Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering.

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.

An immortalized myocyte cell line, HL-1, expresses a functional delta -opioid receptor.

The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [(3)H]bremazocine by selective ligands for the mu (mu), delta (delta), and kappa (kappa) receptors revealed that only the delta -selective ligands could fully displace specific [(3)H]bremazocine binding, indicating the presence of only the delta -receptor in these cells. Saturation binding studies with the delta -antagonist naltrindole afforded a B(max)of 32 fmols/mg protein and a K(D)value for [(3)H]naltrindole of 0.46 n M. The binding affinities of various delta ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastomaxglioma cell line, NG108-15. Finally, various delta -agonists were found to stimulate the binding of [(35)S]GTP gamma S, confirming coupling of the cardiac delta -receptor to G-protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [(35)S]GTP gamma S to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled delta -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors.

The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen-immortalized AT-1, AT-2 and HL-1 cardiomyocytes.

To investigate molecular controls of cardiomyocyte proliferation, we utilized cardiomyocytes induced to proliferate indefinitely by SV40 large T antigen (T-ag). In the T-ag-immortalized AT-1, AT-2 and HL-1 cardiomyocytes, normal cellular proteins associating with T-ag and p53 were identified, isolated and micro-sequenced. Peptide sequencing revealed that proteins of 90, 100 and 160 kDa were homologs of MRE11, NBS1 and RAD50, respectively. These three proteins play critical roles in the detection and repair of DNA double-strand breaks, activation of cell cycle checkpoints and telomere maintenance. In this report, we describe the cDNA cloning and double-strand sequencing of the rat homologs of MRE11, NBS1 and RAD50. We also determined the mRNA and protein levels of MRE11, NBS1 and RAD50 at different stages of heart development and in different tissues. MRE11 mRNA was only detected in the immortalized cardiomyocytes and in the testes. Although the 90 kDa MRE11 protein was seen in most samples examined, it was only detected at extremely low levels in proliferating cardiomyocytes (normal and immortalized). The 6.0 kb MRE11-related mRNA transcript (MRT) was seen in all samples examined. Levels of both NBS1 and RAD50 mRNA transcripts peaked in the heart at postnatal day 10. NBS1 mRNA levels were at very low levels in the T-ag-immortalized AT-1, AT-2 and HL-1 cells but NBS1 protein was observed at extremely high levels. We propose that SV40 large T antigen's interaction with the MRE11-NBS1-RAD50 pathway and with p53 ablates critical cell cycle checkpoints and that this is one of the major factors involved in the ability of this oncoprotein to immortalize cardiomyocytes.

Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes.

Adrenomedullin (AM) is a hypotensive protein expressed in a variety of cells and tissues. We observed previously that the expression of the adrenomedullin gene increases substantially in the developing rat heart and in cultured adult rat ventricular cardiac myocytes in response to hypoxia as a function of time. An adrenomedullin promoter-luciferase reporter construct was used to show that this increase in adrenomedullin mRNA resulted from increased transcription in response to hypoxia. We report here additional evidence documenting that this hypoxia-induced transcription of the adrenomedullin gene is regulated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. We used Northern blot analysis to show an increase in the levels of AM and HIF-1alpha mRNA but not HIF-1beta mRNA in the HL-1 cardiac myocyte cell line in response to hypoxia. Furthermore, Western blot analysis revealed that the levels of both HIF-1alpha and HIF-1beta protein increased under hypoxic conditions. Data from electrophoretic mobility shift assays indicate that the heterodimeric HIF-1 complex binds to the HIF-1-responsive elements. Combined data from these studies demonstrate that the AM gene is regulated by hypoxia-responsive elements localized in the AM promoter region.

Adrenomedullin gene expression is developmentally regulated and induced by hypoxia in rat ventricular cardiac myocytes.

Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at -1095 and -770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5'-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at -1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter.

Cardiomyocyte transplantation in a porcine myocardial infarction model.

