Genetic Deletion of the p66Shc Adaptor Protein Protects From Angiotensin II–Induced Myocardial Damage
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 p66Shc 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 p66Shc knockout mice (p66Shc−/−), and (2) to test the novel hypothesis that disrupting the p66Shc might protect the heart from the damaging action of elevated Ang II levels. Compared with wild-type littermates (p66Shc+/+), p66Shc−/− 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 p66Shc+/+, 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, p66Shc−/− 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 p66Shc+/+ hearts to a greater extent as compared with p66Shc−/− cardiomyocytes. Our results indicate a fundamental role of p66Shc in Ang II–mediated myocardial remodeling. In perspective, p66Shc inhibition may be envisioned as a novel way to prevent the deleterious effects of Ang II on the heart.
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 (p46Shc, p52Shc, and p66Shc). The 3 isoforms share an Src-homology2 (SH2) domain, a collagen-homology (CH1) region, and a phosphotyrosine-binding (PTB) domain. P46Shc and p52Shc arise from the use of alternative translation initiation sites within the same transcript, whereas p66Shc 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 p46Shc and p52Shc promote cell proliferation and differentiation via Ras and MAP kinases,6 p66Shc was proposed as a key factor controlling aging-related processes.6,7 In fact, some of the authors of the present study demonstrated that p66Shc knock-out mice (p66Shc−/−) exhibit enhanced cellular resistance to oxidative stress and a 30% increase in life span.7
In this study, we investigated whether p66Shc is implicated in the damaging effects elicited by Ang II on the heart. To this aim, the cardiovascular phenotype of p66Shc−/− was characterized under basal conditions and after chronic infusion with a sub-pressor dose of Ang II. Wild-type littermates (p66Shc+/+) served as controls. In addition, an in vitro apoptosis test was performed on adult p66Shc−/− and p66Shc+/+ cardiomyocytes that were stimulated with escalating concentrations of Ang II.
Our results document for the first time that p66Shc disruption combats Ang II–induced myocardial hypertrophy and protects cardiomyocytes and endothelial cells from apoptosis. Moreover, in p66Shc−/−, 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
p66Shc−/− were generated from a 129 Sv background.7 p66Shc−/− littermates (p66Shc+/+) 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 p66Shc−/− (n=21) and p66Shc+/+ (n=18) were determined under basal conditions.
After completion of basal measurements, p66Shc−/− and p66Shc+/+ 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 p66Shc−/− and n=8 p66Shc+/+).
BW, SBP, and HR were recorded at weekly intervals. Intra-arterial mean blood pressure (MBP) was measured at 28 days.
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 α-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 p66Shc+/+ (n=15) and p66Shc−/− (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 p66Shc+/+ and p66Shc−/− hearts.
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 α-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 (p66Shc+/+: n=14, p66Shc−/−: n=17), or 10−7 M (n=8 for each strain), or with saline (p66Shc+/+: n=12, p66Shc−/−: n=18). The percentage of apoptotic cardiomyocytes was then evaluated.
Expression of Shc Isoforms in the Hearts
P66Shc+/+ or p66Shc−/− 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. P46Shc and p52Shc levels were also determined in cardiomyocytes harvested from untreated mice of both strains.
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.
Basal Cardiovascular Phenotype of p66Shc−/−
As shown in Table 1, under basal conditions, no difference was recorded between strains in terms of BW, SBP, or HR.
The impact of p66Shc 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 p66Shc−/− and p66Shc+/+ with regard to the weight of LV and RV and linear LV parameters. However, the LV chamber volume was smaller in p66Shc−/−. Myocardial capillary density was similar in both strains.
We then evaluated whether p66Shc 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 p66Shc−/− and p66Shc+/+ 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 p66Shc−/− (122.56±10.00 mm3 versus 105.14±2.46 mm3 in p66Shc+/+, P<0.01). As a consequence, the mathematical calculation of myocyte number per heart indicates higher numbers of mononucleated (1.6×106±9.2×104) and binucleated cardiomyocytes in p66Shc−/− (5.1×106±2.9×104) than in p66Shc+/+ (1.5×106±2×104 and 4.7×106±6×104, P<0.05 for both comparisons).
Effects of Ang II on the Cardiovascular Phenotype of p66Shc+/+ and p66Shc−/−
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 p66Shc+/+ 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 p66Shc−/−, Ang II neither produced myocyte hypertrophy nor altered the LVW/BW and LV cavitary volume (P=N.S. versus saline infusion for all comparisons).
After Ang II, myocardial capillary density decreased without difference between strains. Thus, deletion of p66Shc preserves the heart from Ang II–induced hypertrophic remodeling, but it does not avoid cardiac capillary rarefaction.
Resistance of p66Shc−/− Hearts to Ang II–Induced Apoptosis
As shown in Figure 2A, the number of TUNEL+ cardiomyocytes was similar in saline-infused p66Shc+/+ or p66Shc−/−. Apoptosis was strikingly enhanced by Ang II in p66Shc+/+, but it remained at low levels in p66Shc−/−. Microphotographs in Figure 2B show greater abundance of apoptotic cardiomyocytes (indicated by arrows) in LV sections from Ang II–infused p66Shc+/+ as compared with p66Shc−/−.
