c-Kit–Positive Cardiac Stem Cells Nested in Hypoxic Niches Are Activated by Stem Cell Factor Reversing the Aging MyopathyNovelty and Significance
Rationale: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown.
Objective: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal.
Methods and Results: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy.
Conclusions: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.
In the myocardium, c-kit–positive cardiac stem cells (CSCs) are clustered in interstitial microdomains where they are connected by gap and adherens junctions to the supporting cells, that is, cardiomyocytes and fibroblasts.1 In the young heart, asymmetric growth kinetics of CSCs maintains their pool and promote the generation of an adequate differentiated progeny.2 With aging, the pool of stem cells expressing the senescence-associated protein p16INK4a increases, reducing the number of functionally competent CSCs.3,4 Apoptosis is restricted to p16INK4a-positive CSCs,3 but this process may be inefficient, leading to the accumulation of old CSCs. A shift in the balance between actively dividing and senescent CSCs may generate dysfunctional niches, altering the critical role that CSCs have in the homeostasis of the aged myocardium. Deregulation of niche function may create abnormal sites of cardiomyogenesis; old CSCs form myocytes, which inherit the shortened telomeres of the mother cells, rapidly acquiring the senescent cell phenotype.3,4 These variables are major determinants of the aging myopathy.3–5
Stem cell niches in the bone marrow are characterized by low O2 tension,6 which offers a selective advantage to stem cells favoring their quiescent, undifferentiated state.7,8 A similar mechanism may be operative in the myocardium, and CSC behavior may be regulated by the O2 gradient within the tissue. The long-term preservation of the CSC compartment may require a hypoxic milieu, although physiological normoxia may be necessary for active cardiomyogenesis. A balance between hypoxic and normoxic CSCs may be present in the young heart, ensuring tissue homeostasis and myocyte renewal. Conversely, this balance may be disrupted in the old myocardium in which defects in tissue oxygenation and enhanced fibroblast accumulation9 may increase the number of hypoxic foci. CSCs nested in this microenvironment are unable to re-enter the cell cycle and divide. By necessity, normoxic CSCs are forced to undergo intense proliferation and differentiation with progressive telomere erosion and formation of senescent dysfunctional cardiomyocytes.10 Hypoxic CSCs may comprise a cell population with intact telomeres and significant growth reserve that, after activation, may partly reverse the cardiac senescent phenotype.
A detailed description of the experimental procedures is provided in the Online Data Supplement.
Hypoxic and Normoxic CSCs
Protocols were approved by the Institutional Animal Care and Use Committee. Male C57BL/6 mice at 3, 24, and 30 months of age were injected intraperitoneally with the hypoxic probe pimonidazole (Pimo).11 Two hours later, mice were euthanized and the heart was formalin-fixed. The presence of Pimo in CSCs was determined by immunolabeling.
Nitroreductase Activity, Mitochondrial Content, and ΔΨ
The hearts of Pimo-injected mice at 3 months of age were enzymatically dissociated, and c-kit–positive CSCs were analyzed by flow cytometry. FACS-sorted CSCs were incubated with nitroblue tetrazolium to detect nitroreductase enzymatic activity. The fluorescent probes MitoTracker Green FM and tetramethylrhodamine ethyl ester were used to evaluate mitochondrial content and membrane potential, respectively, in CSCs. The distribution of Pimo in CSCs was assessed by immunolabeling and confocal microscopy.
Functional Properties of Pimo-Positive and Pimo-Negative CSCs
Sixty-micrometer-thick sections were obtained from hearts of mice at 3 and at 26 to 30 months of age. The distance between hypoxic and normoxic CSCs and the closest endothelial cell and the capillary density per mm2 of myocardium were determined.12
Depletion of Hypoxic CSCs
Mice at 3 and 30 months of age were injected daily with the cytotoxic agent tirapazamine (TPZ; 30 mg/kg body weight)13 and received Pimo 2 hours before euthanization. The number of CSCs,12,14 the percentage of Pimo-positive CSCs (Pimo+-CSCs) and Pimo-negative CSCs (Pimo−-CSCs), and the fraction of cycling and committed CSCs were then measured.
Long-Term Label-Retaining Assay
Mice at 3 months of age were injected intraperitoneally with BrdU at 12-hour intervals for 7 days. One group of animals was euthanized 1 hour after the last administration of the halogenated nucleotide (pulse). Two additional groups of mice were similarly treated and euthanized after a chase period of 2 and 5 months.1–3 The fluorescence intensity of BrdU in CSCs was measured by confocal microscopy.
This parameter was evaluated by Q-FISH and confocal microscopy in CSCs and cardiomyocytes isolated from hearts at 3 and 30 months of age.
Hyperoxia and Stem Cell Factor Administration
Mice at 26 months of age were kept in hyperoxic chambers (70% O2) for 1 and 7 days. Pimo+-CSCs and Pimo−-CSCs were measured by flow cytometry. The hearts of mice at 26 to 30 months of age were injected at 3 to 4 different sites with 1 μg stem cell factor (SCF) or PBS and euthanized at 6, 24, and 48 hours and at 21 days. Mice killed at 2 and 21 days received daily injections of BrdU (10 mg/kg body weight), and the percentage of CSCs and cardiomyocytes positive for BrdU was measured by confocal microscopy.1,3
Myocyte Volume and Number
Sections (40-μm-thick) were stained by α-sarcomeric actin, laminin, and connexin 43. Optical sections 1 μm apart were collected and images were converted into 3-dimensional structures to measure the proportion of mononucleated, binucleated, and multinucleated myocytes and their volume and number.12
Data are presented as mean±SD. Significance between 2 groups was determined by unpaired 2-tailed Student t-test. For multiple comparisons, the ANOVA test was used.
