Uptake and Metabolism of the Novel Peptide Angiotensin-(1-12) by Neonatal Cardiac Myocytes

Background Angiotensin-(1–12) [Ang-(1–12)] functions as an endogenous substrate for the productions of Ang II and Ang-(1–7) by a non-renin dependent mechanism. This study evaluated whether Ang-(1–12) is incorporated by neonatal cardiac myocytes and the enzymatic pathways of 125I-Ang-(1–12) metabolism in the cardiac myocyte medium from WKY and SHR rats. Methodology/Principal Findings The degradation of 125I-Ang-(1–12) (1 nmol/L) in the cultured medium of these cardiac myocytes was evaluated in the presence and absence of inhibitors for angiotensin converting enzymes 1 and 2, neprilysin and chymase. In both strains uptake of 125I-Ang-(1–12) by myocytes occurred in a time-dependent fashion. Uptake of intact Ang-(1–12) was significantly greater in cardiac myocytes of SHR as compared to WKY. In the absence of renin angiotensin system (RAS) enzymes inhibitors the hydrolysis of labeled Ang-(1–12) and the subsequent generation of smaller Ang peptides from Ang-(1–12) was significantly greater in SHR compared to WKY controls. 125I-Ang-(1–12) degradation into smaller Ang peptides fragments was significantly inhibited (90% in WKY and 71% in SHR) in the presence of all RAS enzymes inhibitors. Further analysis of peptide fractions generated through the incubation of Ang-(1–12) in the myocyte medium demonstrated a predominant hydrolytic effect of angiotensin converting enzyme and neprilysin in WKY and an additional role for chymase in SHR. Conclusions/Significance These studies demonstrate that neonatal myocytes sequester angiotensin-(1–12) and revealed the enzymes involved in the conversion of the dodecapeptide substrate to biologically active angiotensin peptides.


Endogenous Localization of Ang-(1-12) in Cardiac Myocytes
The endogenous presence of Ang-(1-12) was visualized by fluorescent staining within the cultured cardiac myocytes of both WKY (top panel) and SHR (bottom panel) in three or more specimens obtained from separate preparations of neonatal cardiac myocytes. As shown in Figure

Angiotensinogen and Renin Expression in Cardiac Myocytes
Angiotensinogen protein expression in the neonatal WKY and SHR cardiac myocytes maintained in serum-free medium for 48 hours was determined by Western blot analysis using appropriate antibodies. The angiotensinogen protein (60 KD band size) was present in both WKY and SHR myocytes ( Figure 6). After correcting the angiotensinogen protein expression level with loading control (EF-1a), we observed a tendency for a decreased level of angiotensinogen protein in 48 hours serum deprived neonatal cultured SHR myocytes as compared to WKY. Renin expression in these samples was also analyzed by using a well-characterized renin antibody obtained from Dr.  Tadashi Inagami (Nashville, TN). No active renin protein band (37-40 KD) was detected in both WKY and SHR myocytes. These findings further suggest that Ang-(1-12) generated in myocytes from angiotensinogen by a non-renin dependent pathways.

