Diabetes Promotes Cardiac Stem Cell Aging and Heart Failure, Which Are Prevented by Deletion of the p66shc Gene
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Marcello Rota, Nicole LeCapitaine, Toru
参见附件。
the Cardiovascular Research Institute (M.R., N.L., T.H., A.B., A.D.A., M.E.P.I., G.E., S.V., K.U., C.C., P.A., A.L., J.K.), Department of Medicine, New York Medical College, Valhalla, NY
Department of Experimental Oncology (M.G., P.G.P.), European Institute of Oncology, Milan, Italy
Cardiovascular Center (T.F.L.), University Zürich-Irchel, Switzerland.
Abstract
Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc–/–, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc–/–, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.
Key Words: cardiac stem cells myocyte regeneration replicative senescence telomeric shortening
Introduction
Death of cardiac cells with chronic loss of myocytes and vascular structures has been proposed as the underlying cause of the anatomical and functional alterations of the diabetic heart.1 However, myocyte death and defects in the mechanical behavior, regulatory proteins, and Ca2+ cycling of myocytes with diabetes have left unanswered the question of whether these variables play a primary role in the onset of the myopathy or represent secondary events related to the progression of the cardiac disease. Similar abnormalities occur with myocyte hypertrophy associated with ischemic and nonischemic cardiomyopathy, and myocyte death is commonly found in the failing heart. Accumulating evidence supports the notion that the heart possesses a compartment of multipotent progenitor cells (CPCs) that differentiate into myocytes, endothelial cells, and smooth muscle cells in vitro2–4 and in vivo.2 The heart constantly renews itself and an imbalance between cell death and regeneration may be present with diabetes and could be mediated by defects in growth and survival of CPCs.
Hyperglycemia leads to enzymatic O-glycosylation of proteins, including the transcription factor p53, whose activation upregulates the local renin–angiotensin system and the synthesis of angiotensin II (Ang II).5 Binding to Ang II type 1 (AT1) receptors and p53 phosphorylation by p38 mitogen-activated protein (MAP) kinase stimulate chronically the tumor suppressor and the formation of Ang II. Hormone secretion and receptor binding increase cytosolic Ca2+, promote the generation of reactive oxygen species (ROS) and initiate cell death.5 ROS trigger DNA damage, telomeric shortening, irreversible growth arrest, and cellular senescence.6 Impaired CPC function affects cell turnover, resulting in an excessive number of old, dying, and poorly contracting myocytes and ventricular failure.
Targeted mutation of the mouse p66shc gene decreases ROS generation, increases the resistance of cells to oxidative stress, and prolongs lifespan.7,8 For these reasons, the wild-type (WT) and the p66shc–/– (KO) mouse were selected to test the hypothesis that oxidative stress with diabetes alters cardiac homeostasis by producing premature aging, loss of replicative growth, and death of CPCs. Therefore, the consequences of ROS production with diabetes should be attenuated in the p66shc–/– mouse, delaying the onset of cardiac restructuring and heart failure. If this possibility were to be correct, diabetic cardiomyopathy could be regarded as a stem cell myopathy in which a defective stem cell compartment conditions premature aging and death of the differentiated progeny. Ultimately, we may be able to identify a genetic link between diabetes and ROS, on the one hand, and diabetic heart failure, on the other.
Materials and Methods
WT and KO mice were used. Diabetes was induced by streptozotocin (STZ), and in vivo studies were performed to obtain functional, anatomical, and structural measurements. These analyses were complemented with in vitro assays of cells exposed to high glucose.
An expanded Materials and Methods section can be found in an online data supplement, available at http://circres.ahajournals.org.
Results
Diabetes and Cardiac Anatomy and Function
STZ injection resulted in an increase in blood glucose, which averaged 400 mg/dL at 7 and 28 days in WTs and KOs (supplemental Figure I). An independent effect of STZ cannot be excluded, but the half-life of the drug is &15 minutes, and it is cleared from the organs within 4 hours,9 suggesting that the results reported below are predominantly related to the development of diabetes. One week of diabetes did not affect cardiac weights in WTs and KOs. However, at 4 weeks, heart weight, left ventricular (LV) weight, heart weight-to-body weight ratio, and LV weight-to-body weight ratio decreased in diabetic WTs (Figure 1A). In these animals, LV longitudinal axis and chamber diameter increased, resulting in a significant expansion in cavitary volume. Conversely, wall thickness decreased, resulting in a reduction in the wall thickness-to-chamber radius ratio (Figure 1B). In diabetic WTs at 28 days, ventricular dilation together with the reduction in LV weight led to a decrease in LV mass-to-chamber volume ratio. These variables were not modified in diabetic KOs (Figure 1B).
Hemodynamic measurements were consistent with the anatomical changes. LV end-diastolic pressure (LVEDP), systolic pressure (LVSP), developed pressure (LVDP), +dP/dt, and –dP/dt were not altered with diabetes at 7 days in WTs and KOs. However, at 28 days, diabetes in WTs was characterized by an increase in LVEDP and a decrease in LVSP, LVDP, +dP/dt, and –dP/dt. The decrease in wall thickness-to-chamber radius ratio, in combination with the increase in LVEDP, resulted in an increase in diastolic wall stress (Figure 1C). Abnormalities in ventricular function and myocardial loading were not detected in diabetic KOs at 28 days. Thus, deletion of the p66shc gene prevents/delays defects in cardiac size and shape and diastolic and systolic function with diabetes.
Diabetes, CPCs, and Myocytes
The decrease in LV mass seen in diabetic WTs at 28 days was suggestive of myocyte loss, a phenomenon that appeared to be absent in diabetic KOs. However, myocyte hypertrophy could have masked partly the extent of cell drop out in WTs and KOs, by increasing the muscle compartment of the ventricle. For this reason, the volume and number of ventricular myocytes were determined.10 Consistent with previous observations,10 a nearly 20% increase in myocyte size was found in diabetic WTs at 28 days, whereas the decrease in myocyte number, 23%, exceeded the reduction in LV weight, 16%. Conversely, no changes in these myocyte parameters were present in diabetic KOs (Figure 1D). By inference, diabetes resulted in an imbalance between cell death and regeneration in WTs, but it did not alter cardiomyocyte homeostasis in KOs.