Hypoxic and Normoxic CSC Niches Are Present in the Heart
Oxygen tension in the heart varies from 18 to 35 mm Hg, reflecting physiological normoxia.15 The small size of the niches, their scattered distribution in the myocardium, and the complexity to identify these structures in vivo make it impossible to measure O2 level in cardiac niches using electrodes. Therefore, the uptake of the hypoxic marker Pimo was introduced to define O2 content in CSCs. Pimo diffuses across the plasma membrane and undergoes bioreduction by cytoplasmic nitroreductases when O2 level is <10 mm Hg or 1.3%. Pimo intermediates bind to macromolecules, allowing the detection of intracellular hypoxia by immunocytochemistry.11
Three variables of CSC function were measured to validate the specificity of Pimo labeling: activity of nitroreductases,16 aggregate mitochondrial volume, and transmembrane potential (ΔΨ) of the inner mitochondrial membrane.17,18 The first parameter was determined by the nitroblue tetrazolium chloride assay,11 which detects the activity of hypoxia-dependent and hypoxia-independent reductases. By this approach, it has been possible to test whether Pimo+-CSCs and Pimo−-CSCs had different enzyme activity. The second and third parameters were measured using fluorescent probes and FACS because it has been recognized that differences in the mitochondrial compartment and ΔΨ are strictly related to the rate of mitochondrial respiration, allowing detection of differences in O2 consumption and in the oxidative state of the cell.
Nitroreductase activity was comparable in Pimo+-CSCs and Pimo−-CSCs (Online Figure IA), as was the mitochondrial volume. In the latter assay, CSCs were divided into 2 categories according to the level of intensity of the Mito Tracker probe, and the localization of Pimo in these 2 CSC classes was determined. The fraction of Pimo+ cells was identical regardless of the degree of mitochondrial content (Online Figure IB). However, Pimo+-CSCs were distributed predominantly in the cell group with low ΔΨ (Online Figure IC). Moreover, human CSCs cultured at 1%, 5%, 10%, and 21% O2 were Pimo+ only in the presence of 1% O2 (Online Figure IIA). Thus, Pimo labeling was consistent with the identification of hypoxic CSCs.
The thymus, an organ characterized by a microenvironment with O2 content <10 mm Hg, was used to establish the efficiency of Pimo in labeling hypoxic cells in vivo (Online Figure IIB). Pimo was injected intraperitoneally 2 hours before euthanization, and the proportion of hypoxic and normoxic CSCs in the atria, base mid-region (Base-MR), and apex of the left ventricle was measured by confocal microscopy in mice at 3, 24, and 30 months of age. Pimo+-hypoxic CSCs (Pimo+-CSCs) and Pimo−-normoxic- CSCs (Pimo−-CSCs) were seen in all anatomical areas (Figure 1A and 1B).
Myocytes and fibroblasts operate as supporting cells within the CSC niches1 so that the presence of Pimo in these cells was determined to define the hypoxic state of the microenvironment of the niches. Fibroblasts in proximity of Pimo+-CSCs were consistently labeled, whereas cardiomyocytes were always Pimo− (Figure 1C). This observation raised the question whether cardiomyocytes adjacent to Pimo+-CSCs were normoxic or in a state of relative hypoxia. Two indices of the molecular response to hypoxia, hypoxia-inducible factor-1α (HIF-1α) and its downstream effector carbonic anhydrase IX (CAIX)6, were introduced to identify potential defects in cell oxygenation. Importantly, HIF-1α but not CAIX was detected in cardiomyocytes contiguous to Pimo+-CSCs; however, CAIX was present in Pimo+-CSCs (Figure 1D and 1E). Pimo−-CSCs labeled by HIF-1α were found, suggesting that these cells were relatively hypoxic but did not reach the severe hypoxic state associated with CAIX and Pimo labeling (Figure 1F). The partial overlapping in the distribution of these 3 markers of hypoxia in cardiac niches is consistent with findings in tumor cells.19,20 The upregulation of the nuclear transcription factor HIF-1α has also been observed in cells with O2 content >1%, although the activation of the transmembrane protein CAIX seems to be limited to cells with O2 content <1%. Thus, the mouse heart is characterized by hypoxic and normoxic niches, which may have a distinct role in organ homeostasis, tissue repair, and myocardial aging.
Number of Hypoxic CSC Niches Increases With Age
At 3 months of age, the number of CSCs per mm3 of myocardium was similar in the atria and apex and lower at the Base-MR. From 3 to 30 months, the number of CSCs increased 53%, 103%, and 86% in the atria, Base-MR, and apex, respectively (Online Figure IIC). At 3 months, Pimo+-CSCs comprised only 27% to 35% of the stem cell population but reached a value of 56% to 65% at 30 months. Conversely, the fraction of Pimo−-CSCs decreased with age (Figure 2A). The senescent heart at 30 months contained in all regions a comparable number of Pimo+-CSCs per mm3 of myocardium, which exceeded the pool of Pimo−-CSCs (Figure 2B). At 30 months, the ratio of Pimo+-CSCs to Pimo−-CSCs was 1.4, 1.9, and 1.3 in the atria, Base-MR, and apex, respectively. When possible, mice at 30 months of age were evaluated separately. However, the low survival rate of mice at 30 months forced us to include animals from 24 to 30 months of age in some experiments.
