This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Barlucchi, L. Articles by Anversa, P. Search for Related Content PubMed PubMed Citation Articles by Barlucchi, L. Articles by Anversa, P. Related Collections Congestive ACE/Angiotension receptors Animal models of human disease Gene expression

(Circulation Research. 2001;88:298.)
© 2001 American Heart Association, Inc.

Cellular Biology

Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure

Laura Barlucchi, Annarosa Leri, David E. Dostal, Fabio Fiordaliso, Hideo Tada, Thomas H. Hintze, Jan Kajstura, Bernardo Nadal-Ginard, Piero Anversa

From the Departments of Medicine (L.B., A.L., F.F., J.K., B.N.-G., P.A.) and Physiology (H.T., T.H.H.), New York Medical College, Valhalla, NY; Division of Molecular Cardiology (D.E.D.), The Texas A&M University, Temple, Tex; and Istituto Ricerche Farmacologiche Mario Negri (F.F.), Milano, Italy.

Correspondence to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, NY 10595. E-mail piero_anversa{at}nymc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT₁ receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II–mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT₁ and AT₂ receptors was evaluated by reverse transcriptase–polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT₁ receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT₁. Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT₁ were markedly increased in paced myocytes. Conversely, chymase and AT₂ proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

Key Words: pacing • myocyte renin-angiotensin system • p53 function • heart failure

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocyte death and myocyte hypertrophy characterize dilated cardiomyopathy in humans.^{1 2} Cardiomegaly, ventricular dilation, myocyte apoptosis, and myocyte hypertrophy develop with rapid ventricular pacing,^{3 4 5} mimicking the human disease. Angiotensin (Ang) II is implicated in the activation of several cellular responses, including myocyte apoptosis and hypertrophy.^{6 7 8} Interference with the systemic and local renin-angiotensin system (RAS) limits myocyte growth and the expansion in cavitary volume, improving ventricular performance and the lifespan of patients with cardiac failure.^{9 10 11} Experimentally, similar interventions inhibit apoptotic cell death in the stressed myocardium preventing or attenuating decompensation.^{12 13} Rat ventricular myocytes possess the various components of RAS, and an upregulation of renin, Aogen, Ang I, angiotensin-converting enzyme (ACE), Ang II, and AT₁ and AT₂ receptors occurs in myocytes of the overloaded rat heart.^{14 15 16 17} Whether canine myocytes synthesize Ang II, influencing directly cellular adaptations via an autocrine mechanism, remains to be demonstrated. This is a relevant issue, because other pathways in the formation of Ang II have been identified in the dog myocardium.^{18 19}

Chymostatin-sensitive angiotensin-producing enzyme, ie, heart chymase, has been implicated as the major source of Ang II accumulation in the canine,^{18 19} baboon,²⁰ and human^{21 22} hearts. These observations tend to minimize the role of ACE in the conversion of Ang I to Ang II in large mammals and raise questions on the existence of a myocyte RAS because of the claimed absence of chymase in this cell population.^{18 19 20 21 22} However, opposite results have also been reported.²³ One of the problems with these findings is that little attention has been given to myocytes even though the entire myocardium or the properties of the interstitial fluid have been analyzed.^{18 19 20 21 22 23 24 25} In view of these uncertainties, the expression of the multiple constituents of RAS was measured at the mRNA and protein levels in myocytes of normal and paced dog hearts. Additionally, p53 function was analyzed because the tumor suppressor transactivates Aogen and AT₁ receptor.^{6 7 26 27 28} Aogen is the limiting factor in the synthesis of Ang II,²⁸ and binding to AT₁ conditions myocyte growth and death.^{6 7} Thus, we established whether canine myocytes have the capability of generating Ang II, whether Ang II formation is enhanced in the overloaded ventricle, and whether p53 transcriptional potential correlates with the response of stressed myocytes. Positive results would imply that Ang II plays a critical role in myocyte growth, myocyte death, and the terminal evolution of the failing heart.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ventricular Function
Mongrel dogs were instrumented with corkscrew electrode in the left ventricle (LV) attached to an external pacemaker.^{4 5 26 29} Hearts were paced at 210 bpm for 1 (n=6) and 3 (n=6) weeks and at 240 bpm for an additional week (n=7). Control dogs were instrumented but not paced (n=7). Protocols were approved by New York Medical College. Cardiac function was measured in conscious dogs with the pacemaker turned off.^{26 29}

Myocyte Isolation
Myocytes were enzymatically dissociated (Figure 1) from 6 control hearts and 4, 4, and 6 hearts paced for 1, 3, and 4 weeks, respectively.^{4 5 26} Purity of the preparation and the distinction of myocytes from other cells is described in the online data supplement available at http://www.circresaha.org.

View larger version (65K): [in this window] [in a new window]   Figure 1. Enzymatically dissociated myocytes from the LV of a 4-week paced dog. Myocyte cytoplasm is illustrated by the red fluorescence of -sarcomeric actin antibody staining, whereas nuclei are depicted by the yellow fluorescence of PI.