Cardiomyogenesis in vivo is controlled by activation and commitment of lineage-negative, c-kit–positive CPCs, which, early during differentiation, maintain the stem cell epitope but express the myocyte transcription factor MEF2C, ie, myocyte progenitors, or MEF2C, and the myofilament protein -sarcomeric actin, ie, myocyte precursors (Figure 2A). Subsequently, myocyte precursors lose c-kit, expand their cytoplasm, and become amplifying myocytes that divide rapidly and simultaneously differentiate.11 In the mouse, lineage-negative, c-kit–positive CPCs and their immediate progeny are clustered in the atria and apex, although they permeate the entire LV. Diabetes may affect CPCs, leading to alterations in myocyte turnover, which in turn may result in the pathological manifestations of the diabetic myopathy.
On this premise, the effects of diabetes on these cell classes11 were determined (Figure 2B). Lineage-negative CPCs and myocyte progenitor precursors were reduced by &50% and &90% in diabetic WTs at 7 and 28 days, respectively. In contrast, an opposite response was found in diabetic KOs; in these animals, CPCs and myocyte progenitor precursors increased modestly at 7 days, but a nearly 1.7-fold increase was recognized at 28 days. Thus, diabetes results in a reduction of the CPC pool and myocyte formation, whereas deletion of p66shc interferes with the impact of diabetes on CPCs, which expand and generate new myocytes, preserving cardiac homeostasis.
Diabetes and Oxidative Stress
Diabetes is characterized by an enhanced oxygen toxicity.1 ROS is the distal signal of the cascade of events triggered by diabetes that leads to the initiation of the cell death pathway in the heart.5,10 Moderate levels of ROS activate apoptosis, high levels of ROS induce cell necrosis, and low levels of ROS stimulate cell growth.12,13 These functions may account for the differential response to diabetes of CPCs in WTs and KOs. However, the metabolic alterations affect all CPCs, but only a fraction of them die or regenerate, pointing to a heterogeneous impact of diabetes on CPCs.
To detect ROS-mediated cytoplasmic and DNA damage, nitrotyrosine and 8-OH-deoxyguanosine (8-OH-dG) were evaluated, respectively, in CPCs and myocytes (Figure 3A). In all hearts, cells positive for 8-OH-dG were also positive for nitrotyrosine, but not all cells labeled by nitrotyrosine showed 8-OH-dG. In WTs, diabetes at 28 days resulted in an increase of CPCs and myocytes positive for these markers of oxidative stress. This was not the case in KOs, in which comparable fractions of cells exhibited nitrotyrosine and 8-OH-dG in the absence or presence of diabetes (Figure 3B).
Levels of 8-OH-dG in nuclei varied from 0 to 5600 pixels, so that frequency distributions were obtained in each animal group. In control WTs and KOs, similar distribution profiles for 8-OH-dG were identified for CPCs and myocytes, although, with respect to CPCs, the levels of oxidized guanosine in myocytes were slightly higher and shifted to the right (Figure 4A and 4B). In CPCs and myocytes of control WTs and KOs, 80% to 90% of the labeled cells had values of <500 pixels. At 7 days, 18% CPCs and 22% myocytes in WTs had values of >1000 pixels. In diabetic KOs at 7 days, the CPC curve did not change from baseline, but 34% of myocytes showed labeling of >1000 pixels. At 28 days, the CPC curve moved further to the right in WTs, whereas minimal changes from baseline were detected in KOs. Similarly, the myocyte curve in WTs was shifted to the right, and 68% myocytes had levels of >1000 pixels. Conversely, the myocyte curve in KOs returned to baseline. Therefore, diabetes leads to significant oxidative damage, which is, however, not apparent in p66shc–/– CPCs and only transient in p66shc–/– myocytes.
Diabetes and Cellular Senescence
Oxygen toxicity and DNA damage alter telomeres, resulting in telomere shortening, cellular senescence, and death.6 Mice have long telomeres, but telomere attrition to a critical length promotes chromosome end-to-end fusion and triggers growth arrest and apoptosis.14 Therefore, telomeric shortening was used as a marker of senescence of CPCs and myocytes, together with p53 and p16INK4a (Figure 5A through 5F). p53 and p16INK4a are upregulated by telomere attrition, and p53 modulates growth arrest and apoptotic death.15 Importantly, p53 and p66shc act in concert to create a positive feedback loop that leads to increased ROS formation, cellular aging, and death.7,8 By necessity, the potential synergistic interaction between p53 and p66shc was limited to WTs. p16INK4a inactivates cdk4 and cdk6, which regulate cell-cycle progression in G1; p16INK4a forms binary inhibitor–cdk complexes that are highly stable and cannot be dissociated by cyclins, and cells arrest permanently.
Measurements of telomeric length in CPCs and myocytes by quantitative fluorescence in situ hybridization (Q-FISH) were restricted to control and diabetic hearts at 28 days. Diabetes in WTs led to a shift to the left, toward shorter telomeres, in the frequency distribution of telomere length in CPCs. This phenomenon was not observed in diabetic KOs. Although average telomere length in CPCs of diabetic WTs decreased 23% from 31 to 24 kbp, 25% CPCs in this population had telomeres less than 16-kbp long. Conversely, only 4% CPCs in diabetic KOs showed this degree of telomeric shortening (Figure 5G). Myocytes behaved in a similar manner (Figure 5H), suggesting that diabetes mimics myocardial aging and the impact that cellular senescence has on the growth reserve of the old heart.16
The expression of p53 and p16INK4a in CPCs and myocytes increased only in diabetic WTs at 28 days. In this group, the fraction of p53- and p16INK4a-positive CPCs increased 3-fold and 5-fold, respectively. Smaller increases were noted in the percentage of p53-positive myocytes, 1.8-fold, and p16INK4a-positive myocytes, 2-fold (Figure 5I). Importantly, &70% of p16INK4a-positive CPCs and myocytes had telomeres shorter than 16 kbp. As demonstrated above (Figure 2B), there was a marked decrease in the total number of CPCs in the LV of diabetic WTs at 28 days, and, as shown here, only 131 of the remaining 258 CPCs were functionally competent; 127 were p16INK4a positive, which was indicative of irreversible growth arrest and cellular senescence (Figure 5J). Similar changes occurred in the atria. Conversely, only a small number of CPCs were no longer functional in diabetic KOs. In these animals, the CPC compartment of the atria and LV expanded with diabetes (Figure 5J). Thus, the diabetic heart is characterized by premature senescence of CPCs that, in turn, is responsible for the increase of senescent myocytes. Deletion of p66shc prevents/delays these negative effects of diabetes.
