Carbonic anhydrase II promotes cardiomyocyte hypertrophy

2012 ◽  
Vol 90 (12) ◽  
pp. 1599-1610 ◽  
Author(s):  
Brittany F. Brown ◽  
Anita Quon ◽  
Jason R.B. Dyck ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Ventricular Myocytes ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Carbonic Anhydrase Ii ◽  
Cardiomyocyte Hypertrophy ◽  
Wild Type ◽  
Over Expression ◽  
Pathological Cardiac Hypertrophy ◽  
Neonatal Rat Ventricular Myocytes

Pathological cardiac hypertrophy, the maladaptive remodelling of the myocardium, often progresses to heart failure. The sodium–proton exchanger (NHE1) and chloride–bicarbonate exchanger (AE3) have been implicated as important in the hypertrophic cascade. Carbonic anhydrase II (CAII) provides substrates for these transporters (protons and bicarbonate, respectively). CAII physically interacts with NHE1 and AE3, enhancing their respective ion transport activities by increasing the concentration of substrate at their transport sites. Earlier studies found that a broad-spectrum carbonic anhydrase inhibitor prevented cardiomyocyte hypertrophy (CH), suggesting that carbonic anhydrase is important in the development of hypertrophy. Here we investigated whether cytosolic CAII was the CA isoform involved in hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were transduced with recombinant adenoviral constructs to over-express wild-type or catalytically inactive CAII (CAII-V143Y). Over-expression of wild-type CAII in NRVMs did not affect CH development. In contrast, CAII-V143Y over-expression suppressed the response to hypertrophic stimuli, suggesting that CAII-V143Y behaves in a dominant negative fashion over endogenous CAII to suppress hypertrophy. We also examined CAII-deficient (Car2) mice, whose hearts exhibit physiological hypertrophy without any decrease in cardiac function. Moreover, cardiomyocytes from Car2 mice do not respond to prohypertrophic stimulation. Together, these findings support a role of CAII in promoting CH.

Outside-in Signalling of Fibronectin Stimulates Cardiomyocyte Hypertrophy in Cultured Neonatal Rat Ventricular Myocytes

2000 ◽  
Vol 32 (5) ◽  
pp. 765-776 ◽  
Author(s):  
Emiko Ogawa ◽  
Yoshihiko Saito ◽  
Masaki Harada ◽  
Shigeki Kamitani ◽  
Koichiro Kuwahara ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

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.

Regulation of the human NBC3 Na+/HCO3− cotransporter by carbonic anhydrase II and PKA

2004 ◽  
Vol 286 (6) ◽  
pp. C1423-C1433 ◽  
Author(s):  
Frederick B. Loiselle ◽  
Patricio E. Morgan ◽  
Bernardo V. Alvarez ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Recovery Rate ◽  
Dominant Negative ◽  
Carbonic Anhydrase Ii ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
Transfected Cells ◽  
293 Cells ◽  
Optimal Function

Human NBC3 is an electroneutral Na+/HCO3− cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3− metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O ⇆ HCO3− + H+ in many tissues. We investigated whether NBC3, like some Cl−/HCO3− exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with Kd = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 μM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [γ-32P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3− transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.

scholarly journals PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2)

2002 ◽  
Vol 156 (5) ◽  
pp. 905-919 ◽  
Author(s):  
Julian C. Braz ◽  
Orlando F. Bueno ◽  
Leon J. De Windt ◽  
Jeffery D. Molkentin
Keyword(s):  
Marker Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Leucine Incorporation ◽  
Wild Type ◽  
Rat Cardiomyocytes ◽  
Signal Transducers ◽  
Hypertrophic Growth ◽  
Extracellular Signal

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ε (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ε revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ε, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ε revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCε reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.

scholarly journals Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes

2008 ◽  
Vol 295 (2) ◽  
pp. H561-H568 ◽  
Author(s):  
Hongguang Wei ◽  
Richard S. Vander Heide
Keyword(s):  
Heat Stress ◽  
Focal Adhesion Kinase ◽  
Signaling Pathways ◽  
Focal Adhesion ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Wild Type ◽  
Rat Ventricular Myocytes ◽  
Myocardial Stress ◽  
Neonatal Rat Ventricular Myocytes

Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.

scholarly journals Role of protein kinase C-ε in hypertrophy of cultured neonatal rat ventricular myocytes

