Laurie J. Kerchner

Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias

Background— Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results— We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by ≈50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% (P=0.01). Conclusions— GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Relaxin is essential for renal vasodilation during pregnancy in conscious rats

Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.

Essential Role for Vascular Gelatinase Activity in Relaxin-Induced Renal Vasodilation, Hyperfiltration, and Reduced Myogenic Reactivity of Small Arteries

Abstract— During pregnancy, relaxin stimulates nitric oxide (NO)–dependent renal vasodilation, hyperfiltration and reduced myogenic reactivity of small renal arteries via the endothelial ETB receptor subtype. Our objective in this study was to elucidate the mechanisms by which relaxin stimulates the endothelial ETB receptor/NO vasodilatory pathway. Using chronically instrumented conscious rats, we demonstrated that a specific peptide inhibitor of the gelatinases MMP-2 and −9, cyclic CTTHWGFTLC (cyclic CTT), but not the control peptide, STTHWGFTLS (STT), completely reversed renal vasodilation and hyperfiltration in relaxin-treated rats. Comparable findings were observed with a structurally different and well-established, general antagonist of MMPs, GM6001. In contrast, phosphoramidon, an inhibitor of endothelin-converting enzyme, did not significantly change the renal vasodilatory response to relaxin administration. When small renal arteries were incubated with either of the general MMP inhibitors, GM6001 or TIMP-2 (tissue inhibitor of MMP), or with the specific gelatinase inhibitor, cyclic CTT, the reduced myogenic reactivity of these blood vessels from relaxin-treated nonpregnant and midterm pregnant rats was totally abolished. Moreover, a neutralizing antibody specific for MMP-2 completely abrogated the reduced myogenic reactivity of small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats. In contrast, phosphoramidon did not significantly affect the reduction in myogenic reactivity. Using gelatin zymography, we showed increased pro and active MMP-2 activity in small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats relative to the control animals. Thus, inhibitors of MMPs in general and of gelatinases in particular reverse the renal vascular changes induced by pregnancy or relaxin administration to nonpregnant rats. Finally, the typical reduction in myogenic reactivity of small renal arteries from relaxin-treated nonpregnant rats was absent in ETB receptor–deficient rats, despite an increase in vascular MMP-2 activity. These results indicate an essential role for vascular gelatinase, which is in series with, and upstream of, the endothelial ETB receptor/NO signaling pathway in the renal vasodilatory response to relaxin and pregnancy.

Plasma and 24-h NOx and cGMP during normal pregnancy and preeclampsia in women on a reduced NOx diet

We tested the hypothesis that nitric oxide (NO) biosynthesis increases during normal human pregnancy and decreases in preeclampsia. The major metabolites of NO, nitrate and nitrite (NOx), were measured in both the plasma and 24-h urine of women subjected to a reduced NOx diet. In this way, the plasma and urinary levels mainly reflected endogenous production rather than dietary intake. Moreover, we assessed cGMP, a second messenger of NO, in the same samples. Both NOx and cGMP assays were validated in our laboratory. We first conducted a cross-sectional study of nonpregnant women ( n = 15), normal pregnant women in the first ( n = 9), second ( n = 17) and third ( n = 22) trimesters, as well as women with preeclampsia ( n = 15) and transient hypertension of pregnancy ( n= 7). We also performed a serial study in the same women ( n = 9) before, during, and after pregnancy. Taken together, the results of the two investigations suggested marked increases in cGMP production especially during the first trimester when the maternal circulation is rapidly vasodilating. In contrast, whole body NO production as estimated by the plasma level and urinary excretion of NOx was not elevated during the first trimester. Finally, unequivocal demonstration of reduced NO biosynthesis in preeclampsia was not forthcoming.

Evidence for local relaxin ligand-receptor expression and function in arteries

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin‐1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin‐1 gene‐deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild‐type (WT) and heterozygous mice. Taken together, these findings reveal an arterial‐derived, relaxin ligand‐receptor system that acts locally to regulate arterial function.—Novak, J., Parry, L. J., Matthews, J. E., Kerchner, L. J., Indovina, K., Hanley‐Yanez, K., Doty, K. D., Debrah, D. O., Shroff, S. G., Conrad, K. P. Evidence for local relaxin ligand‐receptor expression and function in arteries. FASEB J. 20, 2352–2362 (2006)

Cardiac Na+ Current Regulation by Pyridine Nucleotides

Rationale: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (INa) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism. Objective: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Nav1.5). Methods and Results: HEK293 cells stably expressing Nav1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A+/− mouse heart. A280V GPD1-L caused a 2.48±0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased INa (0.48±0.09 or 0.19±0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase. NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI6–22. The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A+/− mouse hearts ameliorated the risk of ventricular tachycardia. Conclusions: Our results show that Nav1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and INa. This effect required protein kinase C activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Nav1.5 by altering the oxidized to reduced NAD(H) balance.