Discussion
Prior experimental [1,[3][4][5][6][7][8] and pilot studies in human atrial tissue [9] demonstrated that Ang-(1-12) may serve as an alternate pathway for the generation of angiotensin peptides, a pathway that may be of relevance in situations of suppressed renin activity or secretion, as well as possibly acting as an intracellular precursor for the formation of angiotensin peptides. In this study we report for the first time the presence of Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in neonatal cardiac myocytes from both WKY and SHR together with a time dependent intracellular incorporation of labeled Ang-(1-12) in both strains. Intact uptake of 125 I-Ang-(1-12) was significantly higher in SHR, a finding that agrees with the previous demonstration of increased Ang-(1-12) content in the myocardium of adult hypertensive rats by both immunohistochemistry and RIA. [3] Assessment of the hydrolysis of 125 I-Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in the presence and absence of inhibitors for ACE, NEP, ACE2, and chymase revealed a primary involvement of ACE and NEP in WKY and an additional important contribution of chymase in SHR. This finding is in keeping with reports showing augmented chymase expression and increased chymostatin-inhibitable angiotensin-converting activity in SHR. [10] The increased hydrolytic effects of chymase on 125 I-Ang-(1-12) metabolism in SHR was associated with a greater processing activity of ACE and NEP. These findings agree with previous studies suggesting increased expression and activity of the cardiac renin angiotensin system in SHR. [10] Although further work will be necessary to elucidate potential differences in the processing and incorporation of extracellular Ang-(1-12) between neonatal and adult cardiac myocytes previous  work shows differences in cellular replication and functional maturation of SHR ventricular myocytes compared to WKY rats during the first postnatal week. [11] This is associated with the presence of earlier development of binucleate myocytes and initiation of hypertrophic myocyte growth in SHR. [12] The use of a radiolabeled form of Ang-(1-12) allowed for a precise and sensitive quantification of the products generated by the hydrolysis of Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The drugs were given in doses sufficient to block 125 I-Ang-(1-12) metabolism by at least 90%. As documented elsewhere, [13][14][15] inclusion of amastatin, bestatin, benzyl succinate, and PCMB in all processed samples was an added precaution to prevent the further degradation of angiotensin peptides by aminopeptidases and carboxypeptidases into smaller fragments.
The heart is an organ where local formation of Ang II is implicated to regulate cardiac remodeling due to increased afterload or ischemia. [16,17] Central to this concept is the demonstration that Ang II can be produced locally and function as a paracrine [16,17] or autocrine factor. [18] Through the use of radiolabeled 125 I-Ang I and 125 I-Ang II van Kats et al. [19] concluded that most of the Ang I and Ang II found in the heart was produced locally. These findings are reinforced by the recent demonstration of increased Ang II synthesis in cardiac myocytes from diabetic rats. [20] In the SHR, increased activity of the RAS is found in the heart [21,22] and the kidneys [23] while in cultured newborn SHR heart cells, increased uptake of 3 H-proline by Ang I and Ang II was abolished by blockade with either an ACE inhibitor or an AT 1 receptor antagonist. [24] The latter findings suggest an intrinsic increase in protein uptake by SHR myocytes, a finding that is in keeping with the current observations of differences in the rate of 125 I-Ang-(1-12) incorporation in WKY and SHR myocytes.
A further insight into the functional significance of local RAS is expanded by our finding of an increased incorporation of intact 125 I-Ang-(1-12) in cardiac myocytes from cultured neonatal cells of SHR. The HPLC analysis of cell lysate showed that intact Ang-(1-12) is internalized by cardiac myocytes, although the amount internalized represents a small fraction of total loaded 125 I-Ang-(1-12) (,5.2% in SHR & ,3.6% in WKY). Further, we report around 50% inhibition of cellular internalization of radiolabeled Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in the presence of excess cold Ang-(1-12) [Iodineunlabeled Ang-(1-12); 10 3 mM]. We suggest that the failure to achieve higher inhibition may be due to differences in binding affinity between cold Ang-(1-12) and 125 I-Ang-(1-12) or the potential existence of several uptake mechanisms. This interpretation is in keeping with the finding that no more than only 50% of bound angiotensinogen could be displaced from human renal tubular epithelial cells [25]. As in their study, Ang-(1-12) as an extended form of Ang I, might bind to a low affinity receptor or alternatively to the same angiotensinogen receptor characterized by Yayama et al., [26] and Pan et al. [25]. In addition, Mineo et al., [27] reported a similar 50% inhibition of 125 I-Ang II in the presence of unlabeled Ang II in endothelial cell monolayers. They interpreted these findings as suggesting that about 50% of Ang II was transported via an intracellular pathway while the remainder was accounted for non-specific transport. The augmented incorporation of Ang-(1-12) into SHR myocytes agrees with the reported observation of increased incorporation of 3 H-uradine and 14 C-leucine into 10-day-old SHR heart cells. [28] The process appears to remain present in older animals as adult SHR showed increased Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)   In the controlled environment of a cell culture system, neonatal cardiac myocytes of SHR and WKY metabolized 125 I-Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) into Ang I, leading to sequential production of Ang II, Ang-(1-7), and other smaller angiotensin fragments. Ang-(1-12) metabolism by cardiac myocytes was significantly blocked in the presence of specific enzyme inhibitors for ACE, neprilysin, ACE2, and chymase. While the hydrolysis of 125 I-Ang-(1-12) was found to be primarily due to ACE and NEP in WKY rats, chymase contributed to the conversion of 125 I-Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) to Ang I and Ang II in SHR. This new observation is in keeping with the demonstration of an important role for mast cell derived chymase in Ang II formation following either decreased expression [29] or pharmacological blockade of ACE [30] This finding also agrees with Prosser et al. [29] report that chymostatin completely inhibited Ang II formation from Ang-(1-12). As discussed elsewhere, [5,31] differences in the nature of the enzymatic pathways accounting for Ang-(1-12) metabolism in the studies reported by Nagata et al. [1] and Prosser et al. [8] may be a result of increased cellular permeability associated with the isolated cardiac perfusion procedures use by Prosser et al. (12). The more controlled preparation employing only cardiac myocytes cultures rather than the whole organ or the systemic circulation provided a more precise system for the identification of the differential routes of Ang-(1-12) processing reported in this study. In addition, the higher contribution of chymase in Ang-(1-12) metabolism found in SHR compared to WKY controls is in keeping with previous findings of increased chymase activity in SHR. [32] Immunofluorescence staining using a well-characterized Ang-(1-12) antibody clearly shows the presence of Ang-(1-12) in neonatal cultured SHR and WKY myocytes. In addition, angiotensinogen protein but not renin was also detected in these myocytes by Western blot analysis. These studies further buttress the hypothesis that Ang-(1-12) may be generated intracellularly from angiotensinogen as well as incorporated into the myocyte.

