More in vivo experimentation is needed in cardiovascular physiology.

IN THIS PERSPECTIVE, I present the view that cardiovascular physiologists may do well to reinvent some of our old in vivo experiments to gain a deeper and more useful understanding of physiology and disease. Certainly, many physiological and pathophysiological phenomena were discovered through classic in vivo experiments on cardiovascular function (exemplified particularly by the work of A. C. Guyton and his colleagues in the 1970s), but methodological limitations at that time precluded the elucidation of the underlying molecular mechanisms. During the ensuing four decades, new methodologies and the extensive use of isolated tissues, isolated cells, and cell lines led to the discovery of a myriad of molecular mechanisms that undoubtedly contribute to cardiovascular regulation. Nevertheless, such studies do not necessarily inform us about the relative contributions of these mechanisms in vivo. A case in point: in clinical trials (perhaps the ultimate in vivo experiment) the approval success rate for new cardiovascular drugs is only 8.7% (3). One frequent reason for the poor success is unanticipated complexity of the intact organism. Thus a new, more insightful, in vivo experimental approach is needed for cardiovascular physiologists to elucidate normal physiological and pathophysiological mechanisms. Many new approaches are now possible, but one with exceptional potential in my opinion is the visualization and quantification of signal transduction within cells in the living, and even conscious, animal. This could be considered a reinvention of classic intravital imaging, extended to the molecular level. But, is this really possible? The answer, quite clearly, is “yes,” and our neuroscientist colleagues are showing the way. For example, calcium signaling is now being observed through a cranial window in cerebral neurons of conscious, behaving mice (5), thus providing a means to associate populations of cells, individual cells, and even parts of cells with behavior, a phenomenon that only exists in living, conscious animals. Recently, in this journal, we reported completely noninvasive, quantitative, two-photon imaging of Ca signaling in individual smooth muscle cells of arterioles of (anesthetized) hypertensive optical biosensor mice (7). As a further example, two-photon microscopy (1) has now made it possible to image blood flow in cerebral blood vessels, noninvasively, through the thinned-skulls of anesthetized or conscious mice (4) and to cause thrombi and strokes in the cerebral circulation through optical means (10). It is the fortuitous confluence of genetic engineering (to produce optical Ca -biosensor mice) and two-photon microscopy (which enables high-resolution imaging deep in the brain) that makes this a reality. These techniques make possible serial imaging of an individual over days. This is significant, since it allows longitudinal studies of disease states or pathology with the important improvements in statistical power and reduction in the number of animals used. Although the studies cited above were on readily accessible cerebral blood vessels or subcutaneous arterioles of the mouse ear, these techniques have also been used to visualize [Ca ] and other signaling molecules in exteriorized kidney (9). Visualization at the molecular level has come to the intact animal. It seems clear that new realms of investigation in the areas of neurovascular coupling, the physiology and pathophysiology of blood flow, stroke, and hypertension have been opened up. An uncritical adoption of such in vivo experimentation is not likely to be helpful. Thus it is important to define the expectations for in vivo experiments and to compare them with those for ex vivo experiments. As alluded to above, the key point is that ex vivo systems can tell us what is possible at the molecular level and in vivo experiments can tell us what, among the many possibilities, actually happens in the living animal. Two examples, that of RhoA signaling (which activates Rho-Kinase) and of Ca signaling in vascular smooth muscle, will serve to illustrate the point. Both are, obviously, key players in hypertension. Much is known about the RhoA/ Rho-kinase signaling pathway and its many potential roles in normal physiology and hypertension. RhoA is regulated by several GEFs, including LARG, PDZRhoGEF, p63RhoGEF, and p115RhoGEF. These GEFs are coupled to both G q/11 and G 12/13 G proteins, which, in turn, couple to receptors for endothelin-1, thromboxane, angiotensin II and norepinephrine, and others. Downstream, Rho-kinase modulates myosin light chain phosphatase (to name just one effector), which is involved in cytokinesis, membrane trafficking, cell migration, smooth muscle contraction, and vascular remodeling. Each step of the Rho-kinase signaling pathway is localized; activation of Rho-kinase by RhoA occurs at the cell membrane, and Rho-kinase effectors are spatially localized. Definition of the regulatory pathway, the many effectors, and the location information has required study of isolated cells or tissues; only in that experimental milieu is it possible to manipulate the pathway to the extent required to define it at the molecular level. But which receptors, GEFs, and effectors are most important in hypertension? Since hypertension, like behavior, exists only in the living animal, in vivo experiments seem almost mandated. Now, some of the same imaging techniques that revealed the details of Rho-kinase signaling in isolated cells can be used in living hypertensive animals. If an optical biosensor animal for Rho-kinase activity were to be created (similar to those existing now for Ca ), we could expect to learn exactly in what cells, and where in the cells, Rho-kinase is being activated and how it is different in hypertension. Similar to the case for RhoA signaling, we know already that vascular smooth muscle cells generate an extensive array of spatially localized changes in [Ca ], all with distinct molecular mechanisms: asynchronous propagating Ca waves, synchronous [Ca ] oscillations (associated with oscillatory vasomotion), Ca sparks, sparklets, puffs, junctional Ca tranAddress for reprint requests and other correspondence: W. G. Wier, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (e-mail: [email protected]). Am J Physiol Heart Circ Physiol 307: H121–H123, 2014; doi:10.1152/ajpheart.00326.2014. Perspectives