Rejuvenation of Aging Hearts

Introduction

S pecific, subtle changes in regulation or activity of factors that maintain homeostasis and cell differentiation may play significant roles in mammalian aging. Both extrinsic and intrinsic reversible aging-associated changes have been described that can profoundly influence tissue function.

There are numerous examples of aging-associated extrinsic changes to the extracellular milieu that affect cell function. For example, muscle satellite stem cell dysfunction correlates with loss of NOTCH function and can be restored by blood-borne factors from young animals, forced activation of NOTCH, small hairpin RNA (shRNA) knockdown of Smad3, or by neutralization of transforming growth factor-β (TGF-β) or osteopontin. ^{1 –5}

An example of an intrinsic change is the loss of function in hematopoietic stem cells (HSCs) from old mice. Dysfunction in HSCs correlates with increased reactive oxygen species (ROS) and the down-regulation of sirtuin-3 (SIRT3) in old HSCs. Forced expression of SIRT3 is sufficient to restore HSC function. ^6,7

Whether due to instability in epigenetic-based cell differentiation programs, such as that hypothesized to result from developmental drift, or to accumulation of damage in critical cell populations, the amplification of small differences in levels or activity of key regulators may play a profound role in aging and provide new targets for therapeutic intervention.

Parabiosis Between Old and Young Mice or Growth Differentiation Factor 11 Treatment Rejuvenates Old Mouse Hearts

Diastolic dysfunction in elderly mice and humans often leads to diastolic heart failure and death. In old mice, as well as long-lived humans, heart failure is one of the most frequent direct causes of death, thus a treatment with potential to rejuvenate heart function may prove helpful in extending longevity. In an important study with significant ramifications for mammalian aging, Loffredo et al. ⁸ report that it is possible in old mice to reverse cardiac hypertrophy that may underlie age-associated ventricular stiffening and diastolic dysfunction. ⁹

Loffredo et al. ⁸ hypothesized that extrinsic circulating factors in young mice might reverse cardiac aging. Implicit in this hypothesis is that maladaptive changes in circulating factors occur during aging. Evidence for systemic factors playing a profound role in aging has been found previously for skeletal muscle regeneration ¹ and neurogenesis. ¹⁰ Reversibility of cardiac aging in mice previously has been reported by an intrinsic intervention—the overexpression of catalase. ¹¹ As in the studies on skeletal muscle and neurogenesis, the effects of surgically linking the circulatory systems (“parabiosis”) of an old and young mouse (“heterochronic parabiosis”) are compared with controls in which an old mouse is linked to an old mouse or a young mouse to a young mouse (“isochronic parabiosis”). In this study, male and female C57BL/6 mice were used because they spontaneously develop cardiac hypertrophy with age. ¹¹ After 4 weeks of cross circulation, hearts from old mice paired with young mice were significantly smaller than those from old mice paired with old mice. Furthermore, hearts from the old partner of old/young mouse pairs weighed less than controls when normalized to tibia length (a standard way to control for size differences in mice). Histological studies revealed that cardiac myocyte cross-sectional area was restored to that of young mice or paired young mice. At the molecular level, transcription of cardiac stress–associated brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) were reduced in the hearts of old mice paired with young mice, whereas levels of sarcoplasmic-endoplasmic reticulum Ca2⁺-ATPase (SERCA)-2 mRNA, which is associated with diastolic relaxation, were increased. Altogether, these data suggested that 4 weeks of parabiosis was sufficient to reduce cardiac hypertrophy to youthful levels in old mice. Interestingly, the hearts of the young mice that had been paired with old mice did not show any signs of hypertrophy in these experiments. These results suggest that young mice likely synthesize an anti-hypertrophic factor or factors that can restore age-associated diastolic heart failure in old mice, but that old mice do not synthesize a factor that promotes hypertrophy, since the young hearts were not altered by parabiosis with old animals. ⁸

Because of the possibility of artifactual results due to indirect physical effects, several control studies were performed. The reduced cardiac hypertrophy was not caused by reduced blood pressure or lower heart rate. Moreover circulating levels of angiotensin II and aldosterone, which can affect blood pressure, were not affected by parabiosis. Effects due to altered physical activity or behavior resulting from the surgical connection of the mice was ruled out in sham parabiosis experiments in which mice were sutured together in a similar way to the actual parabiosed mice, but with no connection made between the circulatory systems. ⁸

