Potassium channel Shaker play a protective role against cardiac aging in Drosophila.

Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.

[The mechanism underlying histone deacetylases regulating cardiac hypertrophy].

Cardiac hypertrophy is a compensatory response that occurs as a result of increased hemodynamic requirement in peripheral tissues. In the process of cardiac hypertrophy, the expression of different types of genes in different stages is transcriptionally regulated by multiple-level physiological and pathological signals. Histone acetylation, as the most extensive post-translational modification, is closely controlled by the antagonistic histone acetyltransferases (HAT) and histone deacetylases (HDACs). Recent studies have shown that HDACs, as a family of enzymes that inhibit transcription and contain highly conserved deacetylase domains, regulate gene expression during cardiac hypertrophy through a variety of pathways. In this review, we mainly summarize the research progress on histone deacetylase in cardiac hypertrophy. By elucidating the role and molecular mechanism of different HDACs in cardiac hypertrophy, it provides new ideas for the treatment of different types of cardiac hypertrophy and heart failure, and molecular targets for new drug design.

Lifetime regular exercise affects the incident of different arrhythmias and improves organismal health in aging female Drosophila melanogaster.

We used Drosophila melanogaster as an animal model system to study the impact of exercise training initiated early in life on cardiac function using a well-established model of inherent myogenic properties of the heart and discussed the changes on myosin, a myocardial contractile protein. We also explored the effect of early physical exercise on organismal aging by analyzing the wake-sleep pattern using a Drosophila activity monitor system. We found that a variety of arrhythmias are part of the heart spectrum in old flies after a lifetime of physical exercise as evidenced by reducing the incidence of fibrillations and increasing the occurrence of bradycardias. Maintenance of myocardial myosin levels may be an underlying contributor to these exercise-induced improvements in cardiac function at an advanced age. Moreover, we found that exercise training resulted in improved sleep quality by ameliorating age-related sleep inefficiency, fragmentation and sleep consolidation.

[Construction and assessment of heart-specific green fluorescence zebrafish line].

Using the promoter for cardiac myosin light chain 2 (cmlc2) gene, an expression vector pTol2-cmlc2-IRES- EGFP for making heart-specific expression of exogenous gene in transgenic zebrafish was generated previously. Here, we reported the construction of a transgenic zebrafish line which stably expresses EGFP using this vector, and the effects of EGFP on the heart development and cardiac function of this transgenic zebrafish line were preliminarily analyzed. The results showed that the green fluorescence signal of cmlc2:EGFP line under fluorescence microscopy specifically expressed in heart and faithfully recapitulated both the spatial and temporal expression patterns of endogenous cmlc2 gene revealed by in situ hybridization in the early developmental stages. The cardiac morphology and development of this transgenic zebrafish line remained to be normal. Furthermore, the heart morphology and physiological function of this transgenic line have been analyzed using M-mode analysis. The results showed that there was no significant difference between the cmlc2:EGFP and the wild type lines with respect to heart period, heart rate, diastolic surface area and systolic surface area, and fractional area change. No tachyarrhythmia was observed in the embryos from either line. Thus, the excessive expression of EGFP in this transgenic line seemed to exert no detrimental effects on the function and development of zebrafish hearts during early stages. Our study laid a foundation for the construction of exogenous gene transgenic line using pTol2-cmlc2-IRES-EGFP vector to study the function of genes that expressed in heart.

[Establishment of hypertrophic chondrocytes-specific Cre transgenic mice].