Transplantation of cardiomyocytes into the heart is a potential treatment for replacing damaged cardiac muscle. To investigate the feasibility and efficiency of this technique, either a cardiac-derived cell line (HL-1 cells), or normal fetal or neonatal pig cardiomyocytes were grafted into a porcine model of myocardial infarction. The myocardial infarction was created by the placement of an embolization coil in the distal portion of the left anterior descending artery in Yorkshire pigs (n = 9). Four to 5 wk after creation of an infarct, the three preparations of cardiomyocytes were grafted, at 1 x 10(6) cells/20 microL into normal and into the middle of the infarcted myocardium. The hearts were harvested and processed for histologic examinations 4 to 5 wk after the cell grafts. Histologic evaluation of the graft sites demonstrated that HL-1 cells and fetal pig cardiomyocytes formed stable grafts within the normal myocardium without any detrimental effect including arrhythmia. In addition, a marked increase in angiogenesis was observed both within the grafts and adjacent host myocardium. Electron microscopy studies demonstrated that fetal pig cardiomyocytes and the host myocardial cells were coupled with adherens-type junctions and gap junctions. Histologic examination of graft sites from infarct tissue failed to show the presence of grafted HL-1 cells, fetal, or neonatal pig cardiomyocytes. Cardiomyocyte transplantation may provide the potential means for cell-mediated gene therapy for introduction of therapeutic molecules into the heart.

Effect of basic fibroblast growth factor on angiogenesis in the infarcted porcine heart.

Administration of growth factors is emerging as a new therapeutic approach for the enhancement of collateral vessel formation in the ischemic heart. We have investigated the effects of intramyocardial delivery of FGF-2 in the presence and absence of heparin on angiogenesis in a porcine model of myocardial infarction. Yorkshire pigs were subjected to myocardial infarction by the placement of an embolization coil in the left anterior descending artery (n = 5). Four to five weeks after creation of an infarct, FGF-2 (10 micrograms) alone or in complex with heparin, heparan sulfate, or heparin agarose beads was injected either into the normal myocardium or along the infarct border area. Histologic evaluation of each injection site was performed 4 to 5 weeks post-injection. The effect of FGF-2 on angiogenesis was evaluated by determining the number of capillaries (diameter < 20 microns (and arterioles (> 20 microns with tunica media) in each area observed. The number of capillaries were not affected by the treatment of FGF-2 both in normal myocardium and infarct border area. However, in the normal myocardium, the number of arterioles were increased with the treatment of FGF-2 alone (85 +/- 59%, P < 0.04), FGF-2 plus heparin (281 +/- 193%, P < 0.004) and FGF-2-coated heparin beads (241 +/- 141%, P < 0.01), as compared to control. Delivery of FGF-2 into the infarct border area, also increased the number of arterioles when FGF-2 was given with heparin (736 +/- 154%, P < 0.001) or heparin beads (700 +/- 109%, P < 0.001), as compared to control. FGF-2 administered with heparin was the most effective method of enhancing angiogenesis as compared to FGF-2 alone, FGF-2 plus heparan sulfate, or FGF-2 coated heparin agarose beads.

HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

Transfer of macromolecules into living adult cardiomyocytes by microinjection.

Among techniques commonly used to deliver bioactive molecules into living cells, microinjection is a very efficient method. Microinjection has been used extensively for gene transfer into different cell types. We applied the microinjection technique to the adult rat ventricular cardiac muscle cells (AVC) in primary culture and optimized microinjection parameters and the appropriate cell culture conditions. We also optimized the use of particular agents (i.e. 2,3-butanedione monoxime, verapamil) for the prevention of the cell damage caused by the micropuncture. We obtained the expression of a CMV-beta-galactosidase reporter gene in up to 20% of the injected cells with efficient maintenance of long term cell viability. Under our experimental conditions direct microinjection is a very advantageous technique to transfer macromolecules into living adult cardiac muscle cells and a powerful system to study and manipulate the biochemistry and molecular biology of the cardiac myocyte.

Differential display: identifying genes involved in cardiomyocyte proliferation.

An estimated 15,000 different mRNA species are expressed in a typical mammalian cell. The differential expression of mRNAs in both a temporal and cell-specific manner determines the fate of the cell and creates the organism. Analysis of this differential gene expression has become a central aim of many laboratories attempting to understand the mechanisms underlying various biological processes. Currently, we are using a technique called differential display to analyze the differential expression of genes in cardiomyocytes. Differential display is a rapid and powerful technique that was introduced by Liang and Pardee in 1992. Since that time, it has been successfully applied by several groups, and it is quickly becoming a standard method for studying differential gene expression. Here, we present a detailed article discussing the differential display methodology and how we have utilized it to identify potential genes involved in cardiomyocyte proliferation. Furthermore, we have provided a list of materials and supplied examples of data obtained, in an effort to allow the reader to perform the technique with success in their own laboratory.

Hypoxia stimulates atrial natriuretic peptide gene expression in cultured atrial cardiocytes.

