The naked mole-rat (NMR) is the longest-lived rodent known, with a maximum lifespan potential (MLSP) of >31 years. Despite such extreme longevity, these animals display attenuation of many age-associated diseases and functional changes until the last quartile of their MLSP. We questioned if such abilities would extend to cardiovascular function and structure in this species. To test this, we assessed cardiac functional reserve, ventricular morphology, and arterial stiffening in NMRs ranging from 2 to 24 years of age. Dobutamine echocardiography (3 μg/g ip) revealed no age-associated changes in left ventricular (LV) function either at baseline or with exercise-like stress. Baseline and dobutamine-induced LV pressure parameters also did not change. Thus the NMR, unlike other mammals, maintains cardiac reserve with age. NMRs showed no cardiac hypertrophy, evidenced by no increase in cardiomyocyte cross-sectional area or LV dimensions with age. Age-associated arterial stiffening does not occur since there are no changes in aortic blood pressures or pulse-wave velocity. Only LV interstitial collagen deposition increased 2.5-fold from young to old NMRs (P < 0.01). However, its effect on LV diastolic function is likely minor since NMRs experience attenuated age-related increases in diastolic dysfunction in comparison with other species. Overall, these findings conform to the negligible senescence phenotype, as NMRs largely stave off cardiovascular changes for at least 75% of their MLSP. This suggests that using a comparative strategy to find factors that change with age in other mammals but not NMRs could provide novel targets to slow or prevent cardiovascular aging in humans.
- naked mole-rat
- cardiovascular aging
- cardiac reserve
- arterial stiffness
- negligible senescence
the naked mole-rat (NMR; Heterocephalus glaber) is a novel and intriguing animal model for biomedical research. With a maximum lifespan potential (MLSP) of more than 31 years, NMRs are the longest-lived rodent known, despite being only slightly larger than the average laboratory mouse. NMRs are indigenous to the Horn of Africa. They exist in eusocial colonies where only one female within the colony breeds with one to four males, whereas the other individuals within the colony are hypogonadic workers. Despite sex steroid disparities, there are no evident differences in MLSP between breeders and nonbreeders in captivity (3). In large social groups in sealed burrows deep below ground, NMRs exist in a thermally buffered habitat protected from predation, disease, and inclement weather. This has led to evolved tolerance of hypoxic conditions and a lower basal metabolic rate than mice, traits that may contribute to their exceptional longevity (16, 19).
Notwithstanding this animal's extraordinary lifespan, the NMR appears able to maintain body composition, bone mineral density, gastrointestinal function, and overall good health well into the third decade of life (10, 29). Furthermore, NMRs are resistant to both experimental tumorigenesis and spontaneous neoplasia (24). As such, NMRs display negligible senescence and stave off the majority of age-associated illnesses until very late in life, if they succumb to such diseases at all (3). This delayed and attenuated aging profile has led to the development of the NMR as an exceptional model for aging, since this rodent is likely to hold clues to mechanisms facilitating prolonged good health. Such mechanisms could help to protect against some of the most pressing medical problems of humanity, such as cardiovascular disease and aging.
Mammals ranging from rodents to primates share characteristics of cardiovascular aging in the absence of outright pathologies, including decreased cardiac functional reserve, cardiac structural changes, and increased arterial stiffness (7). Prior studies suggest that NMRs might withstand these age-associated changes. We previously found that NMRs exhibit attenuated age-dependent declines in left ventricular (LV) diastolic function compared with those seen in mice and humans (12). Furthermore, despite showing low basal cardiac function, NMRs possess an exceptionally large cardiac reserve (13). NMRs, in contrast with rats, also maintain vascular relaxation and withstand endothelial cell death for at least 40% of their lengthy MLSP (5). We hypothesized that NMRs, in keeping with their negligible senescence phenotype, maintain cardiovascular function and structure in the face of extreme longevity and assess here the common signs of cardiovascular functional and structural aging in NMRs ranging from 2 to 24 years of age.
