Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium.

Fetal cardiomyocytes isolated from transgenic mice carrying a fusion gene of the alpha-cardiac myosin heavy chain promoter with a beta-galactosidase reporter were examined for their ability to form stable intracardiac grafts. Embryonic day 15 transgenic cardiomyocytes delivered directly into the myocardium of syngeneic hosts formed stable grafts, as identified by nuclear beta-galactosidase activity. Grafted cardiomyocytes were observed as long as 2 months after implantation, the latest date assayed. Intracardiac graft formation did not induce overtly negative effects on the host myocardium and was not associated with chronic immune rejection. Electron microscopy revealed the presence of nascent intercalated disks connecting the engrafted fetal cardiomyocytes and the host myocardium. These results suggest that intracardiac grafting might provide a useful approach for myocardial repair, provided that the grafted cells can contribute to myocardial function.

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 Thymidine uptake studies indicated that virtually all of the grafted skeletal myocytes were withdrawn from the cell cycle by 14 d after grafting.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.

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.

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.

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.

Atrial natriuretic factor and transgenic mice.

Atrial natriuretic factor (ANF) is a peptide hormone that induces potent but transient hypotensive and natriuretic responses on short-term administration. The role of the hormone in long-term cardiovascular regulation has remained elusive in part because of the temporal limitations of long-term infusion models and the extremely short half-life of the molecule in vivo. To circumvent these temporal limitations, a transgenic mouse model was developed that exhibits lifelong elevated plasma ANF levels. These mice are chronically hypotensive, with arterial pressures averaging 20 to 30 mm Hg less than those observed in nontransgenic siblings. In contrast, no obvious natriuretic or diuretic phenotype was observed in transgenic animals housed in metabolic cages. Thus, the mice adequately compensate for the renal effects but not the hemodynamic effects of the hormone. The ANF transgenic mice provide a tractable model system with which to study the consequences of long-term alterations of ANF expression in vivo.

Comorbid Tourette's disorder and bipolar disorder: an etiologic perspective.

OBJECTIVE: Using an epidemiologic approach, the authors attempt to elucidate relationships between Tourette's disorder and bipolar disorder. METHOD: Of 205 patients with Tourette's disorder in the North Dakota Longitudinal Tourette Syndrome Surveillance Project, 15 had comorbid bipolar disorder. A subset of the patients with Tourette's disorder had been included in earlier population-based prevalence studies of Tourette's disorder in children, adolescents, and adults. Minimal risk ratios were calculated for the patients with Tourette's disorder plus bipolar disorder by age group (children/adolescents and adults). This information was used to estimate genetic risk indicators for comorbid Tourette's disorder and bipolar disorder. RESULTS: The estimated risk of developing bipolar disorder among the study group of children, adolescents, and adults with Tourette's disorder was more than four times higher than the level expected by chance, but this finding did not reach statistical significance. It was indicative of trends, however. CONCLUSIONS: Comorbidity between Tourette's disorder and bipolar disorder does not appear to be due to chance co-occurrence of the two disorders. Although a genetic mechanism may play a causal role, in the absence of family studies an explanatory model involving the concept of canalization of basal-ganglia-mediated dysfunctions is offered. In such a construct, Tourette's disorder would be a likely accompaniment to other conditions, including bipolar disorder, whose pathogenic determinants might channel through neural pathways involving the basal ganglia. The presence of significant developmental disabilities may further enhance factors culminating in comorbid Tourette's disorder and bipolar disorder.

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.

Neuropsychiatric genetics: misclassification in linkage studies of phenotype-genotype research.

Research on neuropsychiatric disorders has produced a number of very important findings in the last few decades. However, several problems continue to hinder research in this area. One problem area has been the appropriate classification of disease status for probands and extended family members in linkage studies. In this article, we examine rates of misclassification in a 12-year follow-up study of children previously diagnosed with Tourette syndrome. At the 12-year follow-up, we found a 5 to 12% rate of misclassification of previously diagnosed cases. We present a model of a linkage study with three classification steps. The model demonstrates that an error rate of 5% would result in misclassification of 20% of true cases by step three. Adding additional steps to improve diagnostic accuracy may increase rather than decrease classification error.

Long-term follow-up of an epidemiologically defined cohort of patients with Tourette syndrome.

