Adult-derived stem cells from the liver become myocytes in the heart in vivo.

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth.

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(-/-) mice). In EP4(-/-) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(-/-) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

Percutaneous needle autopsy sampling.

Despite the recognized advantages of a complete postmortem examination, autopsy rates continued to decline in recent decades. This study compares postmortem needle sampling to the complete, conventional autopsy to determine whether needle sampling is a viable alternative when consent for a complete autopsy is denied. A prospective study where postmortem percutaneous biopsies were obtained from the heart, the lungs, the liver, the kidney, and any other clinically relevant tissue or body fluid before the complete autopsy in 20 consecutive patients is presented. Cultures of the lungs, the spleen, and any other suspicious body fluid were also obtained. Liver and heart were recovered from all 20 of the patients, lung from 18 (90%), and kidney from 16 cases (80%). The cause of death was confirmed in 67% of the patients. Needle sampling correlated with the complete autopsy in 87% of the additional major diagnoses and with equally pertinent negative results. Postmortem needle lung cultures correlated with the complete autopsy in 17 (85%) of 20 patients and 16 (80%) of 20 spleen cultures. Cultures of the brain (one patient), cerebrospinal fluid (two patients), peritoneum (two patients), and serum (one patient) correlated 100% when compared to the complete autopsy. A complete autopsy is the goal of every postmortem examination. Postmortem "biopsies" can be an alternative option in certain situations and may be more acceptable to relatives of the deceased when consent for a complete autopsy is declined.

Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart.

Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT1 through cTnT4, numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30-nt exon in the 5' half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT1 through cTnT4. Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT1 and cTnT2) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT1 and cTnT2, containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT2 being expressed at a very low level. cTnT4, lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT3, containing the 5-residue peptide, is the dominant isoform in the adult heart.(ABSTRACT TRUNCATED AT 250 WORDS)

C2C12 cells: biophysical, biochemical, and immunocytochemical properties.

We examined the myofibril biochemical, structural, and biophysical properties of C2C12, a mouse skeletal muscle cell line (American Type Culture Collection), to assess whether force development and the sensitivity of the myofilaments to calcium could be measured in C2C12 myotubes and whether a cardiac contractile protein, troponin T, is expressed and incorporated into C2C12 myofibrils. When myoblasts fused and differentiated into myotubes, expression of myofilament proteins was initiated. Multiple cardiac and skeletal muscle troponin T isoforms were coexpressed. Cardiac troponin T expression increased and then decreased with time. Fluorescence immunocytochemistry demonstrated incorporation of cardiac troponin T isoforms into the myofibrils. At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns). The estimated maximum force developed by chemically skinned myotubes at 6-7 days poststarvation, 0.88 +/- 0.12 microN (mean +/- 95% confidence interval, n = 5), was significantly less (P < 0.05) than that at 10-13 days poststarvation, 1.12 +/- 0.12 microN (n = 7). The force-pCa relation yielded a Hill coefficient of 2.9 +/- 0.6 (n = 7) and half-maximal activation at pCa of 5.77 +/- 0.20. The demonstration that the biophysical properties of C2C12 cells can be measured and that cardiac and skeletal muscle troponin T isoforms are incorporated and colocalized into myofibrils suggest that these cells could be a useful model to assess the effects of exogenous native and mutated cardiac and skeletal contractile protein isoforms on myofilament function.

Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart.

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

An animal model for cystic fibrosis made by gene targeting.

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.

A two-motif isoform of the major calcium channel subunit in skeletal muscle.

Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel alpha 1 subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel alpha 1 cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.

Troponin T isoform expression in the normal and failing human left ventricle: a correlation with myofibrillar ATPase activity.