The male and female NMRs used in this study were second or third generation captive-born animals, descended from animals captured in Kenya in 1980. Animals were maintained at the University of Texas Health Science Center at San Antonio (UTHSCSA) under normoxic conditions in interconnected systems consisting of tubes and cages of varying sizes to approximate the multi-chambered burrow and tunnel systems that NMRs inhabit in the wild. NMRs were housed under climatic conditions simulating their native habitat (30°C; ≥50% relative humidity). They met all their nutrient and water needs through an ad libitum supply of fruit and vegetables, supplemented with a protein- and vitamin-enriched cereal (Pronutro; Bokomo, Rosendal, South Africa). The young cohort was comprised of animals from 2 to 5 years old, the mid-age NMRs were 8 to 13 years old, and the old cohort had animals ranging from 17 to 24 years old. The Institutional Animal Care and Use Committee at UTHSCSA approved this study.
For all in vivo measurements, animals were anesthetized with 1% to 2% isoflurane in a 100% oxygen mix and placed on a temperature-controlled electrocardiogram board. To ensure physiological relevance, heart rate, respiratory rate, and temperature were monitored during data collection, and heart rate was maintained above 180 beats/min. Measurements were acquired with the Vevo 2100 Imaging System and an MS550D transducer (VisualSonics, Toronto, ON, Canada) as previously described (13). Briefly, m-mode short axis and b-mode long axis images of the LV were taken at baseline and then 30 min after intraperitoneal injection with a maximally stimulating dose of dobutamine (3 μg/g body mass). Dobutamine was used since it binds to both β1- and β2-adrenergic receptors and greatly increases myocardial oxygen consumption, making it a good agent to mimic exercise activity (30).
Doppler ultrasound measurement of blood pressures and pulse-wave velocity.
With the use of blunt surgical techniques, the right carotid artery of the anesthetized NMR was isolated and tied off distally and the proximal end temporarily occluded. A small cut was made in the artery and a 1F Millar catheter (SPR1000; Millar Instruments, Houston, TX) was inserted and held in place with a suture tied over the artery-catheter overlap region. The proximal end of the artery was then opened, and the catheter was advanced into the ascending aorta as close as possible to the aortic root. After aortic blood pressure was measured, the catheter was advanced into the LV and intraventricular pressure was measured. LV pressures were measured at baseline and 30 min after dobutamine treatment (3 μg/g body mass ip). Two-second long segments of aortic and LV pressure signals were acquired at a sampling rate of 4 kHz along with the ECG signal using the MouseDoppler system (Indus Instruments, Houston, TX) and stored for analysis offline. The MouseDoppler system was also used to noninvasively evaluate pulse-wave velocity. As described previously, two 20-MHz Doppler probes were used simultaneously (1 at aortic arch and the other at abdominal aorta) with aortic arch waveform displayed up and abdominal aortic waveform down on the spectral display. Pulse transit time was calculated as the difference between the timing of the foot of each waveforms (15).
After blood pressure analyses, NMRs were euthanized with isoflurane anesthesia and their hearts were excised and weighed. LVs were separated from right ventricles and atria. LVs were then sectioned laterally, and midpapillary slices were fixed in 10% zinc formalin. Slices were then embedded in paraffin and sectioned at 5 μm thickness. Sections were stained with hematoxylin and eosin. To quantify cardiomyocyte cross-sectional area, 20 myocytes per section were randomly scanned at ×40 magnification according to previously described methods, such that quantifiable cardiomyocytes were rounded and had central nuclei (25). Areas were quantified using NIS-Elements imaging software (Nikon, Melville, NY). Staining with picrosirius red (PSR; Electron Microscopy Sciences, Hatfield, PA) was conducted to determine LV interstitial collagen density according to previously described procedures (4). Five images were acquired at ×20 for each PSR-stained section, and the collagen content was analyzed with ImageJ (National Institutes of Health, Bethesda, MD). Thresholds were set to show the area occupied by collagen and total myocyte area. The collagen area was then calculated in proportion to myocyte area.
