Michelle L. Nieman

TGF-β1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II

Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-beta1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-beta1-deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang II treatment. In contrast, Ang II-treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II-treated wild-type mice but unchanged in Ang II-treated TGF-beta1-deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II-treated mice. Atrial natriuretic factor expression was approximately 6-fold elevated in Ang II-treated wild-type, but not TGF-beta1-deficient mice. However, the alpha- to beta-myosin heavy chain switch did not occur in Ang II-treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-beta1. The Ang II effect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-beta1 is an important mediator of the hypertrophic growth response of the heart to Ang II.

Phenotype Resembling Gitelman’s Syndrome in Mice Lacking the Apical Na+-Cl− Cotransporter of the Distal Convoluted Tubule

Mutations in the gene encoding the thiazide-sensitive Na+-Cl− cotransporter (NCC) of the distal convoluted tubule cause Gitelman’s syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman’s syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc −/−) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure.Ncc −/− mice retain Na+ as well as wild-type mice when fed a Na+-depleted diet; however, after 2 weeks of Na+ depletion the mean arterial blood pressure of Ncc −/− mice was significantly lower than that of wild-type mice. In addition, Ncc −/−mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg2+ and Ca2+ are altered, as observed in Gitelman’s syndrome.

Mouse Model of Desmin-Related Cardiomyopathy

Background—The consequence of upregulation of desmin in the heart is unknown. Mutations in desmin have been linked to desmin-related myopathy (DRM), which is characterized by abnormal intrasarcoplasmic accumulation of desmin, but direct causative evidence that a desmin mutation leads to aberrant intrasarcoplasmic desmin accumulation, aggregation, and cardiomyopathy is lacking. Methods and Results—Multiple transgenic mouse lines that expressed either murine wild-type desmin or a 7–amino acid deletion (R173 through E179) desmin (D7-des) mutation linked to DRM were made. The distribution of desmin protein was unchanged, and no overt phenotype was detected in the wild-type desmin transgenic mice. In contrast, the D7-des mouse heart showed aberrant intrasarcoplasmic and electron-dense granular filamentous aggregates that were desmin-positive and characteristic of human DRM. The desmin filament network was significantly disrupted, and myofibril alignment was visibly compromised. Although systolic function at the whole-organ level was substantially conserved in the young adult animals, the ability of the heart to respond to &bgr;-agonist stimulation, as measured in the intact animal, was significantly blunted. Conclusions—Upregulation of desmin protein at moderate levels is not detrimental. However, the D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of aggregates that are characteristic of and diagnostic for human desmin-related cardiomyopathy.

FIBROBLAST GROWTH FACTOR-2 MEDIATES PRESSURE-INDUCED HYPERTROPHIC RESPONSE

In vitro, fibroblast growth factor-2 (FGF2) has been implicated in cardiomyocyte growth and reexpression of fetal contractile genes, both markers of hypertrophy. However, its in vivo role in cardiac hypertrophy during pressure overload is not well characterized. Mice with or without FGF2 (Fgf2(+/+) and Fgf2(-/-), respectively) were subjected to transverse aortic coarctation (AC). Left ventricular (LV) mass and wall thickness were assessed by echocardiography preoperatively and once a week postoperatively for 10 weeks. In vivo LV function during dobutamine stimulation, cardiomyocyte cross-sectional area, and recapitulation of fetal cardiac genes were also measured. AC Fgf2(-/-) mice develop significantly less hypertrophy (4-24% increase) compared with AC Fgf2(+/+) mice (41-52% increase). Cardiomyocyte cross-sectional area is significantly reduced in AC Fgf2(-/-) mice. Noncoarcted (NC) and AC Fgf2(-/-) mice have similar beta-adrenergic responses, but those of AC Fgf2(+/+) mice are blunted. A lack of mitotic growth in both AC Fgf2(+/+) and Fgf2(-/-) hearts indicates a hypertrophic response of cardiomyocytes. Consequently, FGF2 plays a major role in cardiac hypertrophy. Comparison of alpha- and beta-cardiac myosin heavy chain mRNA and protein levels in NC and AC Fgf2(+/+) and Fgf2(-/-) mice indicates that myosin heavy chain composition depends on hemodynamic stress rather than on FGF2 or hypertrophy, and that isoform switching is transcriptionally, not posttranscriptionally, regulated.

Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice.

Previous studies using isolated tissues suggest that the colonic H, K-ATPase (cHKA), expressed in the colon and kidney, plays an important role in K+ conservation. To test the role of this pump in K+ homeostasis in vivo, we generated a cHKA-deficient mouse and analyzed its ability to retain K+ when fed a control or K+-free diet. When maintained on a control diet, homozygous mutant (cHKA-/-) mice exhibited no deficit in K+ homeostasis compared to wild-type (cHKA+/+ greater, similar mice. Although fecal K+ excretion in cHKA-/- mice was double that of cHKA+/+ mice, fecal K+ losses were low compared with urinary K+ excretion, which was similar in both groups. When maintained on a K+-free diet for 18 d, urinary K+ excretion dropped over 100-fold, and to similar levels, in both cHKA-/- and cHKA+/+ mice; fecal K+ excretion was reduced in both groups, but losses were fourfold greater in cHKA-/- than in cHKA+/+ mice. Because of the excess loss of K+ in the colon, cHKA-/- mice exhibited lower plasma and muscle K+ than cHKA+/+ mice. In addition, cHKA-/- mice lost twice as much body weight as cHKA+/+ mice. These results demonstrate that, during K+ deprivation, cHKA plays a critical role in the maintenance of K+ homeostasis in vivo.

Uroguanylin knockout mice have increased blood pressure and impaired natriuretic response to enteral NaCl load

Guanylin and uroguanylin, peptides synthesized in the intestine and kidney, have been postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of uroguanylin in the regulation of sodium excretion, we created gene-targeted mice in which uroguanylin gene expression had been ablated. Northern and Western analysis confirmed the absence of uroguanylin message and protein in knockout mice, and cGMP levels were decreased in the mucosa of the small intestine. Ussing chamber analysis of jejunum revealed that Na+/H+ exchanger-mediated Na+ absorption and tissue conductance was not altered in the knockout animals, but short-circuit current, an index of electrogenic anion secretion, was reduced. Renal clearance measurements showed that uroguanylin deficiency results in impaired ability to excrete an enteral load of NaCl, primarily due to an inappropriate increase in renal Na+ reabsorption. Finally, telemetric recordings of blood pressure demonstrated increased mean arterial pressure in uroguanylin knockout animals that was independent of the level of dietary salt intake. Together, these findings establish a role for uroguanylin in an enteric-renal communication axis as well as a fundamental principle of this axis in the maintenance of salt homeostasis in vivo.

Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5−/− mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5−/− mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5−/− male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by ≈1.4-fold in glands from AQP5−/− mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5−/− compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.

Impaired Cardiac Contractility in Mice Lacking Both the AE3 Cl-/HCO3-Exchanger and the NKCC1 Na+-K+-2Cl¯ Cotransporter : EFFECTS ON Ca2+ HANDLING AND PROTEIN PHOSPHATASES

To analyze the cardiac functions of AE3, we disrupted its gene (Slc4a3) in mice. Cl(-)/HCO3(-) exchange coupled with Na+-dependent acid extrusion can mediate pH-neutral Na+ uptake, potentially affecting Ca2+ handling via effects on Na+/Ca2+ exchange. AE3 null mice appeared normal, however, and AE3 ablation had no effect on ischemia-reperfusion injury in isolated hearts or cardiac performance in vivo. The NKCC1 Na+-K+-2Cl(-) cotransporter also mediates Na+ uptake, and loss of NKCC1 alone does not impair contractility. To further stress the AE3-deficient myocardium, we combined the AE3 and NKCC1 knock-outs. Double knock-outs had impaired contraction and relaxation both in vivo and in isolated ventricular myocytes. Ca2+ transients revealed an apparent increase in Ca2+ clearance in double null cells. This was unlikely to result from increased Ca2+ sequestration, since the ratio of phosphorylated phospholamban to total phospholamban was sharply reduced in all three mutant hearts. Instead, Na+/Ca2+ exchanger activity was found to be enhanced in double null cells. Systolic Ca2+ was unaltered, however, suggesting more direct effects on the contractile apparatus of double null myocytes. Expression of the catalytic subunit of protein phosphatase 1 was increased in all mutant hearts. There was also a dramatic reversal, between single null and double null hearts, in the carboxymethylation and localization to the myofibrillar fraction, of the catalytic subunit of protein phosphatase 2A, which corresponded to the loss of normal contractility in double null hearts. These data show that AE3 and NKCC1 affect Ca2+ handling, PLN regulation, and expression and localization of major cardiac phosphatases and that their combined loss impairs cardiac function.