Isolation of Neonatal Cardiac Myocytes
15-18 days pregnant SHR and normotensive WKY mothers were purchased from Charles River Laboratories Inc. (Wilmington, MA). Neonatal rat ventricular cardiac myocytes of WKY and SHR pups (1-3 day old) were isolated by proteolytic digestion and differential plating, as previously described. [33,34] Briefly, neonatal pups were placed under deep isoflurane (4%) anesthesia and their hearts were quickly removed and placed into ice cold Hanks Balanced Salt Solution (HBSS) medium. The myocytes were isolated from minced ventricles by proteolytic digestion and differential plating (to separate the myocytes from fibroblast). Myocytes were collected and maintained in DMEM/F-12 supplemented with 10% FBS, 10 mg/mL insulin, 10 mg/mL holo-transferrin, 100 mM 5-bromodeoxyuridine (to prevent proliferation of non-myocytes), and antibiotics (ampicillin and streptomycin) in a humidified incubator with an atmosphere of 95% O 2 , 5%CO 2 . Myocytes were incubated overnight in the same DMEM/F-12 medium in the absence of FBS and 5-bromodeoxyuridine before use for 125 I-Ang-(1-12) uptake and metabolism studies. Cells isolated by this protocol routinely showed positive staining for anti-sarcomeric myosin (1:100 dilutions, Sigma-Aldrich Co., St Louis, MO) and no immunoreactivity labeling with antibodies to alpha-actin, fibronectin, or vimentin (1:100 dilutions, Sigma-Aldrich Co., St Louis, MO).

Group
Inhibitors added (10 mM each)

All RAS inhibitor group
For 125 I-Ang-(1-12) metabolism study, the cultured myocytes (WKY or SHR) were preincubated for 15 min under various combinations of RAS and peptidases inhibitors (10 mmol/L of each) as described above. After preincubation of myocytes with the inhibitors, 125 I-Ang-(1-12) (1 nmol/L) was added to reaction medium and incubated for 60 min at 37uC in the water bath. At the end of incubation time, exposed medium and cardiac myocytes were collected separately and processed for HPLC analysis as described below.

