16 ), with adenoviral overexpression of HSP20 improving contractility and cardiac function [8]. Interestingly, cardiac overexpression of HSP20 prevented hypertrophic remodelling in response to chronic infusion of the β-agonist isoprenaline, including reducing interstitial fibrosis and cardiomyocyte apoptosis. In this scenario, HSP20 exhibits its protective actions, at least in part, via inhibition of the ASK1 (apoptosis signal-regulating kinase 1) signalling pathway [13]. More recently, investigations from our group have also highlighted an antihypertrophic role for HSP20 and phospho-HSP20 in neonatal cardiomyocytes and using an in vivo model of aortic constriction, as discussed in the next section. In addition to the protective actions mentioned above, HSP20 also plays a role in myofilament contraction. During β-adrenergic stimulation, HSP20 translocates from the cytosol to the myofilaments [12]. The cardiac chaperone is also directly involved in sarcoplasmic reticulum Ca2 + cycling. Overexpression of HSP20 reduced the expression of protein phosphatase 1 associated with phospholamban, leading to enhanced activity of SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2 + -ATPase 2a) and enhanced cardiac function [9]. More recently, HSP20 has been described as a ‘cardiokine’ involved in regulating myocardial angiogenesis [14]. Initial experiments demonstrated that exogenously applied recombinant HSP20 to HUVECs (human umbilical vein endothelial cells) augmented proliferation, migration and tube formation. A protein-binding assay revealed an interaction between HSP20 and VEGF (vascular endothelial growth factor), indicating a role in angiogenesis. Gratifyingly, cardiac-specific overexpression of HSP20 resulted in increased circulating levels of HSP20 and enhanced capillary density in HSP20 overexpressing hearts [14], providing further confirmation of the cardioprotective nature of HSP20. Collectively, these studies support the notion that HSP20 may represent a novel therapeutic target for a myriad of cardiovascular disorders.
Therapeutic potential of HSP20 As mentioned, many of the cardioprotective actions of HSP20 are dependent on PKA phosphorylation of Ser16 located in the N-terminus [7]. Although there has been some experimental
Figure 1 Disruption of the HSP20–PDE4 complex promotes HSP20 phosphorylation on Ser16 to trigger its cardioprotective actions HSP20 interacts with AKAP-Lbc and PDE4s. (A) Under basal conditions, PDE4 bound to HSP20 hydrolyses cAMP, preventing activation of PKA, and HSP20 remains largely unphosphorylated. (B) Upon β-adrenergic stimulation, cAMP levels increase and activate AKAP-Lbc-bound PKA, which phosphorylates HSP20 on Ser16 . Mapping of the HSP20–PDE4 interaction site informed the discovery of a disruptor peptide that specifically dissociates the complex, augmenting phosphorylation of HSP20. Many of the cardioprotective actions of HSP20 are dependent on HSP20 phosphorylation.
success with using cell-permeant peptide analogues of phosphorylated HSP20 against diseases including vasospasm of human umbilical artery [15], airway smooth muscle relaxation [16], subarachnoid haemorrhage [17], platelet aggregation [18] and keloid scarring [19], these peptides may not be useful therapeutically due to the fact that they contain an unstable phosphate group that is easily targeted by phosphatases. A more feasible approach may be to target protein–protein interactions. A direct association of HSP20 and the cytosolic AKAP (A-kinase-anchoring protein) AKAP-Lbc promotes PKA-mediated phosphorylation [20], whereas association with cAMP-specific PDE4 (phosphodiesterase type 4) inhibits this action [21] (Figure 1). PDE4s degrade cAMP by hydrolysis [22]. Those PDEs associated with HSP20 act as local cAMP sinks to create areas of the cell devoid of the cyclic nucleotide, thus preventing phosphorylation of HSP20 by PKA [23]. The action of PDEs can be overcome by increasing the rate of cAMP production, saturating the activity of the available PDE4, or by directly inhibiting PDE4 enzymes. Chronic β-adrenergic stimulation will lead to sustained increases in levels of cAMP, thus saturating the pool of PDE4 associated with HSP20. Conversely, chronic βadrenergic stimulation will similarly augment PKA activity at Ca2 + -handling proteins (for example ryanodine receptor and troponin I), increasing the strength of contraction and C The
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rate of relaxation of the heart to increase cardiac output; a process that can become decompensatory progressing to cardiac hypertrophy and heart failure [24]. Non-specific PDE4 inhibition by pharmacological means may not be feasible due to the ubiquitous nature of PDE4 isoforms and the high expression in the brain, thus leading to off-target side effects (predominately headache, nausea and emesis) at the therapeutic dose required [25]. Targeting specific protein– protein interactions that underpin the compartmentalization of PDEs in a bid to increase phosphorylated HSP20 may be a more rational approach. Recent research from our laboratory has demonstrated that, in fact, HSP20 interacts and forms a complex with PDE4D [26].
