Biomedical subjects

K R Boheler

Publications and source records attributed to K R Boheler.

At least 37 records · Page 2Linked to original sources

PubMed1996

Molecular cloning and analysis of the human cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) gene promoter.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in regulating Ca2+ movements in myocardium. In cardiac hypertrophy and human heart failure, the decrease in mRNA and protein levels of SERCA2 might account for the reduced diastolic Ca2+ re-uptake seen in these conditions. To investigate the regulation of human SERCA2 gene expression, an 18.6-kb human genomic clone that contains exons 1,2 and 3 of the SERCA2 gene has been isolated, and 13 kb of 5' upstream flanking sequence of which the proximal 2.5 kb of the promoter have been sequenced. Similar to the rabbit gene, the human SERCA2 promoter possesses a TATA-like box (-25 bp), a CAAT-box (-78 bp) and a number of consensus cis-regulatory elements including three Sp1 sites, a CACCC-box, and an OTF-1 binding sequence. No CArG box (present in the rabbit SERCA2 promoter) was identified in the human proximal promoter. Two putative thyroid response elements (TRE) are also present, suggesting that the human SERCA2 gene is also regulated by thyroid hormone as are the rat and rabbit genes. To study transcriptional activity of the human SERCA2 promoter in vitro, luciferase reporter plasmids containing a series of 5' deleted promoter constructs from -2577 bp to +170 bp were transfected into neonatal rat cardiomyocytes and C2C12 myotubes. The results suggest that: (a) the sequences from the transcription start site to -263 bp are necessary to obtain maximal transcriptional activity; (b) sequences from the transcription start site to -125 bp are essential for basal transcriptional activity; (c) at least one positive regulatory element is located between -263 bp and -125 bp; and (d) at least one negative regulatory element is present between -1741 bp and -412 bp.

Animals↗

PubMed1995

Clenbuterol induces hypertrophy of the latissimus dorsi muscle and heart in the rat with molecular and phenotypic changes.

BACKGROUND: Skeletal muscle assistance of the circulation for patients in end-stage heart failure requires electrical training of the latissimus dorsi flap to produce fatigue resistance. This process of electrical transformation and the development of postmobilization atrophy results in a profound loss in peak power generated. The beta 2-adrenoceptor agonist clenbuterol was used to investigate its potential to selectively induce skeletal muscle hypertrophy, particularly the latissimus dorsi muscle (LDM), independent of adverse effects on cardiac muscle. METHODS AND RESULTS: Forty-one male Sprague-Dawley rats were divided into four groups and used in this study. Clenbuterol 2 micrograms.g body wt-1.d-1 was administered subcutaneously for a period of either 5 weeks (group A) or 2 weeks (group A1). Groups B and B1 (controls) were injected with 0.5 mL normal saline once daily. At the end of the experimental period, all rats were weighed and terminally anesthetized for removal of the left LDM, left gastrocnemius-plantaris-soleus (GPS) muscles, and heart. The results showed that the increase in body weight did not differ significantly between the clenbuterol-treated and control groups (P > .5). The ratio of LDM to tibial length (hypertrophic index) for groups A and A1 was significantly greater than controls (P < .01), which represented a 20% to 29% increase. The hypertrophy was more pronounced for hindlimb skeletal muscle (21% to 35% for GPS), and the effects of this relatively high dose of clenbuterol on the heart were less marked (18% to 20% hypertrophy). RNA analyses indicate that ventricles of clenbuterol-treated rats express elevated levels of mRNA to atrial natriuretic factor without a concomitant increase in skeletal alpha-actin and beta-myosin heavy chain, consistent with a "physiological" form of cardiac hypertrophy. CONCLUSIONS: Clenbuterol induces significant hypertrophy of the LDM associated with specific changes in cardiac gene expression.

Adipose Tissue↗

PubMed1995

Characterization of a novel transcript of retroviral origin expressed in rat heart and liver.

