Inositol 1,4,5-Trisphosphate Directs Ca2+Flow between Mitochondria and the Endoplasmic/Sarcoplasmic Reticulum: A Role in Regulating Cardiac Autonomic Ca2+ Spiking

The signaling role of the Ca2+ releaser inositol 1,4,5-trisphosphate (IP3) has been associated with diverse cell functions. Yet, the physiological significance of IP3 in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP3 generated by photolysis of caged IP3 or by purinergic activation of phospholipase Cγ slowed down or abolished autonomic Ca2+ spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cγ antibody prevented the IP3-mediated purinergic effect. IP3 triggered a ryanodine- and caffeine-insensitive Ca2+ release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca2+ and potential, IP3 induced transient Ca2+ loading and depolarization of the organelles. These mitochondrial changes were associated with Ca2+ depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca2+ spiking. Thus, IP3 acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP3 in excitable cells, the regulation of cardiac autonomic activity.

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Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+]i levels in neonatal rat cardiomyocytes

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(99)00115-8 ◽

1999 ◽

Vol 1440 (1) ◽

pp. 89-99 ◽

Cited By ~ 7

Author(s):

Pinggang Liu ◽

Yan-Jun Xu ◽

Rob L Hopfner ◽

Venkat Gopalakrishnan

Keyword(s):

Phosphatidic Acid ◽

Neonatal Rat ◽

Rat Cardiomyocytes ◽

Neonatal Rat Cardiomyocytes ◽

Inositol 1,4,5 Trisphosphate

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Role of Calcium-Activated Neutral Protease (Calpain) in Cell Death in Cultured Neonatal Rat Cardiomyocytes During Metabolic Inhibition

Circulation Research ◽

10.1161/01.res.76.6.1071 ◽

1995 ◽

Vol 76 (6) ◽

pp. 1071-1078 ◽

Cited By ~ 57

Author(s):

Douwe E. Atsma ◽

E.M. Lars Bastiaanse ◽

Anastazia Jerzewski ◽

Lizet J.M. Van der Valk ◽

Arnoud Van der Laarse

Keyword(s):

Cell Death ◽

Metabolic Inhibition ◽

Neonatal Rat ◽

Neutral Protease ◽

Rat Cardiomyocytes ◽

Neonatal Rat Cardiomyocytes

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Abstract 16383: Inhibition of Egfr Signalling Promotes Maladaptive Cardiac Remodelling in Hypertensive Heart Disease

Circulation ◽

10.1161/circ.142.suppl_3.16383 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Susanna Cooper ◽

Zoe Haines ◽

Viridiana Alcantara Alonso ◽

Joshua J Cull ◽

Feroz Ahmad ◽

...

Keyword(s):

Heart Failure ◽

Mrna Expression ◽

Left Ventricular ◽

Egfr Inhibitors ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Rat Cardiomyocytes ◽

Egfr Ligands ◽

Cardiac Adaptation

Introduction: Epidermal growth factor (EGF) receptors (EGFRs: ERBB1-4) are activated by a family of ligands (e.g. EGF, Hb-EGF, EREG, TGFa), signaling through ERK1/2 and Akt to promote cell division and cancer. Antibody-based inhibition of ERBB2 in breast cancer can cause heart failure, but the role of other receptors and EGFR ligands in the heart, and potential cardiotoxicity of generic EGFR inhibitors is unclear. Hypothesis: We hypothesize that EGFR ligands play an important role in cardiac adaptation to hypertension, acting through EGFRs to promote adaptive remodelling. Methods & Results: EGF ligand/receptor mRNA expression was assessed in human failing hearts and normal controls (n=12/8). EGFRs were expressed at similar levels, but ligand expression differed with significant up- or downregulation of EGF/Hb-EGF vs EREG/TGFa, respectively, in failing hearts (p<0.05). EGF potently activated ERK1/2 and Akt (assessed by immunoblotting) in neonatal rat cardiomyocytes, leading to hypertrophy (p<0.05, n=4). The anti-cancer drug afatinib inhibits EGFRs. To assess the role of EGF signaling in cardiac adaptation to hypertension in vivo , C57Bl/6J mice (n=6) were treated with 0.8 mg/kg/d angiotensin II (AngII; 7d) ± 0.45 mg/kg/d afatinib. AngII promoted cardiac hypertrophy with increased left ventricular (LV) wall thickness (WT) and decreased LV internal diameter (ID; assessed by echocardiography). Afatinib enhanced AngII-induced hypertrophy with significantly increased WT:ID ratios (1.30-fold and 1.54-fold in diastole and systole, respectively; p<0.05) but inhibited AngII-induced increases in Nppb mRNA expression and cardiomyocyte cross-sectional area (208.80±9.78 vs 161.10±3.87μm 2 ; p<0.05). In contrast, Col1a1 mRNA expression was enhanced by afatinib, along with interstitial and perivascular fibrosis (3.21±0.38 vs 5.61±0.46, 0.98±0.06 vs 1.45±0.18 % area; p<0.05). Conclusion: EGFR signaling is modulated in human heart failure, promotes cardiomyocyte hypertrophy and is required for cardiac adaptation to hypertension. Since EGFR inhibition in hypertension prevents adaptive cardiomyocyte hypertrophy whilst promoting fibrosis, EGFR inhibitors are likely to cause cardiac dysfunction and be cardiotoxic in hypertensive patients.

