Signaling to Class IIa HDACs in the Heart

Amino-terminal extensions unique to class IIa HDACs possess two conserved serine residues that are phosphorylated in response to stress signaling in the heart. Upon phosphorylation, these sites are bound by the intracellular chaperone protein 14-3-3, resulting in the activation of a cryptic, carboxy-terminal nuclear export sequence conserved in HDACs 4, 5, 7 and 9.14-3-3 binding also appears to mask an internal nuclear localization signal in the HDACs (Grozinger and Schreiber 2000; Nishino et al. 2008). Phosphorylated class IIa HDACs are thus removed and excluded from cardiomyocyte nuclei, resulting in derepression of pathological downstream target genes (Fig. 2).

Hdac Downstream

Fig. 2 Signal-dependent regulation of class IIa HDACs in cardiac myocytes. Genes under the control of class IIa HDACs are derepressed by signals that trigger phosphorylation-dependent nuclear export of the HDACs. Gaq-coupled receptors, including the aradrenergic receptor (a1-AR), the endothelin receptor (ET-R) and the angiotensin receptor (AT-R) trigger class IIa HDAC phosphorylation by activating PKD. Inositol trisphosphate (IP3) produced by phospho-lipase C (PLC) can also trigger release of intracellular Ca2+, with subsequent activation of CaMKII (Wu et al. 2006), which selectively phosphorylates HDAC4. Signaling through Gas-coupled b-adrenergic receptors (b-ARs) activates adenylyl cyclase (AC) and subsequent production of cAMP, which activates protein kinase A (PKA). PKA inhibits PKD and also phosphorylates unique sites on class IIa HDACs that result in inhibition of nuclear export. HDAC4 can hetero-dimerize ($) with other class IIa HDACs and thereby transmit CaMKII signals to HDACs 5, 7 and 9. G protein-coupled receptor kinase-5 (GRK5), microtubule affinity-regulating kinase (MARK), salt-inducible kinase (SIK) and AMP-dependent protein kinase (AMPK) have also been demonstrated to phosphorylate class IIa HDACs on the sites that trigger nuclear export. Oxidative stress can promote phosphorylation-independent nuclear export of class IIa HDACs, and this is antagonized by thioredoxin (Trx). HDAC4 can associate with cardiac sarcomeres and has been implicated in the control of contractility

Signal-dependent nuclear export of class IIa HDACs occurs in cardiomyocytes exposed to diverse hypertrophic agonists that stimulate Gaq/Ga11 signaling, including endothelin-1, angiotensin II, and the ^-adrenergic agonist, phenylephrine (Harrison et al. 2004). Increased nuclear export of class IIa HDACs has also been observed in failing human heart explants (Calalb et al. 2009). Blockade of class IIa HDAC nuclear export through substitution of the phospho-acceptor sites with nonphosphorylatable alanine residues creates a "superrepressor" that potently suppresses cardiac hypertrophy (Zhang et al. 2002), suggesting that it may be possible to manipulate cardiac disease with inhibitors of class IIa HDAC kinases.

2.2.1 PKD Is a Class IIa HDAC Kinase

Protein kinase D (PKD) has been shown to function as class IIa HDAC kinase that promotes pathological cardiac remodeling (McKinsey 2007). Three PKD isoforms (1, 2 and 3) make up a family of related serine/threonine kinases. PKD1 was discovered in 1994 and was also referred to as PKCm due to the presence of amino-terminal diacylglycerol binding domains resembling those of PKC (Johannes et al. 1994; Valverde et al. 1994). However, the catalytic domain of PKD1 is divergent from those of PKC isoforms and more closely related to that of Ca2+/calmodulin-dependent protein kinase (CaMK). Each PKD isoform is capable of phosphorylating all class IIa HDACs on the serines that are targeted by 14-3-3 (Huynh and McKinsey 2006; Parra et al. 2005; Vega et al. 2004), suggesting the potential for redundant control of class IIa HDACs by PKD family members. The antihypertrophic transcription factor, YY1, appears to suppress hypertrophy by physically associating with HDAC5 and preventing PKD-mediated phosphory-lation of the HDAC (Sucharov et al. 2008).