Diabetes, Cell Death, and Cell Growth
Because of the effects of diabetes on oxidative stress, telomeric length, and expression of p53 and p16INK4a in CPCs and myocytes, apoptotic and necrotic cell death were measured. In all cases, CPC and myocyte apoptosis and necrosis were found in cells positive for 8-OH-dG and p16INK4a (Figure 6A through 6D). The colocalization of 8-OH-dG and cell death was examined extensively, but the quantitative levels of 8-OH-dG in nuclei of dying cells were collected only in 30 to 40 CPCs and myocytes of the LV of each group of animals (Figure 6E and 6F). The extent of oxidative damage correlated with the pattern of cell death. Apoptosis was detected with values of 8-OH-dG between 500 and 5000 pixels and necrosis between 3000 and 5600 pixels (Figure 6G).
CPC apoptosis in the atria and LV increased nearly 10-fold in diabetic WTs at both 7 and 28 days (Figure 6H). Conversely, CPCs in KOs appeared to be protected by the effects of diabetes. Moreover, cell necrosis was found only in 3 LV CPCs of diabetic WTs at 28 days (Figure 6G). The values of 8-OH-dG were 3600, 4700, and 5400 pixels. The low incidence of CPC necrosis at these stages of diabetes precluded its quantification. Diabetes increased myocyte apoptosis 4- to 6-fold in WTs and KOs at 7 days but remained moderately elevated exclusively in WTs at 28 days (Figure 6H). Diabetes was also accompanied by a 4-fold increase in myocyte necrosis in WTs at 28 days; the level of myocyte necrosis was not altered in KOs.
The decrease in number of CPCs and myocytes in the heart of diabetic WTs, and the increase in CPCs and preservation of myocyte number in diabetic KOs, raised the possibility that diabetes was associated with differences in cell replication between WTs and KOs. Therefore, the expression of Ki67 and MCM5 was determined in these cell categories (Figure 6I). Diabetes negatively affected CPC and myocyte division in WTs, whereas both cell types showed an increased proliferation in KOs (supplemental Figure II). Interestingly, 8-OH-dG in dividing CPCs and small amplifying myocytes was undetectable or <250 pixels (Figure 6G). The quantitative colocalization of 8-OH-dG with Ki67 was restricted to 15 to 50 CPCs and myocytes of the LV of each group of animals. Most likely, the absence and presence of 8-OH-dG in Ki67-positive CPCs and myocytes reflected cell division and DNA repair, respectively. However, we were unable to separate these 2 processes in tissue sections, and Ki67 expression may be independent from DNA repair. Thus, diabetes enhances oxidative damage, which triggers apoptosis, necrosis, or division/DNA repair. Deletion of p66shc favors the activation of cell growth mechanisms.
ROS-Mediated Cellular Responses
To determine the formation of ROS with diabetes, the intracellular levels of hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) together were measured in CPCs and cardiomyocytes freshly isolated from WTs and KOs 7 days after STZ administration. This time point was selected because of the high levels of cell death and the lack of hemodynamic changes, which could have influenced the results. These cells were loaded with 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and fluorescence intensity was evaluated by 2-photon microscopy (Figure 7A and 7B). The signal for H2O2 and ·OH together was low and comparable in CPCs and myocytes of nondiabetic WTs and KOs. However, 1 week of diabetes resulted in a marked increase in the generation of H2O2 and ·OH in cells from WTs, whereas no changes were detected in CPCs and myocytes from diabetic KOs (Figure 7C).
To obtain a direct relationship between oxidative stress (H2O2 and ·OH), on the one hand, and apoptosis (hairpin-1), cell necrosis (hairpin-2), cell growth (Ki67), and DNA repair, on the other, these parameters were evaluated in individual cells. DNA repair was assessed by detection of DNA-polymerase-, which is implicated in the restoration of the 8-OH-dG/cytosine pairs.17 In cells from control and diabetic mice, low levels of ROS were associated with DNA repair and cell replication, whereas higher degrees of reactive oxygen were linked to cell apoptosis and cell necrosis (Figure 7D), pointing to a potential critical role of ROS in cellular responses.
In vitro experiments were then performed to establish whether high glucose per se enhances the generation of H2O2 and ·OH in CPCs and myocytes. For this purpose, cells were obtained from nondiabetic WTs and KOs and exposed to 27.5 mmol/L glucose for 2 days. ROS formation nearly doubled in cells from WTs but did not increase significantly in CPCs and myocytes from KOs (Figure 8A). Similarly, the increases in p53, p16INK4a, and apoptosis of CPCs from WTs with hyperglycemia were not observed in CPCs from KOs (supplemental Figures III and IV). Based on these results, CPCs from WTs were cultured in the presence of 5.5 mmol/L glucose and were exposed to different levels of oxidative stress by various concentrations of H2O2 or xanthine/xanthine oxidase. The latter generates superoxide anion (O2). Low, intermediate, and high concentrations of ROS activated CPC replication, apoptosis, and apoptosis–necrosis, respectively (Figure 8B and 8C). Catalase and Tiron inhibited these responses. By inference, proliferation can be expected to be higher in CPCs from KOs than in WTs, as documented in vivo (Figure 6I). Thus, diabetes increases oxidative stress, but deletion of p66shc attenuates the oxidative challenge and converts death signals into growth pathway (Figure 8D).
Discussion
Findings in the current study indicate that diabetes led to premature myocyte senescence and death, which together resulted in the development of a cardiac myopathy, characterized by a decrease in muscle mass, chamber dilation, and impaired ventricular function. The accumulation of old myocytes was mediated by a dramatic loss of CPCs that markedly attenuated the formation and efficient turnover of parenchymal cells. Importantly, the adaptor protein p66shc played a major role in the effects of diabetes on the heart. Ablation of p66shc had remarkable beneficial consequences on the viability and function of CPCs, positively interfering with death stimuli and inhibition of CPC growth and differentiation. CPC division and myocyte generation were potentiated by the absence of p66shc, which opposed the development of a decompensated diabetic heart. Expansion of the CPC pool and myocyte progenitor precursors was combined with the preservation of cardiac performance, suggesting that an intact stem cell compartment can counteract the impact of uncontrolled diabetes on the myocardium. This might be a time-dependent process in which deletion of p66shc offers an early cardiac protection that is lost during the long-term evolution of the disease.