The number of NMRs in each experiment is denoted in figure legends and tables. Because male and female NMRs were used in this study, preliminary analyses for sex differences were evaluated using unpaired t-tests. No sex differences were evident, and male and female data were thus combined for all assessments. As a quality control assessment, the Grubbs′ test was used to remove outliers (one, if any, per parameter). Data are expressed as means ± SE. For normally distributed data, one-way ANOVA with Tukey's post hoc tests were used to compare values among age cohorts (for echocardiography values, aortic and ventricular blood pressure measurements, and histology). If the variances were significantly different for any of the parameters from these assessments, the Kruskal-Wallis nonparametric ANOVA with a Dunn's multiple comparison post-test was used. For percent change data and pulse-wave velocity, linear regressions were calculated against the ages of individual NMRs. GraphPad Prism 5 (GraphPad Software, San Diego, CA) was used for all statistical calculations.
Cardiac structure and function.
No differences were observed among the three age cohorts in either body or heart mass. Furthermore, the heart mass as a percentage of total body mass was similar for each of the cohorts, and none of the cohorts were significantly different from our previous finding of 4.1 ± 0.3 mg/g for heart-to-body mass ratio (13). M-mode echocardiograms revealed no discernible age-related differences in either dimensions or contractility across the three age cohorts (Fig. 1). Under basal conditions, NMRs did not display age-related changes in LV dimensions or wall thicknesses (Table 1). None of the LV function parameters (heart rate, fractional shortening, ejection fraction, cardiac output, and stroke volume) changed with age in the NMR. Because age-related cardiac contractile dysfunction is unlikely under basal conditions in the absence of outright disease, echocardiography was also conducted under exercise-like stress. Even with dobutamine treatment, there were no significant differences among the three age cohorts in either LV dimensions or function.
Cardiac functional reserve.
Potential age-related changes in NMR cardiac functional reserve were assessed by calculating percent changes in the dobutamine response of each individual NMR. The chronotropic response to dobutamine did not change, since there was no age-related difference in the ability to increase heart rate 30 min after dobutamine treatment (Fig. 2A). Also, the NMR inotropic response was not significantly affected by age: percent changes in fractional shortening (Fig. 2B), ejection fraction (Fig. 2C), cardiac output (Fig. 2D), and stroke volume (Fig. 2E) all were not significantly correlated with the age of the NMRs (Fig. 2E). Together these data suggest that, unlike other mammals, the NMR maintains cardiac functional reserve at least until 24 years of age.
LV pressures at baseline and under stress.
To fully investigate LV function with age in the NMR, invasive techniques were used to determine pressure parameters within the LV (Table 2). At baseline, the cohorts displayed no differences in peak LV pressure, LV end-diastolic pressure, rates of change of contraction (+dP/dtmax) and relaxation (−dP/dtmax), or the τ diastolic relaxation constant. After treatment with the same dobutamine dose used in the echocardiographic measures, there were no significant differences among cohorts. Furthermore, when the percent changes from baseline to post-dobutamine treatment were calculated for each parameter, there were no significant correlations with age.
LV morphology and composition.
To assess age-related hypertrophy at the cellular level, cardiomyocyte cross-sectional area was quantified. There was no significant age-related increase in cardiomyocyte area (Fig. 3). It should be noted that young NMRs have a larger average cardiomyocyte area of 216 ± 10 μm2 than age-matched mice (13). These data substantiate the finding that LV wall thicknesses also did not change with age (Table 1). Furthermore, no correlation was noted between heart weight and cardiomyocyte size.
LV interstitial collagen deposition was quantified in the three age cohorts using a PSR stain to assess if age-related ventricular fibrosis was present in NMRs. Collagen deposition was significantly increased in the old (4.3 ± 0.6%) compared with the young (1.7 ± 0.2%; P < 0.01) and mid-age (2.3 ± 0.5%; P < 0.05) cohorts (Fig. 4).
Arterial blood pressure and compliance.
