Background
The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.
Methods and Results
Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitin-proteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein.
Conclusions
Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.
Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.
Synadenium umbellatum Pax, popularly known as "cola-nota", is a medicinal plant that grows in tropical regions. The latex of this plant is used against various diseases, such as diabetes mellitus, leprosy, tripanosomiasis, leukemia, and several malignant tumors. The mutagenic, antimutagenic, and cytotoxic effects of the latex of this plant were investigated by measuring the frequency of micronuclei in mice bone marrow cells. To evaluate mutagenicity, the animals were treated with four doses of latex (10, 30, 50, and 100 mg/kg body weight). To study the antimutagenic activity, the animals were simultaneously treated with latex and mitomycin C (4 mg/kg). The cytotoxicity was evaluated by polychromatic and normochromatic erythrocytes ratio. Our results showed a significant increase of frequency of micronucleated polychromatic erythrocytes (MNPCE) compared to the negative control group (p < 0.05). Concerning antimutagenicity, the doses of 10 and 30 mg/kg co-administered with mitomycin C showed significant decrease in MNPCE frequency compared to the positive control group (p < 0.05). However, no significant reduction in MNPCE frequency (p > 0.05) was detected at the doses of 50 and 100 mg/kg. Under our experimental conditions, the results obtained indicate strong mutagenic and cytotoxic activity of S. umbellatum latex except the dose of 10 mg/kg and moderate antimutagenic effect at lower doses.
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