Diabetes and p66shc
Data in this study demonstrate that diabetes resulted in an increased formation of ROS and oxidative damage to DNA and cytoplasmic proteins. Ablation of p66shc, however, abolished this negative effect on CPCs and, after an early lack of defensive mechanisms on cardiomyocytes, hindered ROS generation and oxygen toxicity in these cells as well. These observations are consistent with results in other cell systems in which p66shc increases the production of oxidants7 and reduces the resistance of cells to oxidative stress.8 Ultimately, p66shc stimulates the cell death pathway.18
p66shc is a member of the Shc family of proteins, which include 3 splicing isoforms encoded by the same genetic locus.19 p46shc and p52shc activate the Ras oncoprotein,20 MAP kinases, and cell division.19 Conversely, p66shc does not upregulate MAP kinases, inhibits c-fos promoter activity, and contrasts cell replication.7,19 Thus, the Shc proteins exert opposite functions: p46shc and p52shc promote cell proliferation, whereas p66shc negatively affects cell growth and enhances ROS-mediated cell injury. Because of p66shc function, the metabolic dysregulation of diabetes was corrected at least in part by ablation of this gene, which prevented oxidative stress. CPCs modulate myocyte renewal and ventricular hemodynamics11 and recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell myopathy in which alterations in CPCs define the heart phenotype structurally and functionally. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.
Diabetes and Telomere Length
Telomere length reflects the replicative history of cells together with cumulative oxidative damage. Telomeric shortening occurred in CPCs and myocytes with diabetes, but this defect was not detected in diabetic transgenic mice in which the increase in ROS formation was blunted by the deletion of p66shc. Preservation of telomere length in the absence of p66shc strongly suggests that a strict association exists between oxidative stress and loss of telomere integrity with diabetes. Telomerase and telomere-related proteins are responsible for telomere function and cell viability.21 In telomerase-competent cycling cells, the activity of this ribonucleoprotein prevents telomere attrition. However, in pathological conditions and aging, dividing cells are unable to reactivate telomerase and telomeric shortening becomes apparent.22 In telomerase-negative cells, telomeric DNA is lost at a rate of 100 bp per division.23 Furthermore, in terminally differentiated cells, the level of oxidative stress dictates the pace by which telomeric shortening occurs.24
Mild and high concentrations of oxidants induce single-stranded DNA breaks in telomeres, which are more sensitive to ROS formation than genomic DNA or minisatellite regions.6 The most frequent form of DNA damage induced by oxidative stress is dG oxidation. The presence of 8-OH-dG lesions in diabetic CPCs may be mediated by reduction in telomere-related proteins, which lead to telomere dysregulation and critical telomeric shortening.25
Diabetes, Oxidative Stress, Cell Death, and Cell Growth
ROS control cellular lifespan. Oxidative stress inhibits or initiates cell proliferation and promotes cellular senescence and death. The activation of a specific cellular response is linked to the intracellular quantity of free radicals and radical-derived reactive species.12,13 To establish whether CPC and myocyte growth and death were linked to ROS levels in diabetes, we have measured the extent of ROS-mediated DNA damage in vivo and ROS formation in vitro. In both systems, low levels of ROS and DNA damage were found in cells undergoing DNA repair and replication. Progressively higher quantities of oxidative challenge were seen in apoptotic and necrotic cells. Ablation of p66shc restricted the options of CPCs and myocytes. Cells were forced to die by apoptosis or divide. Oxidative stress, however, had a more dramatic effect on cells capable of replicating than on cells permanently withdrawn from the cell cycle. DNA damage accumulated in myocytes but triggered apoptosis in CPCs. The differential adaptation of CPCs and myocytes to diabetes and oxidative stress resulted in 1 month in a reduction of the number of CPCs that was 4-fold larger than in myocytes. Endothelial progenitor cells (EPCs) and the local synthesis of growth factors are equally affected by the diabetic state,26,27 so that other mechanisms contribute to the deterioration of the coronary vasculature and ventricular performance with this disease. EPCs play a major role in vessel repair and formation.28
ROS can induce apoptosis and necrosis, which are separate mechanisms of cell death with distinct biochemical, morphological, and functional characteristics.29 The recognition of these 2 forms of cell death is of great clinical relevance because apoptosis and necrosis have different consequences on the structure and function of the myocardium. Rupture of the plasma membrane with cell necrosis results in the leakage of intracellular contents into the extracellular compartment with stimulation of an inflammatory process and collagen accumulation. Conversely, the rapid clearance of apoptotic bodies by professional and nonprofessional phagocytes prevents inflammation and secondary myocardial scarring. Additionally, apoptotic cells may emit signals that are sensed by the neighboring cells that eventually proliferate in the attempt to restore cell number.30 Typically, diabetes leads to cell apoptosis before necrosis, and, when necrosis occurs, ventricular dysfunction supervenes.
The balance between cardiac cell death and cell regeneration is lost with diabetes, but ablation of p66shc preserves this homeostatic control. ROS generated at low concentrations function as second messengers in the signal transduction cascade of numerous growth factors.12,13 Binding of hematopoietic cytokines to tyrosine kinase receptors increases the intracellular generation of ROS that, in turn, coax quiescent bone marrow stem cells to traverse the cell cycle.31 Growth signals converge on MAP kinases, which are implicated in redox-mediated effector pathways. Moreover, ROS and intracellular calcium act in concert in enhancing cell replication and survival.32 ROS are primarily generated in the mitochondrial compartment, and their production is tightly regulated spatially and temporally.33 The increased formation of ROS is restricted to subcellular localizations and the time required for the transmission of growth signals from membrane receptors to the nucleus. In contrast, high quantities of ROS lack cellular compartmentalization and persist for a long time within the cells; they result in inhibition of cell replication and differentiation, favoring cellular senescence and death.
Acknowledgments
Sources of Funding
This work was supported by NIH grants HL-38132, HL-65577, HL-65573, HL-75480, AG-17042, AG-26107, HL-81737, HL-78825, and AG-23071.
Disclosures
None.
