Abstract 902: Leveraging Natural Cardiomyocyte Variability to Investigate Downregulation of β-1 Adrenoceptors Following Dobutamine Treatment

Abstract

In heart failure, the most consistent effect on the myocardial beta-adrenergic pathway is a downregulation of β-1 adrenoceptors (β1AR). This loss of receptors impairs the ability of the heart to respond to increases in cardiac demand, and can lead to decreased myocyte function even at rest. As a model of the process that leads to downregulation of β1AR, we measured intracellular Ca 2+ transients and unloaded sarcomere shortening in electrically stimulated isolated adult rat cardiomyocytes undergoing 30 minutes of exposure to 40 nM dobutamine. These experiments were facilitated by a custom device containing micro-patterned wells that orient and immobilize the cardiomyocytes, allowing for rapid and consistent measurements over an extended time period. After functional measurements, cells are picked up individually with a computerized micro-aspirator and subjected to single-cell RT-qPCR to quantify abundance of transcripts of interest. In this way, the physiological responses of individual cells to β1AR stimulus can be correlated to gene expression levels. We found a surprising diversity in the single-cell responses to dobutamine exposure. Although average cardiomyocyte contractility increased significantly, several apparently healthy cells registered no change or even a decrease in sarcomere shortening in the presence of dobutamine. Gene expression was quantified in cardiomyocytes representing a spectrum of sensitivities to dobutamine (n = 7). The transcript for β1AR was significantly downregulated in all cells after 30 minutes, indicating consistent adaptation to chronic stimulus. However, abundance of the gene encoding PKA was negatively correlated with dobutamine-triggered augmentation of Ca 2+ release (p-value: 0.0201). In other words, PKA expression was reduced in cells that experienced potent increases in Ca 2+ release after dobutamine treatment. These data suggest the presence of gene regulatory circuits that modulate individual components of the β1AR pathway independent of overall β1AR abundance. These circuits may be critical to restoring β1AR sensitivity in the failing heart.