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

Porter, Michael; Heidkamp, Maria C.; Scully, Brian T.; Patel, Nilamkumar; Martin, J.L.; Samarel, Allen M.

doi:10.1152/ajpcell.00461.2002

Cited by 37 publications

(39 citation statements)

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“…Nevertheless, overexpression of PKCδ did not appear to alter MARCKS phosphorylation. Levels of overexpressed PKCε, PKCα, and PKCδ are also shown, and are consistent with the levels of PKC overexpression obtained in previous studies from our laboratory (21,27). Data summarizing the results of 4-5 experiments following caPKCε expression in NRVM and ARVM are depicted in Fig 2D. …”

Section: Et Treatment Increases Marcks Phosphorylation In Nrvm and Arvmsupporting

confidence: 89%

“…We have also reported cross-talk between PKCα and PKCε. Overexpression of dnPKCε, even at moderate levels, reduced endogenous PKCα levels in NRVM (21). Conversely, overexpression of dnPKCα decreased endogenous PKCε levels.…”

Section: Discussionmentioning

confidence: 89%

“…Replication-defective adenoviruses encoding caPKCε (Adv-caPKCε), caPKCδ (AdvcaPKCδ), wtPKCε (Adv-wtPKCε), wtPKCδ (Adv-wtPKCδ) and wtPKCα (Adv-wtPKCα) were constructed as previously described (10,20,21). Dominant-negative (dn) PKCε adenovirus was kindly provided by Dr. Peipei Ping, University of California-Los Angeles and constructed as previously described (22).…”

Section: Adenoviral Constructsmentioning

confidence: 99%

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Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

Heidkamp

Iyengar

Szotek

et al. 2007

Journal of Molecular and Cellular Cardiology

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The myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a cytoskeletal protein implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. However, its precise role in cardiomyocyte growth, and its PKC-dependent regulation have not been fully explored. In this report, we show that MARCKS is expressed and phosphorylated under basal conditions in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively). The PKC activators phenylephrine, angiotensin II, and endothelin-1 (ET) further increased MARCKS phosphorylation, with ET inducing the greatest response. To determine which PKC isoenzyme was responsible for agonist-induced MARCKS phosphorylation, NRVM and ARVM were infected with replication-defective adenoviruses (Adv) encoding wildtype (wt) and constitutively active (ca) mutants of PKCε, PKCδ, and PKCα. Only PKCε increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKCε reduced basal and ET-stimulated pMARCKS. dnPKCε overexpression also prevented ET-induced, apparent co-localization of pMARCKS with factin staining structures. Adv-mediated overexpression of GFP-tagged, wtMARCKS (wtMARCKS-GFP) increased phosphorylation of focal adhesion kinase (FAK) and also increased NRVM surface area. In constrast, overexpression of a GFP-tagged, non-phosphorylatable (np) MARCKS mutant (npMARCKS-GFP) decreased basal and ET-induced endogenous MARCKS and FAK phosphorylation, and blocked the ET-induced increase in NRVM surface area. We conclude that MARCKS is expressed in cardiomyocytes, is phosphorylated by PKCε, and participates in the regulation of FAK phosphorylation and cell spreading.

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Section: Et Treatment Increases Marcks Phosphorylation In Nrvm and Arvmsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 89%

Section: Adenoviral Constructsmentioning

confidence: 99%

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Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

Heidkamp

Iyengar

Szotek

et al. 2007

Journal of Molecular and Cellular Cardiology

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“…RNA was quantified by absorbance at 260 nm and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. SERCA2, ANF, αMHC, and βMHC mRNAs were then analyzed by real-time RT-PCR, as previously described (5,28). The mixture consisted of 1µL of sample cDNA, 21µL DEPC water, 25µL Platinum Quantitative PCR SuperMix-UDG, and 3µL of a primer/dual-labeled probe combination specific for each gene of interest (Table 1).…”

Section: Mrna Analysismentioning

confidence: 99%

“…All samples were run in triplicate, and the results were averaged. Data were analyzed as previously described (28).…”

Section: Mrna Analysismentioning

confidence: 99%

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

Hart¹,

Heidkamp²,

Iyengar³

et al. 2008

Journal of Molecular and Cellular Cardiology

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PYK2 is a Ca 2+ -dependent, nonreceptor protein tyrosine kinase that is involved in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. We and others have previously investigated PYK2's function in vitro using cultured neonatal and adult rat ventricular myocytes as model systems. However, the function of PYK2 in the in vivo adult heart remains unclear. Here we evaluate the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. First we demonstrate that CRNK functions as a dominant-negative inhibitor of PYK2-dependent signaling, presumably by displacing PYK2 from focal adhesions and costameres. Then, male Sprague-Dawley rats (~300g) underwent permanent left anterior descending coronary artery ligation. One wk post-MI, either Adv-GFP (n=34) or Adv-CRNK (n=28) was administered (10 10 pfu, 0.1ml) via catheter-based, Optison®-mediated gene transfer. LV structure and function were evaluated by echocardiography 1 and 3wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK overexpression was readily detected by Western blotting 1wk following gene transfer. Adv-CRNK improved overall survival (P=0.03; Logrank Test) and LV fractional shortening (23±2% vs. 31±2% for Adv-GFP vs. Adv-CRNK infected animals, respectively; P<0.05). Whereas MI hearts exhibited increased β-, and decreased α-myosin heavy chain (MHC) mRNA expression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3±1.4 fold increase in αMHC; 0.4±0.1 fold decrease in βMHC; P<0.05 for both). In summary, CRNK gene transfer improves survival, increases LV function, and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI.Proofs and Correspondences to:

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PKCε upregulates voltage‐dependent calcium channels in cultured astrocytes

Burgos

Pastor

Gonzalez

et al. 2007

Glia

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Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

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

Cited by 37 publications

References 50 publications

Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

PKCε upregulates voltage‐dependent calcium channels in cultured astrocytes

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