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(Hypertension. 2005;46:433.)
© 2005 American Heart Association, Inc.

Original Articles

Genetic Deletion of the p66^Shc Adaptor Protein Protects From Angiotensin II–Induced Myocardial Damage

Gallia Graiani; Costanza Lagrasta; Enrica Migliaccio; Frank Spillmann; Marco Meloni; Paolo Madeddu; Federico Quaini; Ines Martin Padura; Luisa Lanfrancone; PierGiuseppe Pelicci; Costanza Emanueli

From the Molecular and Cellular Medicine Laboratory (G.G., M.M., C.E.) and Experimental Medicine and Gene Therapy Section of INBB (F.S., P.M.) Alghero and Osilo, Italy; Pathology (G.G., C.L.) and Internal Medicine (F.Q.), University of Parma, Italy; IFOM-FIRC (E.M.), Milan, Italy; the Bristol Heart Institute (P.M., C.E.), University of Bristol, Bristol, UK; and Hematology-Oncology (I.M.P.) and Experimental Oncology (L.L., P.P.), IEO, Milan, Italy.

Correspondence to Costanza Emanueli, PhD, Division of Experimental Cardiovascular Medicine, Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol BS2 8HW, UK. E-mail emanueli{at}yahoo.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II (Ang II), acting through its G protein–coupled AT₁ receptor (AT₁), contributes to the precocious heart senescence typical of patients with hypertension, atherosclerosis, and diabetes. AT₁ was suggested to transactivate an intracellular signaling controlled by growth factors and their tyrosin-kinase receptors. In cultured vascular smooth muscle cells, this downstream mechanism comprises the p66^Shc adaptor protein, previously recognized to play a role in vascular cell senescence and death. The aim of the present study was 2-fold: (1) to characterize the cardiovascular phenotype of p66^Shc knockout mice (p66^Shc–/–), and (2) to test the novel hypothesis that disrupting the p66^Shc might protect the heart from the damaging action of elevated Ang II levels. Compared with wild-type littermates (p66^Shc+/+), p66^Shc–/– showed similar blood pressure, heart rate, and left ventricular wall thickness. However, cardiomyocyte number was increased in mutant animals, indicating a condition of myocardial hyperplasia. In p66^Shc+/+, infusion of a sub-pressor dose of Ang II (300 nmol/kg body weight [BW] daily for 28 days) caused left ventricular hypertrophy and apoptotic death of cardiomyocytes and endothelial cells. In contrast, p66^Shc–/– were resistant to the proapoptotic/hypertrophic action of Ang II. Consistently, in vitro experiments showed that Ang II causes apoptotic death of cardiomyocytes isolated from p66^Shc+/+ hearts to a greater extent as compared with p66^Shc–/– cardiomyocytes. Our results indicate a fundamental role of p66^Shc in Ang II–mediated myocardial remodeling. In perspective, p66^Shc inhibition may be envisioned as a novel way to prevent the deleterious effects of Ang II on the heart.

Key Words: angiotensin II • cardiomyocytes • endothelial growth factors • apoptosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exaggerated activation of the renin–angiotensin system accelerates cardiac senescence, a process consisting of cardiomyocyte hypertrophy and loss, capillary rarefaction, replacement fibrosis, and ventricular remodeling. Ang II produces its multiple actions by activating the G protein–coupled 7 transmembrane domain (7-TMD) AT₁ and AT₂ receptors (AT₁ and AT₂). AT₁ mediates vasoconstriction, vascular smooth muscle cell (VSMC) proliferation, cardiomyocyte hypertrophy, apoptosis, and fibrosis. AT₁ is also involved in oxidative stress.¹

Beside its notorious G protein–mediated effects, in cultured VSMCs, AT₁ was shown to transactivate a pathway downstream of the tyrosin-kinase receptors for epidermal growth factor (EGF) and platelet-derived growth factors (PDGFs) and including the Shc adaptor protein.^2,3

