Biomedical subjects

E Loukianov

Publications and source records attributed to E Loukianov.

10 recordsLinked to original sources

PubMed1999

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates.

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.

Animals↗

PubMed1999

Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene.

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Animals↗

PubMed1999

SERCA1a can functionally substitute for SERCA2a in the heart.

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an approximately 2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889-897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased ( approximately 1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

Adenosine Triphosphate↗

PubMed1998

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

Animals↗

PubMed1997

Expression of mRNA for a short form of heparin-binding EGF-like growth factor.

In this paper we report the cloning and characterization of cDNA encoding a novel, short form of heparin-binding EGF-like growth factor (SF HB-EGF), and show expression of specific mRNA in various tissues and cell types. Our data suggest that SF HB-EGF mRNA is a product of alternative splicing. Like normal HB-EGF, SF HB-EGF contains the signal peptide, the propeptide, the heparin-binding domain and the first two conservative disulfide loops of the EGF unit. Instead of the third disulfide loop, the spacer, the transmembrane and the cytoplasmic domains, SF HB-EGF has a nine amino acid tail.

Amino Acid Sequence↗

PubMed1997

Myosin heavy chain isoforms in smooth muscle.

In recent years, significant progress has been made toward understanding smooth muscle myosin heavy chain (SMHC) structure. Molecular cloning analysis has identified four different MHC isoforms. They are products of a single gene and result from alternative mRNA splicing. In addition, two non-muscle MHC isoforms are also expressed in smooth muscle cells. Studies show that SMHC expression is highly tissue specific and does not appear in cardiac or skeletal muscle cells. Each smooth muscle tissue is characterized by a specific pattern of MHC isoform expression that changes during development and disease. This review essentially focuses on SMHC isoforms and their expression in mammals.

Alternative Splicing↗

PubMed1997

Cloning DNA fragments between two adjacent/overlapping restriction sites using a "positive stuffer".

Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site. The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin. This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.

Binding Sites↗

PubMed1997

Efficient cloning method that selects the recombinant clones.

Directional cloning using cohesive ends is the most efficient cloning method. However, sometimes it is necessary to use blunt ends to clone a DNA fragment into the plasmid vector. Compared with that of cohesive ends, efficiency of blunt-end ligation is low. Compared with the native blunt ends (e.g., SmaI or EcoRV), blunt-end ligation is particularly difficult when blunt ends are derived from overhangs. This results in low efficiency of insertion and high background from self-ligation of the vector. To remedy the problem, we developed a "positive selector" cloning strategy that provides positive selection for the recombinant clones. It is particularly useful when making complex recombinant constructs and the choice of restriction sites is limited.

Cloning, Molecular↗

PubMed1994

Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene.

Despite the importance of smooth muscle cell proliferation in vascular pathophysiological states, the mechanisms regulating smooth muscle cell growth and differentiation are poorly understood. Previous studies have shown that adult rabbit smooth muscles express two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are generated through alternative RNA splicing from a single smooth muscle MHC (SMHC) gene. In the present study, we isolated and characterized the rabbit SMHC gene promoter. DNA sequence analysis of the upstream region of the SMHC gene revealed several putative cis-DNA regulatory elements proximal to the transcription start site. Most notably, cis-acting regulatory elements that closely resemble CC(A/T)6GG (CArG box) and myocyte enhancer binding factor 2 (MEF-2)-type sequence motifs were found in the SMHC 5'-flanking region. In addition, six E-box motifs were found in the 5'-flanking region of the SMHC gene between -374 and -2109 base pairs from the transcription start site. A series of transient transfection assays using SMHC promoter deletion constructs indicated that a promoter fragment extending to 2266 base pairs upstream of the transcription start site has the highest reporter activity in cultured rat aortic smooth muscle cells. Gel mobility shift analyses using the MEF-2-like sequence located at -1540 revealed a specific DNA protein complex, whereas the CArG-like element located at -1275 did not show protein binding. The SMHC promoter construct, p509-CAT, which included neither the CArG- nor MEF-2-type motifs, conferred 32% of chloramphenicol acetyltransferase activity in the same cells, whereas the construct p188-CAT, which contained the minimal promoter elements (TATA box), was significantly less active (7%; 2.0-fold over background). This is the first report describing the promoter elements of a gene whose expression is restricted to smooth muscle cells.

Alternative Splicing↗

PubMedDate not supplied

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts.

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.

Animals↗