Cycling Cardiomyocytes Are More Abundant in p66Shc−/−
In the LV of saline-infused p66Shc+/+, a small fraction of cardiomyocytes was MCM-2+ (circa 0.2% of total), and this figure was doubled in p66Shc−/− (Figure 2C). As shown by Figure 2A and 2C, the number of MCM-2+ cardiomyocytes and TUNEL+ cardiomyocytes was similar in saline-infused p66Shc+/+. At variance, in saline-infused p66Shc−/−, MCM-2+ cardiomyocytes numerically exceeded TUNEL+ cardiomyocytes. Ang II increased the number of cycling cardiomyocytes in both strains. Nevertheless, in p66Shc+/+, apoptosis exceeded proliferation, whereas the opposite figure was observed in p66Shc−/−. 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 p66Shc−/−. One of these cells is a small myocyte, as indicated by its expression of α-sarcomeric actin (in red fluorescence). Nuclei are counterstained in blue.
Altogether, the above results indicate that, in p66Shc+/+, Ang II triggers cardiomyocyte hypertrophy and apoptosis overwhelming concurrent cell cycling. Interestingly, the heart of mice lacking the p66Shc gene is protected from Ang II–induced hypertrophy and apoptosis and enriched with cycling 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 p66Shc+/+ cardiomyocytes, whereas Ang II 10−11 mol/L was not effective. The apoptotic response to Ang II was abrogated in p66Shc−/−. These results suggest that p66Shc is essential for Ang II to induce cardiomyocyte apoptosis. Figure 3B and 3C show representative images of the experiments performed incubating p66Shc+/+ and p66Shc−/− cardiomyocytes with 10−9 mol/L Ang II.
Cardiac Progenitor Cells
CPCs might be implicated in myocardial plasticity. Therefore, we explored whether p66Shc 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 p66Shc+/+ and p66Shc−/−. However, a strain difference was observed with regard to CPC response to Ang II. In fact, Ang II augmented CPC density in p66Shc+/+, but not in p66Shc−/−. The difference is compatible either with a stimulatory action of Ang II on CPCs through p66Shc or with a compensatory activation of CPCs aimed at counteracting the Ang II–induced myocyte loss.
Figure 4C shows a LV section from Ang II–infused p66Shc+/+, 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 α-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 p66Shc−/− than in p66Shc+/+. 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 p66Shc+/+ 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, p66Shc−/− hearts were also protected from Ang II–induced EC apoptosis. Figure 5B shows images of LV sections from Ang II–infused p66Shc+/+ and p66Shc−/−. Apoptotic ECs (indicated by the arrows) can be recognized by concomitant positivity for TUNEL (dark brown) and Factor VIII (purple).
As shown in Table 2 and Figure 5D, Ang II similarly reduced cardiac capillary density in p66Shc+/+ and p66Shc−/−. We hypothesize that p66Shc controls EC proliferation under physiological levels of Ang II, while facilitating EC death as triggered by exaggerated Ang II.
We then evaluated the possibility that p46Shc and p52Shc expression may be affected by p66Shc knock-out. As shown in the representative Western blot bands of Figure 6, the cardiac expression levels of p46Shc or p52Shc were similar in saline-infused p66Shc+/+ and p66Shc−/−. Similar findings were obtained in cardiomyocyte lysates (data not shown). Chronic Ang II did not affect the myocardial expression of p46Shc or p52Shc in any strain. As expected, p66Shc was not detected in p66Shc−/−.
p66Shc 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 p66Shc−/− 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 p66Shc−/−.
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 p66Shc−/−. 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 p66Shc−/− hearts. We know from the literature that apoptosis participates in the intrauterus and early postgestational heart development14,15 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 p66Shc 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 p66Shc 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 p66Shc-comprising pathway. Circumstantial evidence suggests that activation of the p66Shc, which lays downstream to tyrosin kinase receptors, may be implicated in Ang II–induced cardiovascular alterations. For instance, 2 studies showed increased p66Shc expression in Ang II–stimulated VSMCs.18,19 Using genetically modified mice, we specifically challenged the hypothesis that p66Shc 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 p66Shc+/+ but not in p66Shc−/−, thus suggesting a fundamental role of p66Shc in Ang II–induced cardiac damage. Consistently, p66Shc−/− were protected from apoptosis caused by limb ischemia,13 high-fat diet,20 and free radicals generating poisons.7 However, p66Shc−/− 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 p66Shc−/−. However, we also show that Ang II reduced the rate of spontaneous proliferation of myocardial ECs, particularly in p66Shc−/−. 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 p66Shc gene deletion.
In vitro experiments on adult cardiomyocytes definitively documented that Ang II–induced apoptosis is abrogated in the absence of p66Shc. Similarly, primary cells (hematopoietic precursors, fibroblasts, and ECs) isolated from p66Shc−/− are resistant to oxidative stress–induced apoptosis, whereas p66Shc 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 p66Shc was initially shown to be involved in tyrosin-kinase receptor–initiated apoptosis.25 Recent studies indicate that p66Shc regulates apoptosis by controlling mitochondrial transmembrane potential and ROS production/accumulation.12 Accordingly, p66Shc−/− cells have reduced ROS under basal concentrations and after p53-induced apoptosis. Thus, it is possible that p66Shc 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 p66Shc. Thus, another possibility is that the p66Shc−/− 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 p66Shc+/+ hearts. p66Shc 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 p66Shc in the control of cardiac cell maturation and turnover.
Our study newly demonstrates the involvement of p66Shc 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 p66Shc 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 p66Shc as a potential target to combat myocardial remodeling and senescence triggered by exaggerated Ang II. In perspective, p66Shc inhibitors might protect from excessive apoptotic loss of cardiomyocytes, thereby delaying the onset of cardiac decompensation.
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.
Current address for F.S.: Cardiology and Pneumology, Charitè-Medical University, Berlin, Germany.
- Received April 1, 2005.
- Revision received April 27, 2005.
- Accepted June 14, 2005.
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