Invasive pressure measurements were also conducted to assess aortic blood pressure. No significant differences were observed among the three age cohorts in systolic, diastolic, mean, or pulse pressures (Table 3). Of note, NMRs display very low systolic and diastolic blood pressures. This is likely a feature of the species, relating to the overall low basal cardiovascular function, seen in both the echocardiographic and LV pressure data. Although age-associated pulse pressure increases are linked to arterial stiffness, this trend was insignificant in NMRs. There was also no relationship between age and pulse wave velocity (Fig. 5). Thus NMRs maintain arterial compliance in the face of extreme longevity.
In this study we found that many common features of cardiovascular aging were not evident in NMRs ranging in age from 2 to 24 years, despite the fact that these features manifest relatively earlier in other aging mammals (7). No significant differences were evident among young, mid-age, and old NMRs in LV dimensions or function either at baseline or under exercise stress-like conditions (Tables 1 and 2). When individual responses to dobutamine were analyzed, there were also no changes in cardiac functional reserve with age (Fig. 2). Strikingly, these results indicate that this species, unlike other mammals, is able to maintain cardiac reserve for the majority of its lifespan. The lack of change in LV dimensions was further corroborated by no increase in cardiomyocyte cross-sectional area (Fig. 3) with age. When vascular function was examined, similarly there was no evidence of age-associated change in arterial blood pressures (Table 3) or arterial stiffness (Fig. 5). Interestingly, the only parameter indicative of classic cardiovascular aging in the NMR was the 2.5-fold increase in LV interstitial collagen deposition from young to old animals (Fig. 4). Together these results indicate that NMRs are largely able to maintain their cardiovascular function and structure for at least 24 years, an age physiologically equivalent to a 92-year-old human. In contrast, man, macaques, dogs, and mice show pronounced declines in these age-associated variables long before 75% of their MLSP has been attained (Table 4).
Ecophysiology and aging of the NMR.
A large body of data on aging in the NMR, in addition to the results presented here, supports the concept that this species shows negligible senescence. Bone composition, gastrointestinal function, metabolic rate, neuronal integrity, and reproductive capacity all do not change for the majority of the >31-year species MLSP (3, 10, 29). Furthermore, NMRs do not exhibit a significant age-associated increase in mortality (3). The ability to stave off age-associated diseases likely evolved in response to the subterranean habitat of the NMR. In their sealed underground burrows, NMRs are protected from infectious diseases, weather, and predation and thus have low extrinsic mortality. Moreover, burrow atmospheres are hypoxic and hypercapnic, and the soils in which burrows are excavated contain high levels of heavy metals. In response, NMRs evolved to deal with toxins and tolerate hypoxia and have low energy and oxygen consumption associated with low basal metabolic rates. These energy-associated traits may contribute to their exceptional longevity (16, 19) and certainly impact their cardiovascular function. We previously described very low basal cardiac function in NMRs. Their resting heart rates and cardiac output are very low for mammals with an average body size of 45 g. Moreover, their average fractional shortening (Table 1) is low enough to border on what would be considered LV systolic dysfunction in humans (≤25%). However, when both mice and NMRs are stimulated with dobutamine, NMRs are able to enhance their cardiac function nearly twofold more than mice. The low basal function coupled with high cardiac reserve is in keeping with the natural subterranean lifestyle of the NMR. The energetic cost of tunneling in the compacted soils of the arid and semi-arid zones of northeast Africa is extremely high, requiring NMRs to possess a large metabolic scope (27). Existing with such a low basal level of cardiac function yet maintaining the ability to ramp up function when needed might allow for reduced metabolic stress and contribute to the maintenance of cardiac function with age in NMRs.
Aging NMRs have attenuated cardiovascular declines compared with other mammals.
With advancing age, humans, macaques, dogs, and mice display shared characteristics of cardiovascular aging, including reduced cardiac reserve, cardiomyocyte hypertrophy, ventricular fibrosis, and arterial stiffening (Table 4). These age-related changes occur naturally over time in mammals even in the absence of outright cardiovascular diseases (7). Our results indicate that NMRs are an exception to this paradigm, maintaining numerous features of cardiovascular function and structure throughout the majority of their lives.