Eleven cDNA clones of retroviral origin (SORO) have been isolated and characterized from a rat cardiac cDNA library that are highly homologous to the RAL-element family of rat retroviral endogenous sequences. SORO-1, the largest of the clones isolated at approximately 3.5 kb, contains a characteristic long terminal repeat (LTR) that is unique to previously identified retroposons. Its LTR contains elements homologous to the CAAT box, a TATA box, a core enhancer sequence, and potential binding sites for GATA. SORO-1 hybridizes to a mRNA of approximately 7 kb, that is present both in rat heart and liver. This transcript was not, however, detected in other tissues (fast and slow skeletal muscles, brain, kidney, testis, lung, intestine, spleen) or from any other species (man, monkey, mouse, dog, rabbit, chicken, cow, yeast) examined. During postnatal cardiac development, the expression of the transcript appears to be down-regulated and is present at levels 10-fold greater in neonates than in adults. The function of this retroposon that is expressed in rat heart and liver has not yet been determined, but the sequence analysis of its LTR and its expression pattern suggest that it may be regulated by specific trans-acting factors that are present in both rat heart and liver.

Animals↗

PubMed1995

Molecular aspects of myocardial differentiation.

The embryonic heart can pump blood in a single direction without one-way valves. With the development of molecular cell markers specific for contraction and relaxation, functional aspects of myocardial differentiation have been addressed through the use of in situ hybridization. In this study, we report how expression of the cardiac sarcoplasmic reticulum calcium-adenosine triphosphatase (SERCA2) and phospholamban (PLB) in the rat may partly explain why the embryonic atrium and ventricle function essentially as they do in the adult. SERCA2 is expressed in a craniocaudal gradient from as early as 10 embryonic days (ED) of development. PLB is first expressed at 12 ED but in a gradient essentially opposite to that seen for SERCA2. This spatial pattern of expression is maintained throughout much of fetal development. The spatial distribution of skeletal alpha-actin in the developing human heart indicates that alpha-actin isoform gradients or switching are not important in the establishment of unidirectional blood flow in the absence of valves, but it may serve as a marker for cardiac maturation.

Actins↗

PubMed1995

Patterns of expression of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban mRNAs during rat heart development.

This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.

Adenosine Triphosphatases↗

PubMed1993

The molecular biology of heart failure.

In the mammalian heart, the development of cardiac hypertrophy is a common feature that normally precedes all forms of heart failure. This adaptive process involves molecular changes in the myocardium, including the altered expression of several genes encoding proteins for contraction and relaxation. The expression of myosin heavy chain (MHC) and sarcomeric alpha-actin messenger ribonucleic acid (mRNA) changes qualitatively during cardiac hypertrophy; however, their accumulations are not coordinated. Skeletal alpha-actin transcripts accumulate throughout the ventricles and earlier than beta-MHC transcripts, which accumulate primarily around large coronary vessels. Skeletal alpha-actin transcripts also "hyperaccumulate" relative to cardiac alpha-actin mRNA, whose expression does not change. Expression of MHC isomRNA shows an inverse relation; as beta-MHC accumulates, alpha-MHC decreases in abundance. From nuclear run-on assays, we present evidence that the accumulation of these gene products is at least under partial transcriptional control with developmental growth, suggesting that those changes that occur with hypertrophy and heart failure may be primarily transcriptionally regulated. The expression of the mRNA for the calcium-adenosine triphosphate (Ca(2+)-ATPase) of the sarcoplasmic reticulum changes quantitatively with cardiac hypertrophy without the reexpression of a different isoform. The relative mRNA and protein concentrations for this protein diminish with both cardiac hypertrophy and heart failure, a change that may partially explain the delayed relaxation rates seen in hypertrophied and failing hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Isoprenaline stimulates gene transcription of the inhibitory G protein alpha-subunit Gi alpha-2 in rat heart.

