The (Pro)renin Receptor/ATP6AP2 is Essential for Vacuolar H+-ATPase Assembly in Murine Cardiomyocytes
Rationale: The (pro)renin receptor [(P)RR], encoded in ATP6AP2, plays a key role in the activation of local renin-angiotensin system (RAS). A truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H+-ATPase (V-ATPase), implicating a non–RAS-related function of ATP6AP2.
Objective: We investigated the role of (P)RR/ATP6AP2 in murine cardiomyocytes.
Methods and Results: Cardiomyocyte-specific ablation of Atp6ap2 resulted in lethal heart failure; the cardiomyocytes contained RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles, especially in the perinuclear regions. The myofibrils and mitochondria remained at the cell periphery. Cardiomyocyte death was accompanied by numerous autophagic vacuoles that contained undigested cellular constituents, as a result of impaired autophagic degradation. Notably, ablation of Atp6ap2 selectively suppressed expression of the VO subunits of V-ATPase, resulting in deacidification of the intracellular vesicles. Furthermore, the inhibition of intracellular acidification by treatment with bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes.
Conclusions: Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non–RAS-related function that is essential for cell survival.
Activation of the (pro)renin receptor [(P)RR], the gene product of ATP6AP2, plays a key role in the local renin-angiotensin system (RAS). We have shown that (P)RR activation is involved in the development of cardiac fibrosis and proteinuria in hypertension and diabetes. Interestingly, a truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H+-ATPase (V-ATPase), implicating a non–RAS-related function of the gene products of ATP6AP2.1 In the present study, we show that gene products of ATP6AP2 are essential for cardiomyocyte survival via regulating V-ATPase function.
We generated conditional knockout (CKO) mice in which exon 2 of the Atp6ap2 gene was flanked by loxP sites (Online Figure I). Atp6ap2-floxed mice were bred with mice that expressed the Cre recombinase under the control of the cardiomyocyte-specific α-myosin heavy chain (α-MHC) promoter (Online Figure II).2,3 The resulting Atp6ap2lox/Y, αMHC-Cre+/0 mice represent cardiac-specific Atp6ap2 CKO mice. The control male mice were littermates that were heterozygous for α-MHC-Cre (αMHC-Cre+/0; Atp6ap2+/Y), thereby excluding Cre-mediated toxicity as the basis for phenotypic disparity. An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Results and Discussion
The CKO mice were born at the expected mendelian frequency, without any gross cardiac anomalies being noted in the newborn mice, although cardiomyocyte-specific ablation of Atp6ap2 inevitably resulted in heart failure and the mice died within 3 weeks of birth (Figure 1a through 1c). Ventricular functions were severely impaired on postnatal day (PD)18 (fractional shortening, 12.87±4.73% versus 27.69±0.58% for CKO versus control; n=3; P<0.05) (Figure 1d, Online Video I). The CKO mice showed significantly increased ratios of heart weight—to–body weight beginning on PD14 (Figure 1e). The levels of cardiac stress markers, including atrial natriuretic peptide, brain natriuretic peptide, α-skeletal actin, connective tissue growth factor, and monocyte chemoattractant protein-1, were increased as early as PD10 (Figure 1f). Histological examination of the CKO mice on PD18 revealed that clusters of degenerating cardiomyocytes with extensive vacuolation, especially in the perinuclear region, were embedded in areas of replacement fibrosis (Figure 2a and 2b). Electron microscopic examination of the CKO cardiomyocytes revealed perinuclear accumulations of numerous multivesicular vacuoles (Figure 2c and 2d). The myofibrils and mitochondria were located exclusively at the cell periphery. In addition, we observed large, electron-dense autophagic vacuoles that contained partially digested or undigested cellular constituents, such as mitochondria and aberrant vacuoles, scattered in the field of cell debris around the perinuclear region (Online Figure III). The accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II (a phosphatidylethanolamine conjugate) and p62/SQSTM1, as well as the induction of genes in response to amino acid starvation (eg, the genes for asparagine synthetase, activating transcription factor 4, and C/EBP homologous protein) reflected defective autophagic protein degradation in the CKO mice (Figure 2e and 2f).