Shc exists in 3 isoforms with relative molecular masses of 46, 52, and 66 kDa (p46^Shc, p52^Shc, and p66^Shc). The 3 isoforms share an Src-homology2 (SH2) domain, a collagen-homology (CH1) region, and a phosphotyrosine-binding (PTB) domain. P46^Shc and p52^Shc arise from the use of alternative translation initiation sites within the same transcript, whereas p66^Shc contains a unique N-terminal region and is generated as a result of alternative splicing. Shc is tyrosine-phosphorylated in response to growth factors and Ang II. Once phosphorylated, Shc forms a complex with Grb2,^4,5 recruiting the Son-of-sevenless (SOS) exchange protein to the plasma membrane for activation of Ras.⁶

Recent studies have suggested distinct physiological roles for the 3 Shc isoforms. In particular, whereas both p46^Shc and p52^Shc promote cell proliferation and differentiation via Ras and MAP kinases,⁶ p66^Shc was proposed as a key factor controlling aging-related processes.^6,7 In fact, some of the authors of the present study demonstrated that p66^Shc knock-out mice (p66^Shc–/–) exhibit enhanced cellular resistance to oxidative stress and a 30% increase in life span.⁷

In this study, we investigated whether p66^Shc is implicated in the damaging effects elicited by Ang II on the heart. To this aim, the cardiovascular phenotype of p66^Shc–/– was characterized under basal conditions and after chronic infusion with a sub-pressor dose of Ang II. Wild-type littermates (p66^Shc+/+) served as controls. In addition, an in vitro apoptosis test was performed on adult p66^Shc–/– and p66^Shc+/+ cardiomyocytes that were stimulated with escalating concentrations of Ang II.

Our results document for the first time that p66^Shc disruption combats Ang II–induced myocardial hypertrophy and protects cardiomyocytes and endothelial cells from apoptosis. Moreover, in p66^Shc–/–, the number of cycling cardiomyocytes was higher under basal conditions and after Ang II.

Altogether, our findings open new avenues for mechanistic interventions targeting specific molecular pathways of Ang II–induced myocardial damage.

	Materials and Methods

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p66^Shc–/– were generated from a 129 Sv background.⁷ p66^Shc–/– littermates (p66^Shc+/+) served as wild-type controls. Experiments were performed on male mice aged 3 to 5 months.

Details of experimental procedures are provided in the Expanded Methods Online Supplement.

In Vivo Experimental Protocol
The body weight (BW), systolic blood pressure (SBP), and heart rate (HR) of p66^Shc–/– (n=21) and p66^Shc+/+ (n=18) were determined under basal conditions.

After completion of basal measurements, p66^Shc–/– and p66^Shc+/+ were randomized to receive a chronic infusion of Ang II at sub-pressor dosage (300 nmol/kg BW, IP daily for 28 days, n=10 for each strain) or saline (vehicle, n=11 p66^Shc–/– and n=8 p66^Shc+/+).

BW, SBP, and HR were recorded at weekly intervals. Intra-arterial mean blood pressure (MBP) was measured at 28 days.

Heart Histology
After final MBP measurements, hearts of anesthetized mice were arrested in diastole and fixed. Left ventricle (LV) weight (LVW) and right ventricle weight (RVW) were recorded and corrected to the BW. The following parameters were determined: LV transverse diameter, LV free wall thickness, RV wall thickness, LV chamber volume. Transverse LV slices were embedded in paraffin for subsequent histological or immunohistochemical analyses.

Cardiomyocyte transverse diameter and myocardial capillary density were determined in LV sections.