In vitro transcription reactions were performed with isolated ventricular nuclei of adult rats to investigate whether increased mRNA levels of the inhibitory G protein alpha-subunit Gi alpha-2 after prolonged in vivo stimulation with the beta-adrenoceptor agonist isoprenaline are caused by increased transcription. Rats were treated by a 4-day subcutaneous infusion of isoprenaline (2.4 mg/kg per day) or 0.9% NaCl as control. To avoid the influence of developmental expression patterns, adult rats were chosen for all experiments. Signals for Gi alpha-2 and the stimulatory G protein alpha-subunit Gs alpha were specific and due to hybridization of nascent mRNA transcripts. In the isoprenaline group the transcriptional activity of Gi alpha-2 gene increased to 140% of the control value, whereas gene specific hybridization for Gs alpha remained unchanged. These results show that increased Gi alpha-2 mRNA levels after stimulation with isoprenaline are at least partially caused by enhanced transcription of Gi alpha-2 mRNA.

Animals↗

PubMed1993

[Left ventricular hypertrophy: molecular aspects].

The expression of genes changes during left ventricular hypertrophy. These changes are known as mechanogenic transduction. With respect to the two main contractile proteins, myosin and actin, this is due to a differential expression of their respective multigenic families. Changes of isoform are uncoordinated in time and space. It is important to recognise that the genes themselves are normal and that it is their regulation which is disturbed. Recent data shows that, under normal conditions, the regulation occurs at the transcription stage. A fuller understanding of these mechanisms is necessary if we are to comprehend the pathogenesis of cardiac hypertrophy and failure.

Actins↗

PubMed1992

[Molecular bases of cardiac aging].

The total volume occupied by myocytes in the heart decreases with age, and this is accompanied by a loss of myocytes, by hypertrophy and, to a lesser extent, by hyperplasia of the remaining myocytes. There are differences between ventricles: the right ventricular myocytes have a greater capacity for replication than those of the left ventricle. The expression of genes coding for the 2 principal contractile proteins (myosin and actin) is modified, and there are strong resemblances between the phenotype of the aged heart and that of an adult heart with haemodynamic overload.

Actins↗

PubMed1992

Cardiac expressions of alpha- and beta-myosin heavy chains and sarcomeric alpha-actins are regulated through transcriptional mechanisms. Results from nuclear run-on assays in isolated rat cardiac nuclei.

In the heart, mRNA accumulations for sarcomeric actins and myosin heavy chains (MHC) are subject to diverse regulatorial processes. To study cardiac contractile protein transcriptional regulations, an in vitro transcription system using nonenzymatically isolated rat cardiac nuclei was characterized. Transcription was shown to be rapid and continuous during the first 20 min of incubation and 5.4-fold less than that seen from comparably isolated hepatocyte nuclei. Neither RNase nor DNase activities were detectable. Direct transcriptional analyses of the alpha- and beta-MHC and cardiac and skeletal alpha-actin genes from cardiac nuclei were performed. In 23-24-day-old rats, significant levels of transcription were seen for alpha-MHC and for the sarcomeric alpha-actins. beta-MHC was just detectable, and no positive signals were ever seen for fibronectin. We then compared the perecentages of MHC and sarcomeric alpha-actin expressions determined from 1) the transcriptional assays and 2) total isolated RNA (alpha-MHC: 90.1 +/- 4.8% (transcription), 93.0 +/- 4.7% (accumulation); beta-MHC: 9.9 +/- 4.8%, 7.0 +/- 4.7%; cardiac alpha-actin: 84.0 +/- 2.5%, 84.9 +/- 2.5%; skeletal alpha-actin: 16.1 +/- 2.5%, 15.0 +/- 2.5%). The results support the conclusion that the primary mechanisms controlling the accumulations of these gene products are transcriptional. Additionally, we show that an anti-sense mRNA showing strong homology or identity with the 5' end of the beta-MHC gene is transcribed in cardiac nuclei but not in hepatocyte nuclei.

Actins↗

PubMed1992

Contractile proteins and sarcoplasmic reticulum calcium-ATPase gene expression in the hypertrophied and failing heart.

