Low density lipoproteins promote unstable calcium handling accompanied by reduced SERCA2 and connexin-40 expression in cardiomyocytes

Abstract

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 mg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.3060.04 to 0.1760.02 (p,0.05). Moreover, LDLcholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 mg LDL/mL (p,0.05) and SR calcium loading was reduced by 3866% (p,0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDLcholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.760.1 mm/s with 500 mg LDL/mL (p,0.05). This coincided with a reduction in Cx40 expression (by 4463%; p,0.05 for mRNA and by 7962%; p,0.05 for Cx40 protein at 200 mg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDLcholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.