Putative cardiac progenitor cells (CPCs) were identified as those cells expressing both the stem cell marker c-Kit and the cardiac transcription factor GATA-4.⁸ The density of c-kit+/GATA-4+ CPCs per mm³ of myocardium was determined. We also calculated the CPC number every 1000 myocardial cells. Cycle-active cells were recognized by staining for the minichromosome maintenance protein-2 (MCM-2).⁹ MCM proteins, inducible by the E2F transcription factors,¹⁰ are currently used as cell-cycling markers.⁹ The MCM-2-positivity of CPCs and cardiomyocytes (recognized by -sarcomeric actin staining) was differentially calculated and used as an index of cellular cycling for the 2 cardiac populations. Proliferating endothelial cells (ECs) were recognized by double staining for MCM-2 and the EC marker Factor VIII.

Adult Cardiomyocyte Dimensions and Number
The dimensions of cardiomyocytes isolated from untreated p66^Shc+/+ (n=15) and p66^Shc–/– (n=24) were determined. The aggregate number of myocytes in the heart was calculated by combining measurements obtained by the in situ analysis of the myocardium with the estimation of the volume of isolated myocytes.

Additionally, we measured the transverse diameter of myocytes in LV sections from saline- and Ang II–treated p66^Shc+/+ and p66^Shc–/– hearts.

Myocardial Apoptosis
Apoptotic cardiomyocytes and ECs were recognized in LV sections by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining assay. Cardiomyocytes and ECs were identified for their morphology and for being positive to -sarcomeric actin or Factor VIII, respectively. The number of apoptotic cardiomyocytes and apoptotic ECs per square millimeter section was calculated.

In Vitro Ang II–Induced Apoptosis
Adult cardiomyocytes were incubated for 24 hours with Ang II at 10^–11 M (n=5 hearts from each strain), 10^–9 M (p66^Shc+/+: n=14, p66^Shc–/–: n=17), or 10^–7 M (n=8 for each strain), or with saline (p66^Shc+/+: n=12, p66^Shc–/–: n=18). The percentage of apoptotic cardiomyocytes was then evaluated.

Expression of Shc Isoforms in the Hearts
P66^Shc+/+ or p66^Shc–/– received saline or Ang II for 22 days (n=5 mice per each group). The contents of the 3 shc isoforms in heart homogenates were determined by Western blot. P46^Shc and p52^Shc levels were also determined in cardiomyocytes harvested from untreated mice of both strains.

Statistics
Results were expressed as mean±SEM. Multivariate repeated-measures ANOVA was performed. In multiple comparisons in which ANOVA indicated significant differences, the statistical value was determined according to Bonferroni. Differences within and between groups were determined using paired or unpaired Student t test, respectively. A P value <0.05 was interpreted to denote statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Basal Cardiovascular Phenotype of p66^Shc–/–
As shown in Table 1, under basal conditions, no difference was recorded between strains in terms of BW, SBP, or HR.

View this table: [in this window] [in a new window]   TABLE 1. Basal Body Weight and Hemodynamic Values

The impact of p66^Shc gene deletion on cardiac phenotype was evaluated in more detail at the end of a 4-week infusion with saline or Ang II.

As shown in Table 2, no differences were observed between saline-treated p66^Shc–/– and p66^Shc+/+ with regard to the weight of LV and RV and linear LV parameters. However, the LV chamber volume was smaller in p66^Shc–/–. Myocardial capillary density was similar in both strains.

View this table: [in this window] [in a new window]   TABLE 2. Effect of Chronic Angiotensin II on Cardiovascular Parameters

We then evaluated whether p66^Shc deletion alters cardiomyocyte composition and volume. No difference between strains was observed as far as the percentage of mononucleated and binucleated cells is concerned (data not shown). Similarly, no difference was detected between p66^Shc–/– and p66^Shc+/+ in terms of mononucleated cell length (85.0±5.7 µm versus 80.8±4.9 µm) or volume (15201±1245 µm³ versus 14136±1014 µm³), and of binucleated cell length (106.7±3.5 µm versus 106.7±5.9 µm) or volume (19210±919 µm³ versus 17754±1412 µm³) (P=N.S. for all comparisons). The myocardial volume occupied by myocytes was higher in p66^Shc–/– (122.56±10.00 mm³ versus 105.14±2.46 mm³ in p66^Shc+/+, P<0.01). As a consequence, the mathematical calculation of myocyte number per heart indicates higher numbers of mononucleated (1.6x10⁶±9.2x10⁴) and binucleated cardiomyocytes in p66^Shc–/– (5.1x10⁶±2.9x10⁴) than in p66^Shc+/+ (1.5x10⁶±2x10⁴ and 4.7x10⁶±6x10⁴, P<0.05 for both comparisons).