The expression of troponin T, a thin filament regulatory protein, was examined in normal and failing left ventricles. The samples were obtained from the hearts of patients with severe heart failure who were undergoing cardiac transplantation, and from normal adult hearts that could not be used for transplantation. Western blots of the myofibrillar proteins demonstrated two isoforms, troponin T 1 (TnT1) and troponin T 2 (TnT2). TnT2 is expressed at significantly higher levels in failing hearts (p less than 0.004). Western blots of two-dimension SDS-PAGE gels resolved two dominant spots of TnT1 and of TnT2 and several minor troponin T species. Alkaline phosphatase treatment markedly decreased the sizes of the two acidic spots while increasing the two more basic spots by a comparable amount. Myofibrillar ATPase activity had an inverse and negative linear relationship (r = 0.7, p less than 0.02) with the myofibrillar percentage of total troponin T comprised of TnT2. In that heart failure in these transplant patients had multiple bases, we propose that rather than a cause of heart failure, the disease-associated changes in troponin T isoform expression are an adaptation to abnormal myocardial function.

Force-pCa relation and troponin T isoforms of rabbit myocardium.

We have previously reported the existence of at least four troponin T isoforms in rabbit ventricular muscle and described the changes in their distribution with development. In this report we test whether the proportions of the troponin T isoforms are related to the sensitivity of the myofilaments to calcium. We measured the force-pCa relations in 12 detergent-skinned ventricular strands of cardiac muscle from newborn (2-5-day-old) rabbits. We determined from each strand the amount of each troponin T isoform relative to the total amount of troponin T by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometric scans of Western blots probed with a cardiac-specific troponin T monoclonal antibody, MAb 13-11. To assess the presence of different relative amounts of cardiac and slow skeletal troponin I among the strands, we determined the amount of cardiac troponin I relative to tropomyosin. We determined the Hill coefficient and the pCa for half-maximal force, pCa50, for each strand. pCa50 was related directly to the relative amount of troponin T2 (pslope = 0.037). Our results do not indicate a relation between the Hill coefficient and troponin T2. We also did not find a relation between pCa50 and the cardiac troponin I/tropomyosin ratio, which suggests that the correlation between pCa50 and troponin T2 was not a result of changes in the relative amounts of cardiac and slow skeletal muscle troponin I. Our findings indicate that a relation exists between the force-pCa characteristics of rabbit myocardium and the troponin T isoforms that it expresses, suggesting a role for troponin T in modulating the sensitivity of cardiac myofilaments to calcium.

Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle.

The expression of troponin (Tn) T, a thin-filament regulatory protein, was examined in left ventricular myocardium from normal and from failing adult human hearts. The differences in isoform expression between normal and failing myocardium led us to examine the ontogenic expression of TnT in human striated muscle. Left ventricular samples were obtained from patients with severe heart failure undergoing cardiac transplantation and normal adult organ donors. Fetal muscle was obtained from aborted fetuses after 14-15 weeks of gestation, and adult skeletal muscle was obtained from surgical biopsies. Western blots of normal and failing adult heart proteins demonstrated that two isoforms, TnT1 and TnT2, are expressed in different amounts, with TnT2 being significantly greater in failing hearts (p less than 0.004). Western blots of two-dimensional gels of these proteins resolved two predominant spots of both TnT1 and TnT2 and several minor TnT species. Alkaline phosphatase treatment converted the two major spots of each isoform into the single more basic spots. A comparison of the ATPase activities and the TnT2 percentage of total TnT in individual failing and normal adult hearts demonstrated an inverse and negative relation (r = 0.7, p less than 0.02). In the fetal heart, four TnT isoforms were found, two of which had the same electrophoretic mobilities as the adult cardiac isoforms TnT1 and TnT2. Fetal skeletal muscle expressed two of the four fetal cardiac TnT isoforms, one of which comigrated with adult cardiac TnT1. These cardiac isoforms were expressed in low abundance in fetal skeletal muscle relative to seven fast skeletal muscle TnT isoforms. No cardiac isoforms were present in adult skeletal muscle. Because many etiologies caused heart failure in the transplant patients, we propose that the disease-associated increased expression of the TnT isoform TnT2 is an adaptation to the heart failure state and a partial recapitulation of the fetal expression of cardiac TnT isoforms.

Monoclonal antibody specific for the transverse tubular membrane of skeletal muscle activates the dihydropyridine-sensitive Ca2+ channel.

