Calcium/calmodulin dependent protein kinase II δ modulates intracellular CA2+ release and cell proliferation in adult cardiac fibroblasts

M Mohd Salleh, C McCluskey, T Bushell, S Currie

Research output: Contribution to journalMeeting abstract

Abstract

Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5% (AIP) vs 123±6% (control), p<0.001, n=4 and 65±4% (KN-93) vs 123±6% (control) respectively, p<0.001, n=4). Both CaMKII inhibition and IP3R inhibition also inhibit CFs proliferation (∼10% and 30% inhibition, AIP and 2-APB treatments respectively). The possibility that Ca MKIIδ may modulate IP3R2 activity directly via protein-protein interaction has also been explored using co-immunoprecipitation. Collectively, this study reveals for the first time that CaMKIIδ modulates intracellular Ca2+ release and proliferation in adult CFs and more specifically, that this may be modulated via physical interaction between CaMKIIδ and IP3R2.
Original languageEnglish
JournalHeart
Volume100
Issue numberS4
DOIs
Publication statusPublished - 31 Dec 2014
EventBSCR: Cardiovascular Signalling in Health and Disease - University of Reading
Duration: 8 Sep 20159 Sep 2015

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases
Fibroblasts
Cell Proliferation
Ryanodine Receptor Calcium Release Channel
Angiotensin II
Peptides
Inositol 1,4,5-Trisphosphate Receptors
Fura-2
Immunoprecipitation
Cardiac Myocytes
Proteins

Cite this

@article{3919c5e8b5f245dd884fe733414fd178,
title = "Calcium/calmodulin dependent protein kinase II δ modulates intracellular CA2+ release and cell proliferation in adult cardiac fibroblasts",
abstract = "Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5{\%} (AIP) vs 123±6{\%} (control), p<0.001, n=4 and 65±4{\%} (KN-93) vs 123±6{\%} (control) respectively, p<0.001, n=4). Both CaMKII inhibition and IP3R inhibition also inhibit CFs proliferation (∼10{\%} and 30{\%} inhibition, AIP and 2-APB treatments respectively). The possibility that Ca MKIIδ may modulate IP3R2 activity directly via protein-protein interaction has also been explored using co-immunoprecipitation. Collectively, this study reveals for the first time that CaMKIIδ modulates intracellular Ca2+ release and proliferation in adult CFs and more specifically, that this may be modulated via physical interaction between CaMKIIδ and IP3R2.",
author = "{Mohd Salleh}, M and C McCluskey and T Bushell and S Currie",
year = "2014",
month = "12",
day = "31",
doi = "10.1136/heartjnl-2014-306916.48",
language = "English",
volume = "100",
journal = "Heart",
issn = "1355-6037",
number = "S4",

}

Calcium/calmodulin dependent protein kinase II δ modulates intracellular CA2+ release and cell proliferation in adult cardiac fibroblasts. / Mohd Salleh, M; McCluskey, C; Bushell, T; Currie, S.

In: Heart , Vol. 100, No. S4, 31.12.2014.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Calcium/calmodulin dependent protein kinase II δ modulates intracellular CA2+ release and cell proliferation in adult cardiac fibroblasts

AU - Mohd Salleh, M

AU - McCluskey, C

AU - Bushell, T

AU - Currie, S

PY - 2014/12/31

Y1 - 2014/12/31

N2 - Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5% (AIP) vs 123±6% (control), p<0.001, n=4 and 65±4% (KN-93) vs 123±6% (control) respectively, p<0.001, n=4). Both CaMKII inhibition and IP3R inhibition also inhibit CFs proliferation (∼10% and 30% inhibition, AIP and 2-APB treatments respectively). The possibility that Ca MKIIδ may modulate IP3R2 activity directly via protein-protein interaction has also been explored using co-immunoprecipitation. Collectively, this study reveals for the first time that CaMKIIδ modulates intracellular Ca2+ release and proliferation in adult CFs and more specifically, that this may be modulated via physical interaction between CaMKIIδ and IP3R2.

AB - Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5% (AIP) vs 123±6% (control), p<0.001, n=4 and 65±4% (KN-93) vs 123±6% (control) respectively, p<0.001, n=4). Both CaMKII inhibition and IP3R inhibition also inhibit CFs proliferation (∼10% and 30% inhibition, AIP and 2-APB treatments respectively). The possibility that Ca MKIIδ may modulate IP3R2 activity directly via protein-protein interaction has also been explored using co-immunoprecipitation. Collectively, this study reveals for the first time that CaMKIIδ modulates intracellular Ca2+ release and proliferation in adult CFs and more specifically, that this may be modulated via physical interaction between CaMKIIδ and IP3R2.

UR - http://heart.bmj.com/

U2 - 10.1136/heartjnl-2014-306916.48

DO - 10.1136/heartjnl-2014-306916.48

M3 - Meeting abstract

VL - 100

JO - Heart

JF - Heart

SN - 1355-6037

IS - S4

ER -