HPLC Analysis of Ang-(1-12) Metabolic Products in Medium and in Cardiac Myocytes
Cellular uptake of 125 I-Ang-(1-12) by cardiac myocytes and their metabolic products in cell lysate and dosing medium were analyzed by HPLC as previously described. [35,36] The medium from the cell cultures was removed and mixed with 1% of phosphoric acid and stored at 280uC until processing the samples for Ang contents [Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), Ang I, Ang II and Ang-(1-7)] by HPLC. After removing the dosing medium, myocytes were washed three times with ice-cold PBS and one time with 0.05 M glycine-HCl (pH 4) to remove the membrane bound 125 I-Ang. After removing the membrane bound 125 I-Ang-(1-12), the myocytes were scraped using a cell lifter in 1 mL of acid-ethanol (0.1N HCl + 80% ethanol). The scraped cells were immediately frozen and stored at 280uC till processing the samples to detect the cellular 125 I-Ang content by HPLC.
Before using the samples for HPLC analysis, the cell lysate and medium were passed through Sep-Pak C18 cartridge columns (Waters Corp., Milford, MA) to collect the 125 I-labeled Ang-(1-12) metabolic products. Briefly, cells and medium (stored at 280uC) were thawed in ice and the cell lysate were briefly sonicated for 10 sec. The lysed cells were centrifuged at 28,000 rpm for 15 min to remove the cell membrane and debris. The clear supernatant and the medium were diluted with 9x vol. of 0.1% trifluoroacetic acid (TFA) and were passed through activated Sep-Paks by gravity. The columns were first washed with 10 mL of 0.1% TFA and then with 5 mL of MilliQ water. After washing the column, the bound 125 I-labeled Ang-(1-12) metabolic products were eluted with 80% methanol + 0.1% TFA. Finally, Sep-Pak eluted samples were analyzed by HPLC to detect the 125 I-Ang-(1-12) metabolic products. We used a linear gradient from 10% to 50% mobile phase B at a flow rate of 0.35 mL/min at ambient temperature. The solvent system consisted of 0.1% phosphoric acid (mobile phase A) and 80% acetonitriles/0.1% phosphoric acid (mobile phase B). The eluted 125 I products were monitored by an in-line flow-through gamma detector (BioScan). Products were identified by comparison of retention times to synthetic [ 125 I] standard peptides and the data were analyzed with Shimadzu (version 7.2.1) acquisition software. The iodination of rat Ang-(1-12) and other angiotensins was performed as described previously. [35,36] Contribution of specific enzymes (ACE, NEP, ACE or chymase) The contribution of ACE, NEP, ACE2 or chymase to the hydrolysis of 125 I-Ang-(1-12) substrate were analyzed by measuring the amount of Ang products formation in the cultured medium after exposing the cells in the presence of all RAS inhibitors cocktail and in the absence of specific enzyme inhibitors for ACE, NEP, ACE2 or chymase only (as described above). Medium was collected after exposing the WKY and SHR cells for 60 min at 37uC under two different enzyme inhibitors conditions (+All RAS inhibitors versus minus ACE/NEP/ACE2/chymase inhibitor only) and were analyzed by HPLC as described above. The enzyme activity was calculated based on amount of 1 nM of 125 I-Ang-(1-12) substrate added to the reaction mixture and metabolized into final products by specific RAS enzyme. The protein content of each well was determined by Bradford Reagent using BSA as standard protein. Experiments were performed three or more times and the enzyme activity values were reported as fmoles of Ang product formation from 125 I-Ang-(1-12) substrate per min per mg protein (fmol?mg protein 21 ?min 21 ).

Western blot analysis of Angiotensinogen and Renin in Cultured Cardiac Myocytes
The expression of angiotensinogen protein in neonatal cultured cardiac myocytes was assessed by immunoblot. Myocytes were isolated and maintained as describe above. Before collecting the myocytes for angiotensinogen protein expression, the cells were cultured for 48 hours in serum-deprived medium. Angiotensinogen protein expression was assessed in total cell lysate by using an antibody directed against an epitope on the NH 2 -terminus region of the protein (residues 44-56) which detects both Ang Icontaining and des-Ang I forms of the protein. [37] The myocytes cell lysate (50 mg protein) were separated by gel electrophoresis and transferred to polyvinylidene difluoride membranes (PVDF). The PVDF membranes were probed with an antibody against rat angiotensinogen protein (1:1000 dilutions). Densities of the 60-KD immunoreactive bands of WKY and SHR groups were determined with a MCID imaging system (MCID Elite 7.0, Imaging Research Inc., St. Catharine, ON, Canada). The expression of renin in neonatal cultured myocytes was also assessed using an antibody against rat renin (1:1000 dilution) obtained from Dr. Tadashi Inagami as described above.
Chymostatin (chymase inhibitor), amastatin, bestatin and benzyl succinate and PCMB were purchased from Sigma-Aldrich Co. (St. Louis, MO). Radioactive 125 I was purchased from PerkinElmer Life and Analytical Sciences, Inc. (Waltham, Massachusetts). All other chemicals used in this study were of analytical grade and were obtained from Sigma (St. Louis, MO), and Fisher Scientific (Atlanta, GA).