Lynne H. Liu

Balance and Hearing Deficits in Mice with a Null Mutation in the Gene Encoding Plasma Membrane Ca2+-ATPase Isoform 2

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Impaired Renal NaCl Absorption in Mice Lacking the ROMK Potassium Channel, a Model for Type II Bartter's Syndrome

ROMK is an apical K+channel expressed in the thick ascending limb of Henle (TALH) and throughout the distal nephron of the kidney. Null mutations in theROMK gene cause type II Bartters syndrome, in which abnormalities of electrolyte, acid-base, and fluid-volume homeostasis occur because of defective NaCl reabsorption in the TALH. To understand better the pathogenesis of type II Bartters syndrome, we developed a mouse lacking ROMK and examined its phenotype. Young null mutants had hydronephrosis, were severely dehydrated, and ∼95% died before 3 weeks of age. ROMK-deficient mice that survived beyond weaning grew to adulthood; however, they had metabolic acidosis, elevated blood concentrations of Na+ and Cl−, reduced blood pressure, polydipsia, polyuria, and poor urinary concentrating ability. Whole kidney glomerular filtration rate was sharply reduced, apparently as a result of hydronephrosis, and fractional excretion of electrolytes was elevated. Micropuncture analysis revealed that the single nephron glomerular filtration rate was relatively normal, absorption of NaCl in the TALH was reduced but not eliminated, and tubuloglomerular feedback was severely impaired. These data show that the loss of ROMK in the mouse causes perturbations of electrolyte, acid-base, and fluid-volume homeostasis, reduced absorption of NaCl in the TALH, and impaired tubuloglomerular feedback.

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.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 ± 5.6% in wild type, 35.4 ± 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Plasticity and adaptation of Ca2+ signaling and Ca2+‐dependent exocytosis in SERCA2+/− mice

Dariers disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2+/− mouse, to define the adaptation of Ca2+ signaling and Ca2+‐dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2+/− mice, due to an up‐regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused ∼50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist‐stimulated exocytosis was identical in cells from wild‐type and SERCA2+/− mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2+/− mice. Accordingly, exocytosis was ∼10‐fold more sensitive to Ca2+ in cells from SERCA2+/− mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+‐dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.

Physiological functions of plasma membrane and intracellular Ca2+ pumps revealed by analysis of null mutants.

Abstract: It is known that plasma membrane Ca2+‐transporting ATPases (PMCAs) extrude Ca2+ from the cell and that sarco(endo)plasmic reticulum Ca2+‐ATPases (SERCAs) and secretory pathway Ca2+‐ATPases (SPCAs) sequester Ca2+ in intracellular organelles; however, the specific physiological functions of individual isoforms are less well understood. This information is beginning to emerge from studies of mice and humans carrying null mutations in the corresponding genes. Mice with targeted or spontaneous mutations in plasma membrane Ca2+‐ATPase isoform 2 (PMCA2) are profoundly deaf and have a balance defect due to the loss of PMCA2 in sensory hair cells of the inner ear. In humans, mutations in SERCA1 (ATP2A1) cause Brody disease, an impairment of skeletal muscle relaxation; loss of one copy of the SERCA2 (ATP2A2) gene causes Darier disease, a skin disorder; and loss of one copy of the SPCA1 (ATP2C1) gene causes Hailey‐Hailey disease, another skin disorder. In the mouse, SERCA2 null mutants do not survive to birth, and heterozygous SERCA2 mutants have impaired cardiac performance and a high incidence of squamous cell cancers. SERCA3 null mutants survive and appear healthy, but endothelium‐dependent relaxation of vascular smooth muscle is impaired and Ca2+ signaling is altered in pancreatic β cells. The diversity of phenotypes indicates that the various Ca2+‐transporting ATPase isoforms serve very different physiological functions.

Haploinsufficiency of Atp2a2, Encoding the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 Ca2+ Pump, Predisposes Mice to Squamous Cell Tumors via a Novel Mode of Cancer Susceptibility

A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/- mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/- mice.

Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3), an isoform of the intracellular Ca2+ pump that has been shown to mediate endothelium-dependent relaxation of vascular smooth muscle, is also expressed in tracheal epithelium. To determine its possible role in regulation of airway mechanical function, we compared tracheal contractility in gene-targeted mice deficient in SERCA3 (SERCA3-) with that in wild-type tracheae. Cumulative addition of ACh elicited concentration-dependent increases in isometric force (ED50 = 2 μM, maximum force = 8 mN/mm2) that were identical in SERCA3- and wild-type tracheae. After ACh stimulation, substance P (SP) elicited a transient relaxation (42.6 ± 3.2%, n = 28) in both tracheae. However, the rate of relaxation was significantly ( P < 0.04, n = 9) more rapid in the wild-type [half-time ( t ½) = 34.3 s] than in the SERCA3-( t ½ = 61.6 s) trachea. The SP relaxation was reduced by rubbing the trachea, indicative of epithelial cell involvement. This was verified using a perfused trachea preparation. SP in the outside medium had no effect, whereas SP in the perfusate bathing the epithelial side elicited a relaxation. Nitric oxide synthase inhibition (0.2 mM N ω-nitro-l-arginine) reduced the SP relaxation by 36.5 ± 12.5%, whereas the SP effect was abolished by eicosanoid inhibition (10 μM indomethacin). ATP also elicited an epithelium-dependent relaxation similar to SP but with a more rapid relaxation in the SERCA3-trachea than in the wild-type trachea. Our results indicate that SERCA3 gene ablation does not directly affect smooth muscle, which is consistent with the distribution of the isoform, but suggest that SERCA3 plays a role in epithelial cell modulation of airway smooth muscle function.

Altered force–frequency response in non-failing hearts with decreased SERCA pump-level

OBJECTIVE Decreased SERCA2 activity is considered to significantly contribute to the contractile dysfunction of failing hearts. However, it is now known how decreases in SERCA activity affect cardiac function in detail and also if a decrease alone is sufficient to cause heart failure. METHODS SERCA2 (+/-) gene-targeted mice (HET) were generated and heart function was analyzed using the isolated work-performing heart technique. Plasma and cardiac catecholamine levels were determined at three, six and nine months of age and heart sections from twelve months old mice subjected to standard histological analysis. RESULTS We demonstrate that reduced expression of SERCA does not lead to cardiac hypertrophy or fibrosis and does not increase resting plasma-norepinephrine levels in HET mice. However, isolated perfused HET hearts exhibited decreased maximal rates of contraction and relaxation and prolonged time-parameters. The ability of the HET hearts to respond to increases in load (Starling) was not affected and they responded appropriately to beta-adrenergic stimulation. In contrast, the positive force-frequency response found in control hearts was not observed in the HET hearts. The response was flat and three out of five HET hearts failed to maintain work at 550 beats/min. CONCLUSIONS We conclude that the SERCA2 pump level is a critical positive determinant of cardiac contractility and force-frequency relation.