Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA052

Poster Communications

miR-221/222 in cardiac electrical and structural remodeling

M. Knyrim1, S. Binas1, M. Hünerberg1, U. Klöckner1, S. Rabe1, S. Mildenberger1, M. Gekle1, C. Grossmann1, B. Schreier1

1. Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.


Introduction Cardiac remodeling involves structural and electrical alterations that can lead to fatal conditions like heart failure or sudden cardiac arrest. The underlying mechanisms are not fully understood. Previously we could show that epidermal growth factor receptor (EGFR) knockout (KO) mice developed a severe heart hypertrophy and changes in the electrical properties of the heart (prolonged P-duration, QRS- and QTc-intervals). NGS analysis revealed increased levels of the miR-221/222 cluster in KO mice. Additionally, we analysed two mouse models with pharmacologically induced cardiac hypertrophy (chronic angiotensin II (ANGII) or isoprenaline (ISO) treatment). miR-221/222 expression was elevated in ANGII mice but not in ISO mice. RNA-Seq experiments followed by gene enrichment analyses identified differentially regulated ion channels associated to the t-tubule cluster in EGFR KO mice which were also predicted targets of miR-221/222. This implicates a possible role of dysregulated miR-221/222 levels in cardiac electrical remodeling. Therefore, our aim was to evaluate a potential molecular mechanism underlying the miR-221/222 effects on electrical and structural cardiac remodeling in mouse models and in a cardiomyocyte cell line (HL-1). Methods We correlated miR-221/222 levels with expression (qPCR, Western blot) of cardiac ion channels (L-type Ca2+ channel (LTCC) subunits: Cacna1c, Cacnb2, Cacna2d1; voltage gated K+ channel Kv4.2; acetyl choline-activated K+ channel subunit GIRK4) in EGFR KO, ANGII and ISO treated mice. Perivascular and interstitial fibrosis were also evaluated in these models. Dual luciferase assays were performed in HEK-293 cells to validate direct interactions between the miRs and their putative target ion channel 3'-UTRs. In HL-1 cells transfected with mimics for miR-221/222 we analyzed cell size, cardiac Ca2+ and K+ channel expression as well as function (LTCC: whole cell patch clamp recording, GIRK1/4: fluorescence-based Tl+ flux assay). Additionally, as enhanced interstitial fibrosis was only observed in mice without elevated miR-221/222 expression, we further analysed fibrosis markers in these HL-1 cells. Results Cacna1c, Cacnb2, Cacna2d1, Kv4.2 and GIRK4 mRNA expression was decreased in mouse models with increased miR-221/222 levels. Interstitial fibrosis was only enhanced in mice without elevated miR-221/222 expression (ISO), but not in EGFR KO and ANGII mice. Luciferase assays revealed both LTCC and GIRK4 as direct targets of miR-221/222. HL-1 cells transfected with miR-221 mimics showed increased cell size, a decrease in mRNA expression of LTCC subunits, a significant reduction of Cacna1c and GIRK4 protein content as well as LTCC current density. Conclusion Increased expression of miR-221/222 influences electrical remodeling at least in part by downregulation of LTCCs and possibly also GIRK4. We hypothesize that miR-221 may also modulate structural remodeling.

Where applicable, experiments conform with Society ethical requirements