Abstract-Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKII␦ and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5-green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (F nuc /F cyto 3.3Ϯ0.3 vs 7.2Ϯ0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKII␦ C expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP F nuc /F cyto toward control, and simultaneous inhibition restored F nuc /F cyto to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKII␦ B , or CaMKII␦ C reduced baseline HDAC5 F nuc /F cyto in control myocytes (3.4Ϯ0.5, 3.8Ϯ0.5, and 5.2Ϯ0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE), trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP 3 and CaMKII signaling. Thus ␣-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.Various stresses trigger cardiac hypertrophy, remodeling, and functional alterations (1, 2). Prolonged stress can also become maladaptive, leading to heart failure, cardiac arrhythmias, and sudden death. Many of these changes are mediated by altered gene expression, and Class II histone deacetylases (HDACs) 3 (e.g. HDAC5) are recognized as key modulators of this genetic reprogramming. HDAC5 represses transcription by promoting more condensed DNA, and represses transcription factors such as myocyte enhancing factor 2 (MEF2). HDAC5 phosphorylation triggers its nuclear export (allowing gene activation (Fig. 1a)). Both protein kinase D (PKD) and calmodulin-dependent protein kinase II (CaMKII) are key HDAC kinases (1, 2) and accumulating evidence indicates both kinases are more active in heart failure and can contribute directly to cardiac pathogenesis (3-6). Cardiac PKD is activated in response to hypertension, pressure overload, and chronic neurohumoral signaling (7). Overexpression of constitutively active PKD (and CaMKII␦) cause cardiac hypertrophy followed by chamber dilation (8, 9). Moreover there is increased expression of PKD (and CaMKII␦) in failing rat, rabbit, and human myocardium (8, 10). Thus, whereas PKD and CaMKII activation may be involved in a wide variety of cell functions (11-14), they are attractive potential therapeutic targets in cardiac disease. Therapeutic benefit of PKD/CaMKII inhibition may be largely due to prevention of HDAC (class II) phosphorylation, thereby maintaining the repressive effects of HDAC on transcription. Indeed, HDAC5 knock-out mice develop profound pathological cardiac hypertrophy and HDAC5 overexpression can limit progression of cardiac hypertrophy (15,16)...

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII—but not PKA, PKC or PKD—raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

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Within the channel, cargos move in a non-directional manner, consistent with anomalous subdiffusion in a crowded volume, with dimensions of ~55 nm in width and ~68 nm in length. By varying the number of import receptors on the surface of the cargo, we find that the translocation is not governed by simple receptor-NPC binding interactions and that the central channel behaves in accordance with the 'selective phase' model. Finally, in the absence of Ran, cargos still explore the entire volume of the NPC, but have a dramatically reduced probability of exit into the nucleus from the pore, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos.