scholarly journals The MEK/ERK Module Is Reprogrammed in Remodeling Adult Cardiomyocytes

2020 ◽  
Vol 21 (17) ◽  
pp. 6348 ◽  
Author(s):  
Thomas Kubin ◽  
Ayse Cetinkaya ◽  
Natalia Kubin ◽  
Peter Bramlage ◽  
Bedriye Sen-Hild ◽  
...  
Keyword(s):  
Heart Failure ◽  
Confocal Microscopy ◽  
Western Blot ◽  
Human Myocardium ◽  
Mapk Activation ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Resistance Loss ◽  
Fetal Reprogramming

Fetal and hypertrophic remodeling are hallmarks of cardiac restructuring leading chronically to heart failure. Since the Ras/Raf/MEK/ERK cascade (MAPK) is involved in the development of heart failure, we hypothesized, first, that fetal remodeling is different from hypertrophy and, second, that remodeling of the MAPK occurs. To test our hypothesis, we analyzed models of cultured adult rat cardiomyocytes as well as investigated myocytes in the failing human myocardium by western blot and confocal microscopy. Fetal remodeling was induced through endothelial morphogens and monitored by the reexpression of Acta2, Actn1, and Actb. Serum-induced hypertrophy was determined by increased surface size and protein content of cardiomyocytes. Serum and morphogens caused reprogramming of Ras/Raf/MEK/ERK. In both models H-Ras, N-Ras, Rap2, B- and C-Raf, MEK1/2 as well as ERK1/2 increased while K-Ras was downregulated. Atrophy, MAPK-dependent ischemic resistance, loss of A-Raf, and reexpression of Rap1 and Erk3 highlighted fetal remodeling, while A-Raf accumulation marked hypertrophy. The knock-down of B-Raf by siRNA reduced MAPK activation and fetal reprogramming. In conclusion, we demonstrate that fetal and hypertrophic remodeling are independent processes and involve reprogramming of the MAPK.

scholarly journals Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ε to RACK2 in adult rat and mouse

2009 ◽  
Vol 297 (2) ◽  
pp. H718-H725 ◽  
Author(s):  
Richard A. Fenton ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Lynne G. Shea ◽  
James G. Dobson
Keyword(s):  
Confocal Microscopy ◽  
Phospholipase C ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Scanning Confocal Microscopy ◽  
Adult Rat ◽  
Sds Page ◽  
T Tubules ◽  
Isolated Rat ◽  
Stimulation Of

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ε to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ε induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ε and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ε to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ε co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ε with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ε translocation and association with RACK2.

scholarly journals Forced expression of the cyclin B1–CDC2 complex induces proliferation in adult rat cardiomyocytes

2004 ◽  
Vol 382 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Katrina A. BICKNELL ◽  
Carmen H. COXON ◽  
Gavin BROOKS
Keyword(s):  
Cell Division ◽  
Cyclin B1 ◽  
Cell Division Cycle ◽  
Myocardial Regeneration ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Mammalian Myocardium

Repair of the mature mammalian myocardium following injury is impaired by the inability of the majority of cardiomyocytes to undergo cell division. We show that overexpression of the cyclin B1–CDC2 (cell division cycle 2 kinase) complex re-initiates cell division in adult cardiomyocytes. Thus strategies targeting the cyclin B1–CDC2 complex might re-initiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

Reduced expression of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury

2010 ◽  
Vol 298 (2) ◽  
pp. H360-H366 ◽  
Author(s):  
Thane G. Maddaford ◽  
Elena Dibrov ◽  
Cecilia Hurtado ◽  
Grant N. Pierce
Keyword(s):  
Gene Expression ◽  
Nucleotide Sequence ◽  
Contractile Activity ◽  
Ischemic Injury ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Adenoviral Delivery ◽  
Significant Depression ◽  
Short Time

The Na+/Ca2+ exchanger (NCX) is proposed to be an important protein in the regulation of Ca2+ movements in the heart. This Ca2+ regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca2+ overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to ∼60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca2+ homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca2+ and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

Adult rat cardiomyocytes exhibit capacitative calcium entry

2004 ◽  
Vol 286 (3) ◽  
pp. H1124-H1132 ◽  
Author(s):  
Dacia L. Hunton ◽  
LuYun Zou ◽  
Yi Pang ◽  
Richard B. Marchase
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Physiological Responses ◽  
Atpase Inhibitor ◽  
Capacitative Calcium Entry ◽  
Rat Cardiomyocytes ◽  
Inward Current ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Inositol 1,4,5 Trisphosphate