To examine the underlying cellular mechanism responsible for cardiac death, we examined the role of the ATP6AP2 protein in the function of V-ATPase, which maintains a luminal acidic environment in the intracellular vesicular compartments (Figure 3a).4,5 Mouse embryonic fibroblasts (MEFs) were obtained from male mice that were hemizygous for the floxed Atp6ap2 allele. The Atp6ap2 gene was ablated by infecting the MEFs with the Cre adenovirus (Ad-Cre) (Online Figure IVa). Quantitative PCR and Western blot analyses showed that ≥90% of the ATP6AP2 protein was missing in the floxed MEFs after Ad-Cre treatment, as compared with the wild-type (WT) MEFs (Online Figure IVb and Figure 3b). V-ATPase is a large multisubunit complex that is organized into the V1 and VO sectors. In mammals, the V1 sector is composed of 8 different subunits (A through H), whereas the VO sector contains 6 different subunits (a, c, c′, d, e, and the accessory subunit Ac456) (Figure 3a). Western blot and immunohistochemical analyses revealed that the levels of subunits a1, a2, a3, and c were significantly decreased in the floxed MEFs after Ad-Cre infection, as compared with the WT MEFs (Figure 3b and 3c). In contrast, the level of V1 subunit E2 was unaffected. Consistent with these findings, LysoTracker staining revealed that the loss of ATP6AP2 was accompanied by impaired vesicular acidification (Figure 3c). Taken together, these findings suggest that genetic ablation of ATP6AP2 selectively affects the stability and assembly of the VO subunits, thereby compromising vesicular acidification.
Consistent with the findings observed for cultured cells, ATP6AP2-depleted hearts revealed that the characteristic perinuclear vacuoles in the cardiomyocytes were positive for late endosomal/lysosomal markers RAB7 and/or LAMP2 (Figure 3d). The levels of the c-subunit of the VO sector but not the E2 subunit of the V1 sector markedly reduced in the CKO cardiomyocytes.
To investigate whether disruption of intracellular acidification accounts for the phenotype of the ATP6AP2-deficient cardiomyocytes, we treated cultured cardiomyocytes with bafilomycin A1 or chloroquine. Sequential time-lapse microscopic analysis revealed that intracellular vacuoles accumulated over time (Figure 4; Online Video II). These vacuoles were positive for RAB7 (Figure 4). Interestingly, Atp6ap2 mRNA expression in the cultured cardiomyocytes was strikingly upregulated after treatment with either bafilomycin A1 or chloroquine (Online Figure V).
The biogenesis of the multisubunit complex of V-ATPase requires the coordinated association of V1 subunits, which are synthesized in the cytosol, with VO subunits, which are targeted to the vacuolar membrane. Studies in yeast cells have shown that the loss of a V1 subunit has little effect on the stability of the remaining V1 subunit, whereas the loss of any single VO subunit affects the stability and assembly of the remaining VO subunits. In yeast, several additional genes (Vma12p, Vma21p, and Vma22p) that are required for V-ATPase assembly have been identified.7 The VO subunits were detected at greatly reduced levels in the mutant cells that lacked these assembly factors, an effect that is similar to that observed after the loss of a VO subunit.8 Interestingly, there is no known yeast homolog of the mammalian ATP6AP2. It is possible that ATP6AP2 is an assembly chaperone of V-ATPase, representing a function that is unique to mammals. An alternative scenario is that ATP6AP2 is a component of the VO sector itself rather than an assembling factor. Atp6ap2 mRNA expression was upregulated in cells that were treated with bafilomycin A1 or chloroquine, which suggests that ATP6AP2 senses the acidity levels of the intracellular compartments and accordingly regulates V-ATPase activity.
In conclusion, the gene product of Atp6ap2 is considered to act in 2 ways: (1) as (P)RR, exerting an RAS-related function9; and (2) as the V-ATPase–associated protein, exerting a non–RAS-related function that is essential for cell survival.10 The phenotypes observed after genetic ablation of Atp6ap2 are ascribed to V-ATPase loss of function. Further characterizing the function of ATP6AP2 as an assembly chaperone of V-ATPase and the pathological function of (P)RR would require rescue experiments with the WT protein and with mutant proteins that lack the domain responsible for binding renin and prorenin.