To establish whether hypoxia prevented cell-cycle re-entry, the localization of Ki67 was evaluated in Pimo+-CSCs and Pimo−-CSCs. Pimo+-CSCs did not express Ki67. At all ages, in the 3 regions of the heart, Ki67 was restricted to Pimo−-CSCs (Figure 2C). Thus, aging increases the number of hypoxic nondividing CSCs, a process that may interfere with organ homeostasis and repair.
Capillaries and Pimo+-CSCs
The oxygenation of the niches is regulated by the distance between CSCs and the closest capillary.12,21 This parameter was measured by 2-photon microscopy at the left ventricular (LV) Base-MR of 3-month-old and 24-month-old to 30-month-old mice 2 hours after Pimo injection. The myocardium was fixed and 60-μm-thick sections were stained for c-kit and Pimo; coronary capillaries were identified by isolectin. The distance between the closest capillary and Pimo+-CSCs at 3 and 24 to 30 months of age varied from 10.4 to 12.9 μm; these values were significantly higher than those found between the closest capillary and Pimo−-CSCs, ranging from 5.6 to 7.6 μm (Figure 2D). The numerical density of capillaries per mm2 of myocardium12 within a radius of 30 μm from Pimo+-CSCs and Pimo−-CSCs was similar in both CSC subsets in the young heart (not shown). Capillary density decreased with age in a comparable manner in both CSC classes but did not reach statistical significance. The preservation of capillary density in the adjacent myocardium may explain the lack of severe hypoxia in myocytes distributed in proximity to hypoxic niches. Thus, the increase in diffusion distance for O2 is viewed as one of the factors implicated in the induction of CSC quiescence, although cell-autonomous mechanisms may also be involved.
Pimo+-CSCs and Pimo−-CSCs Are Functionally Connected
To define the effects of hypoxia and normoxia on the division and differentiation of CSCs, these cells were isolated from 3-month-old and 30-month-old mice and analyzed by FACS. Cells were depleted with a CD45 antibody to exclude bone marrow cells and mast cells. The small fraction of c-kit-positive CD45-positive cells was Pimo− and normoxic (Online Figure III); this finding is consistent with the increase in O2 content in committed hematopoietic stem cells (HSCs).7
Replicating CSCs were detected by Ki67, and GATA4 and Nkx2.5 were used as markers of the myocyte lineage. At 3 months, largely quiescent, lineage-negative Pimo+-CSCs comprised 38% of the population (Figure 3A and 3B), a value comparable to the results obtained in myocardial sections (Figure 2A). Cycling and early committed CSCs positive for Ki67, GATA4, and Nkx2.5 were predominantly restricted to the Pimo− pool (Figure 3A and 3B). At 30 months, quiescent, lineage-negative Pimo+-CSCs accounted for 65% of the cells. Again, dividing and differentiating cells were primarily confined to Pimo−-CSCs (Figure 3C and 3D). Thus, Pimo+-CSCs increase with age, and intracellular hypoxia conditions their quiescent state, whereas normoxia favors CSC growth and commitment.
A critical question was whether Pimo+-CSCs and Pimo−-CSCs are independent or functionally inter-related cell populations. To determine whether the loss of hypoxic Pimo+-CSCs triggered a response from the pool of normoxic Pimo−-CSCs, the cytotoxic agent TPZ was used.13,22 TPZ kills hypoxic cells with O2 content <1.3%. TPZ is activated by cytoplasmic reductases, resulting in the generation of reactive oxygen species, DNA damage, and apoptosis.13,22 After a single TPZ injection, 3-month-old and 30-month-old mice were euthanized 24 hours later (day 1). Two additional groups of mice received TPZ daily for 4 days and were killed 1 day (day 5) and 8 days (day 12) later. In all cases, Pimo was administered 2 hours before euthanization, and the thymus was used as a positive control (Online Figure IV).
To define whether TPZ had a detrimental effect on ventricular performance, cardiac hemodynamics was measured at day 12 in young and old mice. LV end-diastolic pressure, LV systolic pressure, and positive and negative LV dP/dt were not changed by TPZ in either animal group. However, myocardial aging resulted in a severe depression in systolic and diastolic function (Online Figure V).
In 3-month-old mice at day 1, the fraction of Pimo+-CSCs decreased from 38% to 13%, whereas the fraction of Pimo−-CSCs increased from 62% to 87% (Figure 4A). At day 5, the CSC pool was reduced by 42%, that is, from 152 to 89 CSCs per mm3 of myocardium (P=0.03). However, the percentage of Pimo+-CSCs returned to nearly its baseline value (32%) as did Pimo−-CSCs (68%; Figure 4A). These proportions were maintained at day 12 (Figure 4A). In the senescent heart at 30 months, Pimo+-CSCs and Pimo−-CSCs accounted for 65% and 35% of the population, respectively (Figure 4A). One day after a single injection of TPZ (day 1), the fraction of Pimo+-CSCs decreased 62% and Pimo−-CSCs predominated (Figure 4A). At day 5, a 66% decrease in the CSC class occurred, that is, from 306 to 106 CSCs per mm3 of myocardium (P=0.001). Moreover, at days 5 and 12, the fraction of Pimo+-CSCs remained relatively constant, averaging 29%.
In young mice, there were no changes in the fraction of cycling and differentiating Pimo+-CSCs and Pimo−-CSCs at day 1 (Figure 4B and 4C). However, the percentage of cycling Pimo−-CSCs increased 3-fold at day 5, that is, from 4.8% at day 1 to 15% (Figure 4B). Because the proportion of cycling Pimo+-CSCs did not differ from that seen at baseline and at day 1, these data suggest that the newly formed Pimo−-CSCs contributed to the reconstitution of the compartment of Pimo+-CSCs in the organ. This conclusion is supported by the decrease in lineage specification of Pimo−-CSCs at day 5 (Figure 4C).
