Mouse gene targeting in cardiovascular physiology.

IN THE 1980s a revolution took place in cardiovascular physiology unnoticed by many researchers working in the field. At that time, the rat and the dog were the prime animal models to study integrative mechanisms involved in cardiovascular regulation. The notorious but true saying that cells do not have a blood pressure implies that in vivo experiments will always be critical in the process of discovering new principles of circulatory control. Studies in rats and dogs have helped to unravel many basic principles of cardiovascular physiology, such as autoregulation of blood flow (11, 28), reflex control of blood pressure (3), or blood volume regulation through signals originating from the heart (2), to name just a few. Nevertheless, the lack of techniques to directly link the activity of specific genes to physiological functions—which was then possible already in cellular systems—remained a major drawback. Thus the development of experimental procedures to induce mutations in single genes and the eventual successful generation of mice carrying a targeted mutation in 1989 (7, 19) marked the beginning of a new era area also in cardiovascular physiology. Today, more than 3,000 different knockout mice have been constructed. Many of these mutations affect cardiovascular function, and some of them have helped to solve long-standing open questions in cardiovascular regulation. For example, it has been known for long that the tubuloglomerular feedback (TGF) is a key mechanism of autoregulation of renal blood flow and glomerular filtration and is intimately involved in blood volume homeostasis. In the kidney, the proximal part of the distal tubule gets in close contact with the afferent arteriole to form the juxtaglomerular apparatus. Within this region, the TGF communicates changes in proximal tubular flow rate to afferent arteriolar smooth muscle and renin-secreting cells, causing a decrease in vascular tone and an increase in renin secretion rate if tubular flow rate falls and vice versa. Twenty years ago it was postulated that changes in local adenosine concentrations contribute to the signaling mechanism underlying the TGF response; however, participation of adenosine has remained controversial. Now two independent groups of investigators reported that mice carrying a targeted deletion of the A1-adenosine receptor, which is expressed at a high level in the juxtaglomerular apparatus, completely lack a TGF response (5, 34). These studies clearly demonstrate that adenosine, the major agonist of A1adenosine receptors, plays an essential role in the signaling cascade mediating the TGF. Future studies will have to show whether this role is that of a mediator or a modulator (25, 29). This example illustrates the major strength of the genetic approach compared with the more classical pharmacological and correlative approaches. Although the latter are inherently indirect, pharmacological studies often suffer from uncertain pharmacokinetics and a lack of specificity. Accordingly, the observation of a sevenfold higher salt intake after an overnight fluid restriction in mice carrying a targeted deletion of the oxytocin gene constitutes strong direct evidence for the concept derived from infusion studies of agonists and antagonists that sodium appetite is negatively regulated by central oxytocin (1). The finding that mice lacking tumor necrosis factor(TNF) display threefold elevated renal renin mRNA levels under control conditions as well as after salt depletion provides the first in vivo evidence for a possible physiological function of TNFas a negative regulator of renin synthesis (35). Studies in TNF/ mice also indicate that a strong increase in the local production of TNFinitiates intimal hyperplasia after vascular injury (38). Further examples for important advances in our understanding of cardiovascular regulation obtained by the use of gene-targeted mice are the discovery of a major role of the K channel -subunit KCNE1 in K and renal fluid homeostasis (36), the unmasking of an enhanced central response to dehydration in angiotensin AT1A / mice (24), the identification of multiple physiological functions of 2-adrenergic receptors (27),