Effects of Ang II on the Cardiovascular Phenotype of p66^Shc+/+ and p66^Shc–/–
The cardiac phenotype of the two strains was differentially modified by chronic infusion of Ang II. As shown in Table 2, Ang II did not change tail-cuff SBP or HR or intraarterial MBP in either strain. However, profound differences were observed in heart histomorphology. As shown in Table 2 and Figure 1, Ang II–infused p66^Shc+/+ displayed LV hypertrophic remodeling (as denoted by the 1.11-fold increase in cardiomyocyte transverse diameter, 1.23-fold increase in LVW/BW, and 1.49-fold reduced cavitary volume, P<0.05 versus saline-infused for all comparisons). In contrast, in p66^Shc–/–, Ang II neither produced myocyte hypertrophy nor altered the LVW/BW and LV cavitary volume (P=N.S. versus saline infusion for all comparisons).

View larger version (17K): [in this window] [in a new window]   Figure 1. Bar graphs show the effects of Ang II or vehicle (V) infusion on total ventricular weight (left, LVW; right, RVW) to body weight (BW) ratio (A); LVW to BW ratio (B); LVW to total ventricular weight (LVW+RVW) ratio in p66Shc+/+ (p66+/+) and p66Shc–/– (p66–/–) (C). Values are mean±SEM. *P<0.05 vs vehicle, P<0.05 and P<0.01 vs p66Shc+/+.

After Ang II, myocardial capillary density decreased without difference between strains. Thus, deletion of p66^Shc preserves the heart from Ang II–induced hypertrophic remodeling, but it does not avoid cardiac capillary rarefaction.

Resistance of p66^Shc–/– Hearts to Ang II–Induced Apoptosis
As shown in Figure 2A, the number of TUNEL+ cardiomyocytes was similar in saline-infused p66^Shc+/+ or p66^Shc–/–. Apoptosis was strikingly enhanced by Ang II in p66^Shc+/+, but it remained at low levels in p66^Shc–/–. Microphotographs in Figure 2B show greater abundance of apoptotic cardiomyocytes (indicated by arrows) in LV sections from Ang II–infused p66^Shc+/+ as compared with p66^Shc–/–.

View larger version (56K): [in this window] [in a new window]   Figure 2. Bar graphs show the effects of chronic Ang II or vehicle on cardiomyocyte apoptosis (A) as evidenced by TUNEL assay, and cardiomyocyte cycling (C) as identified by minichromosome maintenance protein 2 (MCM-2) immuno-staining. Values are mean±SEM. *P<0.05 vs vehicle, P<0.05 and P<0.01 vs p66Shc+/+. B, Apoptotic cardiomyocytes (pointed by arrows) in the hearts of Ang II–infused p66Shc+/+ (p66+/+) and p66Shc–/– (p66–/–). Cardiomyocytes are positive for -sarcomeric actin (purple). TUNEL+ nuclei are stained in dark brown. Nuclei are counterstain by hematoxilin. (Optical microscopy. Original magnification 200x). D, Two MCM-2+ (green nuclear dots) cells. One (arrow) is a small myocyte, which expresses -sarcomeric actin (red fluorescence). Nuclei are counterstained by bisbenzimide blue. (Fluorescent microscopy. Original magnification 1000x).