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted with Trizol reagent (Gibco Life Technologies). Total RNA 5 µg was reverse transcribed with 200 U of Superscript II reverse transcriptase (RT) (Gibco Life Technologies) according to the supplier protocol. The resulting cDNA was amplified using 1.5 U Taq DNA polymerase (Boehringer Mannheim) as described in the supplier protocol. Sense and antisense primers and polymerase chain reaction (PCR) amplification cycles used for Aogen, renin, chymase, ACE, AT₁ and AT₂ receptors, and ß-actin are described in the online data supplement. PCR products were separated on a 2% agarose gel.

Electrophoretic Mobility Shift Assay
Oligonucleotides were prepared as recently described.^{6 7 26 27 28} Nuclear extracts, 40 µg, were incubated with 2 µL of [³²P]ATP–end-labeled probe and subjected to electrophoresis. Control for specificity included the exposure of nuclear extracts to anti-p53, 0.5 µg of PAb 240, or an irrelevant antibody, 0.5 µg of mouse anti-c-Jun.

Western Blot
Equivalents of 50 to 75 µg of proteins were separated by SDS-PAGE, transferred on nitrocellulose membranes, and exposed to mouse anti-rat Aogen, mouse anti-rat renin, mouse monoclonal anti-human cathepsin D, mouse monoclonal anti-rat ACE,³⁰ mouse monoclonal anti-human chymase, rabbit polyclonal anti-human AT₁ receptor, and rabbit polyclonal anti-rat AT₂ receptor³¹ antibodies. Rat kidney and purified human ACE were used as positive controls for ACE. Rat adrenal gland was used as a positive control for AT₂.

Ang II Labeling
Myocardial sections were fixed in formaldehyde and incubated with Ang II antiserum.^{6 7} Specificity was determined by preabsorption of 10 µL of antibody with 0.05 mg of antigen. Nonimmune rabbit serum was used as an additional control.^{6 7} Myocyte cytoplasm was identified by -sarcomeric actin antibody.^{6 7} Ang II–positive myocytes and number of Ang II sites per myocyte profile were measured by confocal microscopy.

Ang II Concentration
Ang II levels in myocytes were obtained by ELISA using the methodology previously described.^{9 27}

Data Analysis
Results are mean±SD. Autoradiograms were analyzed by an image analyzer. Each set of samples in each gel included all time points after pacing and controls. Statistical significance was determined by Student’s t test or Bonferroni method.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ventricular Hemodynamics
Alterations in cardiac function in this animal model have been described.^{4 5 26 27} With 4 weeks of pacing, heart rate increased 42% (P<0.001), from 86±12 to 122±15 bpm, and mean arterial pressure decreased 18% (P<0.01), from 106±9 to 87±9 mm Hg. Similarly, LV systolic pressure decreased 24% (P<0.001), from 132±8 to 100±11 mm Hg, and LV positive dP/dt decreased 48% (P<0.001), from 2970±457 to 1546±351 mm Hg/s. Conversely, LV end-diastolic pressure increased 333% (P<0.001), from 6±2 to 26±6 mm Hg. Tachypnea, ascites, pulmonary congestion, and pleural effusion were present in these animals. There were no significant hemodynamic changes at 1 week of pacing, whereas at 3 weeks, LV end-diastolic pressure was increased 133% (P<0.01), to 14±3 mm Hg.

RT-PCR of Aogen, Renin, ACE, Chymase, and AT₁ and AT₂ Receptors
The expression of the components of the local RAS in control and paced canine myocytes was detected by RT-PCR (Figure 2). Four determinations were obtained for each gene. The use of RT-PCR did not permit quantitative measurements of the changes in mRNAs with the duration of pacing. These difficulties in the interpretation of mRNA levels are inherent in this methodology. However, the purpose of this analysis was to document that a myocyte RAS was present at baseline and that ventricular dysfunction and failure with prolonged pacing did not impair the ability of dog myocytes to express these transcripts.

View larger version (65K): [in this window] [in a new window]   Figure 2. Detection of Aogen, renin, ACE, chymase, and AT1 and AT2 receptors mRNAs by RT-PCR in isolated myocytes from nonpaced (N) and paced (P) myocytes for 1 (P-1w), 3 (P-3w), and 4 (P-4w) weeks. ß-actin was used as internal reference band. K indicates dog kidney tissue; H2O, omission of cDNA in the assay; and -Taq, omission of Taq DNA polymerase in the assay.

p53 DNA Binding
p53 is implicated in the upregulation of the myocyte RAS, leading to the synthesis and secretion of Ang II.^{6 7 28} A gel-retardation assay was performed using oligonucleotides, including the consensus site for p53 binding to the Aogen and AT₁ promoter, respectively. On the basis of the results described above, this assay was restricted to myocytes from control and failing hearts at 4 weeks of pacing, to establish whether a correlation existed between p53 function and RAS activation in the terminal phases of the dilated myopathy. Figure 3A illustrates that the Aogen oligonucleotide resulted in the formation of a p53-shifted complex in nuclear extracts from myocytes of sham-operated and failing dogs. However, the optical density (OD) of the p53 band was increased in cells from paced hearts (controls, OD=1±0.2, n=6; paced, OD=2.9±1.1, n=6; P<0.005). The specificity of the assay was confirmed by demonstrating that the addition of an excess of unlabeled self-oligonucleotide or of a monoclonal p53 antibody to nuclear extracts from paced myocytes opposed the appearance of a p53 band. Conversely, preincubation with an irrelevant antibody did not affect p53 DNA binding. The effects of pacing on p53 binding to the promoter of AT₁ receptor are shown in Figure 3B. Two specific bands were identified, but the OD of the shifted complexes was significantly greater in myocytes from paced hearts (OD=1.4±0.4, n=6; paced, OD=3.5±0.8, n=6; P<0.001).