The goal of this study was to collect prospective longitudinal information on the development of an epidemiologically defined cohort of patients with Tourette syndrome. These data may improve prognostic understanding of the condition. This information will also be important for specification of an adult phenotype for genetic marker studies. A prospective longitudinal cohort study was conducted. Fifty-four of 73 patients from our 1986 prevalence study of Tourette syndrome in North Dakota school-aged children were eligible for inclusion. The subjects were diagnosed in 1984 and 1985. We were able to interview 39 of 54 eligible patients for 507 person-years of follow-up. For the cohort, tic severity declined by 59%, global assessment of functioning improved by 50%, and the average number of comorbidities decreased by 42%. Forty-four percent of patients were essentially symptom free at follow-up. Only 22% were on medication as adults. Tourette syndrome is a developmental neuropsychiatric disorder with a long-term course that is favorable for most patients. Males demonstrated substantially more variability in improvement but overall demonstrated more improvement than females.

Prevalence of SIDS risk factors: before and after the "Back to Sleep" campaign in North Dakota Caucasian and American Indian infants.

The objective of this study was to compare rates of infant sleeping position and other risk factors for sudden infant death syndrome from 1991 before the "Back to Sleep" campaign to rates in 1998 after the campaign. We used a cross-sectional risk factor prevalence study of risk factors for the years 1991 and 1998. In North Dakota the prevalence rates of prone sleeping declined 72% for American Indian infants and 62% for Caucasian infants. We were unable to identify a corresponding decline in SIDS in North Dakota for this time period. The relationship between sleeping position and SIDS may be more complex in rural and frontier settings and in American Indian populations than in urban and majority populations. The generalizability of this study is limited by the rural setting and small sample size. Longer term surveillance and additional reports from sites with pre "Back to Sleep" data as a baseline for both SIDS rates and sleeping position will be important to clarify the rate of prone sleeping position and SIDS.

A prevalence methodology for mental illness and developmental disorders in rural and frontier settings.

A prevalence study methodology developed for use in rural and frontier settings is described. The general method was developed over a 15 year period and has been successfully adapted and used in studies of 14 different childhood onset developmental disorders. Subjects were the 168,000 school aged children from North Dakota who were first surveyed for cases of autism--pervasive developmental disorders in 1985 and 1986. The results of the prevalence study were compared with the results of a 12-year ongoing surveillance of the cohort. The 12-year ongoing surveillance identified one case missed by the original prevalence study. Thus the original prevalence study methodology identified 98% of the cases of autism-pervasive developmental disorder in the population. This methodology may also be useful for studies of other developmental disorders in rural and frontier settings.

Aging and cognition: methodological differences in outcome.

A series of longitudinal and cross sectional studies, collected as part of the Seattle Study, were reanalyzed. A longitudinal sequential analysis (N = 232), wherein subjects were measured every seven years for four time periods, was completed on the Primary Mental Abilities test. Cohort differences were at least as strong as age differences; cohorts were generally relatively stable over the measured periods, showing little cognitive decline. A cross-sectional sequential analysis (N = 2813) was completed for the same time periods; decline with age was more evident than with the longitudinal sequential analysis. A cross-sectional analysis for the fourth time period (N = 611) showed the most marked decline of all. Interpretation of outcome was highly dependent on the analysis used.

DNA synthesis and multinucleation in embryonic stem cell-derived cardiomyocytes.

The proliferative capacity of embryonic stem (ES) cell-derived cardiomyocytes was assessed. Enriched preparations of cardiomyocytes were isolated by microdissection of the cardiogenic regions of cultured embryoid bodies. The identity of the isolated cells was established by immunocytology, and mitotic activity was monitored by [3H]thymidine incorporation and pulse-chase experiments. ES-derived cardiomyocytes were mitotically active and predominantly mononucleated at 11 days after cardiogenic induction. By 21 days postinduction, cardiomyocyte DNA synthesis was markedly decreased, with a concomitant increase in the percentage of multinucleated cells. Interestingly, the duration of active cardiomyocyte reduplication in the ES system appeared to be roughly similar to that observed during normal murine cardiogenesis. Given these observations, as well as the genetic tractability of ES cells, ES-derived cardiogenesis might provide a useful in vitro system with which to assess the molecular regulation of the cardiomyocyte cell cycle.

Prenatal and perinatal risk factors for Tourette disorder.

AIMS: To identify pre- and perinatal risk factors for Tourette disorder. METHODS: Case control study. We matched names of patients who met DSM criteria for Tourette disorder with their birth certificates. For each case five controls were selected. The controls were matched by sex, year and month of birth. RESULTS: Univariate analysis of the 92 cases and the 460 matched controls identified 4 risk factors; one categorical variable--trimester prenatal care begun and 3 continuous variables--apgar score at 5 minutes, month prenatal began and number of prenatal visits. Logistic modeling to control for confounding produced a three variable model (apgar score at 5 minutes (OR = 1.31), number of prenatal visits (OR = .904) and fathers age (OR = .909). The model parameters were: chi 2 = 19.76; df = 3; p < .001. CONCLUSIONS: This is an inexpensive methodology to identify potential risk factors of patients with Tourette disorder and other mental illness.