Cycling Cardiomyocytes Are More Abundant in p66^Shc–/–
In the LV of saline-infused p66^Shc+/+, a small fraction of cardiomyocytes was MCM-2+ (circa 0.2% of total), and this figure was doubled in p66^Shc–/– (Figure 2C). As shown by Figure 2A and 2C, the number of MCM-2+ cardiomyocytes and TUNEL+ cardiomyocytes was similar in saline-infused p66^Shc+/+. At variance, in saline-infused p66^Shc–/–, MCM-2+ cardiomyocytes numerically exceeded TUNEL+ cardiomyocytes. Ang II increased the number of cycling cardiomyocytes in both strains. Nevertheless, in p66^Shc+/+, apoptosis exceeded proliferation, whereas the opposite figure was observed in p66^Shc–/–. Figure 2D shows 2 cells in active cell cycling, as revealed by the nuclear expression of MCM-2 (green fluorescent dots) in the LV of Ang II–infused p66^Shc–/–. One of these cells is a small myocyte, as indicated by its expression of -sarcomeric actin (in red fluorescence). Nuclei are counterstained in blue.

Altogether, the above results indicate that, in p66^Shc+/+, Ang II triggers cardiomyocyte hypertrophy and apoptosis overwhelming concurrent cell cycling. Interestingly, the heart of mice lacking the p66^Shc gene is protected from Ang II–induced hypertrophy and apoptosis and enriched with cycling cardiomyocytes.

Isolated Cardiomyocytes
In vitro experiments were conducted to exclude the interference of hemodynamic factors, completely. As shown by Figure 3A, the rate of spontaneous cardiomyocyte apoptosis was similar in the 2 strains. Ang II at 10^–9 to 10^–7 mol/L increased apoptosis of p66^Shc+/+ cardiomyocytes, whereas Ang II 10^–11 mol/L was not effective. The apoptotic response to Ang II was abrogated in p66^Shc–/–. These results suggest that p66^Shc is essential for Ang II to induce cardiomyocyte apoptosis. Figure 3B and 3C show representative images of the experiments performed incubating p66^Shc+/+ and p66^Shc–/– cardiomyocytes with 10^–9 mol/L Ang II.

View larger version (24K): [in this window] [in a new window]   Figure 3. Bar graph in A shows the in vitro apoptotic responses to Ang II of p66Shc+/+ (p66+/+) and p66Shc–/– (p66–/–) cardiomyocytes. Cardiomyocytes were incubated with Ang II (10–11, 10–9M, 10–7M) or vehicle for 24 hours. At the end of incubation, apoptotic cardiomyocytes were identified by in situ TUNEL staining. Values are mean±SEM. **P<0.01 vs vehicle, P<0.01 vs p66Shc+/+. Microphotographs of p66Shc+/+ (B) and p66Shc–/– (C) are representative of the test with 10–9M Ang II. Intense green fluorescence shows apoptotic nuclei (arrows). Nuclei are stained with bisbenzimide (blue). (Original magnification 100x).

Cardiac Progenitor Cells
CPCs might be implicated in myocardial plasticity. Therefore, we explored whether p66^Shc deletion influences the relative abundance and cycling rate of CPCs in the adult heart.

As shown in Figure 4A and 4B, after saline infusion, the relative abundance of c-kit+/GATA-4+ putative CPCs was similar in p66^Shc+/+ and p66^Shc–/–. However, a strain difference was observed with regard to CPC response to Ang II. In fact, Ang II augmented CPC density in p66^Shc+/+, but not in p66^Shc–/–. The difference is compatible either with a stimulatory action of Ang II on CPCs through p66^Shc or with a compensatory activation of CPCs aimed at counteracting the Ang II–induced myocyte loss.

