Effects of sublethal doses of clothianidin and/or V. destructor on honey bee (Apis mellifera) self-grooming behavior and associated gene expression.

Little is known about the combined effects of stressors on social immunity of honey bees (Apis mellifera) and related gene expression. The interaction between sublethal doses of a neurotoxin, clothianidin, and the ectoparasite, Varroa destructor, was examined by measuring differentially expressed genes (DEGs) in brains, deformed wing virus (DWV) and the proportion and intensity of self-grooming. Evidence for an interaction was observed between the stressors in a reduction in the proportion of intense groomers. Only the lowest dose of clothianidin alone reduced the proportion of self-groomers and increased DWV levels. V. destructor shared a higher proportion of DEGs with the combined stressors compared to clothianidin, indicating that the effects of V. destructor were more pervasive than those of clothianidin when they were combined. The number of up-regulated DEGs were reduced with the combined stressors compared to clothianidin alone, suggesting an interference with the impacts of clothianidin. Clothianidin and V. destructor affected DEGs from different biological pathways but shared impacts on pathways related to neurodegenerative disorders, like Alzheimer's, which could be related to neurological dysfunction and may explain their negative impacts on grooming. This study shows that the combination of clothianidin and V. destructor resulted in a complex and non-additive interaction.

Repeated Administrations of Cardiac Progenitor Cells Are Superior to a Single Administration of an Equivalent Cumulative Dose.

BACKGROUND: We have recently found that 3 repeated doses (12×106 each) of c-kitPOS cardiac progenitor cells (CPCs) were markedly more effective than a single dose of 12×106 cells in alleviating postinfarction left ventricular dysfunction and remodeling. However, since the single-dose group received only one third of the total number of CPCs given to the multiple-dose group, it is unknown whether the superior therapeutic efficacy was caused by repeated treatments per se or by administration of a higher total number of CPCs. This issue has major clinical implications because multiple cell injections in patients pose significant challenges, which would be obviated by using 1 large injection. Accordingly, we determined whether the beneficial effects of 3 repeated CPC doses can be recapitulated by 1 large dose containing the same total number of cells. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 3 echo-guided intraventricular infusions, 35 days apart, of vehicle-vehicle-vehicle, 36×106 CPCs-vehicle-vehicle, or 3 equal doses of 12×106 CPCs. Infusion of a single, large dose of CPCs (36×106 cells) produced an initial improvement in left ventricular function, but no further improvement was observed after the second and third infusions (both vehicle). In contrast, each of the 3 doses of CPCs (12×106) caused a progressive improvement in left ventricular function, the cumulative magnitude of which was greater than with a single dose. Unlike the single dose, repeated doses reduced collagen content and immune cell infiltration. CONCLUSIONS: Three repeated doses of CPCs are superior to 1 dose even though the total number of cells infused is the same, possibly because of greater antifibrotic and anti-inflammatory actions.

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

Differential gene expression in Varroa jacobsoni mites following a host shift to European honey bees (Apis mellifera).

BACKGROUND: Varroa mites are widely considered the biggest honey bee health problem worldwide. Until recently, Varroa jacobsoni has been found to live and reproduce only in Asian honey bee (Apis cerana) colonies, while V. destructor successfully reproduces in both A. cerana and A. mellifera colonies. However, we have identified an island population of V. jacobsoni that is highly destructive to A. mellifera, the primary species used for pollination and honey production. The ability of these populations of mites to cross the host species boundary potentially represents an enormous threat to apiculture, and is presumably due to genetic variation that exists among populations of V. jacobsoni that influences gene expression and reproductive status. In this work, we investigate differences in gene expression between populations of V. jacobsoni reproducing on A. cerana and those either reproducing or not capable of reproducing on A. mellifera, in order to gain insight into differences that allow V. jacobsoni to overcome its normal species tropism. RESULTS: We sequenced and assembled a de novo transcriptome of V. jacobsoni. We also performed a differential gene expression analysis contrasting biological replicates of V. jacobsoni populations that differ in their ability to reproduce on A. mellifera. Using the edgeR, EBSeq and DESeq R packages for differential gene expression analysis, we found 287 differentially expressed genes (FDR ≤ 0.05), of which 91% were up regulated in mites reproducing on A. mellifera. In addition, mites found reproducing on A. mellifera showed substantially more variation in expression among replicates. We searched for orthologous genes in public databases and were able to associate 100 of these 287 differentially expressed genes with a functional description. CONCLUSIONS: There is differential gene expression between the two mite groups, with more variation in gene expression among mites that were able to reproduce on A. mellifera. A small set of genes showed reduced expression in mites on the A. mellifera host, including putative transcription factors and digestive tract developmental genes. The vast majority of differentially expressed genes were up-regulated in this host. This gene set showed enrichment for genes associated with mitochondrial respiratory function and apoptosis, suggesting that mites on this host may be experiencing higher stress, and may be less optimally adapted to parasitize it. Some genes involved in reproduction and oogenesis were also overexpressed, which should be further studied in regards to this host shift.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy.