The physiology of myocardial contractility has been studied for over a century, but only recently has molecular biology provided new insights into the mechanisms responsible for the alterations of contraction and relaxation observed during cardiac hypertrophy and heart failure. Pressure and volume overload produce in the myocyte both qualitative changes characterized by protein isoform switches and quantitative changes characterized by modulation of single genes through a mechanogenic transduction the pathways of which are largely unknown. The qualitative changes involve differential expression of multigene families of contractile proteins, especially myosin heavy chain (MHC) and actin. All situations of pressure overload, or of combined pressure and volume overload activate the beta-MHC gene and deactivate the alpha-MHC one, which leads to a slower, more efficient contraction. In rat, pressure overload transitorily activates the alpha-skeletal actin gene, and both the timing and the distribution of the newly formed beta-MHC and alpha-skeletal actin mRNAs differ. We recently found that the isoactin pattern is the same in patients with end-stage heart failure as that of control human hearts. Moreover, both in rat and human, expression of isomyosins and isoactins are not coordinated, neither during ontogeny nor senescence. All this suggests the existence of several regulatory mechanisms activated during normal cardiac growth or by a mechanical trigger, and preliminary results indicate that it is possible to perform nuclear run-on assays in order to analyze the transcriptional step of these isogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed1992

Switches in cardiac muscle gene expression as a result of pressure and volume overload.

In the mammalian heart, the expression of genes encoding proteins responsible for contraction, relaxation, and endocrine function changes in hypertrophy resulting from hemodynamic overload. Different mechanisms are involved in this mechanogenic transduction, including 1) differential expression of myosin and actin multigene families, which may account for the decreased velocity of contractile element shortening in hypertrophied heart, 2) nonactivation of the sarcoplasmic reticulum Ca(2+)-ATPase gene, which may explain the increased duration of isometric relaxation, and finally 3) activation in the ventricle of the atrial natriuretic factor gene that is responsible in part for the high plasma levels of this peptide. It is increasingly apparent that these changes are independently regulated, but little is known about the mechanisms underlying this regulation. Preliminary results indicate that it is now possible to analyze the early time course or transcription for each gene after the imposition of hemodynamic overload. This should significantly enhance our understanding of the regulatory mechanisms involved in the phenoconversions of the hemodynamically overloaded heart.

Actins↗

PubMed1992

Expression of the sarcomeric actin isogenes in the rat heart with development and senescence.

Sarcomeric actin genes, alpha-cardiac and alpha-skeletal, are coexpressed in neonatal rodent hearts and are regulated in response to hormonal and hemodynamic stimuli; however, their precise developmental pattern of expression has not been determined, and it is unknown whether they are coexpressed during senescence. We have, therefore, investigated the accumulation of sarcomeric actin transcripts in rat heart during fetal and postnatal development and with senescence by two different techniques: primer extension analysis with an oligonucleotide common to both sarcomeric actins and RNA hybridization with specific cardiac alpha-actin cRNA probes. We found that at 17-19 days in utero both isogenes are coexpressed and alpha-skeletal actin mRNAs represent 28.0 +/- 0.8% of the sarcomeric actin mRNA total. Skeletal actin mRNAs increase to 40% of the total 1 week after birth (NS, p = 0.15), remain constant for 3 weeks, and decrease to less than 20% of the total in ventricles and atria of 1-month-old rats. The alpha-skeletal actin transcripts further decline to less than 5% of the total at 2 months of age and do not reaccumulate in senescent animals. There was no significant difference between male and female rat ventricles. By comparison with the known accumulations of alpha- and beta-myosin heavy chain mRNAs, our results demonstrate that whatever the developmental stage the kinetics of expression for the sarcomeric myosin and actin multigene families are independent.

Actins↗

PubMed1992

Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy.

