Myocardialinfarction-induced heart failure (16weeks) is accompanied by a decrease in miR-1 levels, and rescue of contractionby in vivo gene transfer of SERCA2a AAV9 reverses miR-1 loss. Bioinformaticsequence analysis links miR-1 to the sodium calcium exchanger (NCX), whose expressionin heart failure is upregulated. Increase of NCX competes for diastolic calciumwith SERCA2a-induced SR loading, contributing to the contractile dysfunction.Therefore restoration of miR-1 (and consequently NCX) expression could counterthese detrimental changes relating to calcium handling. In this study we aimedto establish whether downregulation of miR-1 would produce changes expected forincreased NCX activity.Methods
Cardiomyocytes were enzymatically isolated and transfectedby lipofection with pre miR-1, anti miR-1 (100nM) or scrambled controls, thencultured for 24 or 48 hours. Quantitative RT-PCR was used to assess changes inmiR-1 expression with the video edge detection system (IonOptix) used to study contractility. The fluorescent dye Fluo-4AM was loaded intothe cardiomyocytes with 0.16% Pluronic Acid F127 to visualise intracellularcalcium. A rapid application of caffeine was applied to release SR calciumstores and reveal NCX-dependent effects.Results
QuantitativeRT-PCR confirmed successful transfection, but Pre/ Anti miR-1 treatment toboost/ depress miR-1 expression had no effect on cardiomyocyte contractility inresponse to increasing calcium, isoprenaline (a beta-adrenoceptor agonist) orfrequency of stimulation. However after a caffeine spritz, the rate of calcium removal wasaccelerated for those cells treated with anti miR-1 (n=14) compared to thescrambled control (n=21); decay constant Tau ± SEM 1063 ± 66.2 vs 1481 ± 131.2 milliseconds,*P < 0.05.Conclusion
These results are consistent with an effect ofmiR-1 downregulation to increase NCX activity. Modest changes in NCX-induceddiastolic calcium removal did not affect overall cardiomyocyte function, likelydue to compensatory effects of other calcium handling systems. NCX alterations may be part of a co-ordinatedprogramme of changes initiated by miR-1 during heart failure.