. The ␣ 2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro. Am J Physiol Heart Circ Physiol 288: H477-H485, 2005. First published September 30, 2004 doi:10.1152/ajpheart.00083.2004.-Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the ␣2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the ␣2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive ␣2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the ␣2-isoform of Na-K-ATPase. As the ␣1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in ␣2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the ␣ 2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific ␣ isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the ␣2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive ␣2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the ␣2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.

The primary objective of this study was to examine the functional role of the Na,K-ATPase ␣1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the ␣1 or ␣2 isoform gene exhibit opposite phenotypes. Hearts from ␣2 ؉/؊ animals are hypercontractile, whereas those of the ␣1 ؉/؊ animals are hypocontractile. The cardiac phenotype of the ␣1 ؉/؊ animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive ␣1 isoform and a ouabain-resistant ␣2 isoform of the Na,KATPase, and we analyzed cardiac contractility following selective inhibition of the ␣1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the ␣1 isoform without affecting the activity of the ␣2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified ␣1 and ␣2 isoforms. Pretreatment with 10 M KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the ␣1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The ␣1 isoform coimmunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the ␣1 isoform regulates cardiac contractility, and that both the ␣1 and ␣2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart. Active Na ϩ transport across the cardiac sarcolemma, driven by the Na,K-ATPase, is an important regulator of cardiac function (1, 2). The intracellular Na ϩ concentration affects a number of physiological processes in cardiac myocytes, including intracellular Ca 2ϩ handling, contraction-relaxation processes, pH regulation, energy metabolism, and cell growth (1-2). Alterations in the maintenance of normal intracellular Na ϩ homeostasis result in heart failure (1). Na,K-ATPase is a heterodimer composed of ␣ and ␤ subunits (3). The ␣ subunit is the catalytic subunit, and it binds translocating cations and ATP. The ␣ subunit is also the pharmacological receptor for cardiac glycosides. These compounds inhibit Na,K-ATPase activity and are used in the treatment of congestive heart failure. There are four isoforms of the ␣ subunit, each with a distinct tissue distribution and developmental pattern of expression, suggestive of their differential and tissue-specific functional roles (4 -10). Depending on the species, different combinations of these ␣ isoforms are present in heart. The ␣1 and ␣2 isoforms are expressed in rodent heart, whereas three isoforms (␣1, ␣2, and ␣3) are expressed in human heart (5, 10, 11). As multiple isoforms are expressed in heart, it is possible that they play different biological roles.Previous studies...

The Na,K-ATPase is composed of two subunits, alpha and beta, and each subunit consists of multiple isoforms. In the case of alpha, four isoforms, alpha1, alpha2, alpha3, and alpha4 are present in mammalian cells. The distribution of these isoforms is tissue- and developmental-specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the alpha1 and alpha2 isoform genes. Animals lacking both copies of the alpha1 isoform gene are not viable, while animals lacking both copies of the alpha2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the alpha1 or alpha2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the alpha2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the alpha1 isoform gene. These studies indicate that the alpha1 and alpha2 isoforms carry out different physiological roles. The alpha2 isoform appears to be involved in regulating Ca(2+) transients involved in muscle contraction, while the alpha1 isoform probably plays a more generalized role. While we have not yet knocked out the alpha3 or alpha4 isoform genes, studies to date indicate that the alpha4 isoform is necessary to maintain sperm motility. It is thus possible that the alpha2, alpha3, and alpha4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue- and developmental-specific regulation.

OBJECTIVES Pulmonary hypertension (PH) is a process of lung vascular remodeling which can lead to right heart dysfunction and significant morbidity. The underlying mechanisms leading to PH are not well understoon and therapies are limited. Using Intermittent hypoxia (IH) as a model of oxidant-induced PH, we identified an important role for endothelial cell mitophagy via mitochondrial uncoupling protein 2 (Ucp2) in the development of IH-induced PH. APPROACH AND RESULTS Ucp2 endothelial knockout (VE-KO) and Ucp2 Flox (Flox) mice were subjected to 5 weeks of IH. Ucp2 VE-KO mice exhibited higher RVSP and worse right heart hypertrophy, as measured by increased RV/LV+S ratio, at baseline and after IH. These changes were accompanied by increased mitophagy. Primary mouse lung endothelial cells (MLEC) transfected with Ucp2 siRNA and subjected to cyclical exposures to CoCl2 (chemical hypoxia) showed increased mitophagy, as measured by Pink1 and LC3BII/I ratios, decreased mitochondrial biogenesis and increased apoptosis. Similar results were obtained in primary lung endothelial cells isolated from VE-KO mice. Moreover, silencing Pink1 in the endothelium of Ucp2 KO mice, using endothelial-targeted lentiviral silencing RNA in vivo, prevented IH-induced PH. Human pulmonary artery endothelial cells from people with PH demonstrated changes similar to Ucp2-silenced MLEC. CONCLUSION The loss of endothelial Ucp2 leads to excessive Pink1-induced mitophagy, inadequate mitochondrial biosynthesis and increased apoptosis in endothelium. An endothelial Ucp2-Pink1 axis may be effective therapeutic targets in PH.