We thank Shintaro Morizane, Tomohiro Matsuhashi, Shugo Tohyama, Hisayuki Hashimoto, Yoshiko Miyake, and Toshihiro Nagai for excellent technical assistance. We are also grateful to Noboru Mizushima and Genzou Takemura for their comments on the manuscript. Motoaki Sano and Hiroshi Itoh are core members of the Global Center of Excellence (GCOE) for Human Metabolomics Systems Biology, Ministry of Education, Culture, Sports, Science and Technology.
Sources of Funding
This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology (17390249 to A. I.) and a PRESTO (Metabolism and Cellular Function) grant from the Japanese Science and Technology Agency (to M. S.).
Cruciat CM, Ohkawara B, Acebron SP, Karaulanov E, Reinhard C, Ingelfinger D, Boutros M, Niehrs C. Requirement of prorenin receptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling. Science. 2010; 327: 459–463.
Gaussin V, Van de Putte T, Mishina Y, Hanks MC, Zwijsen A, Huylebroeck D, Behringer RR, Schneider MD. Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3. Proc Natl Acad Sci U S A. 2002; 99: 2878–2883.
Sano M, Izumi Y, Helenius K, Asakura M, Rossi DJ, Xie M, Taffet G, Hu L, Pautler RG, Wilson CR, Boudina S, Abel ED, Taegtmeyer H, Scaglia F, Graham BH, Kralli A, Shimizu N, Tanaka H, Makela TP, Schneider MD. Menage-a-trois 1 is critical for the transcriptional function of PPARgamma coactivator 1. Cell Metab. 2007; 5: 129–142.
Supek F, Supekova L, Mandiyan S, Pan YC, Nelson H, Nelson N. A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules. J Biol Chem. 1994; 269: 24102–24106.
Graham LA, Powell B, Stevens TH. Composition and assembly of the yeast vacuolar H(+)-ATPase complex. J Exp Biol. 2000; 203: 61–70.
Hirata R, Umemoto N, Ho MN, Ohya Y, Stevens TH, Anraku Y. VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae. J Biol Chem. 1993; 268: 961–967.
Ichihara A, Hayashi M, Kaneshiro Y, Suzuki F, Nakagawa T, Tada Y, Koura Y, Nishiyama A, Okada H, Uddin MN, Nabi AH, Ishida Y, Inagami T, Saruta T. Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin. J Clin Invest. 2004; 114: 1128–1135.
Advani A, Kelly DJ, Cox AJ, White KE, Advani SL, Thai K, Connelly KA, Yuen D, Trogadis J, Herzenberg AM, Kuliszewski MA, Leong-Poi H, Gilbert RE. The (Pro)renin receptor: site-specific and functional linkage to the vacuolar H+-ATPase in the kidney. Hypertension. 2009; 54: 261–269.
Novelty and Significance
What is Known?
(Pro)renin receptor [(P)RR], which plays a key role in the local renin-angiotensin system, is encoded by the ATP6AP2 gene.
The gene product of ATP6AP2 is also associated with V-ATPase, which maintains an acidic environment in the lumen of intracellular vesicular compartments.
What New Information Does This Article Contribute?
Cardiomyocyte-specific ablation of Atp6ap2 caused fulminant heart failure.
Ablation of Atp6ap2 selectively suppressed protein expression of the VO subunits of V-ATPase, resulting in deacidification of intracellular vesicles.
The phenotypes observed after genetic ablation of Atp6ap2 are ascribed to V-ATPase loss of function.
The gene products of ATP6AP2 have 2 distinct functions. Their first role in regulating the renin-angiotensin system pathway is well established, but a secondary role in regulating V-ATPase (and other cellular roles) has not been proposed or investigated in vivo. In the present study, we generated a mouse with a cardiac-specific deficiency in Atp6ap2. The Atp6ap2-disrupted cardiomyocytes showed extensive vacuolation, a phenotype that could be reproduced by pharmacologically inhibiting intracellular acidification. We demonstrated for the first time that ATP6AP2 might be an essential assembly chaperone of mammalian V-ATPase and that genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
Original received May 19, 2010; revision received June 1, 2010; accepted June 3, 2010. In May 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.