19 research outputs found
In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics
The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSVβ-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods
Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter
BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2)
activity is believed to play a role in the impairment of diastolic
function of the failing heart. Because the expression ratio of
phospholamban (PL) to SERCA2 may be a target to improve contractile
dysfunction, a PL antisense RNA strategy was developed under the control
of either a constitutive cytomegalovirus (CMV) or an inducible atrial
natriuretic factor (ANF) promoter. The latter is upregulated in
hypertrophied and failing heart, allowing "induction-by-disease" gene
therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense
and sense directions into adenovectors under the control of either a CMV
(Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs,
respectively) promoter. Infection of cultured rat neonatal cardiomyocytes
with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to
24+/-3% within 48 and 72 hours, respectively. The effects were vector dose
dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and
reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL
protein was also achieved by infection with Ad5ANFPLas, and the presence
of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation
of PL. The adenovectors expressing PL sense RNA had no effect on any of
the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA
expression may become a feasible approach to modulate myocyte Ca(2+)
homeostasis in the failing heart. The inducible ANF promoter for the first
time offers the perspective for induction-by-disease gene therapy, ie,
selective expression of therapeutic genes in hypertrophied and failing
cardiomyocytes
Effects of acute exercise and long-term exercise on total Na+,K+-ATPase isoform expression profile in equine muscle.
Objective-To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. Animals-6 Standardbreds. Procedures-Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and western blotting, respectively. Results-6 Na(+),K(+)-ATPase mRNA isoforms were present in equine muscle, but only A2 and B1 proteins were detected. Exercise before training resulted in increases of mRNA isoforms A1, A2, A3, and B2 in VLM and A1 and B3 in PDM. Training increased resting values for mRNA isoforms A3 and B1 in VLM and B3 in PDM. The Na(+),K(+)-ATPase, [(3)H]ouabain binding, and proteins of mRNA A2 and B1 increased in VLM, whereas in PDM, only A2 protein increased as a result of training. After training, effects of strenuous exercise on mRNA expression were no longer detectable. Conclusions and Clinical Relevance-Equine muscle contained all Na(+),K(+)-ATPase mRNA isoforms, but only A2 and B1 proteins could be detected. Expression of these isoforms changed as a result of strenuous exercise and long-term training, representing an adaptive response. Determination of Na(+),K(+)-ATPase gene expression may be relevant for understanding alterations in excitability during neuromuscular diseases
Influence of antenatal glucocorticoid on preterm lamb diaphragm
BackgroundPregnant women at a high risk of preterm delivery receive glucocorticoids to accelerate fetal lung maturation and surfactant synthesis. However, the effect of antenatal steroids on the developing diaphragm remains unclear. We hypothesized that maternal betamethasone impairs the fetal diaphragm, and the magnitude of the detrimental effect increases with longer duration of exposure. We aimed to determine how different durations of fetal exposure to maternal betamethasone treatment influence the fetal diaphragm at the functional and molecular levels.MethodsDate-mated merino ewes received intramuscular injections of saline (control) or two doses of betamethasone (5.7 mg) at an interval of 24 h commencing either 2 or 14 days before delivery. Preterm lambs were killed after cesarean delivery at 121-day gestational age. In vitro contractile measurements were performed on the right hemidiaphragm, whereas molecular/cellular analyses used the left costal diaphragm.ResultsDifferent durations of fetal exposure to maternal betamethasone had no consistent effect on the protein metabolic pathway, expression of glucocorticoid receptor and its target genes, cellular oxidative status, or contractile properties of the fetal lamb diaphragm.ConclusionThese data suggest that the potential benefits of betamethasone exposure on preterm respiratory function are not compromised by impaired diaphragm function after low-dose maternal intramuscular glucocorticoid exposure