RATIONALE: The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE: To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS: This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year.

RATIONALE: Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. OBJECTIVE: To assess the outcome of CPC therapy at 1 year. METHODS AND RESULTS: Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. CONCLUSIONS: The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy.

Testing the kinship theory of intragenomic conflict in honey bees (Apis mellifera).

Sexual reproduction brings genes from two parents (matrigenes and patrigenes) together into one individual. These genes, despite being unrelated, should show nearly perfect cooperation because each gains equally through the production of offspring. However, an individual's matrigenes and patrigenes can have different probabilities of being present in other relatives, so kin selection could act on them differently. Such intragenomic conflict could be implemented by partial or complete silencing (imprinting) of an allele by one of the parents. Evidence supporting this theory is seen in offspring-mother interactions, with patrigenes favoring acquisition of more of the mother's resources if some of the costs fall on half-siblings who do not share the patrigene. The kinship theory of intragenomic conflict is little tested in other contexts, but it predicts that matrigene-patrigene conflict may be rife in social insects. We tested the hypothesis that honey bee worker reproduction is promoted more by patrigenes than matrigenes by comparing across nine reciprocal crosses of two distinct genetic stocks. As predicted, hybrid workers show reproductive trait characteristics of their paternal stock, (indicating enhanced activity of the patrigenes on these traits), greater patrigenic than matrigenic expression, and significantly increased patrigenic-biased expression in reproductive workers. These results support both the general prediction that matrigene-patrigene conflict occurs in social insects and the specific prediction that honey bee worker reproduction is driven more by patrigenes. The success of these predictions suggests that intragenomic conflict may occur in many contexts where matrigenes and patrigenes have different relatednesses to affected kin.

The complete mitochondrial genome of the invasive Africanized Honey Bee, Apis mellifera scutellata (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome from an Africanized honey bee population (AHB, derived from Apis mellifera scutellata) was assembled and analyzed. The mitogenome is 16,411 bp long and contains the same gene repertoire and gene order as the European honey bee (13 protein coding genes, 22 tRNA genes and 2 rRNA genes). ND4 appears to use an alternate start codon and the long rRNA gene is 48 bp shorter in AHB due to a deletion in a terminal AT dinucleotide repeat. The dihydrouracil arm is missing from tRNA-Ser (AGN) and tRNA-Glu is missing the TV loop. The A + T content is comparable to the European honey bee (84.7%), which increases to 95% for the 3rd position in the protein coding genes.

Biased Allele Expression and Aggression in Hybrid Honeybees may be Influenced by Inappropriate Nuclear-Cytoplasmic Signaling.

Hybrid effects are often exhibited asymmetrically between reciprocal families. One way this could happen is if silencing of one parent's allele occurs in one lineage but not the other, which could affect the phenotypes of the hybrids asymmetrically by silencing that allele in only one of the hybrid families. We have previously tested for allele-specific expression biases in hybrids of European and Africanized honeybees and we found that there was an asymmetric overabundance of genes showing a maternal bias in the family with a European mother. Here, we further analyze allelic bias in these hybrids to ascertain whether they may underlie previously described asymmetries in metabolism and aggression in similar hybrid families and we speculate on what mechanisms may produce this biased allele usage. We find that there are over 500 genes that have some form of biased allele usage and over 200 of these are biased toward the maternal allele but only in the family with European maternity, mirroring the pattern observed for aggression and metabolic rate. This asymmetrically biased set is enriched for genes in loci associated with aggressive behavior and also for mitochondrial-localizing proteins. It contains many genes that play important roles in metabolic regulation. Moreover we find genes relating to the piwi-interacting RNA (piRNA) pathway, which is involved in chromatin modifications and epigenetic regulation and may help explain the mechanism underlying this asymmetric allele use. Based on these findings and previous work investigating aggression and metabolism in bees, we propose a novel hypothesis; that the asymmetric pattern of biased allele usage in these hybrids is a result of inappropriate use of piRNA-mediated nuclear-cytoplasmic signaling that is normally used to modulate aggression in honeybees. This is the first report of widespread asymmetric effects on allelic expression in hybrids and may represent a novel mechanism for gene regulation.

