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Donald R Menick

Publications and source records attributed to Donald R Menick.

10 recordsLinked to original sources
PubMed2006

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation.

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.

Adenoviridae↗
PubMed2006

Mutations at leucine 215 of beta-tubulin affect paclitaxel sensitivity by two distinct mechanisms.

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.

Amino Acid Substitution↗
PubMed2005

The role of p38 in the regulation of Na+-Ca2+ exchanger expression in adult cardiomyocytes.

The Na(+)-Ca(2+) exchanger is crucial in the regulation of [Ca(2+)](i) in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy and failure. This upregulation can have an effect on calcium transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Here we use adenovirus mediated gene expression to examine the role of p38 MAP kinase in upregulation of the exchanger in adult cardiac myocytes. We demonstrate that p38 mediates a part of the alpha-adrenergic stimulated upregulation of the Na(+)-Ca(2+) exchanger gene. Overexpression of dominant-negative p38 isoforms and activated MKK3 and MKK6 in isolated adult cardiac myocytes demonstrates that p38 activation is sufficient for NCX1 promoter upregulation and that this is mediated primarily by the p38alpha isoform. Lastly, this work demonstrates that the p38alpha stimulated upregulation of the NCX1 promoter is mediated via the -80 CArG box element. This is the first time that a specific role for p38alpha in gene regulation has been demonstrated in isolated adult cardiomyocytes and provides an important clue to our understanding some of the factors regulating exchanger gene expression in the hypertrophic and failing heart.

Adenoviridae↗
PubMed2004

Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5'-untranslated region of mRNA.

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5'-UTR (5'-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either 'stronger' (less structure in the 5'-UTR) or 'weaker' (more structure in the 5'-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5'-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48+/-13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5'-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

5' Untranslated Regions↗
PubMed2003

Phenotypic consequences of beta1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes.

In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of beta1-tubulin rather than the constitutive beta4-tubulin and of microtubule-associated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of beta-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding beta1-tubulin, beta4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed beta1- or beta4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of beta1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total alpha- and beta-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes.

Adenoviridae↗
PubMed2002

Cardiac-specific expression and hypertrophic upregulation of the feline Na(+)-Ca(2+) exchanger gene H1-promoter in a transgenic mouse model.

The NCX1 gene contains three promoters (H1, K1, and Br1), and as a result of alternative promoter usage and alternative splicing, there are multiple tissue-specific variants of the Na(+)-Ca(2+) exchanger. We have proposed that for NCX1, the H1 promoter regulates expression in the heart, the K1 promoter regulates expression in the kidney, and the Br1 promoter regulates expression in the brain as well as low-level ubiquitous expression. Here, using a transgenic mouse model, we test the role of the DNA region including -1831 to 67 bp of intron 1, encompassing exon H1 of the feline NCX1 gene (NCX1H1). The NCX1H1 promoter was sufficient for driving the normal spatiotemporal pattern of NCX1 expression in cardiac development. The luciferase reporter gene was expressed in a heart-restricted pattern both in early embryos (embryonic days 8 to 14) and in later embryos (after embryonic day 14), when NCX1 is also expressed in other tissues. In the adult, no luciferase activity was detected in the kidney, liver, spleen, uterus, or skeletal muscle; minimal activity was detected in the brain; and very high levels of luciferase expression were detected in the heart. Transverse aortic constriction-operated mice showed significantly increased left ventricular mass after 7 days. In addition, there was a 2-fold upregulation of NCX1H1 promoter activity in the left ventricle in animals after 7 days of pressure overload compared with both control and sham-operated animals. This work demonstrates that the NCX1H1 promoter directs cardiac-specific expression of the exchanger in both the embryo and adult and is also sufficient for the upregulation of NCX1 in response to pressure overload.

Animals↗
PubMed2002

Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor.