View larger version (62K): [in this window] [in a new window]   Figure 3. Gel-mobility shift assay showing p53 binding to the consensus motif on the Aogen promoter (A) and the AT1 promoter (B). Nuclear extracts were obtained from myocytes isolated from paced (P) and normal (N) LVs. Addition of a monoclonal p53 antibody (Ab) to nuclear extracts from paced myocytes opposed the appearance of a p53 complex. Competition with an excess of unlabeled self-oligonucleotide (C) failed to reveal a p53 band. Preincubation with an irrelevant antibody (Irr) did not affect p53 DNA binding. SV-T2 cells were used as positive control. Ao indicates Aogen probe; AT1, AT1 probe. P1 through P3 are 3 dogs paced for 4 weeks.

Western Blot of Aogen, Renin, Cathepsin D, ACE, Chymase, and AT₁ and AT₂ Receptors
The protein levels of the myocyte RAS were analyzed in control and failing hearts at 4 weeks of pacing. Expression of Aogen, renin, and cathepsin D in myocytes is illustrated in Figure 4. Aogen was represented by 2 bands at 54 and 56 kDa, but the higher molecular-weight protein prevailed (Figure 4A). Cardiac failure resulted in a 2.5-fold (P<0.001) increase in Aogen expression in myocytes, from a baseline OD value of 3.3±0.7 (n=6) to a value of 8.1±1.6 (n=6). Similarly, renin was detected as 2 distinct bands at 36 and 37 kDa, respectively (Figure 4B). With pacing, renin increased 3.6-fold (controls, OD=1.2±0.4, n=6; paced, OD=4.3±1.1, n=6, P<0.001), paralleling the changes in Aogen. Myocyte cathepsin D appeared as 3 bands at 52, 43, and 34 kDa (Figure 4C). However, the mature form of cathepsin D corresponds to 43 kDa and is the most visible on Western blot. Quantitatively, only the 43-kDa band was measured, because the 52-kDa protein is an inactive precursor, and the 34-kDa protein is an inactive degradation product. Pacing was associated with a 3-fold (P<0.001) increase of the active 43-kDa enzyme (controls, OD=1.1±0.3, n=6; paced, OD=3.3±0.7, n=6).

View larger version (51K): [in this window] [in a new window]   Figure 4. Western blot of Aogen (A), renin (B), and cathepsin D (C) in normal (N) and paced (P) myocytes. Serum from normal (N) and paced (P) dogs was used as positive control for Aogen. Loading of proteins is illustrated by Coomassie blue staining. P1 through P6 are 6 dogs paced for 4 weeks.

Two forms of ACE were identified at 134 and 170 kDa (Figure 5A). The higher molecular weight reflected the glycosylated enzyme; the other form corresponded to nonglycosylated ACE. Both proteins increased with heart failure. The 134-kDa band increased 1.45-fold (P<0.005), and the 170-kDa band increased 1.44-fold (P<0.002). In contrast, chymase, detected at 29 kDa, did not change (P=0.28) in nonpaced and paced animals (Figure 5A). AT₂ receptor increased 42% (Figure 5B) with pacing, but this change was not significant (P<0.06). Conversely, AT₁ receptor in myocytes increased significantly, 1.7-fold (P<0.001) at 4 weeks after pacing (Figure 6) (controls, OD=3.1±0.75, n=6; paced, OD=5.4±0.98, n=6).

View larger version (59K): [in this window] [in a new window]   Figure 5. A, Western blot of nonglycosylated and glycosylated ACE and chymase in normal nonpaced (N) and paced (P) myocytes. Human purified ACE (HA) and rat kidney (RK) were used as positive controls for ACE. Bar graphs show densitometry of these proteins. Results are presented as mean±SD. n=5 in each group. *P<0.05. B, Western blot of AT2 receptor in N and P myocytes. Rat adrenal gland (RA) was used as positive control. Bar graphs show densitometry data. Results are presented as mean±SD. n=5 in each group. Loading of proteins is illustrated by Ponceau red staining. P1 through P5 are 5 dogs paced for 4 weeks.

View larger version (90K): [in this window] [in a new window]   Figure 6. Western blot of AT1 receptor in normal (N) and paced (P) myocytes. Equal loading of proteins is illustrated by Coomassie blue staining. P1 through P3 are 3 dogs paced for 4 weeks.

Data by Western blot were not always consistent with the levels of expression obtained by RT-PCR. These differences reflected the nonquantitative aspects of RT-PCR and the fact that changes in mRNAs are not necessarily identical to those in the quantity of proteins. Conversely, immunoblotting has inherent limitations in the precise identification of the size of each protein. Different molecular weight markers were used to minimize this possibility.