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Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00716.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. H570-H579 ◽

Cited By ~ 58

Author(s):

Chengqun Huang ◽

Wayne Liu ◽

Cynthia N. Perry ◽

Smadar Yitzhaki ◽

Youngil Lee ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Enhanced Recovery ◽

Ischemia Reperfusion ◽

Dominant Negative ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Kinase C ◽

Rat Hearts

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15 ). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R. Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

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Dominant-Negative Connexin43–EGFP Inhibits Calcium-Transient Synchronization of Primary Neonatal Rat Cardiomyocytes

Experimental Cell Research ◽

10.1006/excr.2001.5411 ◽

2002 ◽

Vol 273 (1) ◽

pp. 85-94 ◽

Cited By ~ 19

Author(s):

Yumiko Oyamada ◽

Wuxiong Zhou ◽

Hideto Oyamada ◽

Tetsuro Takamatsu ◽

Masahito Oyamada

Keyword(s):

Calcium Transient ◽

Dominant Negative ◽

Neonatal Rat ◽

Rat Cardiomyocytes ◽

Neonatal Rat Cardiomyocytes

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Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: Role of GRP78

Life Sciences ◽

10.1016/j.lfs.2007.08.015 ◽

2007 ◽

Vol 81 (13) ◽

pp. 1042-1049 ◽

Cited By ~ 21

Author(s):

Yan-Xia Pan ◽

An-Jing Ren ◽

Juan Zheng ◽

Wei-Fang Rong ◽

Hong Chen ◽

...

Keyword(s):

Hypoxic Preconditioning ◽

Neonatal Rat ◽

Rat Cardiomyocytes ◽

Neonatal Rat Cardiomyocytes

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Adenosine kinase regulation of cardiomyocyte hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00684.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. H1722-H1732 ◽

Cited By ~ 14

Author(s):

John T. Fassett ◽

Xinli Hu ◽

Xin Xu ◽

Zhongbing Lu ◽

Ping Zhang ◽

...

Keyword(s):

Protein Synthesis ◽

Cell Size ◽

Adenosine Receptors ◽

Adenosine Kinase ◽

Dominant Negative ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Rat Cardiomyocytes ◽

Mtorc1 Signaling ◽

Constitutively Active

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKTSer473 phosphorylation but did attenuate sustained phosphorylation of RafSer338 (24–48 h), mTORSer2448 (24–48 h), p70S6kThr389 (2.5–48 h), and ERKThr202/Tyr204 (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6kThr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERKThr202/Tyr204 and AKTSer473. Reduction of Raf-induced p70S6kThr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.

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Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.1.c77 ◽

1991 ◽

Vol 261 (1) ◽

pp. C77-C85 ◽

Cited By ~ 69

Author(s):

D. C. Kem ◽

E. I. Johnson ◽

A. M. Capponi ◽

D. Chardonnens ◽

U. Lang ◽

...

Keyword(s):

Angiotensin Ii ◽

Sarcoplasmic Reticulum ◽

Ventricular Myocytes ◽

Acute Effect ◽

Neonatal Rat ◽

Free Calcium ◽

Receptor Subtype ◽

Ang Ii ◽

Rat Cardiomyocytes ◽

Neonatal Rat Ventricular Myocytes

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.

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Abstract 49: Prolyl Isomerase Pin1 Regulates Cardiac Hypertrophy via Controlling Phosphorylation of Akt and MEK

Circulation Research ◽

10.1161/res.111.suppl_1.a49 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Haruhiro Toko ◽

Mathias Konstandin ◽

Natalie Gude ◽

Mark Sussman

Keyword(s):

Cardiac Hypertrophy ◽

Protein Expression ◽

High Serum ◽

Neonatal Rat ◽

Border Zone ◽

Prolyl Isomerase ◽

Rat Cardiomyocytes ◽

Growth And Survival ◽

Pin1 Protein

Rationale: Kinases and phosphatases regulate crucial aspects of growth and survival through phosphorylation and dephosphorylation of target substrates. Processes of cardiac hypertrophy, myocardial infarction, and heart failure are dictated in part by which kinases or phosphatases are involved and also by the intensity and duration of specific enzymatic activities. While research has identified numerous critical regulatory kinases and phosphatases in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the non-myocyte context, control of folding, activity, stability, and subcellular localization of proteins responsible for growth and survival is mediated by the prolyl isomerase Pin1. Objective: To establish the role of Pin1 in the heart. Method and Results: Initial characterization of myocardial Pin1 involved assessment of expression and localization during postnatal development or pathological challenge. Pin1 protein level was decreased and the location of Pin1 was changed from nucleus to cytoplasm with increasing age. Next, Pin1 protein expression and localization were assessed in the pathological challenged heart. Pin1 protein expression increases with pressure overload and ischemia, particularly in perivascular areas and in border zone myocytes, respectively. To determine the role of Pin1 on cardiac hypertrophy, siRNA to Pin1 (siPin1) was applied to neonatal rat cardiomyocytes. Western blot analysis showed that siPin1 decreased phosphorylation of Akt, and immunohistochemical analysis showed that siPin1 reduced cardiomyocyte size in response to high serum. siPin1 also decreased phosphorylation of MEK and reduced cardiomyocyte size in response to phenylephrine treatment. Furthermore, cardiac hypertrophy induced by transaortic constriction was ameliorated in Pin1 knockout mice, compared with littermate wild type mice. Conclusion: Expression and location of Pin1 during development and in response to pathologic challenge point to an important role for Pin1 in adaptation to myocardial growth or stress. Collective evidence indicates that Pin1 controls cardiac hypertrophy at least in part via regulating phosphorylation of Akt and MEK.

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