The current study tested the hypothesis that hypoxia stimulates atrial natriuretic peptide (ANP) gene expression and secretion in cultured atrial myocytes (AT-1 cells). AT-1 cells were obtained from a transplantable mouse atrial cardiomyocyte tumor lineage. Confluent AT-1 cells were exposed to hypoxia (1% oxygen) or normoxia (21% oxygen) as controls for 6 hours to 7 days. Medium ANP levels were measured by radioimmunoassay, and intracellular ANP gene transcripts were quantified by Northern and slot blot analyses. Exposure to hypoxia resulted in a significant increase in cellular ANP mRNA levels within 36 hours, which peaked (3.6-fold increase) at 2 days after hypoxic exposure, and produced a time-dependent increase in the release of ANP from AT-1 cells for 2 to 7 days. Transfection studies with recombinant DNA constructs that contained fragments of the -3003/+62 sequence of the ANP promoter and the luciferase reporter gene revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located within a region that extends from -638 to -518 bp to the transcriptional start site of the ANP gene. Gel mobility shift assays demonstrated that hypoxia-inducible nuclear proteins that bound to the 120-bp putative hypoxia-responsive elements of the ANP gene were produced during hypoxic exposure. We have thus defined a 120-bp region within the ANP gene promoter that contains hypoxia-responsive elements that might be responsible for the enhancement of ANP gene expression in atrial myocytes during hypoxic exposure.

Corticotropin-releasing hormone receptor expression and functional coupling in neonatal cardiac myocytes and AT-1 cells.

CRH is the principal mediator of the stress response in mammals. In addition to pituitary and central nervous system effects, peripheral effects of CRH have been observed involving the immune and cardiovascular systems. Two CRH receptor subtypes, CRH-R1 and CRH-R2, have been cloned and show significant amino acid homology (69%), but differ in their tissue distribution. CRH-R1 is expressed predominantly in the brain and pituitary, whereas the CRH-R2 subtype is highly expressed in heart and skeletal muscle. To investigate the role of CRH in cardiac signaling, we analyzed the effect of CRH on freshly isolated neonatal rat cardiomyocytes and murine atrial cardiomyocyte tumor cells, AT-1, which express CRH-R2 messenger RNA. We show that stimulation of these cells with CRH and the CRH-related peptides, sauvagine from frog and urotensin I from fish, elicits large increases in the intracellular level of cAMP. This stimulation is transient, reaching a maximum in 5-15 min in neonatal cardiomyocytes and in 2-4 min in AT-1 cells, followed by a rapid decline. We show that stimulation of AT-1 cells by these peptides is specific for CRH receptors, as the CRH antagonist, alpha-helical CRH-(9-41) inhibits cAMP increases. Furthermore, we show that CRH, sauvagine, and urotensin I stimulations are dose dependent in both neonatal cardiomyocytes and AT-1 cells. Sauvagine and urotensin I are more potent than CRH at stimulating an increase in intracellular cAMP in neonatal cardiomyocytes (EC50 = 1.74, 2.61, 6.42 nM, respectively) and AT-1 cells (EC50 = 16.2, 15.8, and 149 nM, respectively). This rank order is consistent with that previously demonstrated in CRH-R2-transfected HEK293 cells and parallels the in vivo vasodilatory activity of these peptides. In summary, this is the first evidence that CRH, sauvagine, and urotensin I act directly on cardiac myocytes to stimulate increases in intracellular cAMP, presumably through CRH-R2. In addition, these results indicate that cardiac myocytes may be an informative in vitro model to investigate the effects of CRH and its role in the cardiovascular response to stress.

Myoblast transplantation in the porcine model: a potential technique for myocardial repair.

The use of transgenic cells transplanted in syngeneic rodents has shown modest success, but allogeneic and xenogeneic transplants have not been uniformly successful. To assess the feasibility of xenogeneic and allogeneic myoblast transplantation, we subjected seven adult swine to transplantation of murine atrial tumor cells (xenogeneic), neonatal porcine myocytes (allogeneic), and human fetal cardiomyocytes into the left ventricular wall. After general anesthesia, isolated cells were injected along the anterior and posterior walls of the porcine left ventricle. All the animals were immuno-suppressed and observed for 1 month after injection, at which time they were killed and analyzed. This report will present results primarily concerned with the success of human cell transfers. In all injected sites examined, the transplanted cells thrived within the host myocardium with no significant rejection. Transplant cells formed close associations with host myocytes that resembled nascent intercalated disks on electron microscopy. These cells also contained myofibrils and other cell architecture resembling the transplanted cell lines. Additionally, these cells appeared to produce an angiogenic influence resulting in the proliferation of the surrounding microvasculature. We believe that these findings indicate successful xenogeneic and allogeneic myoblast cell transplantation in a large animal model. These experiments set the stage for future studies to assess the ability of these cells to form a syncytium, contract, and potentially repair failed myocardium.