Footnotes
Original received April 3, 2006; revision received May 22, 2006; accepted May 24, 2006.
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the Cardiovascular Research Institute (M.R., N.L., T.H., A.B., A.D.A., M.E.P.I., G.E., S.V., K.U., C.C., P.A., A.L., J.K.), Department of Medicine, New York Medical College, Valhalla, NY
Department of Experimental Oncology (M.G., P.G.P.), European Institute of Oncology, Milan, Italy
Cardiovascular Center (T.F.L.), University Zürich-Irchel, Switzerland.
Abstract
Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc–/–, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc–/–, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.
Key Words: cardiac stem cells myocyte regeneration replicative senescence telomeric shortening
Introduction
Death of cardiac cells with chronic loss of myocytes and vascular structures has been proposed as the underlying cause of the anatomical and functional alterations of the diabetic heart.1 However, myocyte death and defects in the mechanical behavior, regulatory proteins, and Ca2+ cycling of myocytes with diabetes have left unanswered the question of whether these variables play a primary role in the onset of the myopathy or represent secondary events related to the progression of the cardiac disease. Similar abnormalities occur with myocyte hypertrophy associated with ischemic and nonischemic cardiomyopathy, and myocyte death is commonly found in the failing heart. Accumulating evidence supports the notion that the heart possesses a compartment of multipotent progenitor cells (CPCs) that differentiate into myocytes, endothelial cells, and smooth muscle cells in vitro2–4 and in vivo.2 The heart constantly renews itself and an imbalance between cell death and regeneration may be present with diabetes and could be mediated by defects in growth and survival of CPCs.
Hyperglycemia leads to enzymatic O-glycosylation of proteins, including the transcription factor p53, whose activation upregulates the local renin–angiotensin system and the synthesis of angiotensin II (Ang II).5 Binding to Ang II type 1 (AT1) receptors and p53 phosphorylation by p38 mitogen-activated protein (MAP) kinase stimulate chronically the tumor suppressor and the formation of Ang II. Hormone secretion and receptor binding increase cytosolic Ca2+, promote the generation of reactive oxygen species (ROS) and initiate cell death.5 ROS trigger DNA damage, telomeric shortening, irreversible growth arrest, and cellular senescence.6 Impaired CPC function affects cell turnover, resulting in an excessive number of old, dying, and poorly contracting myocytes and ventricular failure.
Targeted mutation of the mouse p66shc gene decreases ROS generation, increases the resistance of cells to oxidative stress, and prolongs lifespan.7,8 For these reasons, the wild-type (WT) and the p66shc–/– (KO) mouse were selected to test the hypothesis that oxidative stress with diabetes alters cardiac homeostasis by producing premature aging, loss of replicative growth, and death of CPCs. Therefore, the consequences of ROS production with diabetes should be attenuated in the p66shc–/– mouse, delaying the onset of cardiac restructuring and heart failure. If this possibility were to be correct, diabetic cardiomyopathy could be regarded as a stem cell myopathy in which a defective stem cell compartment conditions premature aging and death of the differentiated progeny. Ultimately, we may be able to identify a genetic link between diabetes and ROS, on the one hand, and diabetic heart failure, on the other.
Materials and Methods
WT and KO mice were used. Diabetes was induced by streptozotocin (STZ), and in vivo studies were performed to obtain functional, anatomical, and structural measurements. These analyses were complemented with in vitro assays of cells exposed to high glucose.
An expanded Materials and Methods section can be found in an online data supplement, available at http://circres.ahajournals.org.
Results
Diabetes and Cardiac Anatomy and Function
STZ injection resulted in an increase in blood glucose, which averaged 400 mg/dL at 7 and 28 days in WTs and KOs (supplemental Figure I). An independent effect of STZ cannot be excluded, but the half-life of the drug is &15 minutes, and it is cleared from the organs within 4 hours,9 suggesting that the results reported below are predominantly related to the development of diabetes. One week of diabetes did not affect cardiac weights in WTs and KOs. However, at 4 weeks, heart weight, left ventricular (LV) weight, heart weight-to-body weight ratio, and LV weight-to-body weight ratio decreased in diabetic WTs (Figure 1A). In these animals, LV longitudinal axis and chamber diameter increased, resulting in a significant expansion in cavitary volume. Conversely, wall thickness decreased, resulting in a reduction in the wall thickness-to-chamber radius ratio (Figure 1B). In diabetic WTs at 28 days, ventricular dilation together with the reduction in LV weight led to a decrease in LV mass-to-chamber volume ratio. These variables were not modified in diabetic KOs (Figure 1B).
Hemodynamic measurements were consistent with the anatomical changes. LV end-diastolic pressure (LVEDP), systolic pressure (LVSP), developed pressure (LVDP), +dP/dt, and –dP/dt were not altered with diabetes at 7 days in WTs and KOs. However, at 28 days, diabetes in WTs was characterized by an increase in LVEDP and a decrease in LVSP, LVDP, +dP/dt, and –dP/dt. The decrease in wall thickness-to-chamber radius ratio, in combination with the increase in LVEDP, resulted in an increase in diastolic wall stress (Figure 1C). Abnormalities in ventricular function and myocardial loading were not detected in diabetic KOs at 28 days. Thus, deletion of the p66shc gene prevents/delays defects in cardiac size and shape and diastolic and systolic function with diabetes.
Diabetes, CPCs, and Myocytes
The decrease in LV mass seen in diabetic WTs at 28 days was suggestive of myocyte loss, a phenomenon that appeared to be absent in diabetic KOs. However, myocyte hypertrophy could have masked partly the extent of cell drop out in WTs and KOs, by increasing the muscle compartment of the ventricle. For this reason, the volume and number of ventricular myocytes were determined.10 Consistent with previous observations,10 a nearly 20% increase in myocyte size was found in diabetic WTs at 28 days, whereas the decrease in myocyte number, 23%, exceeded the reduction in LV weight, 16%. Conversely, no changes in these myocyte parameters were present in diabetic KOs (Figure 1D). By inference, diabetes resulted in an imbalance between cell death and regeneration in WTs, but it did not alter cardiomyocyte homeostasis in KOs.