Ang II Quantity in Myocytes
Ang II was measured in left ventricular myocytes by ELISA. Pacing resulted in a 2.2-fold (P<0.004) increase in the quantity of Ang II in myocytes (controls, 20±11 pg/mg protein, n=5; paced, 44±7 pg/mg protein, n=5). Two additional approaches were used: quantitative evaluation of the percentage of myocytes labeled by Ang II antibody and measurements of Ang II sites per unit area of myocyte cytoplasm by confocal microscopy. Figure 7A illustrates by green fluorescence the discrete sites of Ang II labeling, and Figure 7B depicts by red fluorescence myocytes stained by -sarcomeric actin. These two images are shown together in Figure 7C; the fluorescent dots correspond to the localization of Ang II in the myocardium. Myocyte profiles, defined by laminin staining, contained a minimum of 1 to a maximum of 14 stained sites per cell. Preabsorption of the primary Ang II antibody with Ang II resulted in the absence of immunostaining (Figure 7D). Similarly, substitution of the Ang II antibody with nonimmune rabbit serum was characterized by the lack of staining in the myocardium (not shown). An average of 180 ventricular myocytes were examined at random in each of 6 nonpaced and 6 paced dogs, for a total of 1086 and 1056 cells in the 2 groups of animals. This analysis demonstrated that 22±4% and 45±7% of myocytes were labeled in nonpaced and paced hearts, respectively. Cardiac failure resulted in a 2-fold (P<0.001) increase in the fraction of myocytes containing Ang II. Additionally, the number of Ang II–positive sites per mm² of myocytes was 2310±607 and 6285±659 in control and paced dogs, respectively. The 2.7-fold greater value with pacing was significant (P<0.001). The distribution of Ang II–positive dots in labeled cells and the fraction of negative myocytes are shown in Figure 8. Ang II was increased in all cell categories of paced dogs. Whether the increase in Ang II correlated with an increase in myocyte size with the progression of pacing remains to be investigated.

View larger version (102K): [in this window] [in a new window]   Figure 7. Immunocytochemical detection by confocal microscopy of Ang II in paced myocytes (A through C). Myocyte cytoplasm is illustrated by red fluorescence of -sarcomeric actin, and Ang II is depicted by green fluorescence dots. Myocyte profiles are defined by laminin staining (A, C, and D) Preabsorption of Ang II antibody with Ang II resulted in the lack of staining by green fluorescence of myocytes (D). Magnification x1100.

View larger version (18K): [in this window] [in a new window]   Figure 8. Distribution of Ang II–positive sites in myocytes. Open bars indicate nonpaced; hatched bars, paced. Results are presented as mean±SD. n=6 in each group. *P<0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study indicate that canine ventricular myocytes expressed the various molecular components of RAS and were capable of forming Ang II. Cathepsin D and chymase were detectable in these cells, pointing to the possibility of the conversion of Aogen to Ang I and Ang I to Ang II through pathways independent from renin and ACE. Surface AT₁ and AT₂ receptors were identified in myocytes. Cardiac failure was characterized by an upregulation of Aogen, renin, cathepsin D, ACE, and Ang II, whereas chymase was not increased; AT₁ receptor protein increased, but the quantity of AT₂ was not significantly altered. p53 function was enhanced in myocytes, leading to activation of the p53-regulated genes Aogen and AT₁ receptor. These observations support the notion that the local synthesis of Ang II may be implicated in myocyte death and cellular hypertrophy in experimental dilated cardiomyopathy.

Ventricular Pacing and Generation of Ang II in Myocytes
Analysis of the local RAS in the dog and baboon heart has been restricted to myocardial samples^{18 19 20} or examination of interstitial fluid.^{24 25} These investigations have provided significant information concerning the role of the chymase system in the generation of Ang II in the normal and overloaded heart.^{18 19 20 24 25} Similar findings have been reported in humans, strengthening the contribution of chymase in the formation of Ang II in the nonfailing and failing heart.^{21 22} Although not all studies are in agreement,²³ the major source of chymase was linked to interstitial cells, and ACE was localized to the endothelial cell membrane only.²³ On this basis, myocytes were not identified as a cell population expressing chymase or ACE activity and thereby were assumed of limited importance in the synthesis and secretion of Ang II in the myocardium of large mammals.^{18 19 20 21 22 23 24 25}

Results in this study document that canine myocytes contained Ang II. Contrary to expectation, chymase and ACE were detected in these cells, and the upregulation of the myocyte RAS with cardiac failure was associated with increased ACE without changes in chymase. This does not exclude that both enzymes were involved in the generation of Ang II in the overloaded myocytes. Renin mRNA has been previously identified in rat cardiac myocytes,¹⁴ and its expression increases with heart failure.¹⁶ The present data confirm these observations in canine myocytes. Renin can be synthesized directly in cardiac muscle cells, and its formation is enhanced in the decompensated dog heart. Sarcomere stretching in vitro, which mimics diastolic overload in vivo, results in an upregulation of renin³² and other components of RAS, leading to the production and release of Ang II from the cells.^{6 7} Cathepsin D, which is ubiquitously present in the lysosomes of all cells,³³ was found in myocytes, and a 3-fold increase was measured after ventricular pacing. This aspartyl protease³⁴ may be implicated in combination with renin in the synthesis of Ang I with pacing. In summary, dog myocytes are most likely implicated in the regulation of the myocardial RAS under normal and pathological states.