In the absence of heart disease, humans and other mammals experience age-related declines in cardiac functional reserve that stem from a reduced exercise capacity. Such declines in cardiac reserve are observed across species and are linked to a decrease in the β-adrenergic response of both the heart and vasculature (18, 32, 37). Aortic stiffness and enlargement of cardiomyocytes are also thought to contribute to this process (18). Dobutamine treatment revealed no age-dependent deficits in exercise-mediated increases in NMR heart rate or contractility (Fig. 2). This is in sharp contrast with aging humans, who, in the absence of outright cardiac pathology, experience a blunting of both exercise-induced heart rate and ejection fraction (11). LV pressure measurements also showed no age-related changes during exercise-like stress (Table 2). Furthermore, there was no evidence of ventricular or cardiomyocyte hypertrophy (Table 1 and Fig. 3), nor any increase in arterial stiffness (Fig. 5). Taken together these data are striking, for unlike what is known about other mammals, they suggest NMRs do not display a reduction in cardiac functional reserve for three quarters of their >31-year lifespan.
The aforementioned changes in mammalian cardiac function and structure with age are in part mediated by increased arterial stiffening. Such reduction in elasticity is reflected in systolic blood pressure increases with age, even without a clinical diagnosis of hypertension (18). Additionally, aging in humans and other mammals brings about a widening of pulse pressure, also attributed to increased arterial stiffness (18, 33). NMRs appear to withstand such changes since they show no increase in systolic blood pressure or pulse pressure with age (Table 3). Furthermore, there was no age-associated increase in arterial stiffness, as measured by pulse-wave velocity (Fig. 5). Previous ex vivo experiments conducted on carotid arteries reported that NMRs maintain their vascular compliance for at least 12 years of life, 40% of MLSP, and that this was due to sustained nitric oxide bioavailability (5). The in vivo data collected in the present study extend this time period of unchanged arterial stiffness to at least 24 years. Because stiffening of arteries commonly precedes atherosclerosis, it is likely that these animals also show a resistance to developing atherosclerotic plaques. Given that both humans and other mammals exhibit pronounced increases in arterial stiffness at comparatively lower percentages of their MLSPs (14, 18, 32, 33), the unchanged arterial stiffness of the NMR at ages equivalent to nonagenarians is quite remarkable.
Interestingly, there was an insignificant decline in LV peak pressure and contractility, measured by +dP/dtmax, in the old age cohort with exercise-like stress (Table 2). Together, these data may indicate the beginnings of a reduction in cardiac reserve for NMRs as they enter their last quartile of life. The significant increase in LV interstitial collagen deposition is the only characteristic of cardiovascular aging that was evident in NMRs (Fig. 4). Increased ventricular fibrosis is a hallmark of cardiac aging in mammals (2, 18, 35). Previously, we found increased isovolumic relaxation times and decreased E/A ratios in aging female NMRs (12). These changes are both implicated in age-related declines in diastolic relaxation and are linked to increased LV interstitial fibrosis (4, 18). Thus the increase in collagen deposition likely contributes to the diastolic functional declines with age of the NMRs. However, the rate at which NMRs experience such declines is attenuated in comparison with those of both mice and humans, which experience significant diastolic dysfunction by 50% of their MLSPs (6, 17). Notably, the 2.5-fold increase shown here in NMR LVs is lower than the 3.3-fold change reported for aging mice (4).
We have, on rare occasions, noted indicators of spontaneous cardiac disease in our colony, such as cardiomyocyte damage and lipofuscin accumulation. Yet, these were seen in 29–30-yr-old animals, some of the oldest in our colony (10). The presence of a few age-associated cardiac lesions was also reported in an analysis of 138 NMR necropsies at a zoo in the United States. These lesions were present in such a small portion of the population; however, that cardiac pathology was declared highly unlikely as a cause of death in NMRs (8). Therefore, although NMRs are not completely immune from cardiac dysfunction, they likely either attenuate or delay the onset of cardiac pathologies with the help of various protective mechanisms.