Cardiomyogenesis in vivo is controlled by activation and commitment of lineage-negative, c-kit–positive CPCs, which, early during differentiation, maintain the stem cell epitope but express the myocyte transcription factor MEF2C, ie, myocyte progenitors, or MEF2C, and the myofilament protein -sarcomeric actin, ie, myocyte precursors (Figure 2A). Subsequently, myocyte precursors lose c-kit, expand their cytoplasm, and become amplifying myocytes that divide rapidly and simultaneously differentiate.11 In the mouse, lineage-negative, c-kit–positive CPCs and their immediate progeny are clustered in the atria and apex, although they permeate the entire LV. Diabetes may affect CPCs, leading to alterations in myocyte turnover, which in turn may result in the pathological manifestations of the diabetic myopathy.
On this premise, the effects of diabetes on these cell classes11 were determined (Figure 2B). Lineage-negative CPCs and myocyte progenitor precursors were reduced by &50% and &90% in diabetic WTs at 7 and 28 days, respectively. In contrast, an opposite response was found in diabetic KOs; in these animals, CPCs and myocyte progenitor precursors increased modestly at 7 days, but a nearly 1.7-fold increase was recognized at 28 days. Thus, diabetes results in a reduction of the CPC pool and myocyte formation, whereas deletion of p66shc interferes with the impact of diabetes on CPCs, which expand and generate new myocytes, preserving cardiac homeostasis.
Diabetes and Oxidative Stress
Diabetes is characterized by an enhanced oxygen toxicity.1 ROS is the distal signal of the cascade of events triggered by diabetes that leads to the initiation of the cell death pathway in the heart.5,10 Moderate levels of ROS activate apoptosis, high levels of ROS induce cell necrosis, and low levels of ROS stimulate cell growth.12,13 These functions may account for the differential response to diabetes of CPCs in WTs and KOs. However, the metabolic alterations affect all CPCs, but only a fraction of them die or regenerate, pointing to a heterogeneous impact of diabetes on CPCs.
To detect ROS-mediated cytoplasmic and DNA damage, nitrotyrosine and 8-OH-deoxyguanosine (8-OH-dG) were evaluated, respectively, in CPCs and myocytes (Figure 3A). In all hearts, cells positive for 8-OH-dG were also positive for nitrotyrosine, but not all cells labeled by nitrotyrosine showed 8-OH-dG. In WTs, diabetes at 28 days resulted in an increase of CPCs and myocytes positive for these markers of oxidative stress. This was not the case in KOs, in which comparable fractions of cells exhibited nitrotyrosine and 8-OH-dG in the absence or presence of diabetes (Figure 3B).
Levels of 8-OH-dG in nuclei varied from 0 to 5600 pixels, so that frequency distributions were obtained in each animal group. In control WTs and KOs, similar distribution profiles for 8-OH-dG were identified for CPCs and myocytes, although, with respect to CPCs, the levels of oxidized guanosine in myocytes were slightly higher and shifted to the right (Figure 4A and 4B). In CPCs and myocytes of control WTs and KOs, 80% to 90% of the labeled cells had values of <500 pixels. At 7 days, 18% CPCs and 22% myocytes in WTs had values of >1000 pixels. In diabetic KOs at 7 days, the CPC curve did not change from baseline, but 34% of myocytes showed labeling of >1000 pixels. At 28 days, the CPC curve moved further to the right in WTs, whereas minimal changes from baseline were detected in KOs. Similarly, the myocyte curve in WTs was shifted to the right, and 68% myocytes had levels of >1000 pixels. Conversely, the myocyte curve in KOs returned to baseline. Therefore, diabetes leads to significant oxidative damage, which is, however, not apparent in p66shc–/– CPCs and only transient in p66shc–/– myocytes.
Diabetes and Cellular Senescence
Oxygen toxicity and DNA damage alter telomeres, resulting in telomere shortening, cellular senescence, and death.6 Mice have long telomeres, but telomere attrition to a critical length promotes chromosome end-to-end fusion and triggers growth arrest and apoptosis.14 Therefore, telomeric shortening was used as a marker of senescence of CPCs and myocytes, together with p53 and p16INK4a (Figure 5A through 5F). p53 and p16INK4a are upregulated by telomere attrition, and p53 modulates growth arrest and apoptotic death.15 Importantly, p53 and p66shc act in concert to create a positive feedback loop that leads to increased ROS formation, cellular aging, and death.7,8 By necessity, the potential synergistic interaction between p53 and p66shc was limited to WTs. p16INK4a inactivates cdk4 and cdk6, which regulate cell-cycle progression in G1; p16INK4a forms binary inhibitor–cdk complexes that are highly stable and cannot be dissociated by cyclins, and cells arrest permanently.
Measurements of telomeric length in CPCs and myocytes by quantitative fluorescence in situ hybridization (Q-FISH) were restricted to control and diabetic hearts at 28 days. Diabetes in WTs led to a shift to the left, toward shorter telomeres, in the frequency distribution of telomere length in CPCs. This phenomenon was not observed in diabetic KOs. Although average telomere length in CPCs of diabetic WTs decreased 23% from 31 to 24 kbp, 25% CPCs in this population had telomeres less than 16-kbp long. Conversely, only 4% CPCs in diabetic KOs showed this degree of telomeric shortening (Figure 5G). Myocytes behaved in a similar manner (Figure 5H), suggesting that diabetes mimics myocardial aging and the impact that cellular senescence has on the growth reserve of the old heart.16
The expression of p53 and p16INK4a in CPCs and myocytes increased only in diabetic WTs at 28 days. In this group, the fraction of p53- and p16INK4a-positive CPCs increased 3-fold and 5-fold, respectively. Smaller increases were noted in the percentage of p53-positive myocytes, 1.8-fold, and p16INK4a-positive myocytes, 2-fold (Figure 5I). Importantly, &70% of p16INK4a-positive CPCs and myocytes had telomeres shorter than 16 kbp. As demonstrated above (Figure 2B), there was a marked decrease in the total number of CPCs in the LV of diabetic WTs at 28 days, and, as shown here, only 131 of the remaining 258 CPCs were functionally competent; 127 were p16INK4a positive, which was indicative of irreversible growth arrest and cellular senescence (Figure 5J). Similar changes occurred in the atria. Conversely, only a small number of CPCs were no longer functional in diabetic KOs. In these animals, the CPC compartment of the atria and LV expanded with diabetes (Figure 5J). Thus, the diabetic heart is characterized by premature senescence of CPCs that, in turn, is responsible for the increase of senescent myocytes. Deletion of p66shc prevents/delays these negative effects of diabetes.