The minimal level of expression of GATA4 and Nkx2.5 in Pimo−-CSCs at day 5 is consistent with the notion that these cells divided symmetrically, generating 2 daughter stem cells, which were involved in the restoration of hypoxic niches within the adult heart. However, at day 12, the commitment of Pimo−-CSCs was higher than that seen at the earlier interval, whereas the fraction of Ki67-positive cells decreased by 73%. Thus, Pimo−-CSCs and Pimo+-CSCs were slowly re-establishing their physiological behavior based on the critical role that Pimo−-CSCs seem to have in the restoration of the pool of quiescent CSCs within the healthy myocardium. In old mice, a 39% increase in cycling Pimo−-CSCs was seen at day 5 in the absence of cell commitment (Figure 4B and 4C), reflecting an attempt to expand their own pool that was severely affected by age.
Thus, depletion of Pimo+-CSCs in the young heart seems to activate a population replacement process23 involving replication of Pimo−-CSCs, which partially reconstitute the hypoxic CSC pool. However, depletion of Pimo+-CSCs in the old heart results in growth activation of Pimo−-CSCs, which partly restores the balance between these 2 CSC compartments lost with physiological aging.
Pimo+-CSCs Have Longer Telomeres
With cell multiplication, chromosomal ends lose telomeric repeat sequences, ultimately resulting in replicative senescence and apoptosis.24 Despite the high level of telomerase activity, rapidly dividing CSCs undergo telomere erosion,14 whereas quiescence protects telomere length and CSC growth. Long-term repopulating CSCs may retain relatively intact telomeres that sustain their proliferative reserve with aging. After activation, this stem cell class may partly rejuvenate the old heart, because the long telomeres of the mother cell are inherited by the derived progeny, a parameter that is coupled with the integrity of the mechanical and electrical properties of myocytes.10
To test this possibility, mice at 3 and 30 months of age were exposed to Pimo; after enzymatic digestion of the myocardium, Pimo+-CSCs and Pimo−-CSCs were FACS-sorted (Online Figure VI) and ventricular myocytes were collected by differential centrifugation. Telomere length was then measured in these cells by Q-FISH (Figure 5A). Moreover, the expression of the senescence-associated protein p16INK4a, which prevents the re-entry of stem cells into the cell cycle,25 was determined in CSCs.
In the young heart, the distribution of telomere lengths in Pimo+-CSCs was shifted to the right to values higher than those in Pimo−-CSCs. Cardiomyocytes showed telomeres shorter than in Pimo+-CSCs and Pimo−-CSCs (Figure 5B). The difference in telomere length between Pimo+-CSCs and Pimo−-CSCs increased in the senescent heart; again, cardiomyocytes carried significantly shorter telomeres compared with CSCs. Telomere length in Pimo+-CSCs did not decrease with age, whereas a 43% and a 46% reduction in telomere length occurred in Pimo−-CSCs and myocytes, respectively (Figure 5C).
At 3 and 30 months, the fraction of Pimo−-CSCs expressing p16INK4a was, respectively, 6-fold and 2.7-fold higher than in Pimo+-CSCs (Figure 5D). Thus, Pimo+-CSCs possess long telomeres, a property that largely persists in the senescent heart where 74% of this CSC class can divide and create a specialized progeny. In contrast, the majority of Pimo−-CSCs experience telomere erosion with age, and only a small fraction (30%) retains the ability to replicate and differentiate.
Hypoxic CSCs Have Properties of Long-Term Label-Retaining Cells
To determine whether Pimo+-CSCs represent the compartment of slowly dividing stem cells, a label-retaining assay was performed in 3-month-old mice.1 The pulse period consisted of 14 injections of BrdU administered at 12-hour intervals for 7 days. One group of mice was then euthanized, whereas 2 additional animal groups were killed, respectively, 2 and 5 months later. The fluorescence intensity of BrdU in Pimo+-CSCs and in Pimo−-CSCs were measured by confocal microscopy.1–3 Fluorescent levels >1000 units (pixel×average intensity) were recorded; tissue autofluorescence together with the signal generated by the irrelevant antibody used as a negative control for BrdU staining was at most 900 units (Online Figure VII). BrdU intensities varying from 1000 to 3999 units and from 4000 to 10 000 units were assigned to dimly labeled and brightly labeled cells, respectively (Figure 6A–6C); low fluorescence reflected CSCs that underwent several divisions, whereas high fluorescence indicated CSCs that proliferated rarely.
At 7 days and at 2 and 5 months, the percentage of BrdU-labeled Pimo−-CSCs was similar in atria, Base-MR, and apex, averaging 45% (Figure 6A–6C). At 7 days, the fraction of BrdU-labeled Pimo+-CSCs was comparable in the 3 anatomical regions, averaging 11%. However, at 2 months, BrdU-labeled Pimo+-CSCs reached 30% and 32% at the Base-MR and apex, respectively. From 2 to 5 months, BrdU-labeled Pimo+-CSCs increased to 57% at the apex; there was no further increase at the Base-MR. The fraction of BrdU-positive Pimo+-CSCs in the atria did not increase at 2 and 5 months (Figure 6C); thus, CSCs experienced a minimal number of divisions in this region.
