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 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol 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 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol 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.

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems

“…NRVMs were centrifuged at 990 g at 4°C for 10 min, and cell pellets were resuspended in Dulbecco's modified Eagle's medium ϩ Glu-taMAX (10565-042; ThermoFisher), supplemented with 5% horse serum (16050 -122; ThermoFisher), pyruvic acid (3 mM, P-8574; Sigma), bovine serum albumin (2 g/L, A-7030; Sigma), ampicillin (100 g/mL, A-0166; Sigma), 100ϫ insulin-transferrin-selenium (41400045; ThermoFisher), antibiotic/antimycotic (1%, 15240062; ThermoFisher), linoleic acid (5 g/mL, L5900; Sigma), and ascorbic acid (100 M, Sigma; A4544), and then preplated to remove fibroblasts (2 ϫ 30 min, 37°C, 5% CO 2). Purified NRVMs were seeded on MEAs precoated with fibronectin (10 g/mL, F2006; Sigma) by either coating the whole MEA well (1 mL at 1,400,000 cells/mL, final density of 500,000 cells/cm 2 ) or only the central recording matrix with a 75 l droplet of 300,000 NRVMs. To ensure stable droplet formation critical to establishing a confluent cardiomyocyte monolayer, MEA hydrophobicity was increased by heating to 70°C for Ն2 h before seeding.…”

Physiological and therapeutic regulation of PCSK9 activity in cardiovascular disease

“…These experiments suggest that LDL-C may improve cardiac function. On the other hand, at least in atrial-like cardiomyocytes (HL-1 cells, an immortalized cell line with some cardiac-specific differentiation), LDL-C decreases the expression of sarcoplasmatic reticulum (SR) calcium ATPase (SERCA) 2a, ryanodine receptor 2 (RyR2), and connexin-40 within 24 h. This effect of LDL-C reduced calcium transients and slowed conduction velocity in such cells [ 9 ]. It remains elusive whether or not this difference is due to time differences between the studies (minutes vs. hours) or due to the source of myocytes (atrial vs. ventricular).…”

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes

“…To detect changes in intracellular calcium handling, HL-1 cardiomyocytes were loaded with CAL-520 AM (#21130 AAT Bioquest) and calcium was assessed in a 0.5 × 0.5 mm 2 of the cell culture using a resonance scanning confocal microscope (Leica SP5 AOBS) with a 20× objective as previously described [30]. Briefly, cell cultures were loaded with 2 μM of the calcium indicator CAL-520 AM for 20 min at RT followed by 30 min of deesterification.…”