View larger version (52K): [in this window] [in a new window]   Figure 4. Bar graphs are related to c-kit+/GATA-4+ putative cardiomyocyte progenitor cells (CPCs) in LV sections obtained from saline (V)- or Ang II-infused p66Shc+/+ or p66Shc–/–. A, CPC density per mm2 of myocardium. B, Number of CPCs every 1000 myocardial cells. C, Picture captured from the LV of an Ang II-infused p66Shc–/–. A putative CPC (arrow) is recognized for its double positivity for c-kit- (green fluorescence) and GATA-4- (magenta fluorescent dots in the nucleus). Cardiomyocytes express -sarcomeric actin (red fluorescence). Nuclei are visualized by bisbenzimide blue. (Fluorescent Microscopy. Original magnification 1000x). Bar graph of D shows the percentage of MCM-2+ CPCs in the 4 experimental groups. *P<0.05 vs vehicle, P<0.05 vs p66Shc+/+.

Figure 4C shows a LV section from Ang II–infused p66^Shc+/+, where a CPC (white arrow) is identified by its double positivity for c-kit (green fluorescence) and GATA-4 (magenta fluorescent dots in the nucleus) and cardiac myocytes are recognized by their expression of -sarcomeric actin (red fluorescence). Because of CPC scarcity in the heart, this picture is not representative of the actual CPC density.

As shown by Figure 4D, the percentage of MCM-2+ CPCs was higher in saline-treated p66^Shc–/– than in p66^Shc+/+. Ang II did not affect CPC cycling in any strain. Assuming the CPC proliferation rate to be constant through all the infusion period, the latter finding indirectly suggests that CPC numerical increment in p66^Shc+/+ may be attributable to differentiation from more immature cardiac stem cells¹¹ rather than to CPC proliferation.

Myocardium Capillary Density and Turnover of Cardiac ECs
As shown in Figure 5A, p66^Shc–/– hearts were also protected from Ang II–induced EC apoptosis. Figure 5B shows images of LV sections from Ang II–infused p66^Shc+/+ and p66^Shc–/–. Apoptotic ECs (indicated by the arrows) can be recognized by concomitant positivity for TUNEL (dark brown) and Factor VIII (purple).

View larger version (46K): [in this window] [in a new window]   Figure 5. Bar graphs show the effects of Ang II or vehicle (V) on apoptosis (recognized by TUNEL staining; A) or cycling (recognized by MCM-2 staining; C) of endothelial cells (ECs). Pictures of B are representative of apoptotic ECs (pointed by the arrows) in the hearts of Ang II–infused p66Shc+/+ and p66Shc–/–. Factor VIII staining (purple) identifies ECs. TUNEL staining (dark brown) identifies the apoptotic nuclei. Nuclei are counterstained by hematoxilin (Optical microscopy. Original magnification 200x). Bar graph of D shows the capillary density of the endomyocardium in the 4 groups. Values are mean±SEM. *P<0.05 and **P<0.01 vs vehicle, P<0.05 and P<0.01 vs p66Shc+/+.

As shown in Figure 5C, under saline infusion, the number of MCM-2+ ECs was higher in p66^Shc–/– than in p66^Shc+/+. However, capillary density was similar in the 2 strains (Figure 5D).

As shown in Table 2 and Figure 5D, Ang II similarly reduced cardiac capillary density in p66^Shc+/+ and p66^Shc–/–. We hypothesize that p66^Shc controls EC proliferation under physiological levels of Ang II, while facilitating EC death as triggered by exaggerated Ang II.

Shc Isoforms
We then evaluated the possibility that p46^Shc and p52^Shc expression may be affected by p66^Shc knock-out. As shown in the representative Western blot bands of Figure 6, the cardiac expression levels of p46^Shc or p52^Shc were similar in saline-infused p66^Shc+/+ and p66^Shc–/–. Similar findings were obtained in cardiomyocyte lysates (data not shown). Chronic Ang II did not affect the myocardial expression of p46^Shc or p52^Shc in any strain. As expected, p66^Shc was not detected in p66^Shc–/–.