Subsequently, the proportion of highly proliferating (dim) and rarely dividing (bright) Pimo−-CSCs and Pimo+-CSCs was determined. At 7 days, BrdU-bright Pimo−-CSCs in the atria, Base-MR, and apex averaged 30%. BrdU-dim Pimo−-CSCs were higher in number at Base-MR and apex (19%) than in the atria (5%; Figure 6D). At 2 and 5 months, BrdU-bright Pimo−-CSCs were essentially undetectable in all regions. However, BrdU-dim Pimo−-CSCs increased to 44% at 2 and 5 months. The population of BrdU-negative Pimo−-CSCs remained relatively constant at 7 days and at 2 and 5 months, averaging 55% (Figure 6D). Thus, a large fraction of Pimo−-CSCs divided frequently over a period of 5 months.
The growth kinetics of Pimo+-CSCs differed from that of Pimo−-CSCs. At 7 days, throughout the heart, BrdU-bright Pimo+-CSCs included 11% of the population, and BrdU-dim Pimo+-CSCs were undetectable (Figure 6E). At 2 months, BrdU-bright Pimo+-CSCs remained constant in the atria, Base-MR, and apex, averaging 10%. BrdU-dim Pimo+-CSCs were not found in the atria but appeared at the Base-MR and apex, comprising 24% of the cells. From 2 to 5 months, BrdU-bright Pimo+-CSCs in the atria did not change, but a small pool (9%) of BrdU-dim Pimo+-CSCs became apparent (Figure 6E). Conversely, at Base-MR, BrdU-bright Pimo+-CSCs increased 2-fold, from 7% to 14%, whereas BrdU-dim Pimo+-CSCs remained constant (≈20%). From 2 to 5 months, no changes occurred in the fraction of BrdU-bright Pimo+-CSCs in the apex, although BrdU-dim Pimo+-CSCs increased from 24% to 52% (Figure 6E).
Importantly, BrdU-negative Pimo+-CSCs did not vary in the atria from 7 days to 2 and 5 months, averaging 88%. However, from 7 days to 5 months, BrdU-negative Pimo+-CSCs decreased from 86% to 68% at the Base-MR, and from 92% to 43% at the apex (see Figure 6C). Collectively, these data suggest that Pimo+-CSCs divide significantly less compared with Pimo−-CSCs, and atrial Pimo+-CSCs possess properties consistent with long-term repopulating stem cells.
Pimo+-CSCs, SCF, and Myocardial Aging
Myocardial aging manifests itself with the accumulation of poorly contracting, p16INK4a-positive myocytes, despite an increase in their turnover rate and the addition of chronologically younger cells.3,5,10 The CSCs being activated lose their phenotypic integrity, a process dictated by telomere dysfunction. CSCs with shortened telomeres divide frequently, conditioning the characteristics of the myocyte progeny that inherits the partially eroded telomeres of the mother cells.3,5
To define whether hypoxic CSCs can be coaxed to re-enter the cell cycle and generate mechanically efficient myocytes, 2 strategies were used: 26-month-old mice were kept in an atmosphere of 70% O2 to increase O2 content in hypoxic CSCs; and SCF was delivered to multiple areas of the LV in 26-month-old to 30-month-old mice to stimulate c-kit, the receptor of SCF, in hypoxic CSCs. Hyperoxia at 70% O2 was used because this level of O2 does not result in symptomatic lung injury.26
Hyperoxia decreased the fraction of Pimo+-CSCs by 18% and 19% at 1 and 7 days, respectively. Accordingly, the pool of Pimo−-CSCs increased 29% at 1 day and 32% at 7 days (Online Figure VIIIA and VIIIB), indicating that prolonged hyperoxia did not change the acute effects of this protocol. This strategy has no clinical relevance but provided preliminary information on the reversibility of the hypoxic state of CSCs in the old heart.
Subsequently, we tested whether c-kit activation by SCF promoted cell-cycle re-entry of Pimo+-CSCs. Intramyocardial delivery of 2.5 μg SCF over a 6-hour period did not increase the blood level of c-kit–positive HSCs (Online Figure IX). A cumulative dose of 1.0 μg was injected in 3 to 4 separate sites of the left ventricle. Six hours and 24 hours later, the myocardium was enzymatically dissociated and the proportions of Pimo+-CSCs and Pimo−-CSCs were measured by FACS. At 6 hours, Pimo+-CSCs decreased 55% and Pimo−-CSCs increased 97% (Figure 7A). These cells were c-kit–positive and CD45-negative, excluding the contribution of bone marrow cells and mast cells. The proportion of Pimo+-CSCs and Pimo−-CSCs returned to baseline at 24 hours. Thus, SCF has an acute effect on Pimo+-CSCs, a response consistent with the short half-life of this cytokine.27
To establish whether SCF led to CSC division, BrdU was administered daily and the fraction of BrdU-positive CSCs was measured at 2 days. SCF resulted in a 95% increase in the percentage of BrdU-labeled CSCs (Figure 7B), a value consistent with the 97% increase in Pimo−-CSCs. Thus, SCF activates the growth of Pimo+-CSCs.
The impact of SCF and CSC growth on the anatomy and function of the heart was measured in 26-month-old mice 3 weeks after echo-guided cytokine injection. Importantly, all data were collected blindly. SCF led to a significant decrease in LV diastolic and systolic diameters, together with a dramatic reduction in LV diastolic and systolic volumes. Moreover, the thickness of the anterior and posterior regions of the LV wall consistently increased (Figure 7C), indicating a remarkable change in ventricular remodeling. Invasive cardiac hemodynamics did not differ in control and SCF-treated animals (Online Figure X); however, when the echocardiographic parameters of ventricular anatomy were combined with pressure measurements, a 56% reduction in diastolic anterior (P<0.008) and posterior (P<0.008) wall stress was found with SCF. Similarly, systolic anterior and posterior wall stress decreased 45% (P<0.004) and 43% (P<0.006), respectively (Figure 7D); notably, LV mass-to-chamber volume ratio increased 84% in diastole (P<0.003) and 2.2-fold in systole (P<0.04; Figure 7E; Online Figure XI). Thus, division and differentiation of Pimo+-CSCs via c-kit receptor activation result in a dramatic decrease in load on the senescent heart.