Capacitative Ca2+ entry (CCE) refers to the influx of Ca2+ through plasma membrane channels activated on depletion of endoplasmic-sarcoplasmic reticulum Ca2+ stores. We utilized two Ca2+-sensitive dyes (one monitoring cytoplasmic free Ca2+ and the other free Ca2+ within the sarcoplasmic reticulum) to determine whether adult rat ventricular myocytes exhibit CCE. Treatments with inhibitors of the sarcoplasmic endoplasmic reticulum Ca2+-ATPases were not efficient in releasing Ca2+ from stores. However, when these inhibitors were coupled with either Ca2+ ionophores or angiotensin II (an agonist generating inositol 1,4,5 trisphosphate), depletion of stores was observed. This depletion was accompanied by a significant influx of extracellular Ca2+ characteristic of CCE. CCE was also observed when stores were depleted with caffeine. This influx of Ca2+ was sensitive to four inhibitors of CCE (glucosamine, lanthanum, gadolinium, and SKF-96365) but not to inhibitors of L-type channels or the Na+/Ca2+ exchanger. In the whole cell configuration, an inward current of ∼0.7 pA/pF at –90 mV was activated when a Ca2+ chelator or inositol (1,4,5)-trisphosphate was included in the pipette or when Ca2+ stores were depleted with a Ca2+-ATPase inhibitor and ionophore. The current was maximal at hyperpolarizing voltages and inwardly rectified. The channel was relatively permeant to Ca2+ and Ba2+ but only poorly to Mg2+ or Mn2+. Taken together, these data support the existence of CCE in adult cardiomyocytes, a finding with likely implications to physiological responses to phospholipase C-generating agonists.

Uncoupling protein 2 modulates cell viability in adult rat cardiomyocytes

2007 ◽  
Vol 293 (1) ◽  
pp. H829-H835 ◽  
Author(s):  
Natalya Bodyak ◽  
Debra L. Rigor ◽  
Yee-Shiuan Chen ◽  
Yuchi Han ◽  
Egbert Bisping ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Myocytes ◽  
Rat Heart ◽  
Uncoupling Protein ◽  
Atp Depletion ◽  
Uncoupling Protein 2 ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Rat Cardiac Myocytes ◽  
Hypoxia Reoxygenation

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane proton carrier that uncouples ATP synthesis. The aim of this study was to determine whether UCP2 plays a role in survival of adult rat cardiac myocytes. We first studied the effects of UCP2 overexpression in vitro. Overexpression of UCP2 in primary cardiomyocytes led to a significant decline in ATP level and the development of acidosis but had no observable effect on cell survival. When cardiomyocytes were challenged with hypoxia-reoxygenation, cells overexpressing UCP2 survived significantly less compared with control. This finding was associated with upregulation of proapoptotic protein Bcl-2 and 19-kDa interacting protein 3 (BNIP3). Furthermore, UCP2 short interfering RNA prevented both the increase in cell death and BNIP3 expression. To examine the in vivo role of UCP2 in the heart, we used the Dahl salt-sensitive rat heart-failure model. Northern blot analysis revealed that UCP2 mRNA level was significantly upregulated in rat heart failure along with BNIP3 protein level. In conclusion, UCP2 increases sensitivity of adult rat cardiac myocytes to hypoxia-reoxygenation by way of ATP depletion and acidosis, which in turn causes accumulation of prodeath protein BNIP3.

Regulation of myocardial function by histidine-rich, calcium-binding protein

2004 ◽  
Vol 287 (4) ◽  
pp. H1705-H1711 ◽  
Author(s):  
Guo-Chang Fan ◽  
Kimberly N. Gregory ◽  
Wen Zhao ◽  
Woo Jin Park ◽  
Evangelia G. Kranias
Keyword(s):  
Heart Failure ◽  
Ryanodine Receptor ◽  
Calcium Binding ◽  
Recombinant Adenovirus ◽  
Contractile Function ◽  
Binding Protein ◽  
Human Heart Failure ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Protein Levels

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

2006 ◽  
Vol 290 (4) ◽  
pp. H1493-H1502 ◽  
Author(s):  
Rumi Maruyama ◽  
Genzou Takemura ◽  
Noritsugu Tohse ◽  
Tomoko Ohkusa ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Electron Microscopic Examination ◽  
Immunohistochemical Analysis ◽  
Calcium Transient ◽  
Cytoskeletal Proteins ◽  
Adrenergic Stimulation ◽  
Electron Microscopic ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Adult Cardiomyocytes ◽  
Intracellular Ca

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca2+ transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe β-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca2+ concentration and cell length. Trains of Ca2+ transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca2+ transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca2+ transients declined and diastolic intracellular Ca2+ concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca2+ channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca2+-ATPase (thapsigargin), and Na+/Ca2+ exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (α-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca2+ transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.

scholarly journals Oxidative stress and adenosine A1 receptor activation differentially modulate subcellular cardiomyocyte MAPKs

2008 ◽  
Vol 294 (1) ◽  
pp. H263-H271 ◽  
Author(s):  
Cherry Ballard-Croft ◽  
Adam C. Locklar ◽  
Byron J. Keith ◽  
Robert M. Mentzer ◽  
Robert D. Lasley
Keyword(s):  
Oxidative Stress ◽  
P38 Mapk ◽  
Membrane Fraction ◽  
Receptor Activation ◽  
Mapk Signaling ◽  
Scaffolding Protein ◽  
Mapk Activation ◽  
Rat Cardiomyocytes ◽  
Main Site ◽  
Adult Rat

The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine A1 receptor agonist 2-chloro- N6-cyclopentyladenosine (CCPA; 500 nM) or H2O2 (100 μM) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and H2O2 activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, H2O2 activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between H2O2 and CCPA was in the Triton-insoluble membrane fraction, where H2O2 increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 μM) reduced H2O2-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. H2O2 but not CCPA increased reactive oxygen species (ROS). Thus the adenosine A1 receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A1 receptor-mediated MAPK activation does not involve ROS formation.

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