Nkx2.5 and serum response factor (SRF) are critically important transcription factors in cardiac morphogenesis. They are also widely expressed in adult cardiomyocytes, but there is little data to indicate their possible role in adult cardiac cells. In this paper we demonstrate that the interaction of Nkx2.5 and SRF in cardiac-specific gene regulation is different between neonatal and adult cardiomyocytes. Our experimental model utilizes transient transfection and adenovirus mediated gene transfer of the proximal promoter fragment of the cardiac isoform of the sodium-calcium exchanger gene (NCX1). This promoter construct (NCX184) contains a single Nkx2.5-response element (NKE) and a single serum response element (CArG). In rat neonatal cardiomyocytes NCX184 activity is substantially induced with Nkx2.5 or SRF and additively with both. Mutagenesis of these NKE and CArG elements demonstrated the specificity of the interactions, which was confirmed with gel retardation analysis of cardiac ventricular tissue. In contrast, in adult cardiomyocytes, co-infection of Nkx2.5 and SRF adenovirus vectors showed Nkx2.5 induction but SRF did not have additive effects on NCX1 promoter regulation. As opposed to NCX1, the proximal atrial natriuretic factor (ANF) promoter was regulated identically in response to SRF and Nkx2.5 in both adult and neonatal cardiomyocytes. These results show that Nkx2.5-SRF interactions are capable of producing different transcriptional responses in adult versus neonatal cardiomyocytes, implying important differences in NCX1 promoter tertiary complex formation dependent on developmental stage.

Animals↗
PubMed2002

Pathways regulating Na+/Ca2+ exchanger expression in the heart.

The Na(+)/Ca(2+) exchanger (NCX1) is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. Following pressure overload, activation of MAPKs coincides with the kinetics of NCX1 gene upregulation in adult cardiocytes. Using adenoviral gene delivery, we begin to identify the molecular pathways responsible for upregulation of the exchanger gene. Inhibition of ERK with the MEK inhibitor UO126, the ERK protein phosphatase MKP-3, inhibited ERK activation, but only inhibited alpha-adrenergic-induced NCX1 upregulation by 30%. Overexpression of DN-JNK lowered basal NCX1 expression. Overexpression of activated MKK-3 was sufficient for alpha-adrenergic-stimulated upregulation of the reporter gene. Together, this data indicates that (1) JNK mediates basal cardiac expression of the NCX1 gene, (2) ERK and p38 play a role in alpha-adrenergic-stimulated NCX1 upregulation, and (3) p38 activation alone is sufficient for NCX1 upregulation.

5' Untranslated Regions↗
PubMed2002

Role of sodium-calcium exchanger (Ncx1) in embryonic heart development: a transgenic rescue?

Na(+)/Ca(2+) exchanger (Ncx-1) is highly expressed in cardiomyocytes, is thought to be required to maintain a low intracellular Ca(2+) concentration, and may play a role in excitation-contraction coupling. Significantly, targeted deletion of Ncx-1 results in Ncx1-null embryos that do not have a spontaneously beating heart and die in utero. Ultrastructural analysis revealed gross anomalies in the Ncx1-null contractile apparatus, but physiologic analysis showed normal field-stimulated Ca(2+) transients, suggesting that Ncx-1 function may not be critical for Ca(2+) extrusion from the cytosol as previously thought. Using caffeine to empty the intracellular Ca(2+) stores, we show that the sarcoplasmic reticulum is not fully functional within the 9.5-dpc mouse heart, indicating that the sarcoplasmic reticulum is unlikely to account for the unexpected maintenance of intracellular Ca(2+) homeostasis. Using the Ncx1-lacZ reporter, our data indicate restricted expression patterns of Ncx1 and that Ncx1 is highly expressed within the conduction system, suggesting Ncx1 may be required for spontaneous pacemaking activity. To test this hypothesis, we used transgenic mice overexpressing one of the two known adult Ncx1 isoforms under the control of the cardiac-specific a-myosin heavy chain promoter to restore Ncx1 expression within the Ncx1-null hearts. Results indicate that the transgenic re-expression of one Ncx1 isoform was unable to rescue the lethal null mutant phenotype. Furthermore, our in situ results indicate that both known adult Ncx1 isoforms are coexpressed within the embryonic heart, suggesting that effective transgenic rescue may require the presence of both isoforms within the developing heart.

Animals↗