Diabetes, Cell Death, and Cell Growth
Because of the effects of diabetes on oxidative stress, telomeric length, and expression of p53 and p16INK4a in CPCs and myocytes, apoptotic and necrotic cell death were measured. In all cases, CPC and myocyte apoptosis and necrosis were found in cells positive for 8-OH-dG and p16INK4a (Figure 6A through 6D). The colocalization of 8-OH-dG and cell death was examined extensively, but the quantitative levels of 8-OH-dG in nuclei of dying cells were collected only in 30 to 40 CPCs and myocytes of the LV of each group of animals (Figure 6E and 6F). The extent of oxidative damage correlated with the pattern of cell death. Apoptosis was detected with values of 8-OH-dG between 500 and 5000 pixels and necrosis between 3000 and 5600 pixels (Figure 6G).
CPC apoptosis in the atria and LV increased nearly 10-fold in diabetic WTs at both 7 and 28 days (Figure 6H). Conversely, CPCs in KOs appeared to be protected by the effects of diabetes. Moreover, cell necrosis was found only in 3 LV CPCs of diabetic WTs at 28 days (Figure 6G). The values of 8-OH-dG were 3600, 4700, and 5400 pixels. The low incidence of CPC necrosis at these stages of diabetes precluded its quantification. Diabetes increased myocyte apoptosis 4- to 6-fold in WTs and KOs at 7 days but remained moderately elevated exclusively in WTs at 28 days (Figure 6H). Diabetes was also accompanied by a 4-fold increase in myocyte necrosis in WTs at 28 days; the level of myocyte necrosis was not altered in KOs.
The decrease in number of CPCs and myocytes in the heart of diabetic WTs, and the increase in CPCs and preservation of myocyte number in diabetic KOs, raised the possibility that diabetes was associated with differences in cell replication between WTs and KOs. Therefore, the expression of Ki67 and MCM5 was determined in these cell categories (Figure 6I). Diabetes negatively affected CPC and myocyte division in WTs, whereas both cell types showed an increased proliferation in KOs (supplemental Figure II). Interestingly, 8-OH-dG in dividing CPCs and small amplifying myocytes was undetectable or <250 pixels (Figure 6G). The quantitative colocalization of 8-OH-dG with Ki67 was restricted to 15 to 50 CPCs and myocytes of the LV of each group of animals. Most likely, the absence and presence of 8-OH-dG in Ki67-positive CPCs and myocytes reflected cell division and DNA repair, respectively. However, we were unable to separate these 2 processes in tissue sections, and Ki67 expression may be independent from DNA repair. Thus, diabetes enhances oxidative damage, which triggers apoptosis, necrosis, or division/DNA repair. Deletion of p66shc favors the activation of cell growth mechanisms.
ROS-Mediated Cellular Responses
To determine the formation of ROS with diabetes, the intracellular levels of hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) together were measured in CPCs and cardiomyocytes freshly isolated from WTs and KOs 7 days after STZ administration. This time point was selected because of the high levels of cell death and the lack of hemodynamic changes, which could have influenced the results. These cells were loaded with 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and fluorescence intensity was evaluated by 2-photon microscopy (Figure 7A and 7B). The signal for H2O2 and ·OH together was low and comparable in CPCs and myocytes of nondiabetic WTs and KOs. However, 1 week of diabetes resulted in a marked increase in the generation of H2O2 and ·OH in cells from WTs, whereas no changes were detected in CPCs and myocytes from diabetic KOs (Figure 7C).
To obtain a direct relationship between oxidative stress (H2O2 and ·OH), on the one hand, and apoptosis (hairpin-1), cell necrosis (hairpin-2), cell growth (Ki67), and DNA repair, on the other, these parameters were evaluated in individual cells. DNA repair was assessed by detection of DNA-polymerase-, which is implicated in the restoration of the 8-OH-dG/cytosine pairs.17 In cells from control and diabetic mice, low levels of ROS were associated with DNA repair and cell replication, whereas higher degrees of reactive oxygen were linked to cell apoptosis and cell necrosis (Figure 7D), pointing to a potential critical role of ROS in cellular responses.
In vitro experiments were then performed to establish whether high glucose per se enhances the generation of H2O2 and ·OH in CPCs and myocytes. For this purpose, cells were obtained from nondiabetic WTs and KOs and exposed to 27.5 mmol/L glucose for 2 days. ROS formation nearly doubled in cells from WTs but did not increase significantly in CPCs and myocytes from KOs (Figure 8A). Similarly, the increases in p53, p16INK4a, and apoptosis of CPCs from WTs with hyperglycemia were not observed in CPCs from KOs (supplemental Figures III and IV). Based on these results, CPCs from WTs were cultured in the presence of 5.5 mmol/L glucose and were exposed to different levels of oxidative stress by various concentrations of H2O2 or xanthine/xanthine oxidase. The latter generates superoxide anion (O2). Low, intermediate, and high concentrations of ROS activated CPC replication, apoptosis, and apoptosis–necrosis, respectively (Figure 8B and 8C). Catalase and Tiron inhibited these responses. By inference, proliferation can be expected to be higher in CPCs from KOs than in WTs, as documented in vivo (Figure 6I). Thus, diabetes increases oxidative stress, but deletion of p66shc attenuates the oxidative challenge and converts death signals into growth pathway (Figure 8D).
Discussion
Findings in the current study indicate that diabetes led to premature myocyte senescence and death, which together resulted in the development of a cardiac myopathy, characterized by a decrease in muscle mass, chamber dilation, and impaired ventricular function. The accumulation of old myocytes was mediated by a dramatic loss of CPCs that markedly attenuated the formation and efficient turnover of parenchymal cells. Importantly, the adaptor protein p66shc played a major role in the effects of diabetes on the heart. Ablation of p66shc had remarkable beneficial consequences on the viability and function of CPCs, positively interfering with death stimuli and inhibition of CPC growth and differentiation. CPC division and myocyte generation were potentiated by the absence of p66shc, which opposed the development of a decompensated diabetic heart. Expansion of the CPC pool and myocyte progenitor precursors was combined with the preservation of cardiac performance, suggesting that an intact stem cell compartment can counteract the impact of uncontrolled diabetes on the myocardium. This might be a time-dependent process in which deletion of p66shc offers an early cardiac protection that is lost during the long-term evolution of the disease.