View larger version (50K): [in this window] [in a new window]   Figure 6. Western blot bands of p66Shc, p52Shc, and p46Shc in lysates from total hearts of saline- or Ang II–infused p66Shc+/+ and p66Shc–/–. ß-tubulin was used as a reference protein.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
p66^Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress acting as a downstream target of the tumor suppressor p53.¹²

Here, we show that p66^Shc–/– display a normal basal phenotype with regards to BW, systemic hemodynamics, and left ventricular wall thickness. CPC density was also similar in the two strains. At variance with what reported for the limb skeletal muscle,¹³ myocardial capillary density was normal in p66^Shc–/–.

However, there were also peculiar differences that could be related to the mutation. In particular, the number of cardiomyocytes and the cycling index of both cardiomyocytes and CPCs were all increased in p66^Shc–/–. This was associated with a reduced LV chamber volume in mutant animals. The combination of these characteristics is compatible with a condition of myocardial hyperplasia. We hypothesize that these features may derive from increased cell proliferation together with reduced apoptosis during the development of p66^Shc–/– hearts. We know from the literature that apoptosis participates in the intrauterus and early postgestational heart development^14,15 and that all the components of the renin–angiotensin system are expressed by the fetal heart and participate in its normal development.^16,17 Interestingly, a recent study from a coauthor of this article showed that the 3 Shc isoforms are activated in the human fetal heart, in a period that is characterized by apoptotic and proliferative processes.¹⁵ In our opinion, the p66^Shc knock-out may interfere with the heart development by inhibiting the apoptotic and hypertrophic signaling emanating from AT₁ or other factors as well as enhancing the proliferation of cardiomyocyte and CPCs.

The major objective of this study was to ascertain whether p66^Shc modulates the cardiac effects of moderate increments of circulating Ang II. A large body of evidence supports the notion that Ang II, independently of its blood pressure-elevating effect, plays a central role in the pathophysiology of heart remodeling and failure. The underlying molecular mechanisms are complex and not yet completely clarified. Here, we report for the first time that Ang II produces damaging effects on the heart through a p66^Shc-comprising pathway. Circumstantial evidence suggests that activation of the p66^Shc, which lays downstream to tyrosin kinase receptors, may be implicated in Ang II–induced cardiovascular alterations. For instance, 2 studies showed increased p66^Shc expression in Ang II–stimulated VSMCs.^18,19 Using genetically modified mice, we specifically challenged the hypothesis that p66^Shc participates in the cascade leading to Ang II–induced ventricular remodeling. To eliminate the confounding effects of hypertension, Ang II was chronically infused at a dose known to be devoid of pressor activity. Accordingly, systemic hemodynamics remained unchanged in both strains. Nevertheless, relevant differences were observed with regard to the cardiac effects of the agent: chronic Ang II caused myocardial hypertrophy and apoptosis in p66^Shc+/+ but not in p66^Shc–/–, thus suggesting a fundamental role of p66^Shc in Ang II–induced cardiac damage. Consistently, p66^Shc–/– were protected from apoptosis caused by limb ischemia,¹³ high-fat diet,²⁰ and free radicals generating poisons.⁷ However, p66^Shc–/– were not spared from Ang II–induced rarefaction of myocardial microcirculation. This fact is apparently difficult to reconcile with our own findings of reduced EC apoptosis in p66^Shc–/–. However, we also show that Ang II reduced the rate of spontaneous proliferation of myocardial ECs, particularly in p66^Shc–/–. Therefore, the capillary rarefaction in response to Ang II may derive from differential effects on ECs in the 2 strains.

Capillary destabilization in response to chronic Ang II is apparently discrepant with neo-angiogenesis which reportedly occurs in ischemic limb muscles of Ang II–infused animals.²¹ One major difference, however, consists of the presence or absence of the hypoxic environment as a determinant of microvascular responses to Ang II. Another possible explanation deals with expressional and functional differences of vascular endothelium from different organs.²² Whatever is the interpretation, microvascular factors are not accountable for the cardiac protection ensured by p66^Shc gene deletion.