A Search for Parent-of-Origin Effects on Honey Bee Gene Expression.

Parent-specific gene expression (PSGE) is little known outside of mammals and plants. PSGE occurs when the expression level of a gene depends on whether an allele was inherited from the mother or the father. Kin selection theory predicts that there should be extensive PSGE in social insects because social insect parents can gain inclusive fitness benefits by silencing parental alleles in female offspring. We searched for evidence of PSGE in honey bees using transcriptomes from reciprocal crosses between European and Africanized strains. We found 46 transcripts with significant parent-of-origin effects on gene expression, many of which overexpressed the maternal allele. Interestingly, we also found a large proportion of genes showing a bias toward maternal alleles in only one of the reciprocal crosses. These results indicate that PSGE may occur in social insects. The nonreciprocal effects could be largely driven by hybrid incompatibility between these strains. Future work will help to determine if these are indeed parent-of-origin effects that can modulate inclusive fitness benefits.

Genomic correlates of recombination rate and its variability across eight recombination maps in the western honey bee (Apis mellifera L.).

BACKGROUND: Meiotic recombination has traditionally been explained based on the structural requirement to stabilize homologous chromosome pairs to ensure their proper meiotic segregation. Competing hypotheses seek to explain the emerging findings of significant heterogeneity in recombination rates within and between genomes, but intraspecific comparisons of genome-wide recombination patterns are rare. The honey bee (Apis mellifera) exhibits the highest rate of genomic recombination among multicellular animals with about five cross-over events per chromatid. RESULTS: Here, we present a comparative analysis of recombination rates across eight genetic linkage maps of the honey bee genome to investigate which genomic sequence features are correlated with recombination rate and with its variation across the eight data sets, ranging in average marker spacing ranging from 1 Mbp to 120 kbp. Overall, we found that GC content explained best the variation in local recombination rate along chromosomes at the analyzed 100 kbp scale. In contrast, variation among the different maps was correlated to the abundance of microsatellites and several specific tri- and tetra-nucleotides. CONCLUSIONS: The combined evidence from eight medium-scale recombination maps of the honey bee genome suggests that recombination rate variation in this highly recombining genome might be due to the DNA configuration instead of distinct sequence motifs. However, more fine-scale analyses are needed. The empirical basis of eight differing genetic maps allowed for robust conclusions about the correlates of the local recombination rates and enabled the study of the relation between DNA features and variability in local recombination rates, which is particularly relevant in the honey bee genome with its exceptionally high recombination rate.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Fine-scale linkage mapping reveals a small set of candidate genes influencing honey bee grooming behavior in response to Varroa mites.

Populations of honey bees in North America have been experiencing high annual colony mortality for 15-20 years. Many apicultural researchers believe that introduced parasites called Varroa mites (V. destructor) are the most important factor in colony deaths. One important resistance mechanism that limits mite population growth in colonies is the ability of some lines of honey bees to groom mites from their bodies. To search for genes influencing this trait, we used an Illumina Bead Station genotyping array to determine the genotypes of several hundred worker bees at over a thousand single-nucleotide polymorphisms in a family that was apparently segregating for alleles influencing this behavior. Linkage analyses provided a genetic map with 1,313 markers anchored to genome sequence. Genotypes were analyzed for association with grooming behavior, measured as the time that individual bees took to initiate grooming after mites were placed on their thoraces. Quantitative-trait-locus interval mapping identified a single chromosomal region that was significant at the chromosome-wide level (p<0.05) on chromosome 5 with a LOD score of 2.72. The 95% confidence interval for quantitative trait locus location contained only 27 genes (honey bee official gene annotation set 2) including Atlastin, Ataxin and Neurexin-1 (AmNrx1), which have potential neurodevelopmental and behavioral effects. Atlastin and Ataxin homologs are associated with neurological diseases in humans. AmNrx1 codes for a presynaptic protein with many alternatively spliced isoforms. Neurexin-1 influences the growth, maintenance and maturation of synapses in the brain, as well as the type of receptors most prominent within synapses. Neurexin-1 has also been associated with autism spectrum disorder and schizophrenia in humans, and self-grooming behavior in mice.

High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees.

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene 'no receptor potential A' and a dopamine receptor. 'No receptor potential A' is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis-regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

A genetic analysis of the stinging and guarding behaviors of the honey bee.

