Dr. Ferid Murad is examining the role of soluble guanylyl cyclase (sGC), a cardiac regulation enzyme, in adaptation to weightlessness. Using genetically-engineered mice, Murad will observe the effects of disrupted levels of sGC on heart function and on the development of orthostatic intolerance. He also will identify the sGC-dependent pathways necessary to adapt to weightlessness and re-adapt to gravity.
A Soluble Guanylyl Cyclase Mouse Knock-Out Model
Ferid Murad, M.D., Ph.D.
The University of Texas Health Science Center at Houston
To investigate this hypothesis, we will use a gene-targeted mouse animal model with myocardium-specific and smooth muscle-specific disruption of gene expression to study the specific role of sGC in microgravity/gravity adaptation. In specific aim 1, a mouse animal model with myocardium-specific and vascular smooth muscle-specific disruption of sGC beta 1 gene will be developed. Following development, we will characterize the effects of myocardial-specific and vascular smooth muscle-specific gene disruption on general appearance, embryonic development, histology of the heart and hemodynamic changes in knockout mice versus wild-type mice. In specific aim 2, we will determine the role of sGC pathway deficiency on the development of orthostatic intolerance that occurs during re-adaptation to gravity using the established tail-suspended rodent model to stimulate microgravity conditions. The time course of changes in cardiovascular function induced by re-adaptation to gravity in knockout mice versus wild-type mice will be studied. Finally, the cardiovascular responsiveness in knockout mice versus wild-type mice following de-suspension will be assessed using vaso- and cardio-active agents.
Comparisons between knockout mice and wild-type mice in simulating weightlessness conditions will aid to identify sGC-dependent pathways necessary for adaptation to microgravity and re-adaptation to gravity. In order to fulfill specific aim 1, we are developing a mouse animal model with myocardium-specific and vascular smooth muscle-specific disruption of sGC gene. During last year, we used Lox-Beta 1 sGC targeted vector generated previous year to produce the gene-targeted mouse ES line. Targeted ES cells were introduced into mouse blastocysts and chimeric male mice were obtained. In order to identify animals with a transmission of the targeted ES cells into the germ line, chimeric males were bred with wild-type females. Presently, we are at the stage of genotyping F1 offsprings to identify Lox-targeted heterozygous mice.
Homozygotes with Lox-targeted Beta1 sGC gene will be obtained by crossing identified heterozygotic mice. In the upcoming year, Beta1 sGC-lox mice will be bred with alpha-MyHC-Cre mice containing a myocardium-specific Cre-recombinase to generate myocardium specific Beta1-sGC-Cre/lox knockout mice and SM-CreER(T2)(ki) containing smooth muscle-specific Cre recombinase to produce smooth muscle-specific Beta1-sGC-Cre/lox knockout mice. Following their development, we will characterize the effects of myocardial-specific and vascular smooth muscle-specific gene disruption on general appearance, embryonic development, histology of the heart and hemodynamic changes in knockout mice versus wild-type mice.
While the role of sGC in the vasculature was first identified 20 years ago, the physiological significance of sGC-cGMP signaling in the myocardium is not yet fully understood. Indeed, the significance of cGMP signaling in cardiomyocytes is controversial with conflicting reports in the literature. Several problems are associated with the study of sGC-mediated physiology. Distinctions between cGMP-dependent and -independent actions of NO and between the physiological contributions of cGMP produced by the particulate and soluble forms of the enzyme are difficult to determine due to the lack of selective sGC inhibitors. Furthermore, at least two sGC isoforms demonstrate very similar pharmacological and functional properties. A more precise understanding of sGC-cGMP signaling and regulatory events would have profound pathophysiological significance and could potentially help to develop novel therapeutic strategies with minimized negative side effects. A gene-targeted mouse model represents a unique opportunity to study sGC-cGMP action and to clarify the role of sGC in these processes.