Hypertrophic chondrocytes, which are the terminally differentiated form of chondrocytes, play a key role in endochondral ossification. In order to investigate the functions of hypertrophic chondrocytes during bone development, we generated a new transgenic line expressing Cre recombinase under the control of a 8.2 kb mouse type X collagen gene promoter (Col10a1(8.2)-Cre). Microinjection was employed to introduce the 11.5 kb transgenic fragment into 328 oocytes, from which 51 progenies were obtained. Three mice carrying the transgene in genome were identified by PCR genotyping. PCR detected expression of Col10a1(8.2)-Cre transgene within tissues containing hypertrophic chondrocytes. To examine the activity and specificity of Cre recombinase in vivo, transgenic line was crossed with ROSA26 report line. As indicated by LacZ staining, ROSA26; Col10a1(8.2)-Cre double transgenic mice showed efficient expression of Cre recombinase within hypertrophic chondrocytes. In situ hybridization analyses further confirmed the transcription of Col10a1(8.2)-Cre transgene within the upper zone of hypertrophy, indicating a better activity and specificity in contrast to the previously constructed Col10a1(1.0)-Cre transgenic line. These results showed that this Col10a1(8.2)-Cre transgenic line could be used as a powerful tool to achieve conditional gene knockout in hypertrophic chondrocytes.

Role of svp in Drosophila pericardial cell growth.

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DAI muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.

[Inhibitory effects of human papilloma virus 18 E6 gene in HeLa cell transfected with shRNA].

OBJECTIVE: To investigate the inhibition of HPV18E6 gene in HeLa cell transfected with plasmid expressing human papilloma virus 18 E6 (HPV18E6) short hairpin RNA (shRNA). METHODS: We synthesized two HPV18E6 shRNA frames and sub-cloned them into pSUPER which can express shRNA in mammalian cells to construct pE6-1shRNA and pE6-2shRNA which were mutant in E6 shRNA frame. The pE6-1shRNA, pE6-2shRNA and pcDNA3.1 were co-transfected into HeLa cells by cationic liposome respectively and the positive transfectants were selected by G418. The HPV18E6 mRNA and protein expression level was detected by semi-quantitative RT-PCR and streptavidin-peroxidase conjugated method (SP) to assay the inhibitory effects of pE6shRNA. RESULTS: We successfully constructed several new HeLa cell lines transfected with pE6-1shRNA and pE6-2shRNA. In the HeLa cells without transfection and the HeLa cells transfected with pE6-1shRNA plasmid, the HPV18E6 mRNA levels were 1.14 +/- 0.45, 0.76 +/- 0.28 respectively, and the difference of HPV18E6 mRNA levels was significant (P < 0.05). The inhibition efficiency of HPV18E6 gene mRNA was 33.3% and the HPV18E6 protein levels were declined after transfection with pE6-1shRNA. In the HeLa cells transfected with pE6-2shRNA and pSUPER plasmids, HPV18E6 mRNA and protein expression levels were not different from those in wild HeLa cells. CONCLUSIONS: The pE6-1shRNA plasmid can inhibit HPV18E6 expression in HeLa cells, which is persistent, specific and heritable.

Fatiguing exercise initiated later in life reduces incidence of fibrillation and improves sleep quality in Drosophila.

As the human body ages, the risk of heart disease and stroke greatly increases. While there is evidence that lifelong exercise is beneficial to the heart's health, the effects of beginning exercise later in life remain unclear. This study aimed to investigate whether exercise training started later in life is beneficial to cardiac aging in Drosophila. We examined 4-week-old wild-type virgin female flies that were exposed to exercise periods of either 1.5, 2.0, or 2.5 h per day, 5 days a week for 2 weeks. Using M-mode traces to analyze cardiac function by looking at parameters including heart rate, rhythmicity, systolic and diastolic diameter, and interval and fractional shortening, we found that cardiac function declined with age, shown by an increase in the number of fibrillation events and a decrease in fractional shortening. About 2.0 and 2.5 h of exercise per day displayed a reduced incidence of fibrillation events, and only physical exercise lasting 2.5-h period increased fractional shortening and total sleep time in Drosophila. These data suggested that training exercise needs to be performed for longer duration to exert physiological benefits for the aging heart. Additionally, climbing ability to assess the exercise-induced muscle fatigue was also measured. We found that 2.0 and 2.5 h of exercise caused exercise-induced fatigue, and fatiguing exercise is beneficial for cardiac and healthy aging overall. This study provides a basis for further study in humans on the impact of beginning an exercise regimen later in life on cardiac health.

[Functional analysis of the human T-type calcium channel alpha 1H subunit gene in cellular proliferation].