Diabetes and p66shc
Data in this study demonstrate that diabetes resulted in an increased formation of ROS and oxidative damage to DNA and cytoplasmic proteins. Ablation of p66shc, however, abolished this negative effect on CPCs and, after an early lack of defensive mechanisms on cardiomyocytes, hindered ROS generation and oxygen toxicity in these cells as well. These observations are consistent with results in other cell systems in which p66shc increases the production of oxidants7 and reduces the resistance of cells to oxidative stress.8 Ultimately, p66shc stimulates the cell death pathway.18
p66shc is a member of the Shc family of proteins, which include 3 splicing isoforms encoded by the same genetic locus.19 p46shc and p52shc activate the Ras oncoprotein,20 MAP kinases, and cell division.19 Conversely, p66shc does not upregulate MAP kinases, inhibits c-fos promoter activity, and contrasts cell replication.7,19 Thus, the Shc proteins exert opposite functions: p46shc and p52shc promote cell proliferation, whereas p66shc negatively affects cell growth and enhances ROS-mediated cell injury. Because of p66shc function, the metabolic dysregulation of diabetes was corrected at least in part by ablation of this gene, which prevented oxidative stress. CPCs modulate myocyte renewal and ventricular hemodynamics11 and recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell myopathy in which alterations in CPCs define the heart phenotype structurally and functionally. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.
Diabetes and Telomere Length
Telomere length reflects the replicative history of cells together with cumulative oxidative damage. Telomeric shortening occurred in CPCs and myocytes with diabetes, but this defect was not detected in diabetic transgenic mice in which the increase in ROS formation was blunted by the deletion of p66shc. Preservation of telomere length in the absence of p66shc strongly suggests that a strict association exists between oxidative stress and loss of telomere integrity with diabetes. Telomerase and telomere-related proteins are responsible for telomere function and cell viability.21 In telomerase-competent cycling cells, the activity of this ribonucleoprotein prevents telomere attrition. However, in pathological conditions and aging, dividing cells are unable to reactivate telomerase and telomeric shortening becomes apparent.22 In telomerase-negative cells, telomeric DNA is lost at a rate of 100 bp per division.23 Furthermore, in terminally differentiated cells, the level of oxidative stress dictates the pace by which telomeric shortening occurs.24
Mild and high concentrations of oxidants induce single-stranded DNA breaks in telomeres, which are more sensitive to ROS formation than genomic DNA or minisatellite regions.6 The most frequent form of DNA damage induced by oxidative stress is dG oxidation. The presence of 8-OH-dG lesions in diabetic CPCs may be mediated by reduction in telomere-related proteins, which lead to telomere dysregulation and critical telomeric shortening.25
Diabetes, Oxidative Stress, Cell Death, and Cell Growth
ROS control cellular lifespan. Oxidative stress inhibits or initiates cell proliferation and promotes cellular senescence and death. The activation of a specific cellular response is linked to the intracellular quantity of free radicals and radical-derived reactive species.12,13 To establish whether CPC and myocyte growth and death were linked to ROS levels in diabetes, we have measured the extent of ROS-mediated DNA damage in vivo and ROS formation in vitro. In both systems, low levels of ROS and DNA damage were found in cells undergoing DNA repair and replication. Progressively higher quantities of oxidative challenge were seen in apoptotic and necrotic cells. Ablation of p66shc restricted the options of CPCs and myocytes. Cells were forced to die by apoptosis or divide. Oxidative stress, however, had a more dramatic effect on cells capable of replicating than on cells permanently withdrawn from the cell cycle. DNA damage accumulated in myocytes but triggered apoptosis in CPCs. The differential adaptation of CPCs and myocytes to diabetes and oxidative stress resulted in 1 month in a reduction of the number of CPCs that was 4-fold larger than in myocytes. Endothelial progenitor cells (EPCs) and the local synthesis of growth factors are equally affected by the diabetic state,26,27 so that other mechanisms contribute to the deterioration of the coronary vasculature and ventricular performance with this disease. EPCs play a major role in vessel repair and formation.28
ROS can induce apoptosis and necrosis, which are separate mechanisms of cell death with distinct biochemical, morphological, and functional characteristics.29 The recognition of these 2 forms of cell death is of great clinical relevance because apoptosis and necrosis have different consequences on the structure and function of the myocardium. Rupture of the plasma membrane with cell necrosis results in the leakage of intracellular contents into the extracellular compartment with stimulation of an inflammatory process and collagen accumulation. Conversely, the rapid clearance of apoptotic bodies by professional and nonprofessional phagocytes prevents inflammation and secondary myocardial scarring. Additionally, apoptotic cells may emit signals that are sensed by the neighboring cells that eventually proliferate in the attempt to restore cell number.30 Typically, diabetes leads to cell apoptosis before necrosis, and, when necrosis occurs, ventricular dysfunction supervenes.
The balance between cardiac cell death and cell regeneration is lost with diabetes, but ablation of p66shc preserves this homeostatic control. ROS generated at low concentrations function as second messengers in the signal transduction cascade of numerous growth factors.12,13 Binding of hematopoietic cytokines to tyrosine kinase receptors increases the intracellular generation of ROS that, in turn, coax quiescent bone marrow stem cells to traverse the cell cycle.31 Growth signals converge on MAP kinases, which are implicated in redox-mediated effector pathways. Moreover, ROS and intracellular calcium act in concert in enhancing cell replication and survival.32 ROS are primarily generated in the mitochondrial compartment, and their production is tightly regulated spatially and temporally.33 The increased formation of ROS is restricted to subcellular localizations and the time required for the transmission of growth signals from membrane receptors to the nucleus. In contrast, high quantities of ROS lack cellular compartmentalization and persist for a long time within the cells; they result in inhibition of cell replication and differentiation, favoring cellular senescence and death.
Acknowledgments
Sources of Funding
This work was supported by NIH grants HL-38132, HL-65577, HL-65573, HL-75480, AG-17042, AG-26107, HL-81737, HL-78825, and AG-23071.
Disclosures
None.
Footnotes
Original received April 3, 2006; revision received May 22, 2006; accepted May 24, 2006.
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