Biomedical Engineering Reference
In-Depth Information
2.8 Statistical Analysis
Pooled data are presented as mean ± standard error of the mean (SEM). Comparisons
between groups were performed using one-way ANOVA.
P
-values less than 0.05
were deemed significant.
3 Results
3.1 Neuregulin and cAMP Increase mRNA Levels of Connexin 40
To investigate whether neuregulin or cAMP might induce differentiation of
embryonic cardiomyocytes towards cells with a pacemaker-like phenotype RNA
levels of Cx-40, a marker of conduction cells, were analyzed by northern
blot experiments. Embryonic cardiomyocytes, isolated from 13-dpc mouse embryos
were treated with neuregulin-1-Į, neuregulin-1-
ȕ
and cAMP. Untreated cells served
as negative control. Two blots with RNA of each aliquot were prepared
simultaneously and were hybridized with riboprobes specific for Cx-40 or cardiac
troponin I. After signal detection by autoradiography, both blots were hybridized
with
ȕ
-actin specific probe to allow nor-malization of the amounts of loaded RNA.
The differences in Cx-40 expression were calculated by the densitometric Cx-40
value with reference to the amount of tested RNA (
ȕ
-actin) divided by the cardiac
troponin I signal which itself was adjusted to the RNA content to only detect
myocardial RNA without any interference of fibroblasts (fibroblasts make up to
50-70% of myocardial cells). Mean data of repeated series show that the
normalized values of Cx-40 expression were more than two-fold increased by
both pretreatment with neuregulin Į and
ȕ
compared to the control group (Fig. 1a).
Moreover, cAMP exposure resulted in a three-fold upregulation of Cx-40 mRNA
levels compared to control values (Fig. 1b).
3.2 Protein Levels of Connexins Are Not Modified by Neuregulin or cAMP
Since changes of mRNA levels may not readily reflect alterations of membrane
proteins we performed additional analysis of connexin expression levels by
flowcytometry following immunostaining. Day 13 fetal cardiomyocytes were
treated with neuregulin-1-Į, neuregulin-1-
ȕ
and cAMP whereas time-matched
untreated cells served as negative control. To omit any interference by
non-myocardial cells only Į-actinin expressing cells were analyzed. In contrast to
our northern blot experiments showing a clear increase of Cx-40 mRNA levels
upon neuregulin or cAMP exposure, we did not obtain any effect by either
substance on Cx-40 protein expression compared to control cells (Table 1).
Moreover, there was no change of the protein levels of Cx-43 and Cx-45 indicating
that also the relative expression levels of different connexin subtypes remained
unaffected (Table 1).