In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive Ca2+ channels are preferentially localized in the transverse tubular membranes. Starting with an antigenic membrane fraction enriched in rabbit muscle transverse tubules (T-tubules), several monoclonal antibodies were produced by a fusion of spleen cells from an immunized BALB/c mouse with P3 X 63Ag.8.6.5.3 mouse myeloma cells. Antibodies were screened according to a scheme designed to select IgG immunoglobins that recognized a determinant specifically associated with the T-tubule membrane. Antibodies that fulfilled the screening criteria were used in in vitro planar bilayer recording of the activity of the dihydropyridine-sensitive Ca2+ channel present in T-tubules. Cells producing one antibody (Ab 21) survived cloning dilution and stably produced a monoclonal antibody (mAb21-4) that increased the rate of single channel opening when interacting with the internal side of the channel protein. mAb21-4 immobilized by covalent crosslinking on beads (Affi-Gel 10) consistently immunoprecipitated polypeptide bands with the following electrophoretic mobility: Mr values of greater than or equal to 175,000; 90,000; 55,000; and 34,000.

Renal-hepatic-pancreatic dysplasia: a syndrome reconsidered.

Five infants, three dying neonatally and two later in the first year of life, had renal, hepatic, and pancreatic dysplasia, a combination of abnormalities first described by Ivemark et al [1959]. The renal malformation consisted of cystic dysplasia, with abnormally differentiated ducts, deficient nephron differentiation, and glomerular cysts. The hepatic abnormality consisted of enlarged portal areas containing numerous elongated biliary "profiles," with a tendency to perilobular fibrosis. Serial liver biopsies in one child with cholestasis from birth showed a progression from bile duct paucity at 1 1/2 wk to typical biliary "dysgenesis" at 7 mo. Four of the five children had intrahepatic ductal dilatation, diagnosed ante mortem in the two older children as Caroli disease. The pancreatic abnormality consisted of fibrosis and cysts, with a diminution of parenchymal tissue. The clinical and functional reflection of these abnormalities in the two children surviving the newborn period included renal insufficiency, chronic jaundice, and insulin-dependent diabetes mellitus. Similar renal, hepatic, and pancreatic abnormalities occur in other syndromes, including trisomy 9, Meckel syndrome, Jeune, Saldino-Noonan, and Elejalde types of chondrodysplasia, and glutaric aciduria II. After exclusion of identifiable syndromes, the remaining cases of renal-hepatic-pancreatic dysplasia do not necessarily constitute a homogeneous group.

Proliferation of the surface-connected intracytoplasmic membranous network in skeletal muscle disease.

A surface-connected intracytoplasmic membranous (SCIM) network proliferates in skeletal muscle diseases and in myotubes grown in vitro. The authors observed frequent occurrence of "coated" microdomains in the form of budding vesicles in the proliferated components of this network and suspected a potential role the proliferated membranes might have in the endocytosis of molecules into myotubes undergoing repair or regeneration. Five-day-old myotubes in culture were incubated at 37 C and between 2 and 4 C with two tracers, Lucifer yellow and ferritin, both known to enter other types of cells via a fluid-phase endocytotic pathway. The differential penetration of Lucifer yellow at 37 C and below 2-4 C was examined by fluorescence microscopy and by electron microscopy. Lucifer yellow was rendered electron-opaque by photoreacting it with an intense light in the presence of DAB. Ferritin penetration at 37 C and between 2 and 4 C was compared and quantitated ultrastructurally. The authors found that endocytosis of the tracers into myotubes and eventually into lysosomes took place after the tracers had diffused into the lumen of the proliferated SCIM network. These processes were inhibited below 4 C. This finding, coupled with the presence of "coated" microdomains in the proliferated membranes, led us to suspect that the SCIM network may have a role in membrane turnover of metabolically active diseased muscle cells undergoing regeneration.

Monoclonal antibody specific for the T-tubule of skeletal muscle.

