Regulation of the L-type Ca2+ channel expression in cardiomyocytes by corticosteroids and insulin

Abstract Intracellular calcium handling plays an important role in regulating cardiac function. The L-type Ca2+ channel is hereby crucial for the Ca2+ induced Ca2+ release in cardiomyocytes necessary for cardiac muscle contraction. Excitation-contraction coupling in cardiomyocytes is hereby critical in maintaining the synchronous heart rhythm. Electrophysiological studies have shown that an increase of the Ca2+ cur-rent in cardiomyocytes (ICaL) is a first step in the process of electrical and mechanical remodelling in heart pathologies increasing the risk of sudden cardiac death, hypertrophy and heart failure. In previous studies we saw an increase of ICaL and L-type Ca2+ mRNA (cacna1c) after treatment of primary rat cardiomyocytes with the corticosteroids dexamethasone and aldosterone for 24 h. This effect was enhanced additively by adding insulin into the culture medium. The aim of this work was to identify possible improvements for primary cell culture conditions and investigate the still unknown molecular pathways involved in the increase of the cacna1c mRNA after corticosteroid and insulin treatment. Since adult cardiomyocytes are only viable for a couple of days in cell culture and undergo a variety of morphological and functional changes, we aimed to reduce some of these negative effects by using continuous electrical pacing. Since the cacna1c gene undergoes extended alternative splicing, we looked at different exons (1a-c) found in the heart and the influence of pacing during cell culture on the mRNA expression levels. Our results showed a significant decrease of overall cacna1c mRNA after 24 h incubation which could not be prevented by electrical pacing at either 1 Hz or 3 Hz in comparison to freshly isolated cells. However, we found an increase after 24 h incubation in the expression of the exons 1b and 1c in non-paced cells, showing the impact of cell culture on alternative splicing patterns. Since the overall mRNA levels showed a signifi-cant decrease after pacing, we concluded that non-paced cardiomyocyte cell culture will be used in future experiments. The corticosteroids dexamethasone and aldosterone can both activate the glucocorticoid as well as the mineralocorticoid receptor dose dependant. Therefore, we used dose-response curves of different concentrations of aldosterone and dexamethasone with 100 nM insulin to investigate which receptor is predominantly in-volved in mediating the increase of mRNA expression and found the glucocorti-coid receptor to be primarily responsible. This was further confirmed using inhibi-tors for the mineralo- and glucocorticoid receptor. Moreover, we used luciferase assays to determine the interaction between the corticosteroid and insulin signal-ling pathways of the cacna1c mRNA upregulation. Our results showed that insulin influences the activity at the glucocorticoid response elements subsequent possibly leading to different gene expression patterns. Additionally, we used time-course experiments confirming, that secondary regulators seem to be necessary for the dexamethasone and insulin effect on cacna1c, as an increase in mRNA expression was observed between 18 to 24 h. We excluded Ca2+ influx by adding verapamil and the involvement of the translation of other proteins using cycloheximide, as main regulators for the mRNA increase. Further downstream targets involved the PI3K/Akt/mTOR and Ras/Raf/Erk1/2/MAPK insulin activated pathways. We used different kinase inhibitors for either pathway individually and both collectively but found no significant effect on cacna1c upregulation by dexame-thasone and insulin. However, the transcription factor NF-κB and the GR have been found to interact and regulate their respective signalling pathways. Therefore, we used pyrrolidinedithiocarbamate (PDTC) to inhibit NF-κB activity. Our results identified NF-κB as an important player in mediating the dexamethasone and insulin effect on cacna1c mRNA and we further validated these results with different NF-κB inhibitors. Furthermore, we utilized westernblotting to investigate, if the upregulation of mRNA expression could as well be seen on protein level but were not able to find significant differences between control and treated cells. To investigate if our results are transferable from the animal model to human hearts, we used a new technique for longterm human myocyardium tissue culture. As we were not able to isolate viable cardiomyocytes from human tissue, we used biometic cultivation chambers. We could show a similar effect of dexamethasone and insulin on the human L-type Ca2+ channel mRNA after 48 h and up to 8 days of long-term functional and structural tissue culture in this specific setup. These results solidify the importance of our studies in the rat model and future experiments might validate our findings regarding the molecular signalling pathway behind the cacna1c mRNA upregulation by dexamethasone and insulin in human heart tissue. Further identifying the role of NF-κB in regulating the L-type Ca2+ channel mRNA expression during dexamethasone treatment might be especially of interest regarding the influence on excitation-contraction coupling as well as pathologies in human tissue like inflammation and remodelling.