2001 ◽  
Vol 280 (2) ◽  
pp. H756-H766 ◽  
Author(s):  
James B. Strait ◽  
Jody L. Martin ◽  
Allison Bayer ◽  
Ruben Mestril ◽  
Diane M. Eble ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Area ◽  
Total Protein ◽  
Ventricular Myocytes ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Protein Accumulation ◽  
Cell Surface Area ◽  
Kinase C ◽  
Neonatal Rat Ventricular Myocytes

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-ε, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-ε did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-ε also increased atrial natriuretic factor (ANF) and β-myosin heavy chain (MHC) mRNA levels 2.5 ± 0.3- and 2.1 ± 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector ( P < 0.05 for both). Conversely, Adv-dnPKC-ε did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of β-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-ε overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-ε activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.

scholarly journals Lysosomal-Associated Protein Transmembrane 5 Functions as a Novel Negative Regulator of Pathological Cardiac Hypertrophy

2021 ◽  
Vol 8 ◽  
Author(s):  
Lu Gao ◽  
Sen Guo ◽  
Rui Long ◽  
Lili Xiao ◽  
Rui Yao ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Therapeutic Potential ◽  
Negative Regulator ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Pathological Cardiac Hypertrophy

Lysosomal-associated protein transmembrane 5 (LAPTM5) is mainly expressed in immune cells and has been reported to regulate inflammation, apoptosis and autophagy. Although LAPTM5 is expressed in the heart, whether LAPTM5 plays a role in regulating cardiac function remains unknown. Here, we show that the expression of LAPTM5 is dramatically decreased in murine hypertrophic hearts and isolated hypertrophic cardiomyocytes. In this study, we investigated the role of LAPTM5 in pathological cardiac hypertrophy and its possible mechanism. Our results show that LAPTM5 gene deletion significantly exacerbates cardiac remodeling, which can be demonstrated by reduced myocardial hypertrophy, fibrosis, ventricular dilation and preserved ejection function, whereas the opposite phenotype was observed in LAPTM5 overexpression mice. In line with the in vivo results, knockdown of LAPTM5 exaggerated angiotensin II-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes, whereas overexpression of LAPTM5 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, LAPTM5 directly bound to Rac1 and further inhibited MEK-ERK1/2 signaling, which ultimately regulated the development of cardiac hypertrophy. In addition, the antihypertrophic effect of LAPTM5 was largely blocked by constitutively active mutant Rac1 (G12V). In conclusion, our results suggest that LAPTM5 is involved in pathological cardiac hypertrophy and that targeting LAPTM5 has great therapeutic potential in the treatment of pathological cardiac hypertrophy.

scholarly journals NAP1L5 Promotes Nucleolar Hypertrophy and Is Required for Translation Activation During Cardiomyocyte Hypertrophy

2021 ◽  
Vol 8 ◽  
Author(s):  
Ningning Guo ◽  
Di Zheng ◽  
Jiaxin Sun ◽  
Jian Lv ◽  
Shun Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Nucleosome Assembly ◽  
Translation Control ◽  
Incorporation Assay ◽  
Necessary And Sufficient ◽  
Neonatal Rat Ventricular Myocytes ◽  
Upregulated Genes

Pathological growth of cardiomyocytes during hypertrophy is characterized by excess protein synthesis; however, the regulatory mechanism remains largely unknown. Using a neonatal rat ventricular myocytes (NRVMs) model, here we find that the expression of nucleosome assembly protein 1 like 5 (Nap1l5) is upregulated in phenylephrine (PE)-induced hypertrophy. Knockdown of Nap1l5 expression by siRNA significantly blocks cell size enlargement and pathological gene induction after PE treatment. In contrast, Adenovirus-mediated Nap1l5 overexpression significantly aggravates the pro-hypertrophic effects of PE on NRVMs. RNA-seq analysis reveals that Nap1l5 knockdown reverses the pro-hypertrophic transcriptome reprogramming after PE treatment. Whereas, immune response is dominantly enriched in the upregulated genes, oxidative phosphorylation, cardiac muscle contraction and ribosome-related pathways are remarkably enriched in the down-regulated genes. Although Nap1l5-mediated gene regulation is correlated with PRC2 and PRC1, Nap1l5 does not directly alter the levels of global histone methylations at K4, K9, K27 or K36. However, puromycin incorporation assay shows that Nap1l5 is both necessary and sufficient to promote protein synthesis in cardiomyocyte hypertrophy. This is attributable to a direct regulation of nucleolus hypertrophy and subsequent ribosome assembly. Our findings demonstrate a previously unrecognized role of Nap1l5 in translation control during cardiac hypertrophy.

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.

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