Monoclonal antibodies were raised against a triad-enriched (sarcoplasmic reticulum-T-tubule complex) microsomal membrane fraction of rabbit skeletal muscle. The avidin-biotin complex (ABC) immunoperoxidase staining method was used to screen hybrid colonies. Positive antibodies exhibited a granular doublet pattern at the A-I junction, consistent with the location of triads in rabbit muscle. One monoclonal antibody, M171, was further characterized by ultrastructural and immunoadsorption techniques. Postembedding electron immunocytochemistry was performed on tissue sections embedded in Lowicryl K4M. Goat anti-mouse immunoglobulin absorbed to 10 nm colloidal gold particles was used as an ultrastructural label. In these studies, M171 recognized an epitope at the triads and at periodic openings along the plasmalemma. Immunoadsorption on protein transfers of isolated sarcoplasmic reticulum, surface membrane (plasmalemma and T-tubule), and triad-enriched fractions showed that M171 reacts with a surface membrane component. Taken together, these studies suggest that M171 recognizes an epitope associated with the T-tubule at the triad and at the "mouth" of the T-system at the plasmalemma.

Biochemical and cytochemical comparison of surface membranes from normal and dystrophic chickens.

Cytochemical and biochemical characteristics of the surface membrane components of avian dystrophic muscle were examined. A Mg2+- or Ca2+-activated ("basic") adenosine triphosphate (ATPase) was localized cytochemically in fixed, intact dystrophic muscle slices in a medium containing Mg2+ or Ca2+, adenosine triphosphate (ATP), and 1 microM free Pb2+ to capture enzymatically released phosphate ions. Electron-dense staining precipitates were found to be associated with the plasmalemma and its tortuous invaginations, and the transverse components of the T-system membrane and its associated proliferated networks. Enzymatic analysis of microsomal fractions isolated from 7-day-old and 90-day-old normal and dystrophic muscle showed a complex behavior. Specific activity of "basic" ATPase decreased with maturity in normal and dystrophic animals. The specific activities of the surface membrane associated enzymes, leucyl beta-naphthylamidase, adenylate cyclase, and guanylate cyclase, remained at various elevated levels in the mature dystrophic animals, in contrast to the normal muscle, which showed decreases in the specific activity of all three enzymes with maturation. The persistent high levels in some but not all enzyme activities in 90-day-old dystrophic muscle indicates a complicated developmental pattern in the dystrophic chicken muscle.

Cytochemical localization of a "basic" ATPase to canine myocardial surface membrane.

Enzymatic properties of a canine cardiac muscle microsomal fraction were determined to localize in situ a "basic," divalent cation dependent adenosine triphosphatase (ATPase) by ultrastructural cytochemistry. The microsomal fraction had a buoyant density of 1.08--1.13 (20--30% [w/w] sucrose) and hydrolyzed adenosine triphosphate in the presence of Mg2+, Ca2+, Mn2+, or Co2+, but not in that of Sr2+ or Ni2+, under conditions that inhibited interfering (Na+ + K+)-ATPase and sarcoplasmic reticulum Ca2+-ATPase activities. "Basic" ATPase was localized in paraformaldehyde-fixed tissue in a medium containing Mg2+ or a high Ca2+ concentration (4 mM). A free Pb2+ concentration of less than 1 microM was used to capture enzymatically released phosphate anions. Electron-dense lead precipitates were present at the plasmalemma, T-system, and intercalated disc membranes with the exception of the nexus. These studies suggest that "basic" ATPase activity is associated with surface membrane structures of canine cardiac muscle.

Chicken dystrophy. The geometry of the transverse tubules.

The pectoral muscle of chickens afflicted with muscular dystrophy, when examined with the electron microscope, contains a) numerous, often quite large vesicles with and without caveolar evaginations, b) tubules with caveolar evaginations, and c) tubular networks. We have demonstrated that all these structures are derivatives of the transverse tubles as revealed by tracer studies and freeze-fracture complementary replicas. The membranes of transverse tubular origin show a small number of intramembranous particles on both P and E faces with no complementary geometry. The membranes of the free sacrcoplasmic reticulum (SR) and the junctional SR of normal and dystrophic muscle appear identical in complementary freeze-fracture replicas. Vesicles that carry only a small number of particles on both E and P faces exposed by freeze-fracturing in isolated SR preparations can be taken as presumptive evidence and serve as a morphologic marker for transverse tubular origin of such vesicles when mitochondrial and lysosomal contamination has been excluded.