In rodents, cardiac PKD is activated in response to chronic hypertension, pressure overload mediated by aortic constriction and infusion of agonists such as norepinephrine (Avkiran et al. 2008; Harrison et al. 2006; Haworth et al. 2000). Knockdown of endogenous PKD1 expression with siRNA blunts agonist-dependent nuclear export of class IIa HDACs and associated hypertrophy of cultured neonatal rat cardiac myocytes (Harrison et al. 2006). Conversely, ectopic overexpression of constitutively active PKD1 in these cells has been reported to induce hypertrophy (Iwata et al. 2005). In vivo, cardiac-specific expression of constitutively active PKD1 in mice causes a brief phase of cardiac hypertrophy, followed by chamber dilation and impaired systolic function (Harrison et al. 2006). These mice do not have significant fibrosis (Massare et al. 2010). Conversely, mice in which the PKD1 gene was conditionally deleted in cardiac myocytes showed dramatically reduced cardiac hypertrophy and cardiac fibrosis in response to pressure overload or chronic administration of adrenergic or angiotensin receptor agonists (Fielitz et al. 2008).

In addition to class IIa HDACs, cardiac PKD has also been shown to phosphor-ylate the CREB transcription factor as well as sarcomeric proteins such as troponin I (Bardswell et al. 2010; Haworth et al. 2004; Ozgen et al. 2009). PKD may also have beneficial effects on the heart by preventing lipoprotein lipase driven accumulation of triglycerides in the diabetic state (Kim et al. 2008, 2009). However, with regard to heart failure, the genetic gain- and loss-of-function data described above suggest a pathological role for PKD signaling. Consistent with this, PKD is hyperactivated in ventricular cardiac myocytes from humans with heart failure (Bossuyt et al. 2008), and efficacy of an HMG-CoA reductase inhibitor in a hypertensive rat model of heart failure was linked to attenuation of PKD signaling

(Geng et al. 2010). Effects of small molecule inhibitors of PKD on cardiac growth have recently been described. PKD inhibition blocked agonist-dependent phosphorylation of class IIa HDACs and hypertrophy of cultured cardiac myocytes (Monovich et al. 2010), but failed to alter pathologic growth of the heart in vivo (Meredith et al. 2010a, b). Additional work with these and chemically distinct PKD inhibitors is required to fully address the potential therapeutic benefit of inhibiting PKD in the context of heart failure.

Studies by the Avkiran Lab recently elucidated a role for protein kinase A (PKA) as a negative regulator of PKD-mediated phosphorylation of class IIa HDACs in cardiac myocytes. ß-Adrenergic receptor (ß-AR) signaling, which stimulates cAMP production and downstream activation of PKA, blocks PKD activation in cardiomyocytes in response to a-adrenergic receptor signaling (Haworth et al. 2010). PKA also blocks PKD activation in response to endothelin stimulation (Haworth et al. 2007), and inhibition of cAMP-directed phosphodiesterases 3 and 4 is sufficient to antagonize PKD in myocytes (Haworth et al. 2010).

2.2.2 Other Class IIa HDAC Kinases

Additional kinases including salt-inducible kinase (SIK) (Berdeaux et al. 2007), microtubule affinity-regulating kinase (MARK) (Chang et al. 2005; Dequiedt et al. 2006) and AMP-dependent protein kinase (AMPK) (McGee et al. 2008) have been implicated as class IIa HDAC kinases. A connection between CaMKII and class IIa HDACs has also been described (Backs et al. 2006, 2008; Zhang et al. 2007), and G protein-coupled receptor kinase-5 (GRK5) was shown to translocate to the nucleus and phosphorylate HDAC5 in cardiomyocytes in response to Gaq signaling (Martini et al. 2008). This novel function of GRK5 is associated with enhanced cardiac remodeling due to pressure overload. All of these redundant pathways converge on the 14-3-3 target phospho-acceptors on class IIa HDACs. In contrast, protein kinase C-related kinase and Dyrk phosphorylate sites in or near the class IIa HDAC nuclear localization signal, and thereby impair nuclear import of the enzymes (Deng et al. 2005; Harrison et al. 2010).

Some signaling pathways appear to enhance the nuclear function of class IIa HDACs by blocking their export from the nucleus. In addition to suppressing PKD activity, PKA was also shown to directly phosphorylate HDAC5, resulting in repression of agonist-dependent nuclear export of the HDAC and suppression of cardiac hypertrophy (Ha et al. 2010). Thus, PKA utilizes dual mechanisms for suppressing class IIa HDAC nuclear export in cardiac myocytes.

A kinase-independent mechanism for regulation of class IIa HDAC nuclear export was also described (Ago et al. 2008; Oka et al. 2009). HDAC4 harbors two cysteine residues that become oxidized in response to hypertrophic stimuli. In the oxidized state, HDAC4 undergoes nuclear export. However, reduction of these cysteines through a thioredoxin-dependent mechanism results in inhibition of HDAC4 nuclear export. These findings suggest alternative therapeutic strategies based on inhibiting class IIa HDAC nuclear export via modulation of redox state.

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