CARP Plays a Key Role in Cellular Growth Under Hypertrophic Stimuli in Neonatal Rat Ventricular Myocytes

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Ventricular Myocytes ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Dual Nature ◽  
Hypertrophic Growth ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes ◽  
Over Time

Cardiac hypertrophy is a growth process that occurs in response to stress stimuli or injury, and leads to the induction of several pathways to alter gene expression. Under hypertrophic stimuli, sarcomeric structure is disrupted, both as a consequence of gene expression and local changes in sarcomeric proteins. Cardiac-restricted ankyrin repeat protein (CARP) is one such protein that function both in cardiac sarcomeres and at the transcriptional level. We postulate that due to this dual nature, CARP plays a key role in maintaining the cardiac sarcomere. GATA4 is another protein detected in cardiomyocytes as important in hypertrophy, as it is activated by hypertrophic stimuli, and directly binds to DNA to alter gene expression. Results of GATA4 activation over time were inconclusive; however, the role of CARP in mediating hypertrophic growth in cardiomyocytes was clearly demonstrated. In this study, Neonatal Rat Ventricular Myocytes were used as a model to detect changes over time in CARP and GATA4 under hypertrophic stimulation by phenylephrine and high serum media. Results were detected by analysis of immunoblotting. The specific role that CARP plays in mediating cellular growth under hypertrophic stimuli was studied through immunofluorescence, which demonstrated that cardiomyocyte growth with hypertrophic stimulation was significantly blunted when NRVMs were co-treated with CARP siRNA. These data suggest that CARP plays an important role in the hypertrophic response in cardiomyocytes.

Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes

2003 ◽  
Vol 285 (1) ◽  
pp. C39-C47 ◽  
Author(s):  
Michael J. Porter ◽  
Maria C. Heidkamp ◽  
Brian T. Scully ◽  
Nehu Patel ◽  
Jody L. Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Rat Ventricular Myocytes ◽  
Kinase C ◽  
Neonatal Rat Ventricular Myocytes

Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKCα, PKCϵ, and PKCδ), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKCα, PKCϵ, and PKCδ to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKCϵ and wtPKCδ, but not wtPKCα, was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RT-PCR (69 ± 7 and 61 ± 9% of control levels for wtPKCϵ and wtPKCδ, respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKCδ > dnPKCϵ > dnPKCα). dnPKCδ overexpression produced the largest increase (2.8 ± 1.0-fold; n = 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKCϵ and PKCδ selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated.

scholarly journals AFos inhibits phenylephrine-mediated contractile dysfunction by altering phospholamban phosphorylation

2010 ◽  
Vol 298 (6) ◽  
pp. H1719-H1726 ◽  
Author(s):  
Mark Y. Jeong ◽  
John S. Walker ◽  
R. Dale Brown ◽  
Russell L. Moore ◽  
Charles S. Vinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Ventricular Myocytes ◽  
Calcium Atpase ◽  
Calcium Transients ◽  
Neonatal Rat ◽  
Gene Profile ◽  
Rat Ventricular Myocytes ◽  
Plasmic Reticulum ◽  
Neonatal Rat Ventricular Myocytes

Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing β-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.

scholarly journals Prostaglandin F2αStimulates Hypertrophic Growth of Cultured Neonatal Rat Ventricular Myocytes

1996 ◽  
Vol 271 (2) ◽  
pp. 1179-1186 ◽  
Author(s):  
John W. Adams ◽  
Darren S. Migita ◽  
Maggie K. Yu ◽  
Robert Young ◽  
Mark S. Hellickson ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Hypertrophic Growth ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

Aldosterone modulates If current through gene expression in cultured neonatal rat ventricular myocytes

2007 ◽  
Vol 293 (5) ◽  
pp. H2710-H2718 ◽  
Author(s):  
Takao Muto ◽  
Norihiro Ueda ◽  
Tobias Opthof ◽  
Tomoko Ohkusa ◽  
Kohzo Nagata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Ventricular Myocytes ◽  
Evaluation Study ◽  
Neonatal Rat ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Protein Levels ◽  
Channel Gene ◽  
Neonatal Rat Ventricular Myocytes

Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current ( If) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17–24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. If current elicited by hyperpolarization from −50 to −130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 μM eplerenone or 0.1 μM spironolactone) abolish the increase of If channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 μM aldosterone downregulated If channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of If current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates If channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.

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