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Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy.

Sin YY, Martin TP, Wills L, Currie S, Baillie GS - Cell Commun. Signal (2015)

Bottom Line: This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation.In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex.These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical sciences, CMVLS, University of Glasgow, Glasgow, G128QQ, UK. angie.sin@queensu.ca.

ABSTRACT

Background: Nuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, however, little is known about the molecular events that govern this event. We have identified a novel complex between PKD1 and a heat shock protein (Hsp), Hsp20, which has been implicated as cardioprotective. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation.

Results: In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex. We use this peptide to show that formation of the Hsp20-PKD1 complex is essential for PKD1 nuclear translocation, signaling mechanisms leading to hypertrophy, activation of the fetal gene programme and pathological cardiac remodeling leading to cardiac fibrosis.

Conclusions: These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

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Related in: MedlinePlus

Disruption of the Hsp20-PKD1 complex attenuates hypertrophic signaling in neonatal cardiac myocytes. A. A cell permeable disruptor peptide (DIS), but not scrambled control (CTR), ablates interaction of PKD1 and Hsp20 in cardiac myocytes. B. and C. The Hsp20-PKD1 disruptor peptide reduces cardiac myocyte cell size following chronic β-adrenergic (isoprenaline; ISO) stimulation. D. The Hsp20-PKD1 disruptor peptide reduces relative protein/DNA ratio of cells following chronic β-adrenergic stimulation (n = 9). E. The Hsp20-PKD1 disruptor peptide reduces the expression of hypertrophic markers atrial natruiretic peptide (ANP), brain natruiretic peptide (BNP) and β-myosin heavy chain (β-MHC) following chronic β-adrenergic stimulation (n = 7). F. The Hsp20-PKD1 disruptor peptide reduces actin cytoskeletal rearrangement following chronic β-adrenergic stimulation. Data representative of n = 3 independent experiments.
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Fig2: Disruption of the Hsp20-PKD1 complex attenuates hypertrophic signaling in neonatal cardiac myocytes. A. A cell permeable disruptor peptide (DIS), but not scrambled control (CTR), ablates interaction of PKD1 and Hsp20 in cardiac myocytes. B. and C. The Hsp20-PKD1 disruptor peptide reduces cardiac myocyte cell size following chronic β-adrenergic (isoprenaline; ISO) stimulation. D. The Hsp20-PKD1 disruptor peptide reduces relative protein/DNA ratio of cells following chronic β-adrenergic stimulation (n = 9). E. The Hsp20-PKD1 disruptor peptide reduces the expression of hypertrophic markers atrial natruiretic peptide (ANP), brain natruiretic peptide (BNP) and β-myosin heavy chain (β-MHC) following chronic β-adrenergic stimulation (n = 7). F. The Hsp20-PKD1 disruptor peptide reduces actin cytoskeletal rearrangement following chronic β-adrenergic stimulation. Data representative of n = 3 independent experiments.

Mentions: Gratifyingly, a disruptor peptide comprising of PKD1 residues 606 to 630 (DIS), but not a control peptide (CTR) with three of the seven key residues replaced by alanine (GAAVAIKIIAKLRFPTKQESQLRNE), ablated co-immunopreciptation of endogenous Hsp20 and PKD1 from neonatal cardiac myocyte lysates (Figure 2A). As we could successfully disrupt the Hsp20-PKD1 complex with disruptor peptide (Figure 2A), we were interested to see if it would be able to attenuate the hypertrophic response induced by chronic β-agonist (isoprenaline; ISO) stimulation of neonatal cardiac myocytes. To this end, we utilised 3 different assay methods to measure the effectiveness of the peptide to confer protection against β-adrenergic receptor-triggered hypertrophic signalling. Firstly, we employed a novel approach utilizing xCELLigence technology, which we have previously developed [15] to measure the size of cardiac myocytes (Figure 2B and C). Secondly, we measured the relative protein/DNA ratio of cells (Figure 2D) and thirdly, in order to investigate whether the peptide affected increased fetal gene expression that characterises the hypertrophic response, we determined the mRNA levels of 3 hypertrophy marker genes atrial naturietic peptide (ANP), brain naturietic peptide (BNP) and β-myosin heavy chain (β-MHC) (Figure 2E). All three techniques suggested that the disruptor peptide (but not control peptide) could attenuate the hypertrophic response. Note that differences seen in BNP between ISO and ISO plus control peptide are not significant (p = 0.46) Cell size, measured in real-time by cell index (CI; Figure 2B and C) and protein/DNA ratio (Figure 2D) of cells increased following chronic ISO treatment and were significantly reduced by pre-treatment with disruptor peptide but not with control peptide. The Hsp20-PKD1 disruptor peptide also significantly reduced the upregulation of fetal gene expression (Figure 2E) triggered by chronic β-agonist stimulation. Another key characteristic of hypertrophy is the reorganization of actin filaments [20] and we were able to observe the expected formation of β-adrenergic–stimulated stress fibers when compared with untreated cells (Figure 2F). Interestingly, the actin cytoskeletal rearrangement stimulated by hypertrophy induction was visibly reduced in cells pre-treated with disruptor peptide, but not control peptide, (Figure 2F) suggesting that the Hsp20-PKD1 association is also likely to have the capacity to regulate the integrity of cellular actin filaments.Figure 2


Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy.