Because the parabiosis experiments suggested the existence of an anti-hypertrophic factor or factors synthesized by young mice, an attempt was made to identify candidate factors. Metabolomic profiling of 69 amino acids and amines revealed no differences between plasma from heterochronic or isochronic parabiosed mice. Similarly, lipidomic analysis of 142 lipids from nine lipid classes revealed no differences. However, a proteomic analysis using RNA aptamers that specifcally bind over ∼1100 targets revealed 13 peptides that distinguish young mice from old mice. More extensive analysis of plasma for levels of each of these analytes identified growth differentation factor 11 (GDF-11) as having the expected expression pattern for an anti-hypertrophic factor. GDF-11 levels were higher in young mice, lower in old mice, and increased in old mice paired with young mice. GDF-11 expression is widespread in mouse tissues, but highest in the spleen. Old mice express much less GDF-11 in the spleen, compared with young mice, consistent with the observed plasma levels. ⁸

In cultured rat neonatal cardiomyocytes treated with phenylephrine to stimulate hypertrophy, which is measured by increases in [³H]leucine incorporation, GDF-11 inhibits the hypertrophic effects of phenylephrine. Interestingly, myostatin, a protein very closely related structurally to GDF-11, which inhibits skeletal muscle hypertrophy, has no effect on phenylephrine-stimulated [³H]leucine incorporation, indicating that GDF-11 may exert a specialized anti-hypertrophic effect on cardiomyocytes. In a preliminary experiment to assess how GDF-11 may differ from myostatin in signal transduction, human induced pluripotent stem cell (iPSC)-derived cardiomyocytes were treated with either GDF-11 or myostatin. However, no differences in activation of downstream pSMAD2, pSMAD3, or suppression of Forkhead transcription factor were observed, suggesting uncharacterized signaling differences between the two factors are yet to be elucidated. ⁸

Daily intra-peritoneal injection of GDF-11 for 30 days into old mice resulted in many of the same effects seen in the heterochronic parabiosis experiments. Treated mouse hearts were smaller and weighed less (after tibia standardization; see above), and cross area was reduced compared to untreated controls. Transcription levels of ANP and BNP were again reduced, and conversely SERCA-2 levels were increased. It is important to note that the magnitude of these effects was not nearly as strong as that seen in the parabiosis experiments, suggesting a contribution by other anti-hypertrophic factors. The effects of GDF-11 were observed to be specific to age-associated hypertrophy and not pressure overload–induced hypertrophy: In young mice subjected to aortic constriction, 30 days of treatment with GDF-11 had no effect on hypertrophy. ⁸

Altogether, these results suggest that dysregulation of anti-hypertrophic factors such as GDF-11 may play an important role in cardiac aging. However, these results are only preliminary. It will be essential that these studies be extended to assess actual changes in cardiac function, as well as repeated in other model systems by other researchers.

Medical Implications

Anti-hypertrophic factors such as GDF-11 may be candidates for therapeutics to prevent or reverse cardiac aging. Reversal of cardiac aging may extend longevity, as was reported for by Dai et al. for the overexpression of catalase in mouse mitochondria. ¹¹ This may prove of significant benefit in elderly populations. However, the need for replication in animal models and, if confirmed, subsequent human clinical trials highlights the preliminary status of GDF-11 as a rejuvenation agent.

One of the potential problems with GDF-11 is that it may play significant roles in other tissues. Knockout mice lacking GDF-11 die at birth, with skeletal and renal abnormalities, suggesting that GDF-11's role in these tissues should be examined. GDF-11 may also play a role in pancreatic β-cell function. ^12,13 Furthermore, GDF-11 has been observed to inhibit neurogenesis, ¹⁴ a potential unwanted side effect. These pitfalls suggest that modulation of the diverse activities of GDF-11 may be required for development of effective therapeutics.

The identification of drugs or nutriceuticals that stimulate native GDF-11 expression may have some utility and should be pursued. For example, it has been reported that the histone deacetylase inhibitor trichostatin A stimulates transcriptional expression of GDF-11. ¹⁵ Although trichostatin A has broad physiological effects, and is probably not suitable for inducing GDF-11 clinically, there is reasonable hope that more specific drugs that stimulate GDF-11 can be identified.

Conclusion

Identification of an anti-hypertrophic factor such as GDF-11 that appears to rejuvenate aging murine hearts raises exciting prospects for the development of anti-aging therapeutics. Similar rejuvenating factors for diverse tissues are expected to exist as well and will hopefully be identified in the near future. However, much work remains to be done to evaluate the utility of GDF-11 as a therapeutic rejuvenation factor.