An increase in the functional expression of the T-type calcium (Ca) channel previously has been proposed to be associated with vascular smooth muscle cell proliferation although direct evidence for the channel causing these effects remains to be demonstrated. In this study, we provide evidence that stable over-expression of the alpha 1H subunit of the T-type Ca channel (CACNA1H) in HEK-293 cells confers a significant growth advantage. Over-expression of the alpha 1H subunit of the T-channel was confirmed in stable transfects by RT-PCR analysis using specific primer pairs and also by electrophysiology. Growth curve assays showed population doubling time for alpha 1H stable transfects was 14.0 +/- 0.4 h, whereas control cultures had a population doubling time of 22.1 +/- 1.1 h (n = 3, P < 0.05). In addition, total cell numbers significantly increased in stable transfects at all time points investigated from 48-120 h after plating (5 x 10(3) cells/well) compared with control cultures. Consistent with these findings flow cytometry showed that 53.9% of control cells were in G1, 34.5% in S and 11.6% in G2/M whereas alpha 1H transfectants displayed 45.6% of cells in G1, 44.6% in S and 9.8% in G2/M. Finally, the Western blotting results verified that the levels of protein expression of CDK2, cyclin A and cyclin E were relatively high in alpha 1H transfectants compared to control cultures. Our results demonstrate that the T-type Ca channel provides a growth advantage to HEK-293 cells when stably expressed and that it probably exerts these effects via control of the G1/S cell cycle mechanism.

[The use of RNAi as a technique to study the functions of heart-related genes in Drosophila].

RNAi is a recently developed method to block the activity of cellular genes by artificially providing sense and anti-sense RNA corresponding to a target gene. By inducing rapid degradation of the corresponding endogenous mRNA and blocking new mRNA synthesis, RNAi leads to post-transcriptional gene silencing. Now this phenomenon has been claimed to exist in C. elegans, Drosophila, buds, fungi and plants and is being used to study the functions of some special genes or the known genes at specific time point. It is extremely useful for those genes or organisms that their mutants are not easily obtained. The Drosophila heart related genes, tinman and wingless, have been shown to play an important role in coordinating the early formation of heart progenitor cells and precursors, yet the late function is still unexplored. In this experiment, we took the advantage of RNAi technique, microinjected tinman and wingless dsRNA into the early embryos in Drosophila respectively and got these two genes' RNAi phenotypes, which were very similar to that of their mutant, showing heart tube defects or no heart precursors formation. tinman dsRNA even caused visceral mesoderm defects and the somatic muscles disruption, yet wingless dsRNA only affected heart precursors and had no effect on visceral mesoderm and somatic muscles, indication that the heart-related genes dsRNA interference worked effectively and exclusively in Drosophila.

[Screening of the genes in controlling heart development of Drosophila].

It is becoming increasingly evident that remarkable similaries of heart development are revealed in Drosophila and vertebrate, therefore Drosophila can be used as a prototype to explore the vertebrate. This can in accelerate to revealing of the machanisms of human heart development. In order to screen and clone new genes that control the heart development,we have established the balanced-lethal lines by chemical mutagen and performed the heart-specific antibody. Ten of lines showed mutant phenotype,of which 6 were determined the smaller genetic sites for gene location.

[Progress of the calcium signal pathway during cardiomyogenesis and cardiomyocyte differentiation].

The process of cardiomyogenesis and cardiomyocyte differentiation in vertebrates is complex, which is controlled by a series of cardiac embryonic genes. The underlying calcium signaling pathways are important to the expression of these genes. The role of calcium dependent transcription process, Ca2+/CaM/CaN/NF AT/GATA 4 transcription pathway and Ca2+/CaM/ CaMK/HDAC/MEF2 transcription pathway, in cardiomyogenesis and cardiomyocyte differentiation are reviewed.

[Establishment and application of double color and fiber-FISH].