Sin YY, Martin TP, Wills L, Currie S, Baillie GS - Cell Commun. Signal (2015)

Disruption of the Hsp20-PKD1 complex attenuates hypertrophic signaling in neonatal cardiac myocytes. A. A cell permeable disruptor peptide (DIS), but not scrambled control (CTR), ablates interaction of PKD1 and Hsp20 in cardiac myocytes. B. and C. The Hsp20-PKD1 disruptor peptide reduces cardiac myocyte cell size following chronic β-adrenergic (isoprenaline; ISO) stimulation. D. The Hsp20-PKD1 disruptor peptide reduces relative protein/DNA ratio of cells following chronic β-adrenergic stimulation (n = 9). E. The Hsp20-PKD1 disruptor peptide reduces the expression of hypertrophic markers atrial natruiretic peptide (ANP), brain natruiretic peptide (BNP) and β-myosin heavy chain (β-MHC) following chronic β-adrenergic stimulation (n = 7). F. The Hsp20-PKD1 disruptor peptide reduces actin cytoskeletal rearrangement following chronic β-adrenergic stimulation. Data representative of n = 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356135&req=5

Fig2: Disruption of the Hsp20-PKD1 complex attenuates hypertrophic signaling in neonatal cardiac myocytes. A. A cell permeable disruptor peptide (DIS), but not scrambled control (CTR), ablates interaction of PKD1 and Hsp20 in cardiac myocytes. B. and C. The Hsp20-PKD1 disruptor peptide reduces cardiac myocyte cell size following chronic β-adrenergic (isoprenaline; ISO) stimulation. D. The Hsp20-PKD1 disruptor peptide reduces relative protein/DNA ratio of cells following chronic β-adrenergic stimulation (n = 9). E. The Hsp20-PKD1 disruptor peptide reduces the expression of hypertrophic markers atrial natruiretic peptide (ANP), brain natruiretic peptide (BNP) and β-myosin heavy chain (β-MHC) following chronic β-adrenergic stimulation (n = 7). F. The Hsp20-PKD1 disruptor peptide reduces actin cytoskeletal rearrangement following chronic β-adrenergic stimulation. Data representative of n = 3 independent experiments.
Mentions: Gratifyingly, a disruptor peptide comprising of PKD1 residues 606 to 630 (DIS), but not a control peptide (CTR) with three of the seven key residues replaced by alanine (GAAVAIKIIAKLRFPTKQESQLRNE), ablated co-immunopreciptation of endogenous Hsp20 and PKD1 from neonatal cardiac myocyte lysates (Figure 2A). As we could successfully disrupt the Hsp20-PKD1 complex with disruptor peptide (Figure 2A), we were interested to see if it would be able to attenuate the hypertrophic response induced by chronic β-agonist (isoprenaline; ISO) stimulation of neonatal cardiac myocytes. To this end, we utilised 3 different assay methods to measure the effectiveness of the peptide to confer protection against β-adrenergic receptor-triggered hypertrophic signalling. Firstly, we employed a novel approach utilizing xCELLigence technology, which we have previously developed [15] to measure the size of cardiac myocytes (Figure 2B and C). Secondly, we measured the relative protein/DNA ratio of cells (Figure 2D) and thirdly, in order to investigate whether the peptide affected increased fetal gene expression that characterises the hypertrophic response, we determined the mRNA levels of 3 hypertrophy marker genes atrial naturietic peptide (ANP), brain naturietic peptide (BNP) and β-myosin heavy chain (β-MHC) (Figure 2E). All three techniques suggested that the disruptor peptide (but not control peptide) could attenuate the hypertrophic response. Note that differences seen in BNP between ISO and ISO plus control peptide are not significant (p = 0.46) Cell size, measured in real-time by cell index (CI; Figure 2B and C) and protein/DNA ratio (Figure 2D) of cells increased following chronic ISO treatment and were significantly reduced by pre-treatment with disruptor peptide but not with control peptide. The Hsp20-PKD1 disruptor peptide also significantly reduced the upregulation of fetal gene expression (Figure 2E) triggered by chronic β-agonist stimulation. Another key characteristic of hypertrophy is the reorganization of actin filaments [20] and we were able to observe the expected formation of β-adrenergic–stimulated stress fibers when compared with untreated cells (Figure 2F). Interestingly, the actin cytoskeletal rearrangement stimulated by hypertrophy induction was visibly reduced in cells pre-treated with disruptor peptide, but not control peptide, (Figure 2F) suggesting that the Hsp20-PKD1 association is also likely to have the capacity to regulate the integrity of cellular actin filaments.Figure 2

Bottom Line: This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation.In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex.These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cardiovascular and Medical sciences, CMVLS, University of Glasgow, Glasgow, G128QQ, UK. angie.sin@queensu.ca.

ABSTRACT

Background: Nuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, however, little is known about the molecular events that govern this event. We have identified a novel complex between PKD1 and a heat shock protein (Hsp), Hsp20, which has been implicated as cardioprotective. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation.

Results: In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex. We use this peptide to show that formation of the Hsp20-PKD1 complex is essential for PKD1 nuclear translocation, signaling mechanisms leading to hypertrophy, activation of the fetal gene programme and pathological cardiac remodeling leading to cardiac fibrosis.

Conclusions: These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

Show MeSH
Related in: MedlinePlus