Cardiac fibroblasts are responsible for the production of the extracellular matrix of the heart, with alterations of fibroblast function implicated in myocardial infarction and cardiac hypertrophy. Here the role of heterotrimeric GTP-binding proteins (G proteins) in the mechanotransduction of strain in rat cardiac fibroblasts was investigated. Cells in an equibiaxial stretch device were incubated with the photoreactive GTP analog azidoanalido [α-32P]GTP (AAGTP) and were subjected to various regimens of strain. Autoradiographic analysis showed a 42-kDa protein labeled for cells exposed to 12 cycles of 3% strain or 6 cycles of 6% strain over 60 s (strain rate of 1.2%/s), whereas 6 cycles of 3% strain (0.6%/s) elicited no measurable response. To further investigate the role of strain rate, a single 6% cycle over 10 or 60 s (1.2% and 0.2%/s, respectively) was applied, with the more rapid cycle stimulating AAGTP binding, whereas the lower strain rate showed no response. In cells subjected to a single 6% cycle/10 s, immunoprecipitation identified the AAGTP-labeled 42-kDa band as the G protein subunits Gαq and Gαi1. These results demonstrate that G protein activation represents one of the early mechanotransduction events in cardiac fibroblasts subjected to mechanical strain, with the rate at which the strain is applied modulating this response.

Fluid shear stimulates endothelial cells, with the external hemodynamic forces transduced across the plasma membrane to modulate intracellular events. We report the first direct evidence that identifies specific GTP binding proteins (G proteins) activated within 1 second of flow onset, representing one of the earliest mechanochemical signal transduction events reported to date in shear-stimulated endothelium. A nonhydrolyzable GTP photoreactive analogue, azidoanilido [alpha-32P]GTP (AAGTP), allowed irreversible labeling of flow-stimulated G proteins, with two protein bands (42 kD and 31 kD) identified in human umbilical vein endothelial cells (HUVECs) subjected to laminar flow (10 dyne/cm2) in a parallel-plate flow chamber. Immunoprecipitation of labeled whole-cell lysates identified the specific G-protein subunits G q zero/alpha 11 and G alpha i3/alpha 0) as being activated by flow. Endothelial cell membrane vesicles were sheared in a cone-and-plate viscometer, with the 42-kD protein band labeled by AAGTP, but the 31-kD protein absent, indicating that the 42-kD G protein is membrane associated and activated independently of intact cytoskeletal or cytosolic components. Our results describe one of the earliest flow-induced signaling events reported in HUVECs, providing insight into the primary mechanosensing and signal transduction mechanisms.

On the basis of these findings, we conclude that acidosis-induced coronary arteriolar dilation is mediated by the opening of smooth muscle KATP channels through the activation of PTX-sensitive G proteins.

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