Ventricular Pacing and Ang II–Receptor Subtypes
Two isoforms of Ang II receptors, AT₁ and AT₂, have been identified in neonatal myocytes.³⁴ With maturation, AT₂ progressively decreases and AT₁ predominates.^{35 36 37} AT₂ receptors are upregulated in the myocardium after pressure overload or infarction,^{17 38 39} but their role remains unclear. In neonatal myocytes, Ang II–mediated hypertrophy is potentiated when AT₂ blocking agents are used.⁴⁰ Similarly, deletion of the AT₂ gene leads to an abnormal elevation in arterial blood pressure, coupled with alterations in resistance vessels and smooth muscle cell hyperplasia.⁴¹ By autoradiography of the human heart, AT₂ receptors have been found in fibroblasts; AT₂ expression is increased with heart failure, and this may oppose fibroblast proliferation induced by AT₁–receptor activation.³⁷ Myocytes show minimal amount of AT₂ at baseline, and this quantity is not affected by cardiac decompensation.³⁷

In this study, AT₁ and AT₂ were detected in pure preparations of myocytes from nonpaced and paced hearts. The documentation of AT₂ in adult canine myocytes has no precedent. Cardiac failure was characterized by an increase in AT₁, but the changes in AT₂ were not significant. This differential adaptation suggests that AT₁ receptor activation is implicated in the cellular responses that condition remodeling of the paced ventricle: myocyte apoptosis^{5 26} and cellular enlargement.⁴ Increases in Ang II and AT₁ receptors with tachycardia may promote hypertrophy⁸ or trigger death in myocytes susceptible to growth or apoptotic signals.^{6 7}

Ventricular Pacing and p53
Although stretching and the secretion of Ang II from myocytes may be the immediate consequence of the elevation in left ventricular end-diastolic pressure with pacing,⁴² it is more difficult to understand the mechanism by which the local RAS is activated, maintaining the formation of Ang II. p53 binding to the promoter of Aogen and AT₁ receptor was enhanced in the failing heart, resulting in an upregulation of Aogen and AT₁ proteins in myocytes. Because renin and cathepsin D, as well as ACE and chymase, are normally present in canine myocytes, the formation of Aogen may be the limiting factor in the generation of Ang II. Overexpression of p53 in myocytes in vitro enhances transcription of Aogen and AT₁ receptor, leading to the production and release of Ang II.⁴³ Similarly, sarcomere elongation increases the quantity and functional activity of p53, which are coupled with the induction of multiple p53-regulated genes, including Aogen and AT₁ receptor.^{6 7} p53 binding to the bax promoter is enhanced, and the amount of Bax protein in myocytes increases several fold.⁶ Conversely, Bcl-2 is decreased. These cellular responses are inhibited by infection of myocytes with an inactive form of p53.²⁸ Thus, upregulation of p53 function with pacing may play a critical role in the chronic synthesis of Ang II and Ang II–mediated myocyte hypertrophy and death in this model.^{4 26}

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-38132, HL-39902, HL-43023, AG-15756, HL-65577, HL-69923, AG-17042 and by grant JDFI 1-2000-62.