Knockout of the Na,K-ATPase α2-isoform in the cardiovascular system does not alter basal blood pressure but prevents ACTH-induced hypertension

The α(2)-isoform of Na,K-ATPase (α(2)) is thought to play a role in blood pressure regulation, but the specific cell type(s) involved have not been identified. Therefore, it is important to study the role of the α(2) in individual cell types in the cardiovascular system. The present study demonstrates the role of vascular smooth muscle α(2) in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model utilizing the Cre/LoxP system to generate a cell type-specific knockout of the α(2) in vascular smooth muscle cells using the SM22α Cre. We achieved a 90% reduction in the α(2)-expression in heart and vascular smooth muscle in the knockout mice. Interestingly, tail-cuff blood pressure analysis reveals that basal systolic blood pressure is unaffected by the knockout of α(2) in the knockout mice. However, knockout mice do fail to develop ACTH-induced hypertension, as seen in wild-type mice, following 5 days of treatment with ACTH (Cortrosyn; wild type = 119.0 ± 6.8 mmHg; knockout = 103.0 ± 2.0 mmHg). These results demonstrate that α(2)-expression in heart and vascular smooth muscle is not essential for regulation of basal systolic blood pressure, but α(2) is critical for blood pressure regulation under chronic stress such as ACTH-induced hypertension.

Loss of NHE1 activity leads to reduced oxidative stress in heart and mitigates high-fat diet-induced myocardial stress

Acute inhibition of the NHE1 Na(+)/H(+) exchanger protects against ischemia-reperfusion injury and chronic inhibition attenuates development of cardiac hypertrophy and failure. To determine the cardiac effects of chronic inhibition of NHE1 under non-pathological conditions we used NHE1-null mice as a model of long-term NHE1 inhibition. Cardiovascular performance was relatively normal in Nhe1(-/-) mice although cardiac contractility and relaxation were slightly improved in mutant mice of the FVB/N background. GSH levels and GSH:GSSG ratios were elevated in Nhe1(-/-) hearts indicating an enhanced redox potential. Consistent with a reduced need for antioxidant protection, expression of heat shock proteins Hsp60 and Hsp25 was lower in Nhe1(-/-) hearts. Similarly, expression of mitochondrial superoxide dismutase 2 was reduced, with no increase in expression of other ROS scavenging enzymes. GLUT1 levels were increased in Nhe1(-/-) hearts, the number of lipid droplets in myocytes was reduced, and PDK4 expression was refractory to high-fat diet-induced upregulation observed in wild-type hearts. High-fat diet-induced stress was attenuated in Nhe1(-/-) hearts, as indicated by smaller increases in phosphorylation of Hsp25 and α-B crystallin, and there was better preservation of insulin sensitivity, as evidenced by PKB/Akt phosphorylation. Plasma glucose and insulin levels were lower and high-fat diet-induced hepatic lipid accumulation was reduced in Nhe1(-/-) mice, demonstrating extracardiac effects of NHE1 ablation. These data indicate that long-term ablation of NHE1 activity increases the redox potential, mitigates high-fat diet-induced myocardial stress and fatty liver disease, leads to better preservation of insulin sensitivity, and may alter both cardiac and systemic metabolic substrate handling in mice.