Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.     

Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element. These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.     

The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro

Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.     

Profiling of cAMP and cGMP phosphodiesterases in isolated ventricular cardiomyocytes from human hearts: comparison with rat and guinea pig

AimsPhosphodiesterases (PDEs) are key enzymes controlling cAMP and cGMP levels and spatial distribution within cardiomyocytes. Despite the clinical importance of several classes of PDE inhibitor there has not been a complete characterization of the PDE profile within the human cardiomyocyte, and no attempt to assess which species might best be used to model this for drug evaluation in heart disease.Main methodsVentricular cardiomyocytes were isolated from failing human hearts of patients with various etiologies of disease, and from rat and guinea pig hearts. Expression of PDE isoforms was determined using RT-PCR. cAMP- and cGMP-PDE hydrolytic activity was determined by scintillation proximity assay, before and after treatment with PDE inhibitors for PDEs 1, 2, 3, 4, 5 and 7. Functional effects of cAMP PDEi were determined on the contraction of single human, rat and guinea pig cardiomyocytes.Key findingsThe presence and activity of PDE5 were confirmed in ventricular cardiomyocytes from failing and hypertrophied human heart, as well as PDE3, with ventricle-specific results for PDE4 and a surprisingly large contribution from PDE1 for hydrolysis of both cAMP and cGMP. The total PDE activity of human cardiomyocytes, and the profile of inhibition by PDE1, 3, 4, and 5 inhibitors, was modelled well in guinea pig but not rat cardiomyocytes.SignificanceOur results provide the first full characterisation of human cardiomyocyte PDE isoforms, and suggest that guinea pig myocytes provide a better model than rat for PDE levels and activity.     

Adenine incorporation in human and rat endothelium

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1+/-0.3, 82.8+/-11.1 and 115.1+/-11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7+/-23.2 and 262.0+/-30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7+/-3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.     

The expression pattern of nitric oxide-sensitive guanylyl cyclase in the rat heart changes during postnatal development.

Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat beta(1)-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohistochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart.     

Cathepsin B in osteoblasts

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.     

Re-expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the 'embryonic' alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (αSKA) and a final predominant expression of alpha cardiac actin (αCA). Our objective was to detect whether re-expression of αSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of αCA, αSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro . Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for αCA and αSKA. Up-regulation of αSKA was observed in human ventricular cardiomyocytes showing down-regulation of αCA and cardiotin. A patchy re-expression pattern of αSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of αSKA. Comparable αSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of αSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.     

Steering signal transduction pathway towards cardiac lineage from human pluripotent stem cells: A review

In humans injured myocardium cannot avert the onset and progression of ventricular dysfunction because of limited regenerative ability of myocytes. Although limited renaissance of cardiomyocytes has been reported in human infarcted hearts, it is generally accredited that non-functional fibrous tissue replaces the dead myocardium. High cardiovascular morbidity and dearth of donor hearts warrant a constant hunt for radically different approach to treat heart failure. Pluripotent stem (PS) cells possess the ability to produce functional cardiomyocytes for clinical applications and drug development, which may provide the answer to this problem. Although progress has been made in differentiating human PS cells into cardiomyocytes, however, the in vitro differentiation of pluripotent cells into cardiomyocytes involves a poorly defined, inefficient and relatively non-selective process. A thorough understanding of signaling pathways would tender a roadmap for the streamlined development of in vitro cardiac differentiation strategies. The ability to obtain unlimited numbers of human cardiomyocytes would improve development of cell-based therapies for cardiovascular diseases, facilitate the study of cardiovascular biology and improve the early stages of drug discovery. Here in this review, we highlight the interacting endogenous cellular signals and their modulators involved in directing the human PSCs towards cardiac differentiation.     

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21^Cip1 and p27^Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.     

Evidence for a membrane-bound carbonic anhydrase in the heart of an ancient vertebrate,the sea lamprey (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

In order to gain insight into the early evolution of carbonic-anhydrase (CA) isozymes in vertebrates, the main objective of the present study was to determine whether the hearts of an ancient vertebrate species, Petromyzon marinus, possess a membrane-bound CA isozyme. Since a significant amount of CA activity appeared to be strongly associated with the heart membrane fraction after differential centrifugation and washing, further experiments were conducted to examine the inhibitor properties of the CA from the membrane fraction in comparison with lamprey cytoplasmic CA from the red blood cell (rbc) fraction. These experiments showed that the inhibitor properties of the CA from the heart membranes were significantly different from those of the cytoplasmic CA from lamprey rbcs. A final series of experiments showed that the membrane-bound CA in the lamprey heart is not anchored via a glycosylphosphatidylinositol (GPI) linkage. Taken together, the results of these studies indicate that a membrane-bound CA does appear to be present in the hearts of lamprey, but the properties of the membrane-bound CA isozyme in these ancient vertebrates appear to differ from those in more recently evolved groups.Abbreviations Az acetazolamide - CA carbonic anhydrase - GPI glycosylphosphatidylinositol - PI-PLC phosphatidylinositol specific phospholipase C - Rbc red blood cell     

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.     

Diphtheria toxin-induced autophagic cardiomyocyte death plays a pathogenic role in mouse model of heart failure

It is still not clear whether loss of cardiomyocytes through programmed cell death causes heart failure. To clarify the role of cell death in heart failure, we generated transgenic mice (TG) that express human diphtheria toxin receptor in the hearts. A mosaic expression pattern of the transgene was observed, and the transgene-expressing cardiomyocytes (17.3% of the total cardiomyocytes) were diffusely scattered throughout the ventricles. Intramuscular injection of diphtheria toxin induced complete elimination of the transgene-expressing cardiomyocytes within 7 days, and approximately 80% of TG showed pathophysiological features characteristic of heart failure and were dead within 14 days. Degenerated cardiomyocytes of the TG heart showed characteristic features indicative of autophagic cell death such as up-regulated lysosomal markers and abundant autophagosomes containing cytosolic organelles like cardiomyocytes of human dilated cardiomyopathy. The heart failure-inducible TG are a useful model for dilated cardiomyopathy, and provided evidence indicating that myocardial cell loss through autophagic cell death plays of a causal role in the pathogenesis heart failure.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.