Rejuvenation of the Senescent Heart
To evaluate the formation of cardiomyocytes and CSCs over the 3-week period after the delivery of SCF, BrdU was administered daily and the percentage of cells positive for the thymidine analog was measured at euthanization. In comparison with control hearts, the fraction of BrdU-positive myocytes increased 114% after SCF administration (Figure 8A). Interestingly, the percentage of BrdU-labeled CSCs was similar in the 2 groups, despite the striking increase in proliferating cells observed at 2 days (see Figure 7B). However, the number of CSCs per mm3 of myocardium was 32% higher in mice treated with SCF (Figure 8B). Thus, SCF exerted an integrated effect on the senescent heart: repopulating the organ with chronologically younger CSCs and myocytes.
To define the mechanisms of ventricular remodeling, myocyte size, number, and the proportion of mononucleated and binucleated cells were determined. Sections (40-μm-thick) were stained by α-sarcomeric actin, connexin 43, and laminin, and the volume of mononucleated and binucleated myocytes and their relative contribution were measured by optical section reconstruction and confocal microscopy (Online Figure XII). These parameters allowed the computation of the number of LV mononucleated and binucleated myocytes. The 29% expansion in LV mass 3 weeks after SCF administration was characterized by a 2.1-fold increase in the number of mononucleated myocytes, which were 55% smaller than in control myocardium (Figure 8C). Conversely, binucleated myocytes decreased 12% in SCF-treated hearts. The volume of binucleated myocytes was similar in both groups of animals, but the number of LV myocytes was 39% higher after treatment with SCF (Figures 8C).
After SCF, telomere length in newly formed BrdU-positive myocytes was 75% longer than in pre-existing BrdU-negative cells, supporting the notion that regenerated myocytes were derived from activation of CSCs, which possessed a younger cell phenotype. In PBS-injected mice, BrdU-positive myocytes carried telomeres only 31% longer than in BrdU-negative cells (Figure 8D). Moreover, in these control hearts, the distribution of telomere lengths in BrdU-positive myocytes was shifted to the left toward shorter values, a phenomenon opposite to that observed in SCF-treated hearts (Figure 8D). As expected, telomere length in BrdU-negative cardiomyocytes did not differ in mice injected with PBS or SCF. Thus, the myocyte progeny derived from growth and differentiation of CSCs with relatively intact telomeres has profound consequences on the cellular composition of the myocardium, partly repopulating the heart with a pool of younger cardiomyocytes.
Currently, we have little understanding of the causes of myocardial aging. Rarely, aging has been considered an independent event and time is the major cause of the senescent cardiac phenotype. Aging has been interpreted as a variable that cooperates with a variety of diseases to define the old, poorly functional heart.28–30 Results in this study suggest that myocardial aging has to be viewed as a stem cell disease, dictated partly by lack of activation of quiescent hypoxic CSCs with intact telomeres, which are present in the senescent heart. This defect has a notable impact on cellular age that differs significantly from chronological age. Physiologically, myocyte renewal in the aged organ is controlled by a class of CSCs with shortened telomeres that, by necessity, are destined to differentiate into myocytes that are chronologically young but phenotypically old, impacting on the myocardium structurally and functionally. Telomere dysfunction dictates their actual age and mechanical behavior.10 However, the residual compartment of quiescent phenotypically young CSCs can be stimulated in situ by SCF reversing the aging myopathy. Our findings support the notion that strategies targeting CSCs may interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of experimental findings to human beings, our observations suggest that the protocol used here may be beneficial for the aging myopathy improving health span in the elderly people.
CSCs represent a heterogeneous cell category that is particularly apparent in the old heart in which CSCs with extremely short telomeres coexist with CSCs carrying long, intact telomeres. In analogy to HSCs in the bone marrow,7 hypoxic CSCs, particularly in the atria, correspond to quiescent long-term label-retaining cells that rarely divide, whereas normoxic CSCs reflect cells that proliferate frequently and generate a specialized progeny. The balance between these 2 stem cell classes is critical for the regulation of myocardial homeostasis and the preservation of CSC compartment during the organ lifespan. Aging, however, expands dramatically the pool of quiescent CSCs so that the smaller pool of cycling CSCs sustains cell turnover.
The combination of insulin-like growth factor-1 and hepatocyte growth factor has been shown to activate resident CSCs in the infarcted31,32 and senescent3 myocardium, resulting in tissue regeneration and restoration of a younger organ phenotype. SCF preferentially operates at the level of hypoxic CSCs with long telomeres, suggesting that this strategy may be important in conditions associated with defects in tissue oxygenation. Moreover, the derived myocyte progeny typically shows long telomeres, a property that is associated with superior contractile performance.11 However, a direct comparison between insulin-like growth factor-1/hepatocyte growth factor and SCF remains to be made to establish potential therapeutic differences of these 2 modalities of intervention for the injured and old mammalian heart.
Consistent with previous results,3 the number of CSCs increases with age. However, p16INK4a is also increased together with the level of cell apoptosis. These variables decrease the number of functionally competent CSCs and the growth reserve of the old myocardium. A similar phenomenon has been observed in the number and activity of HSCs in the bone marrow and crypt stem cells in the small intestine.33,34 Collectively, our results emphasize the critical role that resident CSCs have in the homeostatic control of the myocardium as a function of age. These findings are in contrast with recent reports questioning the relevance of c-kit–positive CSCs in myocyte turnover and regeneration.35,36 These differences may reflect the methodology used and the inherent limitations of lineage tracing studies with promoters of genes encoding contractile proteins.