Disruption of HSP20–PDE4D Peptide array analyses were undertaken where a library of immobilized peptides (overlapping peptides of up to 25mers, each shifted by five amino acids) of the PDE4 sequence was probed with recombinant HSP20. From these analyses, insight was gained into the sites of interaction, and it was apparent that HSP20 forms a complex with PDE4 within its conserved catalytic domain. A cell-permeant peptide on the basis of this interaction site was shown to disrupt HSP20– PDE4 complexes and induce HSP20 phosphorylation under basal conditions. The disruptor peptide, but not a scrambled control peptide, attenuated chronic β-adrenergic-induced cardiac hypertrophy in neonatal cardiomyocytes, increasing phosphorylation of HSP20 at Ser16 [26]. Intriguingly, aortic banded mice treated with the HSP20–PDE4 disruptor peptide, but not scrambled control peptide, exhibited improved left ventricular function (percentage fractional shortening), attenuated hypertrophic remodelling and reduced interstitial and perivascular fibrosis (T.P. Martin, S. Currie and G.S. Baillie, unpublished work). High-throughput drug screening of small-molecule libraries could be applied to harness the cardioprotective potential of HSP20. Identification of a small molecule that can mimic the actions of disruptor peptide represents a logical and achievable therapeutic goal. A similar approach has been used recently to discover a small-molecule modulator of HSP20 that promotes relaxation of human airway smooth muscle cells and intact tissue ex vivo [21].
Conclusions Over the last decade, a wealth of knowledge has been gathered highlighting HSP20 as a multifunctional cardioprotective agent. In this sense, HSP20 represents a novel therapeutic target for treatment of a myriad of cardiovascular disorders. Currently, a cell-permeant phosphopeptide analogue of HSP20, AZX100, is in Phase II clinical trials [19]. Nevertheless, our novel approach in targeting the interaction of HSP20 with an inhibitory protein (PDE4D) may represent a new strategy to liberate the cardioprotective properties of this cardiac chaperone. C The
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Funding T.P.M., S.C. and G.S.B. are funded by Heart Research UK [grant number RG2610/12/14] to undertake research on HSP20.
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Targeting cAMP Signalling to Combat Cardiovascular Diseases
18 Kanno, Y. and Matsuno, H. (2006) The possibility of novel antiplatelet peptides: the physiological effects of low molecular weight HSPs on platelets. Curr. Pharm. Des. 12, 887–892 19 Lopes, L.B., Furnish, E.J., Komalavilas, P., Flynn, C.R., Ashby, P., Hansen, A., Ly, D.P., Yang, G.P., Longaker, M.T., Panitch, A. and Brophy, C.M. (2009) Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-β1-induced CTGF expression in keloid fibroblasts. J. Invest. Dermatol. 129, 590–598 20 Edwards, H.V., Scott, J.D. and Baillie, G.S. (2012) The A-kinase-anchoring protein AKAP-Lbc facilitates cardioprotective PKA phosphorylation of Hsp20 on Ser16 . Biochem. J. 446, 437–443 21 An, S.S., Askovich, P.S., Zarembinski, T.I., Ahn, K., Peltier, J.M., von Rechenberg, M., Sahasrabudhe, S. and Fredberg, J.J. (2011) A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening. Respir. Res. 12, 8 22 Conti, M. and Beavo, J. (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76, 481–511
23 Lee, L.C.Y., Maurice, D.H. and Baillie, G.S. (2013) Targeting protein–protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease. Future Med. Chem. 5, 451–464 24 Vidal, M., Wieland, T., Lohse, M.J. and Lorenz, K. (2012) β-Adrenergic receptor stimulation causes cardiac hypertrophy via a Gβγ /Erk-dependent pathway. Cardiovasc. Res. 96, 255–264 25 Zhang, K.Y., Ibrahim, P.N., Gillette, S. and Bollag, G. (2005) Phosphodiesterase-4 as a potential drug target. Expert Opin. Ther. Targets 9, 1283–1305 26 Sin, Y.Y., Edwards, H.V., Li, X., Day, J.P., Christian, F., Dunlop, A.J., Adams, D.R., Zaccolo, M., Houslay, M.D. and Baillie, G.S. (2011) Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)–HSP20 complex attenuates the β-agonist induced hypertrophic response in cardiac myocytes. J. Mol. Cell. Cardiol. 50, 872–883
Received 10 December 2013 doi:10.1042/BST20130272
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