Identification and antisense inhibition of a renin-angiotensin system in transgenic cardiomyocytes.

Cardiac myocytes (AT-1 cells) derived from heart tumors of mice transgenic for an atrial natriuretic factor promoter, SV40 large T-antigen DNA transgene, demonstrate properties consistent with normal cardiac myocytes but retain the capacity to proliferate in culture. We studied the renin-angiotensin system (RAS) and related growth regulation of these cells because AT-1 cells (or transgenically similar cells) may be useful to repair injured myocardium. This study reveals two separate and distinct findings: 1) AT-1 cells proliferate or hypertrophy in response to angiotensin II (ANG II), depending on their competence to proceed through the cell cycle; and 2) AT-1 cells possess components of a RAS, and angiotensinogen antisense experiments suggest that the RAS is functional in these cells. Specifically, AT-1 cells proliferate in response to ANG II in low-serum medium but hypertrophy in response to ANG II when first treated with mitomycin C (at a concentration that inhibits DNA replication but is not cytotoxic). The ANG II-mediated proliferative and hypertrophic responses are inhibited by DuP 753. In addition, there is a significant increase in the protein-to-DNA ratio of cells, which are proliferation-inhibited in the absence of ANG II treatment (20%, P < 0.05). DuP 753 also inhibits this hypertrophy, suggesting that these cells possess a functional RAS. AT-1 cells contain mRNAs for angiotensin-converting enzyme, renin, angiotensinogen, and the AT1 receptor as determined by sequence analysis of polymerase chain reaction amplification products. Antisense oligonucleotides complementary to the angiotensinogen mRNA specifically inhibit angiotensinogen mRNA accumulation and proliferation of AT-1 cells. In summary, these cells contain a growth-regulating RAS, suggesting that such a system may play a significant role in left ventricular hypertrophy.

Spontaneous activity in transgenic mouse heart: comparison of primary atrial tumor with cultured AT-1 atrial myocytes.

INTRODUCTION: We have generated transgenic animals that heritably develop atrial tumors composed of differentiated proliferating cardiomyocytes. Experiments were initiated to characterize the electrical properties of these cells. METHODS AND RESULTS: We show that the primary atrial tumors are composed of discrete foci that exhibit spontaneous automatic activity. A direct correlation was observed between tumor size and firing rate of these foci. In addition to the primary atrial tumors, we examined the properties of cultured cardiomyocytes isolated from a transplantable transgenic tumor lineage (designated AT-1 cells). Cultured AT-1 cells are also spontaneously automatic. The action potential configuration from these preparations is similar to that observed in nontransgenic atrial cardiomyocytes, albeit somewhat more depolarized and of longer duration. As would be expected for cardiomyocytes of atrial origin, the transgenic cardiomyocyte preparations hyperpolarize during muscarinic stimulation due to increased K+ conductance mediated by a pertussis toxin sensitive G-protein. Assessment of pharmacologic blockage of the "if" pacemaker current suggests that the automaticity of both transgenic cardiomyocyte preparations may be of novel origin. In this context, the cultured AT-1 cells showed spontaneous behavior that was clearly of cellular origin; this activity was manifest as transient bursts of electrical activity followed by periods of electrical quiescence. This bursting pattern is unusual for normal adult cardiomyocytes, but has been observed in several other cell types. In the primary tumors, automatic behavior may arise from a similar cellular origin or alternatively from a microreentrant phenomena. CONCLUSION: Primary tumors and AT-1 cells show essential atrial electrophysiology with important novel features.

Identification of SV40 large T-antigen-associated proteins in cardiomyocytes from transgenic mice.

A cell line derived from transgenic mice expressing the SV40 large T-antigen oncogene in the heart was used to identify cardiomyocyte targets for T-antigen binding. A novel protein of molecular mass of 193 kDa was identified as an associated protein by virtue of its ability to be co-immunoprecipitated with multiple anti-T-antigen antibodies. Two previously described proteins, p120 and p53, were also observed to complex with T-antigen in transformed cardiomyocytes. In addition, several proteins that cross-reacted with either anti-T-antigen or anti-p53 antibodies were identified. Two of these proteins, of apparent molecular masses of 250 and 110 kDa, were only observed in cardiomyocytes. Expression of a third cross-reacting protein of a molecular mass of 180 kDa appeared to be dependent on the growth status of the cells. These proteins may be important constituents of the cardiomyocyte cell cycle, as well as potential cellular targets for myocardial regeneration.

Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes.

Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5' end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3',5'-cyclic monophosphate (cAMP). It was determined by column chromatography that cAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met5-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-1-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Met5-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells.