Rho, a Ser-Thr
kinase identified as a member of the
RAS GTPase super family, is highly expressed in the heart, and has been implicated in the development of
heart failure.
GTPase Rho is located downstream of Gq, and Rho and the associated
kinase (
Rho kinase) regulate myofibril organization, apoptosis, and myofibrillar sensitivity to
calcium. Myocardial injury and dysfunction occur after major
burn injury, and this phenomenon has been linked to cardiac myocyte synthesis and the secretion of proinflammatory
cytokines. Whether
Rho-associated kinase modulates any aspect of cardiomyocyte synthesis of inflammatory mediators, contributing to myocardial dysfunction, has not been studied and was the focus of this study. Hearts were collected at several times postburn to determine if an acute injury such as thermal
trauma altered myocardial
Rho kinase expression. In addition, cardiomyocytes were isolated (
collagenase digestion) from adult control Sprague Dawley rats, plated (5 x 10 cells/microtiter well), incubated with medium alone or in the presence of
burn serum (collected 24 h after
burn over 40% total body surface area in rats) in a CO2 incubator at 37 degrees C in the presence/absence of specific
Rho-kinase inhibitors (HA1077, 10 microM or
Y27632, 10 microM). After 18 h, supernatants were collected to measure secreted
cytokines (
enzyme-linked immunoabsorbant assay), cells were loaded with
Fura-2AM (2 microg) or
sodium-binding benzofuran isophthalate (2 microg) for 45 min at 37 degrees C, and fluorescence was measured with an InCyt IM2 fluorescence imaging system to measure myocyte
calcium and
sodium. In parallel studies, cells were examined to determine if
burn serum challenge increased
Rho kinase in this cell population. In vivo
burn injury or in vitro
burn serum challenge of isolated myocytes increased
Rho-kinase expression and promoted cardiomyocyte secretion of
tumor necrosis factor-alpha,
interleukin 1beta, and
interleukin 6, and increased cardiomyocyte
calcium and
sodium levels compared with values measured when myocytes were incubated in medium alone (P < 0.05). Pretreating cardiomyocytes with
Rho-kinase inhibitor (HA1077 or
Y27632) prevented
burn serum-related upregulation of
Rho-kinase and attenuated the associated inflammatory
cytokine responses, and attenuated myocyte
calcium and
sodium loading. Our data suggest that the
Rho-kinase pathway is one potential upstream regulator of cardiac inflammatory response to
burn injury.