Prenatal and perinatal risk factors for autism.

AIM: To identify pre- and perinatal risk factors for autism. METHOD: Case control study. We matched names of patients from North Dakota who met DSM criteria for autism, a pervasive developmental disorder, and autistic disorder with their birth certificates. Five matched controls were selected for each case. RESULTS: Univariate analysis of the 78 cases and 390 controls identified seven risk factors. Logistic modeling to control for confounding produced a five variable model. The model parameters were chi 2 = 36.6 and p < 0.001. The five variables in the model were decreased birth weight, low maternal education, later start of prenatal care, and having a previous termination of pregnancy. Increasing father's age was associated with increased risk of autism. CONCLUSION: This methodology may provide an inexpensive method for clinics and public health providers to identify risk factors and to identify maternal characteristics of patients with mental illness and developmental disorders.

Involvement of caspase 3- and 8-like proteases in ceramide-induced apoptosis of cardiomyocytes.

Ceramides are the metabolic products of sphingolipids of the eukaryotic cell membranes and are believed to function as signaling molecules in a variety of biological processes. Ceramide induces apoptosis in cultured cardiomyocytes. However, the molecular pathway underlying ceramide-induced apoptosis is not clear. In this study, we investigated the role of the cysteinyl aspartate-specific proteases (caspases) in cardiomyocyte apoptosis induced by ceramide. Treatment of in vitro cultured rat neonatal cardiomyocytes with ceramide results in robust cell death, of which the majority is apoptotic, as shown by positive staining for terminal deoxyribonuclease transferase-mediated deoxyuridine triphosphate nick end-labeling and the appearance of pyknotic nuclei with Hoechst staining. Caspase 3- and 8-like protease activities are induced in cardiomyocytes by ceramide treatment. Addition of the tetrapeptide inhibitors for caspases attenuated ceramide-induced apoptosis. The nonselective caspase inhibitor (B-D-FMK) and the caspase 3 (Z-DEVD-FMK) and caspase 8 (Z-IETD-FMK) inhibitors reduced ceramide-induced cardiomyocyte death and significantly inhibited the activation of caspase 3. However, the inhibitors specific for caspases 1, 2, 4, 6, and 9 have no significant effects on cardiomyocyte survival under the same conditions. These data suggest that caspases 3- and 8-related proteases are involved in ceramide-induced cardiomyocyte apoptosis.

Strategies for myocardial repair.

The therapeutic recourse for end-stage heart disease is currently limited to cardiac transplantation. The ability to augment cardiomyocyte number in an end-stage heart might facilitate myocardial function. Augmentation of cardiomyocyte number may be achievable by the targeted expression of cell cycle regulatory genes to the myocardium. Alternatively, intracardiac grafting of exogenous cardiomyocytes might also provide a viable approach to augment cardiomyocyte number. Potential strategies for heart muscle regeneration via gene therapy and cellular transplantation are discussed.

Prevalence of psychotropic and anticonvulsant drug use among North Dakota group home residents.

In a previous study, the present authors reported on the prevalence of psychoactive (psychotropic and anticonvulsant) medication use among people with intellectual disability residing in community settings in the state of North Dakota, USA. The present study replicates the earlier survey. A questionnaire was sent to all group homes serving people with developmental disabilities. Questionnaires were obtained for 100% of North Dakota group home residents. Psychoactive medications (anticonvulsants included) were used by 38% of the 1384 residents represented. The results are discussed in relation to the previous study from North Dakota.

Long-term survival of AT-1 cardiomyocyte grafts in syngeneic myocardium.

The long-term viability of cardiomyocyte grafts in the adult myocardium was tested. AT-1 cardiomyocytes, a differentiated tumor line derived from transgenic mice expressing an atrial natriuretic factor-simian virus 40 T antigen fusion gene, were grafted directly into the myocardium of syngeneic animals. Viable grafts were detected as long as 4 mo postimplantation. Thymidine uptake studies suggested that the grafted cardiomyocytes retained mitotic activity. The presence of AT-1 cardiomyocyte grafts and the associated myocardial remodeling were not accompanied by overt cardiac arrhythmia. Electron microscopic analyses showed that the majority of the grafts were juxtaposed directly to the host myocardium and were not encapsulated. This study indicates that the myocardium can serve as a stable platform for cells that have been manipulated in vitro and suggests that cardiomyocyte grafts may provide a useful means for the local delivery of recombinant molecules to the heart. The long-term survival of the AT-1 cardiomyocytes in the heart also raises the possibility that similar grafting approaches may be used to replace diseased myocardium.