In vitro experiments on adult cardiomyocytes definitively documented that Ang II–induced apoptosis is abrogated in the absence of p66^Shc. Similarly, primary cells (hematopoietic precursors, fibroblasts, and ECs) isolated from p66^Shc–/– are resistant to oxidative stress–induced apoptosis, whereas p66^Shc overexpression induces apoptosis in the same cells.²³ Ang II, via AT₁ receptors, upregulates the expression of a variety of redox-sensitive factors and NADPH oxidase, thus increasing the generation of reactive oxygen species (ROS) and cellular damage.²⁴ p66^Shc was initially shown to be involved in tyrosin-kinase receptor–initiated apoptosis.²⁵ Recent studies indicate that p66^Shc regulates apoptosis by controlling mitochondrial transmembrane potential and ROS production/accumulation.¹² Accordingly, p66^Shc–/– cells have reduced ROS under basal concentrations and after p53-induced apoptosis. Thus, it is possible that p66^Shc modulates Ang II–induced apoptosis via regulation of ROS. Furthermore, Ang II induces the expression of heat shock protein 70,²⁶ whose antiapoptotic action is constitutively inhibited when complexed with mitochondrial p66^Shc. Thus, another possibility is that the p66^Shc–/– may unveil the protective action of counter-regulatory mechanisms activated by Ang II.

Recent evidence suggests that the heart is not a postmitotic organ and that a continuous turnover of cardiac cells may result in a heterogeneous population of immature, adult, and senescent cardiomyocytes.¹¹ The contribution of resident cardiac stem cells and CPCs in physiological and pathological turnover of adult cardiomyocyte is also a matter of intense debate. Here, we report that Ang II stimulates the cycling of adult cardiomyocytes and increases the number of putative CPCs. These compensatory responses might not be sufficient to counteract ongoing cell loss by apoptosis in the p66^Shc+/+ hearts. p66^Shc deficiency did not affect the cycling of adult cardiomyocytes under Ang II stimulation, but it prevented the increase in c-kit+/GATA-4+ putative CPCs. These results may reflect the fact that in the absence of significant cardiomyocyte apoptosis there is no need for activation of compensatory responses. Alternatively, we might hypothesize a direct effect of p66^Shc in the control of cardiac cell maturation and turnover.

Perspectives
Our study newly demonstrates the involvement of p66^Shc in the deleterious cardiac responses to increased Ang II, namely hypertrophy of cardiomyocytes and LV and apoptotic death of cardiomyocytes and ECs. These results may have important clinical and therapeutic implications. It is possible that p66^Shc expression and activity differ between individuals, thus accounting for inter-individual variation in the susceptibility to Ang II–induced organ damage.

The present study also points to p66^Shc as a potential target to combat myocardial remodeling and senescence triggered by exaggerated Ang II. In perspective, p66^Shc inhibitors might protect from excessive apoptotic loss of cardiomyocytes, thereby delaying the onset of cardiac decompensation.

	Acknowledgments

 
This project was mainly supported by a grant from the Italian Ministery of Health (Ricerca Finalizzata, 2002) to Drs Emanueli and Lanfrancone and by the Telethon grant GP0300/01. INBB and University of Bristol are partners in the European Vascular Genomic Network of Excellence (EVGN). Dr. Emanueli holds a British Heart Foundation (BHF) Basic Science Lectureship. Dr Martin Padura is supported by grants from AIRC, Istituto Superiore di Sanità, and by the EU integrated project "Angiotargeting." Drs Graiani, Lagrasta, Madeddu, Quaini, and Emanueli dedicate this study to the memory of Prof Giorgio Olivetti and Dr Elena Cigola.

	Footnotes

 
Current address for F.S.: Cardiology and Pneumology, Charitè-Medical University, Berlin, Germany.

Received April 1, 2005; first decision April 27, 2005; accepted June 14, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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