Expression of myosin heavy chain (MHC) and actin multigene families changes in mammals during cardiac growth and hypertrophy, but whether or not there is a common regulatory pathway is unclear. To address this question, we have looked at the alpha- and beta-MHC, and at the alpha-skeletal and alpha-cardiac actin (alpha-skel act and alpha-card act) isomRNA transitions during development and senescence, both in rat and human hearts. Since the precise amounts of each isoactin mRNA were not precisely known in the above situations, we first analyzed the time- course of accumulations of the two sarcomeric transcripts by primer extension assays, which allow an umambiguous quantification of the ratios of the two actin transcripts. In rats, both isogenes are expressed in-utero. alpha-skel act represents 40% of the total one week after birth, remains constant for 3 weeks, decreases to less than 5% at two months and does not re-accumulate thereafter. In humans, in contrast, alpha-skel act represents < 20% in-utero and in neonates, increases to 48% during the first decade after birth and becomes the predominant isoform of adult hearts. In rats beta-MHC mRNAs accumulate at birth, become undetectable at 3 weeks and reaccumulate to as much as 80% during senescence, and in humans beta-MHC mRNAs predominate throughout all developmental stages. These data show that in both species, the multigene families encoding the major contractile proteins are not coordinately regulated during development and aging.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1991

Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts.

Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.

Actins↗

PubMed1991

[Improvement of an in vitro transcription method in nuclei isolated from heart myocytes from control and hemodynamically overloaded rats].

The expression of genes crucial for different cardiac functions changes during ontogenic development and during hypertrophy due to hemodynamic overload. To determine if these changes are transcriptional or post-transcriptional, we describe here an optimized method to analyze in vitro the transcriptional activity of nuclei isolated from control and hemodynamically overloaded hearts of 23-day-old rats. Similar results were obtained with both types of hearts: (1) 3 to 6 million nuclei were isolated per gram of ventricular tissue; (2) nuclear incorporation of UTP32P increases with time, plateaus after about 10 to 20 min. and attains an average of 0.17 cpm per nucleus; (3) alpha-amanitin decreases total incorporation by 56%; (4) the sizes of the majority of transcribed RNA are between 0.2 and 4 kb, although a significant fraction of high molecular weight transcripts are also seen (between 4 and 6 kb). All this indicates that mRNAs are transcribed to a significant extent, suggesting that it is now possible, given appropriate nucleotide probes, to analyze the transcription of any given gene during cardiac growth.

Animals↗

PubMed1990

Altered sarcoplasmic reticulum Ca2(+)-ATPase gene expression in the human ventricle during end-stage heart failure.

A decrease in the myocardial level of the mRNA encoding the Ca2(+)-ATPase of the sarcoplasmic reticulum (SR) has been recently reported during experimental cardiac hypertrophy and failure. To determine if such a deficit occurs in human end-stage heart failure, we compared the SR Ca2(+)-ATPase mRNA levels in left (LV) and right ventricular (RV) specimens from 13 patients undergoing cardiac transplantation (6 idiopathic dilated cardiomyopathies; 4 coronary artery diseases with myocardial infarctions; 3 diverse etiologies) with control heart samples using a rat cardiac SR Ca2(+)-ATPase cDNA probe. We observed a marked decrease in the mRNA for the Ca2(+)-ATPase relative to both the 18S ribosomal RNA and the myosin heavy chain mRNA in LV specimens of patients with heart failure compared to controls (-48%, P less than 0.01 and -47%, P less than 0.05, respectively). The LV ratio of Ca2(+)-ATPase mRNA to 18S RNA positively correlated with cardiac index (P less than 0.02). The RV ratio correlated negatively with systolic, diastolic and mean pulmonary arterial pressures (P less than 0.02, P less than 0.02, and P less than 0.01, respectively). We suggest that a decrease of the SR Ca2(+)-ATPase mRNA in the myocardium plays an important role in alterations of Ca2+ movements and myocardial relaxation reported during human end-stage heart failure.

Adult↗

PubMed1989

Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA.

Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end.

Amino Acid Sequence↗