Abstract
Cholesterol regulates numerous cellular processes. Depleting its synthesis in skeletal myofibers induces vacuolization and contraction impairment. However, little is known about how cholesterol reduction affects cardiomyocyte behavior. Here, we deplete cholesterol by incubating neonatal cardiomyocytes with methyl-beta-cyclodextrin. Traction force microscopy shows that lowering cholesterol increases the rate of cell contraction and generates defects in cell relaxation. Cholesterol depletion also increases membrane tension, Ca2+ spikes frequency and intracellular Ca2+ concentration. These changes can be correlated with modifications in caveolin-3 and L-Type Ca2+ channel distributions across the sarcolemma. Channel regulation is also compromised since cAMP-dependent PKA activity is enhanced, increasing the probability of L-Type Ca2+ channel opening events. Immunofluorescence reveals that cholesterol depletion abrogates sarcomeric organization, changing spacing and alignment of α-actinin bands due to increase in proteolytic activity of calpain. We propose a mechanism in which cholesterol depletion triggers a signaling cascade, culminating with contraction impairment and myofibril disruption in cardiomyocytes.
Highlights
Extracellular Ca2+ entry is an important step for Ca2+ cycling within the cardiomyocyte and it is mediated by LTCC
The quantification of the strain energy per cell area from the traction stress maps demonstrates that control cardiomyocytes exhibit periodic contraction-relaxation patterns (Fig. 1c), with a majority of the power exerted at a frequency of 0.3 Hz (Fig. 1d)
In non-muscle cells, cholesterol regulates actin cytoskeleton architecture and cortical mechanical properties[2,3,4,5] whereas in smooth and skeletal muscle cells, cholesterol is important for modulating the contraction of these cells[6,7,8,56]
Summary
Extracellular Ca2+ entry is an important step for Ca2+ cycling within the cardiomyocyte and it is mediated by LTCC. Cholesterol depleted cardiomyocytes show a reduction in mean band spacing ((1.49 ± 0.29) and (1.47 ± 0.31) μmfor 5.0 and 7.5 mmol/L MβCD treated cells respectively) and with a much larger distribution around the mean value for both conditions (Fig. 3c).
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