To determine whether genetic material in 5q13.3 breakpoint region changed in the course of establishing the cell line with a constitutional chromosomal inversion inv(5)(p13.1q13.3) associated with hairy cell leukemia, double color fluorescence in situ hybridization (FISH) on metaphase, interphase and DNA fibers were performed in cells of the cell line with the cosmids cCI5-216 and cCI5-267 DNA probes labeled by either biotinylate or digoxigenin. It showed that the cell line gave the same results as those of the original cells, which indicated that no change of the genetic material at 5q13.3 breakpoint region occurred. The cell line is valuable to reveal the molecular pathogenesis of hairy cell leukemia.

WAVE1 regulates P-glycoprotein expression via Ezrin in leukemia cells.

For children with acute myeloblastic leukemia (AML), multidrug resistance (MDR) reduces treatment effectiveness, and often leads to poor patient survival. While a number of factors have been described that affect MDR, the mechanisms underlying this effect remain unclear. In this study, the role of WAVE1 in MDR was investigated. Among 62 children with AML, high levels of WAVE1 were associated with poor patient outcomes. Proteomic techniques were used to identify novel WAVE1-interacting proteins from leukemia cells, one of which was the cytoskeleton regulator Ezrin. In leukemia cells, WAVE1 co-localized with both Ezrin and P-glycoprotein (P-gp), a critical regulator of the MDR phenotype. Overexpression of WAVE1 in K562 leukemia cells up-regulated P-gp and Ezrin, and reduced K562 cells' sensitivity to the chemotherapy drug adriamycin. The opposite effect was seen when WAVE1 expression was reduced via RNA interference. Critically, overexpression of WAVE1 in the absence of Ezrin did not affect P-gp levels or MDR. These data suggest that WAVE1 affects P-gp and MDR of leukemia cells through Ezrin.

[Interaction of WAVE1 and genes involved in multiple drug resistance in children with acute myeloblastic leukemia].

OBJECTIVE: Multidrug resistance (MDR) is one of the primary causes of suboptimal outcomes in chemotherapy of children with acute myeloblastic leukemia (AML). The mechanisms of drug transport resistance may chiefly contribute to MDR. Expression and/or activity of P-glycoprotein (P-gp), multiple resistance-associated protein-1 (MRP1), lung-resistance related protein (LRP) and breast cancer resistance protein (BCRP) have been considered to be associated with unfavourable outcomes in pediatric AML patients. In previous studies, we found WASP-family verprolin-homologous protein-1 (WAVE1) was involved in the MDR mechanisms in K562/A02 leukemia cells. To investigate the expression of WAVE1, P-gp, MRP1, LRP/MVP and BCRP; and if WAVE1 is involved in MDR of human leukemia cell. METHODS: WAVE1, P-gp, MRP1, LRP, BCRP mRNA and protein expression in bone marrow mononuclear cells (BMMCs) were measured by real-time fluorescence quantitative PCR (RQ-PCR) and Western blot in a cohort of 52 children with acute myeloblastic leukemia. During follow-up, of the 52 patients, 21 were documented as being relapsing or refractory, and 31 were induced into complete continuous remission. Furthermore, HL60 cells and HL60/ADR cells were transiently transfected with PCDNA3.1-WAVE1 reconstructed plasmid and specifically siRNA to WAVE1 respectively, and the expression of WAVE1, MRP1 and BCRP before and after transfection was assessed by real-time PCR and Western blot analysis. RESULTS: (1) The expression levels of WAVE1, P-gp, MRP, LRP and BCRP in refractory/relapsing group were much higher than that in complete continuous remission (CCR) group. (2) WAVE1 mRNA and protein expression in BMMCs of children were at higher levels when they were newly diagnosed or relapsed, compared with complete continuous remission. (3) The WAVE1 expression at mRNA and protein level in HL60/ADR cells was increased by about 353% and 95% respectively as compared with that in HL60 cells. (4) Overexpression of WAVE1 in HL60 cell lines upregulated the expression levels of MRP and BCRP (MRP mRNA and protein level were increased by about 16.54 times and 129% respectively, BCRP was increased by 4.93 times and 96%); whereas suppression of WAVE1 expression by RNA interference downregulated the expression levels of MRP1 and BCRP (MRP mRNA and protein level was only 11% and 43% of pre-disturbance respectively, BCRP was 14% and 71%). CONCLUSIONS: Higher levels of WAVE1 in the BM indicate an unfavorable prognosis in children with AML. WAVE1 is related to the development of AML and involved in the MDR mechanisms, and regulates the level of MRP1 and BCRP.