Potential cardioprotective mechanisms in the NMR.
The natural subterranean habitat of the NMR is thought to have shaped the species′ upregulated cellular stress resistance and protection. Indeed, in vitro experiments have shown that the NMR is resistant to a surfeit of toxins, including oxidative stressors and heavy metals (23, 34). A master regulator of cytoprotective factors, nuclear factor erythroid 2-related factor 2 (Nrf2), is expressed highly in NMRs and is thought to play a large role in the longevity and extended healthspan of the NMR. Nrf2 is triggered by oxidative stress and regulates the transcription of numerous antioxidants, detoxicants, molecular chaperones, and proteasome components (22). Furthermore, NMRs maintain high levels of neuregulin-1 in their brains over the majority of their lifespans (9). This protein is also known to be cardioprotective and has anti-adrenergic and pro-hypertrophic effects (20). If neuregulin-1 is also sustained at high levels in NMR hearts, it could explain the low basal cardiac function and relatively large cardiomyocyte size seen in young NMRs (13). Neuregulin-1 may additionally offer NMRs high levels of cardioprotection well into their last quarter of life. The mechanisms by which neuregulin-1 and Nrf2 protect the heart are multifaceted and are likely to play a key role in sustained cardiac function. These and other cardioprotective pathways are needed since cardiac aging itself is a multifaceted process. For instance, current evidence suggests that age-related cardiovascular changes can occur in response to elevation in matrix metalloproteinase 9 (36), dysregulated calcium signaling (26), deficiencies in sirtuin signaling (31), and even altered mitophagy (28) within the heart.
Pronounced stress resistance, particularly resistance to the harmful effects of oxidative stress, is a key component in the longevity of the NMR (23). From an early age the NMR heart has more protein and lipid oxidative damage than observed in the short-lived mouse (21). Furthermore, NMRs exhibit an unexceptional antioxidant defense despite being so long-lived (1). This is at odds with the attenuated age-related cardiovascular declines we have found here and previously (12). Thus it is likely that NMR stress resistance extends to the heart and plays a pivotal role in maintaining cardiovascular structure and function in the face of advanced age and high oxidative damage.
The in vivo and histological data acquired in this study show that the NMR does not undergo many of the common cardiovascular changes that occur during aging in other mammals. Strikingly, unlike other mammalian species, NMRs appear to hold off age-related changes in cardiovascular function at least until their last quartile of life. These findings merit further study into the putative protective mechanisms that may contribute to the attenuated age-related cardiovascular dysfunction in NMRs and which may provide novel targets to delay or prevent cardiovascular aging in humans.
This work was supported primarily by the American Heart Association Grant-in-Aid 12030299 (to R. Buffenstein); National Institutes of Health/National Heart, Lung, and Blood Institute HHSN 268201000036C (N01-HV-00244) for the San Antonio Cardiovascular Proteomics Center and HL-075360; and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award 5I01BX000505 (to M. L. Lindsey).
A. K. Reddy is employed part-time as an Assistant Professor at Baylor College of Medicine and part-time as a Senior Scientist at Indus Instruments.
Author contributions: K.M.G., A.K.R., M.L.L., and R.B. conception and design of research; K.M.G. and A.K.R. performed experiments; K.M.G. and A.K.R. analyzed data; K.M.G., A.K.R., M.L.L., and R.B. interpreted results of experiments; K.M.G. prepared figures; K.M.G. and R.B. drafted manuscript; K.M.G., A.K.R., M.L.L., and R.B. edited and revised manuscript; K.M.G., A.K.R., M.L.L., and R.B. approved final version of manuscript.
We thank Yael Edrey, Kaitlyn Lewis, Miranda Orr, and Karl Rodriguez for help with tissue harvesting and Megan Smith for animal care.