	Footnotes

 
Original received October 2, 2000; revision received November 27, 2000; accepted December 13, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Beltrami CA, Finato N, Rocco M, Feruglio GA, Puricelli C, Cigola E, Sonnenblick EH, Olivetti G, Anversa P. The cellular basis of dilated cardiomyopathy in humans. J Mol Cell Cardiol. 1995;27:291–305.[Medline] [Order article via Infotrieve]
2. Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng W, Nitahara JA, Quaini E, Di Loreto C, Beltrami CA, Krajewski S, Reed JC, Anversa P. Apoptosis in the failing human heart. N Engl J Med. 1997;336:1131–1141.[Abstract/Free Full Text]
3. Spinale FG, Zellner JL, Tomita M, Crawford FA, Ziles MR. Relation between ventricular and myocyte remodeling with the development and regression of supraventricular tachycardia-induced cardiomyopathy. Circ Res. 1991;69:1058–1067.[Abstract/Free Full Text]
4. Kajstura J, Zhang X, Liu Y, Szoke E, Cheng W, Olivetti G, Hintze TH, Anversa P. The cellular basis of pacing-induced dilated cardiomyopathy: myocyte cell loss and myocyte cellular reactive hypertrophy. Circulation. 1995;92:2306–2317.[Abstract/Free Full Text]
5. Liu Y, Cigola E, Cheng W, Kajstura J, Olivetti G, Hintze TH, Anversa P. Myocyte nuclear mitotic division and programmed myocyte cell death characterize the cardiac myopathy induced by rapid ventricular pacing in dogs. Lab Invest. 1995;73:771–787.[Medline] [Order article via Infotrieve]
6. Leri A, Claudio PP, Li Q, Wang X, Reiss K, Wang S, Malhotra A, Kajstura J, Anversa P. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell. J Clin Invest. 1998;101:1326–1342.[Medline] [Order article via Infotrieve]
7. Leri A, Liu Y, Claudio PP, Kajstura J, Wang X, Wang S, Kang P, Malhotra A, Anversa P. Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis. Am J Pathol. 1999;154:567–580.[Abstract/Free Full Text]
8. Liu Y, Leri A, Li B, Wang X, Cheng W, Kajstura J, Anversa P. Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes. Circ Res. 1998;82:1145–1159.[Abstract/Free Full Text]
9. The SOLVD Investigators. Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fractions. N Engl J Med. 1992;327:685–691.[Abstract]
10. The CONSENSUS Trial Study Group. Effects of enalapril on mortality in severe congestive heart failure: results of the Cooperative North Scandinavian Enalapril Study Group. N Engl J Med. 1987;316:1429–1435.[Abstract]
11. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R Dagenais G. Effects of an angiotensin-converting enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients: the Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. 2000;342:145–153.[Abstract/Free Full Text]
12. Li Z, Bing OHL, Long X, Robinson KG, Lakatta EG. Increased cardiomyocyte apoptosis during the transition of heart failure in the spontaneously hypertensive rat. Am J Physiol. 1997;272:H2313–H2319.[Abstract/Free Full Text]
13. Goussev A, Sharov VG, Shimoyama H, Tanimura M, Lesch M, Goldstein S, Sabbah HN. Effects of ACE inhibition on cardiomyocyte apoptosis in dogs with heart failure. Am J Physiol. 1998;275:H626–H631.[Abstract/Free Full Text]
14. Dostal DE, Rothblum KN, Chernin MI, Cooper GR, Baker KM. Intra-cardiac detection of angiotensinogen and renin: a localized renin-angiotensin system in neonatal rat heart. Am J Physiol. 1992;263:C838–C850.[Abstract/Free Full Text]
15. Sadoshima J, Xu J, Slater HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977–984.[Medline] [Order article via Infotrieve]
16. Zhang X, Dostal DE, Reiss K, Cheng W, Kajstura J, Li P, Huang H, Sonnenblick EH, Meggs LG, Baker KM, Anversa P. Identification and activation of autocrine renin-angiotensin system in adult ventricular myocytes. Am J Physiol. 1995;269:H1791–H1802.[Abstract/Free Full Text]
17. Leri A, Liu Y, Li B, Fiordaliso F, Malhotra A, Latini R, Kajstura J, Anversa P. Up-regulation of AT₁ and AT₂ receptors in postinfarcted hypertrophied myocytes and stretch-mediated apoptotic cell death. Am J Pathol. 2000;156:1663–1672.[Abstract/Free Full Text]
18. Dell’Italia LJ, Meng QC, Balcells E, Straeter-Knowlen IM, Hankes GH, Dillon R, Cartee RE, Orr R, Bishop SP, Oparil S, Elton TS. Increased ACE and chymase-like activity in cardiac tissue of dogs with chronic mitral regurgitation. Am J Physiol. 1995;269:H2065–H2073.[Abstract/Free Full Text]
19. Balcells E, Meng QC, Hageman G, Palmer RW, Durand J, Dell’Italia LJ. Angiotensin II formation in dog heart is mediated by different pathways in vivo and in vitro. Am J Physiol. 1996;271:H417–H471.[Abstract/Free Full Text]
20. Hoit BD, Shao Y, Kinoshita A, Gabel M, Husain A, Walsh RA. Effects of angiotensin II generated by an angiotensin converting enzyme-independent pathway on left ventricular performance in the conscious baboon. J Clin Invest. 1995;95:1519–1527.
21. Urata H, Boehm KD, Philip A, Kinoshita A, Gabrovsek J, Bumpus FM, Husain A. Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart. J Clin Invest. 1993;91:1269–1281.
22. Wolny A, Clozel JP, Rein J, Mory P, Vogt P, Turino M, Kiowski W, Fischli W. Functional and biochemical analysis of angiotensin II–forming pathways in the human heart. Circ Res. 1997;80:219–227.[Abstract/Free Full Text]