Although measurements of coronary perfusion in aging mice are lacking, myocardial aging typically induces reduction in coronary blood flow reserve in rats,37 which, together with the decrease in capillary density, would result in defects in the parameters controlling tissue oxygenation.12 These age-dependent variables create hypoxic microdomains38,39 that may account for the increase in the number of inactive CSCs within the myocardium. This view is consistent with our results showing an increase in the diffusion distance for O2 between hypoxic CSCs and the nearest capillary. The colocalization of Pimo, CAIX, and HIF-1α in this subset of CSCs strongly supports the contention of their hypoxic state. The expression of HIF-1α in cardiomyocytes surrounding hypoxic CSCs indicates that these tissue microdomains are in a state of relative hypoxia.
Survival in the hypoxic niche imposes on HSCs the use of glycolysis for energy production. This metabolic adaptation is regulated by Meis1 through transcriptional activation of HIF-1α and upregulation of Cripto/GRP78 signaling.8,17 Whether hypoxic CSCs are characterized by a similar metabolic signature remains to be defined. Anaerobic glycolysis and quiescence reduce the generation of free radicals and the incidence of oxidative DNA damage,6 whereas frequent division increases the intracellular content of reactive oxygen species. The selective advantage associated with limitations in the formation of reactive oxygen species may be operative in quiescent CSCs, which are characterized by longer telomeres. Moreover, attenuation in oxidative stress opposes the upregulation of p16INK4a, replicative senescence, and growth arrest, which are significantly decreased in hypoxic CSCs.
The heterogeneity in CSCs raises the question of whether preservation of the number of CSCs within hypoxic and normoxic myocardium depends on the classic modality of self-renewal at the individual cell level or on a population replacement process.23 Stem cell self-renewal implies that each niche constitutes an independent unit controlled by an intrinsic homeostatic mechanism that maintains the number of CSCs constant within the niche. The population replacement process entails that the turnover of CSCs is based on a feedback system between hypoxic quiescent niches and normoxic active niches with exchange of CSCs, replenishing depleted or dysfunctional niches. Our findings suggest that quiescent and active CSCs may have a cooperative role, pointing to a new model of stem cell turnover in the adult heart.
Although the claim is commonly made that age-associated changes predispose older adults to develop chronic heart failure, its cause is essentially unknown. Morbidity and mortality for chronic heart failure continue to increase40 and parallel the extension in median lifespan of the population,41 pointing to aging as the major risk factor for disease in humans. At present, there are 5.1 million patients with chronic heart failure in the United States, with an annual incidence of 670 000 new cases.40 The overwhelming majority of individuals with chronic heart failure are ≥65 years of age.30,42 However, good CSCs may be locally activated to replace old, poorly contracting myocytes with young, mechanically powerful cells. Autologous CSCs have recently been introduced in the experimental treatment of patients with chronic heart failure, showing encouraging results.43–45
There are some technical aspects that need to be acknowledged because they preclude, in part, answering a few important questions related to the interpretation of the collected results. Cardiomyocytes with long telomeres can only derive from CSCs with similar or longer telomeres, suggesting that they represent the progeny of hypoxic CSCs, which transiently acquire a normoxic state. This possibility is consistent with the relative increase in normoxic CSCs 6 hours after the injection of SCF. But, whether the number of normoxic CSCs increased as a result of a decrease in the pool of hypoxic CSCs was not demonstrated directly. Notably, the cell cycle of CSCs is significantly >6 hours.46 Additionally, whether the reconstitution of hypoxic niches after the delivery of TPZ involves normoxic CSCs with initially shorter telomeres was not shown. Moreover, whether these new hypoxic CSCs are able to increase the length of their telomeres with time remains to be addressed in future studies.
FACS analysis of freshly isolated CSCs does not allow a valid measurement of the absolute number of any cell population in the myocardium. Too many variables are involved in the process interfering with the evaluation of the aggregate pool. This limitation has precluded us from expressing FACS data in absolute terms.47 However, flow cytometry is considered the gold standard for the characterization of the stem cell phenotype.
We do recognize that the measurement of the diffusion distance for oxygen is only 1 of the factors implicated in the formation of hypoxic niches. Intracellular processes may be more relevant to the myocardium in view of the high number of capillary structures per unit area of tissue. In this study, both components seem to be operative in hypoxic niches, as shown by the increase in capillary distance, the lower ΔΨ, and the modest effect of hyperoxia.
We used Pimo to obtain data documenting the functional state of CSCs at organ death. However, this approach did not permit us to distinguish whether hypoxia is a transient or a more prolonged cellular phenomenon. This interesting concept will be studied in the future by sequential injection of nitroimidazole probes with distinct pharmacokinetics and intracellular accumulation rates.17,48,49
It might come as a surprise that, despite the high number of capillaries in the myocardium, hypoxic interstitial cells are present within the tissue. Hypoxic stem cell niches are routinely detected in various organs by Pimo together with HIF-1α and its downstream targets.50 It has recently been shown that HSCs with a hypoxic profile are distributed in the bone marrow independently from the proximity to the endosteal surface and adjacency to vascular profiles.51
Sources of Funding
This work was supported by National Institutes of Health grants R01 HL065573, R01 AG026107, R01 AG037490, R01 HL114346, R01 AG017042, R01 AG037495, R01 HL039902, R01 HL105532, R37 HL081737, R01 HL102897, P01 AG043353, P01 AG043353, P01 AG043353, R01 HL111183, P01 HL092868, and R01 HL091021.
