Gou Young Koh

Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.

This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.

Differentiation and long-term survival of C2C12 myoblast grafts in heart.

We have assessed the ability of skeletal myoblasts to form long-term, differentiated grafts in ventricular myocardium. C2C12 myoblasts were grafted directly into the heart of syngeneic mice. Viable grafts were observed as long as 3 mo after implantation. Immunohistological analyses revealed the presence of differentiated myotubes that stably expressed the skeletal myosin heavy chain isoform. Thymidine uptake studies indicated that virtually all of the grafted skeletal myocytes were withdrawn from the cell cycle by 14 d after grafting. Graft myocytes exhibited ultrastructural characteristics typical of differentiated myotubes. Graft formation and the associated myocardial remodeling did not induce overt cardiac arrhythmia. This study indicates that the myocardium can serve as a stable platform for skeletal myoblast grafts. The long-term survival, differentiated phenotype, and absence of sustained proliferative activity observed in myoblast grafts raise the possibility that similar grafting approaches may be used to replace diseased myocardium. Furthermore, the genetic tractability of myoblasts could provide a useful means for the local delivery of recombinant molecules to the heart.

Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice.

D-type cyclin/cyclin-dependent kinase (CDK) complexes regulate transit through the restriction point of the cell cycle, and thus are required for the initiation of DNA synthesis. Transgenic mice which overexpress cyclin D1 in the heart were produced to determine if D-type cyclin deregulation would alter myocardial development. Cyclin D1 overexpression resulted in a concomitant increase in CDK4 levels in the adult myocardium, as well as modest increases in proliferating cell nuclear antigen and CDK2 levels. Flow cytometric and morphologic analyses of dispersed cell preparations indicated that the adult transgenic cardiomyocytes had abnormal patterns of multinucleation. Histochemical analyses confirmed a marked increase in number of cardiomyocyte nuclei in sections prepared from the transgenic mice as compared with those from control animals. Tritiated thymidine incorporation analyses revealed sustained cardiomyocyte DNA synthesis in adult transgenic hearts.

Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs.

This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals.

PRESENCE AND BIOLOGICAL ACTIVITY OF C-TYPE NATRIURETIC PEPTIDE-DEPENDENT GUANYLATE CYCLASE-COUPLED RECEPTOR IN THE PENILE CORPUS CAVERNOSUM

PURPOSEnTo investigate the presence of C-type natriuretic peptide 1-22 (CNP)-dependent guanylyl cyclase (GC)-coupled receptor and its biological function in the penile erectile smooth muscle.nnnMATERIALS AND METHODSnExperiments have been done in rabbit and rat to detect cyclic GMP (cGMP) generation by the activation of particulate GC by natriuretic peptides (NPs) in cavernosal membrane, to localize precise receptor using a quantitative in vitro autoradiography of the snap frozen sections, to define natriuretic peptide receptor (NPR) mRNA using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique and to monitor changes of erectile smooth muscle tone by NPs in the penile tissue strips.nnnRESULTSnProductions of cGMP by particulate GC in the corpus cavernosum membranes of rabbit and rat were stimulated by CNP, atrial natriuretic peptide 1-28 (ANP) and brain natriuretic peptide 1-26 (BNP) with a rank order of potency of CNP > BNP > ANP. HS-142-1, a selective antagonist for the GC-coupled NPR, inhibited the CNP-stimulated cGMP production in corpus cavernosal membrane of rabbit and rat. Specific 125I-(Tyr[0])-CNP bindings were localized in the corpus cavernosal smooth muscle of rabbit with Kd of 19.92+/-3.38 nM. and Bmax of 734.64+/-139.63 amol./mm2. B-subtype of NPR mRNA was detected in the penile corpus cavernosum of rat using RT-PCR technique. CNP relaxed the smooth muscle contracted by Nomega-nitro-L-arginine methylester (L-NAME).nnnCONCLUSIONSnThese results suggest for the first time that CNP modulates the erectile smooth muscle tone of penis by predominant activation of B-subtype of NPR with augmentation of cGMP production via particulate GC.

Targeted expression of transforming growth factor-beta 1 in intracardiac grafts promotes vascular endothelial cell DNA synthesis.