23. Zisman LS, Abraham WT, Meixell GE, Vamvakias BN, Quaife RA, Lowes BD, Roden RL, Peacock SJ, Groves BM, Raynolds MV, Bristow MR, Perryman MB. Angiotensin II formation in the intact human heart: predominance of the angiotensin-converting enzyme pathway. J Clin Invest. 1995;95:1490–1498.
24. Dell’Italia LJ, Meng QC, Balcells E, Wei CC, Palmer R, Hageman GR, Durand J, Hankes GH, Oparil S. Compartmentalization of angiotensin II generation in the dog heart: evidence for independent mechanisms in intravascular and interstitial spaces. J Clin Invest. 1997;100:253–258.[Medline] [Order article via Infotrieve]
25. Kokkonen JO, Saarinen J, Kovanen PT. Regulation of local angiotensin II formation in the human heart in the presence of interstitial fluid: inhibition of chymase by protease inhibitors of interstitial fluid and of angiotensin-converting enzyme by Ang-(1-9) formed by heart carboxypeptidase A–like activity. Circulation. 1997;95:1455–1463.[Abstract/Free Full Text]
26. Leri A, Liu Y, Malhotra A, Li Q, Stiegler P, Claudio PP, Giordano A, Kajstura J, Hintze TH, Anversa P. Pacing-induced heart failure in dogs enhances the expression of p53- and p53-dependent genes in ventricular myocytes. Circulation. 1998;97:194–203.[Abstract/Free Full Text]
27. Leri A, Liu Y, Wang X, Kajstura J, Malhotra A, Meggs LG, Anversa P. Overexpression of IGF-1 attenuates the myocyte renin-angiotensin system in transgenic mice. Circ Res. 1999;84:752–762.[Abstract/Free Full Text]
28. Leri A, Fiordaliso F, Setoguchi M, Limana F, Bishopric NH, Kajstura J, Webster K, Anversa P. Inhibition of p53 function prevents renin-angiotensin system activation and stretch-mediated myocyte apoptosis. Am J Pathol. 2000;157:843–857.[Abstract/Free Full Text]
29. Wang J, Seyedi N, Xu XB, Wolin MS, Hintze TH. Defective endothelium-mediated control of coronary circulation in conscious dogs after heart failure. Am J Physiol. 1994;266:H670–H680.[Abstract/Free Full Text]
30. Auerbach R, Alby L, Grieves J, Joseph J, Lindgren C, Morrissey LW, Sidky YA, Tu M, Watt SL. Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells. Proc Natl Acad Sci U S A. 1982;79:7891–7895.[Abstract/Free Full Text]
31. Wang ZQ, Moore AF, Ozono R, Siragy HM, Carey RM. Immunolocalization of subtype 2 angiotensin II (AT₂) receptor protein in rat heart. Hypertension. 1998;32:78–83.[Abstract/Free Full Text]
32. Malhotra R, Sadoshima J, Brosius FC, Izumo S. Mechanical stretch and angiotensin II differentially upregulate the renin-angiotensin system in cardiac myocytes in vitro. Circ Res. 1999;85:137–146.[Abstract/Free Full Text]
33. Braulke T. Origin of lysosomal proteins. In: Lloyd JB, Mason RW, eds. Subcellular Biochemistry: Biology of the Lysosome. New York, NY: Plenum Press; 1996:15–49.
34. Katwa LC, Sun Y, Campbell SE, Tyagi SC, Dhalla AK, Kandala JC, Weber KT. Pouch tissue and angiotensin peptide generation. J Mol Cell Cardiol. 1998;30:1401–1413.[Medline] [Order article via Infotrieve]
35. Matsubara H, Inada M. Molecular insights into angiotensin II type 1 and type 2 receptors: expression, signaling and physiological function and clinical application of its antagonists. Endocr J. 1998;45:137–150.[Medline] [Order article via Infotrieve]
36. Meggs LG, Coupet J, Huang H, Cheng W, Li P, Capasso JM, Homcy CJ, Anversa P. Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats. Circ Res. 1993;72:1149–1162.[Abstract/Free Full Text]
37. Tsutsumi Y, Matsubara H, Ohkubo N, Mori Y, Nozawa Y, Murasawa S, Kijima K, Maruyama K, Masaki H, Moriguchi Y, Shibasaki Y, Kamihata H, Inada M, Iwasaka T. Angiotensin II type 2 receptor is upregulated in human heart with interstitial fibrosis, and cardiac fibroblasts are the major cell type for its expression. Circ Res. 1998;83:1035–1046.[Abstract/Free Full Text]
38. Lopez JJ, Lorell BH, Ingelfinger JR, Weinberg EO, Schunkert H, Diamant D, Tang SS. Distribution and function of cardiac angiotensin AT₁- and AT₂-receptor subtypes in hypertrophied rat hearts. Am J Physiol. 1994;267:H844–H852.[Abstract/Free Full Text]
39. Nio Y, Matsubara H, Murasawa S, Kanasaki M, Inada M. Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction. J Clin Invest. 1995;95:46–54.
40. Booz GW, Baker KM. Role of type 1 and type 2 angiotensin receptors in angiotensin II–induced cardiomyocyte hypertrophy. Hypertension. 1996;28:635–640.[Abstract/Free Full Text]
41. Ichiki T, Labosky PA, Shiota C, Okuyama S, Inagawa Y, Fogo A, Nimura F, Ichikawa I, Hogan BLM, Inagami T. Effects on blood pressure and exploratory behavior of mice lacking angiotensin II type-2 receptor. Nature. 1995;377:748–750.[Medline] [Order article via Infotrieve]
42. Shannon RP, Komamura K, Stambler BS, Bigaud M, Manders T, Vatner SF. Alterations in myocardial contractility in conscious dogs with dilated cardiomyopathy. Am J Physiol. 1991;260:H1903–H1911.[Abstract/Free Full Text]
43. Pierzchalski P, Reiss K, Cheng W, Cirielli C, Kajstura J, Nitahara JA, Rizk M, Capogrossi MC, Anversa P. p53 induces myocyte apoptosis via the activation of the renin-angiotensin system. Exp Cell Res. 1997;234:57–65.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				W.-Q. Tan, J.-X. Wang, Z.-Q. Lin, Y.-R. Li, Y. Lin, and P.-F. Li Novel Cardiac Apoptotic Pathway: The Dephosphorylation of Apoptosis Repressor With Caspase Recruitment Domain by Calcineurin Circulation, November 25, 2008; 118(22): 2268 - 2276. [Abstract] [Full Text] [PDF]