In September 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
This manuscript was sent to Steven Houser, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.114.302500/-/DC1.
- Nonstandard Abbreviations and Acronyms
- carbonic anhydrase IX
- chronic heart failure
- cardiac stem cell
- hypoxia-inducible factor-1α
- hematopoietic stem cell
- stem cell factor
- Received August 28, 2013.
- Revision received October 26, 2013.
- Accepted October 29, 2013.
- © 2013 American Heart Association, Inc.
- Urbanek K,
- Cesselli D,
- Rota M,
- Nascimbene A,
- De Angelis A,
- Hosoda T,
- Bearzi C,
- Boni A,
- Bolli R,
- Kajstura J,
- Anversa P,
- Leri A
- Hosoda T,
- D’Amario D,
- Cabral-Da-Silva MC,
- et al
- Gonzalez A,
- Rota M,
- Nurzynska D,
- et al
- Kajstura J,
- Gurusamy N,
- Ogórek B,
- et al
- Torella D,
- Rota M,
- Nurzynska D,
- Musso E,
- Monsen A,
- Shiraishi I,
- Zias E,
- Walsh K,
- Rosenzweig A,
- Sussman MA,
- Urbanek K,
- Nadal-Ginard B,
- Kajstura J,
- Anversa P,
- Leri A
- Frangogiannis NG
- Rota M,
- Hosoda T,
- De Angelis A,
- Arcarese ML,
- Esposito G,
- Rizzi R,
- Tillmanns J,
- Tugal D,
- Musso E,
- Rimoldi O,
- Bearzi C,
- Urbanek K,
- Anversa P,
- Leri A,
- Kajstura J
- Page E,
- Fozzard HA,
- Solaro RJ
- Anversa P,
- Olivetti G
- Bearzi C,
- Rota M,
- Hosoda T,
- et al
- Khan M,
- Meduru S,
- Mostafa M,
- Khan S,
- Hideg K,
- Kuppusamy P
- Takubo K,
- Nagamatsu G,
- Kobayashi CI,
- Nakamura-Ishizu A,
- Kobayashi H,
- Ikeda E,
- Goda N,
- Rahimi Y,
- Johnson RS,
- Soga T,
- Hirao A,
- Suematsu M,
- Suda T
- Li L,
- Clevers H
- Aubert G,
- Lansdorp PM
- Clark JM,
- Lambertsen CJ
- Dimmeler S,
- Leri A
- Urbanek K,
- Rota M,
- Cascapera S,
- et al
- Linke A,
- Müller P,
- Nurzynska D,
- Casarsa C,
- Torella D,
- Nascimbene A,
- Castaldo C,
- Cascapera S,
- Böhm M,
- Quaini F,
- Urbanek K,
- Leri A,
- Hintze TH,
- Kajstura J,
- Anversa P
- Hachamovitch R,
- Wicker P,
- Capasso JM,
- Anversa P
- Go AS,
- Mozaffarian D,
- Roger VL,
- et al
- Makkar RR,
- Smith RR,
- Cheng K,
- Malliaras K,
- Thomson LE,
- Berman D,
- Czer LS,
- Marbán L,
- Mendizabal A,
- Johnston PV,
- Russell SD,
- Schuleri KH,
- Lardo AC,
- Gerstenblith G,
- Marbán E
- Chugh AR,
- Beache GM,
- Loughran JH,
- Mewton N,
- Elmore JB,
- Kajstura J,
- Pappas P,
- Tatooles A,
- Stoddard MF,
- Lima JA,
- Slaughter MS,
- Anversa P,
- Bolli R
- Ferreira-Martins J,
- Ogórek B,
- Cappetta D,
- et al
- Mandy F,
- Brando B
- Bennewith KL,
- Durand RE
- Nombela-Arrieta C,
- Pivarnik G,
- Winkel B,
- Canty KJ,
- Harley B,
- Mahoney JE,
- Park SY,
- Lu J,
- Protopopov A,
- Silberstein LE
Novelty and Significance
What Is Known?
In the myocardium, c-kit–positive cardiac stem cells (CSCs) are clustered in interstitial microdomains with the properties of stem cell niches.
With aging, the fraction of CSCs expressing the senescence-associated protein p16INK4a increases, reducing the number of functionally competent CSCs.
A pool CSCs with long telomeres persists in the senescent myocardium.
What New Information Does This Article Contribute?
CSC niches are functionally heterogeneous and are composed of active and quiescent niches.
Cellular hypoxia preserves the quiescent undifferentiated state of CSCs, which are characterized by long telomeres, low expression of p16INK4a, and a high growth reserve, typically present in CSCs with a young cellular phenotype.
Hypoxic CSC niches increase with age but can be activated by stem cell factor (SCF), generating a large progeny of chronologically and phenotypically young cardiomyocytes.
Currently, we have little understanding of the causes of myocardial aging. Rarely, aging has been considered as an independent event and time is the major cause of the senescent cardiac phenotype. Results in this study suggest that myocardial aging has to be viewed as a stem cell disease, dictated partly by lack of activation of quiescent hypoxic CSCs with intact telomeres, which are present in the senescent heart. This defect has a notable impact on cellular age, which differs significantly from chronological age. Myocyte renewal in the aged organ is controlled by a class of CSCs with shortened telomeres that differentiate into old, poorly contracting myocytes. However, the residual compartment of quiescent, phenotypically young CSCs can be stimulated in situ by SCF reversing the aging myopathy. Our findings support the notion that strategies targeting CSCs may interfere with the manifestations of myocardial aging in an animal model.