In order to identify genes that are influencing defensive behaviors, we have taken a new approach by dissecting colony-level defensive behavior into individual behavioral measurements using two families containing backcross workers from matings involving European and Africanized bees. We removed the social context from stinging behavior by using a laboratory assay to measure the stinging response of individual bees. A mild shock was given to bees using a constant-current stimulator. The time it took bees to sting in response to this stimulus was recorded. In addition, bees that were seen performing guard behaviors at the hive entrance were collected. We performed QTL mapping in two backcross families with SNP probes within genes and identified two new QTL regions for stinging behavior and another QTL region for guarding behavior. We also identified several candidate genes involved in neural signaling, neural development and muscle development that may be influencing stinging and guarding behaviors. The lack of overlap between these regions and previous defensive behavior QTL underscores the complexity of this behavior and increases our understanding of its genetic architecture.

Multiple routes of pesticide exposure for honey bees living near agricultural fields.

Populations of honey bees and other pollinators have declined worldwide in recent years. A variety of stressors have been implicated as potential causes, including agricultural pesticides. Neonicotinoid insecticides, which are widely used and highly toxic to honey bees, have been found in previous analyses of honey bee pollen and comb material. However, the routes of exposure have remained largely undefined. We used LC/MS-MS to analyze samples of honey bees, pollen stored in the hive and several potential exposure routes associated with plantings of neonicotinoid treated maize. Our results demonstrate that bees are exposed to these compounds and several other agricultural pesticides in several ways throughout the foraging period. During spring, extremely high levels of clothianidin and thiamethoxam were found in planter exhaust material produced during the planting of treated maize seed. We also found neonicotinoids in the soil of each field we sampled, including unplanted fields. Plants visited by foraging bees (dandelions) growing near these fields were found to contain neonicotinoids as well. This indicates deposition of neonicotinoids on the flowers, uptake by the root system, or both. Dead bees collected near hive entrances during the spring sampling period were found to contain clothianidin as well, although whether exposure was oral (consuming pollen) or by contact (soil/planter dust) is unclear. We also detected the insecticide clothianidin in pollen collected by bees and stored in the hive. When maize plants in our field reached anthesis, maize pollen from treated seed was found to contain clothianidin and other pesticides; and honey bees in our study readily collected maize pollen. These findings clarify some of the mechanisms by which honey bees may be exposed to agricultural pesticides throughout the growing season. These results have implications for a wide range of large-scale annual cropping systems that utilize neonicotinoid seed treatments.

Human cardiac stem cells isolated from atrial appendages stably express c-kit.

The in vivo studies of myocardial infarct using c-ki⁺/Lin⁻ cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit⁺/Lin⁻ cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit⁺/Lin⁻ cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit⁺/Lin⁻ cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit⁺/Lin⁻ cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit⁺/Lin⁻ cell population. Following induced differentiation, c-kit⁺/Lin⁻ CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit⁺ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit⁺/Lin⁻ cells for clinical trials in repairing myocardial infarction.

Atorvastatin therapy during the peri-infarct period attenuates left ventricular dysfunction and remodeling after myocardial infarction.

Although statins impart a number of cardiovascular benefits, whether statin therapy during the peri-infarct period improves subsequent myocardial structure and function remains unclear. Thus, we evaluated the effects of atorvastatin on cardiac function, remodeling, fibrosis, and apoptosis after myocardial infarction (MI). Two groups of rats were subjected to permanent coronary occlusion. Group II (n = 14) received oral atorvastatin (10 mg/kg/d) daily for 3 wk before and 4 wk after MI, while group I (n = 12) received equivalent doses of vehicle. Infarct size (Masson's trichrome-stained sections) was similar in both groups. Compared with group I, echocardiographic left ventricular ejection fraction (LVEF) and fractional area change (FAC) were higher while LV end-diastolic volume (LVEDV) and LV end-systolic and end-diastolic diameters (LVESD and LVEDD) were lower in treated rats. Hemodynamically, atorvastatin-treated rats exhibited significantly higher dP/dt(max), end-systolic elastance (Ees), and preload recruitable stroke work (PRSW) and lower LV end-diastolic pressure (LVEDP). Morphometrically, infarct wall thickness was greater in treated rats. The improvement of LV function by atorvastatin was associated with a decrease in hydroxyproline content and in the number of apoptotic cardiomyocyte nuclei. We conclude that atorvastatin therapy during the peri-infarct period significantly improves LV function and limits adverse LV remodeling following MI independent of a reduction in infarct size. These salubrious effects may be due in part to a decrease in myocardial fibrosis and apoptosis.