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.

scholarly journals Mammalian Orc1 Protein Is Selectively Released from Chromatin and Ubiquitinated during the S-to-M Transition in the Cell Division Cycle

2002 ◽  
Vol 22 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Cong-Jun Li ◽  
Melvin L. DePamphilis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Half Life ◽  
S Phase ◽  
Cell Division Cycle ◽  
26S Proteasome ◽  
Selective Dissociation

ABSTRACT Previous studies have shown that changes in the affinity of the hamster Orc1 protein for chromatin during the M-to-G1 transition correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites. Here we show that Orc1 is selectively released from chromatin as cells enter S phase, converted into a mono- or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G1 transition. Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination. In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination. Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase. Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs.

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.

scholarly journals Immunolocalization of ubiquitin conjugates at Z-bands and intercalated discs of rat cardiomyocytes in vitro and in vivo.

1992 ◽  
Vol 40 (7) ◽  
pp. 1037-1042 ◽  
Author(s):  
L L Hilenski ◽  
L Terracio ◽  
A L Haas ◽  
T K Borg
Keyword(s):  
Striated Muscle ◽  
Intercellular Junctions ◽  
Heart Tissue ◽  
Rat Cardiomyocytes ◽  
Macromolecular Assemblies ◽  
Adult Rat ◽  
Intercalated Discs ◽  
Ubiquitin Conjugation

Ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells, can be covalently bound to a wide variety of proteins in the nucleus, cytosol, cytoskeleton, and plasmalemma. This diversity of target proteins reflects a diversity of functions for ubiquitin conjugation. Previous studies have showed enhanced localization of ubiquitin conjugates to Z-bands of normal skeletal muscle and increased ubiquitination in atrophic muscles. These results have implicated a ubiquitin-mediated pathway in protein turnover and degradation in striated muscle. To investigate whether such a pathway might also exist in cardiac striated muscle, we used an affinity-purified polyclonal antibody (conjugate specific) and indirect immunofluorescence to localize ubiquitin conjugates in neonatal and adult rat cardiac myocytes both in vitro and in vivo. In both cultured myocytes and heart tissue, fluorescent ubiquitin conjugates were found in the nucleus as aggregates, in the cytoplasm in a striated pattern indicative of Z-bands, and in intercellular junctions at the intercalated discs between myocytes. Although the acceptor proteins and the physiological significance of ubiquitination at these locations are unknown, the targeting of ubiquitin to specific sites within the nucleus, myofibrils, and sarcolemma could provide a means for selective processing of individual components within these larger macromolecular assemblies, thus implying a regulatory role for ubiquitin conjugation in turnover or stability of proteins in the heart.

scholarly journals Identification and Verification of the Effect of miR-143 on the Cardioprotective Role of Phosphocreatine in Doxorubicin- Induced Cardiotoxicity by Integrated Bioinformatics Analysis

2021 ◽  
Author(s):  
Chi Zhou ◽  
Zi-Mo Zhou ◽  
Ling Hu ◽  
Ya-Yuan Yang ◽  
Xiang-Wen Meng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Mrna Translation ◽  
Rat Cardiomyocytes ◽  
Adult Rat

Abstract Purpose MicroRNAs (miRNAs) have been reported to play pivotal role in drugs-induced cardiotoxicity act as biomarkes, diagnostic tools and endogenous repressors of gene expression by lowering mRNA stability and interfering with mRNA translation. However, the effect of miRNAs on doxorubicin-induced cardiotoxicity still not clear. In the present study, we identified several key candidate miRNAs involving doxorubicin (DOX)-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes from the Gene Expression Omnibus (GEO) database via integrated bioinformatics analysis, and the possible effect of miR-143 in the protection of DOX-induced cardiotoxicity by phosphocreatine was subsequently investigated in vivo and in vitro. Methods GSE36239 miRNA expression profiles of DOX-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes (ARC) were extracted fromGEO datasets. |log2FC| > 1 and P < 0.05 were set as screening criteria, miRNAs expressed in myocardial tissues or ARC were selected as different expression miRNA (DEMs), and subsequently the key miRNAs were obtained from candidate DEMs between myocardial tissues and ARC with Venny 2.1 software. Target genes of miR-143 were predicted with Targetscan and miRBase in the species of homo sapiens, and candidate genes were obtained with Venny 2.1. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were carried out. Final, the expression and potential role of miR-143 were verified in DOX-induced cardiotoxicity of rat and cardiomyocytes H9c2. Results A total 24 DEMs were captured , including 15 up-regulated and 9 down-regulated genes in rat myocardial tissues and 42 DEMs were discovered, including 13 up-regulated and 29 down-regulated in ARC. Ultimately, 6 DEMs were determined in rat myocardial tissues and ARC by venny 2.1 software. 46 target genes of miR-143, one of the 6 DEMs, were captured from the predict results of Targetscan and miRBase with venny 2.1. The target genes were notably enriched in biological processes (BP) such as cell proliferation and migration. KEGG pathway analysis showed the target genes were enriched in HIF-1 and PI3K-Akt signaling pathway, which closely related to the oxidative stress and cardiomyocytes apoptosis. Further, western blot and RT-PCR results showed DOX-induced oxidative stress down-regulated the expression of miR-143 and Nrf2, SOD and BCL2, and up-regulated Bax and Cleaved caspase 3, while they could been reversed by the intervention of phosphocreatine (PCr) or N-acetyl-L-cystine (NAC) in DOX-induced cardiotoxicity in vivo and in vitro.Conclusion Our data showed that DOX-induced oxidative stress could decrease the expression of miR-143, promote apoptosis of cardiomyocytes, while PCr or NAC mediated antioxidation could reverse the expression down-regulation of miR-143, alleviated apoptosis in DOX-induced cardiotoxicity. Our findings elucidated the regulatory network involving miR-143 in DOX-induced cardiotoxicity, and might unveiled a potential biomarker and molecular mechanisms, which could be helpful to the diagnosis and treatment of DOX-induced cardiotoxicity.

In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy

2004 ◽  
Vol 286 (1) ◽  
pp. H68-H75 ◽  
Author(s):  
Jorge Suarez ◽  
Darrell D. Belke ◽  
Bernd Gloss ◽  
Thomas Dieterle ◽  
Patrick M. McDonough ◽  
...  
Keyword(s):  
Diabetic Cardiomyopathy ◽  
Cardiac Myocyte ◽  
Viral Vector ◽  
Contractile Function ◽  
Cardiac Contractility ◽  
Rat Cardiomyocytes ◽  
Adenoviral Gene Transfer ◽  
Adult Rat ◽  
Adenoviral Transfer

In many types of heart failure cardiac myocyte Ca2+ handling is abnormal because of downregulation of key Ca2+-handling proteins like sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca2+ transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular Ca2+ release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the Ca2+ transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy.

scholarly journals In Vivo Robustness Analysis of Cell Division Cycle Genes in Saccharomyces cerevisiae

PLoS Genetics ◽  
2006 ◽  
Vol 2 (7) ◽  
pp. e111 ◽  
Author(s):  
Hisao Moriya ◽  
Yuki Shimizu-Yoshida ◽  
Hiroaki Kitano
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Robustness Analysis ◽  
Cell Division Cycle

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.

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