Intracardiac grafts comprised of genetically modified skeletal myoblasts were assessed for their ability to effect long-term delivery of recombinant transforming growth factor-beta (TGF-beta) to the heart. C2C12 myoblasts were stably transfected with a construct comprised of an inducible metallothionein promoter fused to a modified TGF-beta 1 cDNA. When cultured in medium supplemented with zinc sulfate, cells carrying this transgene constitutively secrete active TGF-beta 1. These genetically modified myoblasts were used to produce intracardiac grafts in syngeneic C3Heb/FeJ hosts. Viable grafts were observed as long as three months after implantation, and immunohistological analyses of mice maintained on water supplemented with zinc sulfate revealed the presence of grafted cells which stably expressed TGF-beta 1. Regions of apparent neovascularization, as evidenced by tritiated thymidine incorporation into vascular endothelial cells, were observed in the myocardium which bordered grafts expressing TGF-beta 1. The extent of vascular endothelial cell DNA synthesis could be modulated by altering dietary zinc. Similar effects on the vascular endothelial cells were not seen in mice with grafts comprised of nontransfected cells. This study indicates that genetically modified skeletal myoblast grafts can be used to effect the local, long-term delivery of recombinant molecules to the heart.

Potential approaches for myocardial regeneration

Cardiomyocytes in the adult mammal retain little or none of their developmental capacity for hyperplastic growth. As a consequence of this differentiated, nonproliferative phenotype, cardiomyocyte loss due to injury or disease is irreversible. Therapeutic intervention in end-stage diseased hearts is currently limited to cardiac transplantation. An increase in cardiomyocyte number in diseased hearts could improve function. Augmentation of the cardiomyocyte population may be achievable by the expression of regulatory proteins in the myocardium, or by intracardiac grafting of exogenous cardiomyocytes.

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.

Plasma Concentration of Atrial Natriuretic Peptide in Different Phases of Korean Hemorrhagic Fever

Korean hemorrhagic fever (KHF) is an epidemic viral disease characterized by high fever, hemorrhagic tendency and renal failure, and by hemorrhages of right atrium and renal medulla as well as necrosis of anterior hypophysis. Plasma immunoreactive atrial natriuretic peptide (irANP) levels of 15 patients in the oliguric phase was 94.8 +/- 8.4 pg/ml (mean +/- SEM), 80% higher than of the normal control group (53.0 +/- 4.7 pg/ml; n = 28). In the diuretic phase it declined to 63.7 +/- 5.3 pg/ml (n = 26). Plasma renin activity (PRA) in the oliguric phase was 19.0 +/- 1.3 ng AI/ml/h, and in the diuretic phase 5.3 +/- 0.9 ng AI/ml/h, significantly higher than the control value (2.5 +/- 0.1 ng AI/ml/h). Elevations of irANP and PRA were not correlated in each group. Also systemic blood pressure as well as heart beats were significantly increased in the oliguric phase. These findings suggest that the increased irANP may have resulted from increased circulatory volume and that the ANP secretory process may not be affected by the disease.

Reduction volume dependence of immunoreactive atrial natriuretic peptide secretion in isolated perfused rabbit atria

A new technique to permit gradual changes in atrial distension has been developed in an isolated perfused rabbit atrium preparation. Graded volume reduction in the atrium was induced by changing the elevation of the outflow catheter tip. Pressure reduction from 6 cm H2O atrial distension resulted in a decrease in atrial distension volume. Atrial distension by 6 cmH2O did not change the release of immunoreactive atrial natriuretic peptide (irANP). The graded reduction in atrial distension from 0.11 +/- 0.03 (1.5 cm H2O) to 1.36 +/- 0.19 microliters/mg wet weight (6.0 cm H2O) resulted in 1.7 (6.76 +/- 2.05 versus 3.83 +/- 1.18 pg/mg per min, n = 9, P less than 0.025) to 40.1-fold (77.66 +/- 17.82 versus 3.0 +/- 1.14 pg/mg per min, n = 11, P less than 0.025) increases in irANP release. IrANP release in response to the reduction of atrial distension was volume dependent. The relation of percentage increase in irANP release with the percentage reduction of atrial distension was exponential. The data suggest that the atrial muscle shortening, but not stretch per se, may be a potent direct stimulus for the regulation of irANP secretion.