				A. Whaley-Connell, J. Habibi, S. A. Cooper, V. G. DeMarco, M. R. Hayden, C. S. Stump, D. Link, C. M. Ferrario, and J. R. Sowers Effect of renin inhibition and AT1R blockade on myocardial remodeling in the transgenic Ren2 rat Am J Physiol Endocrinol Metab, July 1, 2008; 295(1): E103 - E109. [Abstract] [Full Text] [PDF]

				V. P. Singh, K. M. Baker, and R. Kumar Activation of the intracellular renin-angiotensin system in cardiac fibroblasts by high glucose: role in extracellular matrix production Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1675 - H1684. [Abstract] [Full Text] [PDF]

				I. Murtaza, H.-X. Wang, X. Feng, N. Alenina, M. Bader, B. S. Prabhakar, and P.-F. Li Down-regulation of Catalase and Oxidative Modification of Protein Kinase CK2 Lead to the Failure of Apoptosis Repressor with Caspase Recruitment Domain to Inhibit Cardiomyocyte Hypertrophy J. Biol. Chem., March 7, 2008; 283(10): 5996 - 6004. [Abstract] [Full Text] [PDF]

				B. Burstein and S. Nattel Atrial Fibrosis: Mechanisms and Clinical Relevance in Atrial Fibrillation J. Am. Coll. Cardiol., February 26, 2008; 51(8): 802 - 809. [Abstract] [Full Text] [PDF]

				Y.-Z. Li, D.-Y. Lu, W.-Q. Tan, J.-X. Wang, and P.-F. Li p53 Initiates Apoptosis by Transcriptionally Targeting the Antiapoptotic Protein ARC Mol. Cell. Biol., January 15, 2008; 28(2): 564 - 574. [Abstract] [Full Text] [PDF]

				B. Burstein, X.-Y. Qi, Y.-H. Yeh, A. Calderone, and S. Nattel Atrial cardiomyocyte tachycardia alters cardiac fibroblast function: A novel consideration in atrial remodeling Cardiovasc Res, December 1, 2007; 76(3): 442 - 452. [Abstract] [Full Text] [PDF]

				S. A. Cooper, A. Whaley-Connell, J. Habibi, Y. Wei, G. Lastra, C. Manrique, S. Stas, and J. R. Sowers Renin-angiotensin-aldosterone system and oxidative stress in cardiovascular insulin resistance Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2009 - H2023. [Abstract] [Full Text] [PDF]

				V. P. Singh, B. Le, V. B. Bhat, K. M. Baker, and R. Kumar High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H939 - H948. [Abstract] [Full Text] [PDF]

				A. Whaley-Connell, G. Govindarajan, J. Habibi, M. R. Hayden, S. A. Cooper, Y. Wei, L. Ma, M. Qazi, D. Link, P. R. Karuparthi, et al. Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E355 - E363. [Abstract] [Full Text] [PDF]

				C.-P. Cheng, H.-J. Cheng, C. Cunningham, Z. K. Shihabi, D. C. Sane, T. Wannenburg, and W. C. Little Angiotensin II Type 1 Receptor Blockade Prevents Alcoholic Cardiomyopathy Circulation, July 18, 2006; 114(3): 226 - 236. [Abstract] [Full Text] [PDF]

				A. Modesti, I. Bertolozzi, T. Gamberi, M. Marchetta, C. Lumachi, M. Coppo, F. Moroni, T. Toscano, G. Lucchese, G. F. Gensini, et al. Hyperglycemia Activates JAK2 Signaling Pathway in Human Failing Myocytes via Angiotensin II-Mediated Oxidative Stress Diabetes, February 1, 2005; 54(2): 394 - 401. [Abstract] [Full Text] [PDF]

				K. Funabiki, K. Onishi, K. Dohi, T. Koji, K. Imanaka-Yoshida, M. Ito, H. Wada, N. Isaka, T. Nobori, and T. Nakano Combined angiotensin receptor blocker and ACE inhibitor on myocardial fibrosis and left ventricular stiffness in dogs with heart failure Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2487 - H2492. [Abstract] [Full Text] [PDF]

				B. Rondelet, F. Kerbaul, R. Van Beneden, S. Motte, P. Fesler, I. Hubloue, M. Remmelink, S. Brimioulle, I. Salmon, J.-M. Ketelslegers, et al. Signaling Molecules in Overcirculation-Induced Pulmonary Hypertension in Piglets: Effects of Sildenafil Therapy Circulation, October 12, 2004; 110(15): 2220 - 2225. [Abstract] [Full Text] [PDF]

				R. B. Silver, A. C. Reid, C. J. Mackins, T. Askwith, U. Schaefer, D. Herzlinger, and R. Levi Mast cells: A unique source of renin PNAS, September 14, 2004; 101(37): 13607 - 13612. [Abstract] [Full Text] [PDF]

				Y. Shimoni and X.-F. Liu Gender differences in ANG II levels and action on multiple K+ current modulation pathways in diabetic rats Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H311 - H319. [Abstract] [Full Text] [PDF]

H. Nakajima, H. O. Nakajima, S.-C. Tsai, and L. J. Field Expression of Mutant p193 and p53 Permits Cardiomyocyte Cell Cycle Reentry After Myocardial Infarction