Kavin Desai

Cardiovascular Regulation in Mice Lacking α2-Adrenergic Receptor Subtypes b and c

α2-Adrenergic receptors (α2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific α2AR subtypes (α2a, α2b, and α2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either α2b or α2c receptors. Stimulation of α2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating α2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the α2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective α2AR drugs.

Increased Fibulin-5 and Elastin in S100A4/Mts1 Mice With Pulmonary Hypertension

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months “recovery” in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months “recovery” in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

Alterations in dynamic heart rate control in the β1-adrenergic receptor knockout mouse

beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.β1-Adrenergic receptors (β1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a β1-AR gene knockout model to test the hypothesis that β1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in β1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in β1-AR knockouts is markedly limited, with peak heart rates ∼200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in β1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the β1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

Phospholamban deficiency does not compromise exercise capacity

Deficiency of phospholamban (PLB) results in enhancement of basal murine cardiac function and an attenuated response to β-adrenergic stimulation. To determine whether the absence of PLB also reduces the reserve capacity of the murine cardiovascular system to respond to stress, we evaluated the heart rate (HR), blood pressure, and metabolic responses of PLB-deficient (PLB-/-) mice to graded treadmill exercise (GTE). PLB-/- mice were hypertensive at rest (125 ± 19 vs. 109 ± 16 mmHg, P < 0.05) but had normal tachycardic and hypotensive responses to isoproterenol. The HR response to GTE was normal; however, the hypertension in PLB-/- mice normalized at peak exercise. Their exercise capacities, as measured by duration of exercise and peak oxygen consumption (V˙o 2), were normal. The oxygen pulse (V˙o 2/HR) curve was also normal in PLB-/- mice, suggesting an ability to appropriately increase stroke volume and oxygen extraction during GTE, despite an inability to increase β-adrenergically stimulated cardiac contractility. Thus deficiency of PLB, although resulting in diminished β-adrenergic inotropic reserve, does not compromise cardiac performance during exercise.

The developmental and physiological consequences of disrupting genes encoding beta 1 and beta 2 adrenoceptors.

Publisher Summary The role that individual β AR subtypes play in specific physiological processes has been traditionally defined using subtype-specific ligands. Unfortunately, many of these ligands either possess poor selectivity or are used at concentrations in vivo that can lead to occupation of nonspecific subtypes. The advent of gene disruption techniques in the mouse now enables to selectively delete or alter cloned genes as a means of identifying the specific function(s) of their gene products. To better understand β AR subtype-specific functions in the context of either the whole animal or isolated organs and cells, the genes encoding both β 1 - and β 2 ARs have been disrupted. Gene targeting vectors containing β 1 AR or β 2 AR gene sequences interrupted or partially replaced by a bacterial neomycin resistance gene cassette were flanked by a viral thymidine kinase gene cassette. A standard positive-negative selection strategy (G418 + gancyclovir) has been used to isolate R1 embryonic stem (ES) cells, having undergone homologous recombination at β 1 AR or β 2 AR loci. The pharmacological profile of β 1 AR and β 2 AR knockouts is consistent with the loss of these specific receptor subtypes. When the nonspecific β AR antagonist [ 125 I]cyanopindolol is used in competition binding studies, excess unlabeled CGP 20712A ( β 1 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 1 AR knockout heart or lung membranes while excess unlabeled ICI 118,551 ( β 2 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 2 AR knockout lung membranes. These results provide further evidence that the β 1 AR and β 2 AR gene disruptions have abolished β AR protein expression. The physiological impact of knocking out either β 1 - or β 2 ARs has been studied in instrumented animals and in isolated tissues.

Exercise and Oxygen Consumption in the Mouse

The mouse remains the most commonly used species for gene overexpression studies and continues to be the only species in which targeted genetic alterations are possible. The thorough phenotypic evaluation of genetically manipulated murine models of human cardiovascular disease requires the ability to study all components of murine cardiovascular physiology. The recent development of techniques allowing the evaluation of awake, unrestrained mice has advanced the study of cardiovascular dynamics during stressed states such as exercise. These studies provide valuable information about how individual organ systems respond under maximally stimulated conditions, with the possibility of uncovering subtle phenotypes not observed in at rest.

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95