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Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

Cipolletta E, Rusciano MR, Maione AS, Santulli G, Sorriento D, Del Giudice C, Ciccarelli M, Franco A, Crola C, Campiglia P, Sala M, Gomez-Monterrey I, De Luca N, Trimarco B, Iaccarino G, Illario M - PLoS ONE (2015)

Bottom Line: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction.Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses.The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy.

ABSTRACT

Aims: Activation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and results: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion: These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.

No MeSH data available.


Related in: MedlinePlus

Effects of CaMKII inhibition on CaMKII/ERK pathway in vivo in SHR.A: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. B: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. C: Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. D: To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. E: The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. F: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *P < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.
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pone.0130477.g008: Effects of CaMKII inhibition on CaMKII/ERK pathway in vivo in SHR.A: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. B: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. C: Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. D: To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. E: The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. F: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *P < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.

Mentions: To confirm the role of the cross talk between CaMKII and ERK in cardiac hypertrophy, we evaluated CaMKII and ERK phosphorylation and subcellular localization in left ventricles. We observed that CaMKII expression levels were increased regardless of treatment in SHR compared to WKY (Fig 8A). Importantly, CaMKII phosphorylation levels were enhanced, and this effect was prevented by AntCaNtide and tat-CN17β (Fig 8B). As expected, increased levels of ERK phosphorylation were observed in sham treated SHR left ventricles. Once again treatment with CaMKII selective inhibitors markedly reduced ERK1/2 phosphorylation levels (Fig 8C). In the hypertrophic hearts, the nuclear accumulation of CaMKII and ERK was also enhanced (Fig 8D and 8E), as well as the interaction between both kinases (Fig 8F). Strikingly, CaMKII inhibition by AntCaNtide or tat-CN17β blocked both ERK1/2 phosphorylation (Fig 8C) and CaMKII/ERK interaction (Fig 8F). These inhibitory events were mirrored by the reduction of the nuclear content of both kinases (Fig 8D and 8E). These data indicate that CaMKII inhibitors, AntCaNtide and tat-CN17β, are both able to reduce hypertrophy of cardiac myocytes and remodeling of the heart, by a mechanism that involves the crosstalk between the ERK and CaMKII pathways and their nuclear accumulation.


Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

Cipolletta E, Rusciano MR, Maione AS, Santulli G, Sorriento D, Del Giudice C, Ciccarelli M, Franco A, Crola C, Campiglia P, Sala M, Gomez-Monterrey I, De Luca N, Trimarco B, Iaccarino G, Illario M - PLoS ONE (2015)

Effects of CaMKII inhibition on CaMKII/ERK pathway in vivo in SHR.A: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. B: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. C: Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. D: To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. E: The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. F: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *P < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4481531&req=5

pone.0130477.g008: Effects of CaMKII inhibition on CaMKII/ERK pathway in vivo in SHR.A: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. B: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. C: Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. D: To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. E: The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, P < 0.05 vs. WKY. F: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *P < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.
Mentions: To confirm the role of the cross talk between CaMKII and ERK in cardiac hypertrophy, we evaluated CaMKII and ERK phosphorylation and subcellular localization in left ventricles. We observed that CaMKII expression levels were increased regardless of treatment in SHR compared to WKY (Fig 8A). Importantly, CaMKII phosphorylation levels were enhanced, and this effect was prevented by AntCaNtide and tat-CN17β (Fig 8B). As expected, increased levels of ERK phosphorylation were observed in sham treated SHR left ventricles. Once again treatment with CaMKII selective inhibitors markedly reduced ERK1/2 phosphorylation levels (Fig 8C). In the hypertrophic hearts, the nuclear accumulation of CaMKII and ERK was also enhanced (Fig 8D and 8E), as well as the interaction between both kinases (Fig 8F). Strikingly, CaMKII inhibition by AntCaNtide or tat-CN17β blocked both ERK1/2 phosphorylation (Fig 8C) and CaMKII/ERK interaction (Fig 8F). These inhibitory events were mirrored by the reduction of the nuclear content of both kinases (Fig 8D and 8E). These data indicate that CaMKII inhibitors, AntCaNtide and tat-CN17β, are both able to reduce hypertrophy of cardiac myocytes and remodeling of the heart, by a mechanism that involves the crosstalk between the ERK and CaMKII pathways and their nuclear accumulation.

Bottom Line: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction.Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses.The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy.

ABSTRACT

Aims: Activation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and results: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion: These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.

No MeSH data available.


Related in: MedlinePlus