[Allelic loss of 6q16.3 microsatellite DNA in childhood acute lymphoblastic leukemia and bioinformatics analysis].

OBJECTIVE: To locate the cluster region of loss of heterozygosity (LOH) in children with acute lymphoblastic leukemia (ALL), and explore the new tumor suppressor gene. METHODS: Allelic loss was analyzed by PCR with 15 microsatellite markers mapping on 6q16.3. The LOH was analyzed by bioinformatics. The relationship between LOH and clinical factors was further analyzed. RESULTS: The frequency of LOH at least at one loci on 6q16.3 was 32.7%. The LOH in relapsed patients was higher than those in not relapsed. The higher frequency of LOH was observed in two regions of D6S1709-D6S1028 and D6S2160-D6S1580 at 6q16.3. GRIK2 may be a candidate of tumor suppressor gene. There are 12 ESTs may carry out new anti-oncogene. Patients with 6q LOH had higher WBC counts (P < 0.01), blast cells percentage (P < 0.01), relapse rate (P < 0.05) and chromosomal aberration (P < 0.05). CONCLUSION: D6S1709-D6S1028 and D6S2160-D6S1580 are two regions of minimus deletion on 6q16.3 in which tumor suppressor gene may exist. The LOH on 6q16.3 may be a prognostic index of children with ALL.

[Functional analysis of the alpha1G subunit of the T-type calcium channel in cellular proliferation].

Increases of functional T-type calcium channel (T-channel) expression have been associated with cellular proliferation although evidence for this remains controversial. In the present study, we have used a variety of cellular, molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation. The results showed that stable over-expression of alpha1G T-channel subunit in HEK-293 cells conferred a significant growth advantage. Thus, cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants, compared to control cultures (22.1 +/- 1.1 h) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/G1 phases of the cell cycle compared to controls. The selective T-type calcium channel blocker, mibefradil, induced a dose-dependent inhibition of proliferation in alpha1G tansfectants. Furthermore, the Western blotting results proved that the level of protein expression of CDK2, cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures. Our results demonstrate that the T-type calcium channel provides a significant growth advantage to HEK-293 cells that might occur via effects on the G1/S cell cycle mechanism.

[Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs].

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.

Polymorphisms of four bone mineral density candidate genes in Chinese populations and comparison with other populations of different ethnicity.

Studies on polymorphisms of candidate genes and their association with bone mineral density (BMD) have been reported in many populations, but few have been reported in Chinese populations. We investigated polymorphisms of the following five commonly used markers of four prominent BMD candidate genes with the purpose of identifying useful genetic markers for osteoporosis genetic research in Chinese: the Sp1 and RsaI polymorphisms of the collagen type 1 alpha l (Col1a1) gene, the -174G/C promoter polymorphism of the interleukin 6 (IL-6) gene, the Asn363Ser polymorphism of the glucocorticoid receptor (GR) gene, and the T --> C polymorphism in intron 5 of the transforming growth factor beta(1) (TGF-beta(1)) gene. We evaluated these polymorphisms using PCR-RFLP in samples of at least 124 random individuals. We compared the polymorphisms of these five markers with other populations using the chi(2) test and Fisher's exact two-tailed test. For the RsaI polymorphism, only three heterozygotes but no variant homozygote were identified. For the -174G/C polymorphic site, only one GC heterozygote and no CC homozygote were found. Alleles s, Ser, and A(1) at the Sp1, Asn363Ser, and T --> C marker sites that have been found to be polymorphic in other populations were not found in Chinese. Significant differences of allele and genotype frequency distributions were observed at these polymorphisms ( P < 0.001) after comparing with other populations. Our results suggest that variant alleles of the five markers are absent or too rare to be useful genetic makers in Chinese, despite the fact that they have been commonly used as polymorphic markers in osteoporosis genetic research in other populations.