Renal Handling of Homocysteine During Normal Pregnancy and Preeclampsia

Objective: Maternal plasma homocysteine decreases in normal pregnancy and is significantly increased in preeclampsia. The goal of this study was to investigate the role of the maternal kidney in the changes of plasma homocysteine during normal pregnancy and preeclampsia. Methods: Plasma and 24-hour urine samples were collected in the same women before, during (first, second, and third trimesters), and after normal pregnancy; and in a separate cross-sectional study of normal pregnant, preeclamptic and nonpregnant women and homocysteine concentrations were measured. Results: Longitudinally, maternal plasma homocysteine decreased significantly by the first trimester compared with prepregnancy and postpartum levels (5.6 ± 1.8 versus 6.8 ± 0.5 and 7.4 ± 0.4 μM, respectively, P < .05 by analysis of variance) and paralleled a significant increase in the renal clearance of homocysteine (2.9 ± 0.4 versus 1.8 ± 0.2 and 1.6 ± 0.2 L/24 hours, respectively, < < 001). In addition plasma homocysteine was significantly elevated in preeclampsia compared with normal pregnancy (4.4 ± 0.6 versus 3.2 ± 0.2 μM, P < .04); however, renal clearance was not different (1.2 ± 0.1 versus 1.0 ± 0.1 L/24 hours, P = .55). Conclusion: Increases in renal clearance contribute to the decrease in plasma homocysteine during normal pregnancy. However, changes in renal handling do not appear to contribute to the increase in plasma homocysteine in preeclampsia.

Role of Relaxin in Maternal Renal Vasodilation of Pregnancy

Abstract: The remarkable hemodynamic changes of normal pregnancy are briefly reviewed. In addition, new findings and current concepts related to the underlying hormonal and molecular mechanisms are presented. Finally, work that is in progress as well as future directions is briefly discussed.

Mechanisms of Renal Vasodilation and Hyperfiltration During Pregnancy: Current Perspectives and Potential Implications for Preeclampsia

A thorough understanding of the renal and cardiovascular adaptations to normal gestation is essential for proper diagnosis and management of hypertensive disorders and renal diseases during pregnancy. Here, we briefly review the renal hemodynamic changes of normal pregnancy. In addition, we present new findings and current concepts related to the underlying hormonal and molecular mechanisms. Finally, we speculate on the potential contribution of these insights from normal pregnancy to the pathogenesis of preeclampsia.

Is relaxin the “elusive” vasodilator of pregnancy?

Among the most striking changes seen in biology are those associated with pregnancy. In particular, profound vasodilation of nonreproductive organs epitomizes the maternal cardiovascular adaptation to early gestation in women. Cardiac output, renal blood flow and glomerular filtration rate rise by 40 to 80%, while vascular resistances plummet and blood pressure declines modestly [reviewed in ref.1]. Global arterial compliance markedly increases, thus preserving the efficiency of ventricular and arterial coupling, as well as preventing excessive decline of diastolic blood pressure in the face of the marked fall in systemic vascular resistance [SVR; 2]. All of these alterations begin immediately after conception, peak by the end of the first or beginning of the second trimester, and persist throughout most of gestation. It is likely that the circulations of nonreproductive organs such as the kidney serve as “arteriovenous” shunts during early gestation, thereby reducing ventricular afterload which initiates the enormous increase in cardiac output, and subsequently, the expansion of plasma volume — maternal adaptations associated with healthy pregnancies. Furthermore, pressor response to administration of angiotensin II and vascular reactivity to infusion of norepinephrine become attenuated [1]. Insight into the mechanisms responsible for these vasodilatory phenomena may be particularly critical, because in preeclampsia, the attenuation of pressor responsiveness to angiotensin II, the reduced vascular reactivity to norepinephrine, as well as the systemic and renal vasodilation are compromised [1].