Autophagy and cardiovascular aging

Abstract

The biological aging process is commonly associated with increased risk of cardiovascular diseases. Several theories have been put forward for aging-associated deterioration in ventricular function, including attenuation of growth hormone (insulin-like growth factors and insulin) signaling, loss of DNA replication and repair, histone acetylation and accumulation of reactive oxygen species. Recent evidence has depicted a rather unique role of autophagy as another important pathway in the regulation of longevity and senescence. Autophagy is a predominant cytoprotective (rather than self-destructive) process. It carries a prominent role in determination of lifespan. Reduced autophagy has been associated with aging, leading to accumulation of dysfunctional or damaged proteins and organelles. To the contrary, measures such as caloric restriction and exercise may promote autophagy to delay aging and associated comorbidities. Stimulation of autophagy using rapamycin may represent a novel strategy to prolong lifespan and combat aging-associated diseases. Rapamycin regulates autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR). Inhibition of mTOR through rapamycin and caloric restriction promotes longevity. The purpose of this review is to recapitulate some of the recent advances in an effort to better understand the interplay between rapamycin-induced autophagy and decelerating cardiovascular aging.

Keywords: :

• MTOR
• aging
• autophagy
• cardiovascular
• rapamycin

Background

Human life expectancy has risen significantly over the last century, and consequently, the elderly represent the fastest-growing segment of the population. It is projected that the elderly population (> 65 y of age) will increase from 10% of the total population in 2000 to ~22% by 2050 and 32% in 2100.¹ Not surprisingly, the prevalence of aging-related chronic diseases is on the horizon. Aging is a major independent risk factor for cardiovascular disease, the leading cause of morbidity and mortality in the United States and rest of the world.^2-4 The prevalence of cardiovascular disease increases rapidly with age and ranks the highest in the elderly population.⁵ Together with the adverse health implications, cardiovascular disease also represents a huge economic burden on the society.^6,7 It is therefore pertinent to understand the molecular mechanisms behind declined cardiac function with biological aging, with an ultimate goal to develop effective strategies to manage or halt the progression of cardiac aging.

Aging is a rather complicated pathophysiological process accompanied by a wide array of biological adaptations, including progressive myocardial remodeling and deteriorated cardiac reserve.^8-11 With aging, myocardial contractile capacity progressively declines as manifested by increased left ventricular wall thickness and chamber size as well as change in diastolic filling pattern, such as prolonged diastole.^11,12 In addition, aging leads to stiffening and loss of elasticity of the ventricular wall and coronary vasculature, possibly due to increase in after-load in the elderly.^10,13,14 This increased workload for the heart often triggers unfavorable remodeling, leading to a much greater heart mass (or cardiac hypertrophy).¹⁵ Although an initial adaptive process develops, the aging heart will eventually transit from compensatory to decompensatory stage, resulting in further impairment of contractile function. In addition, aging is also associated with increased cardiomyocyte cell death via apoptosis. Furthermore, with aging there is a decline in the response of myocardium to certain endogenous autonomic (e.g., adrenergic) stimulation. Advanced age is accompanied with overt calcification and fibrosis of ventricular structure (such as free walls and valves), further deteriorating cardiac geometry and contractile function. In addition to the aforementioned physiological changes, many factors have been identified to play a role in the increased incidence of cardiovascular diseases with aging. For example, sympathetic over-activation, hypertension, dyslipidemia, oxidative stress, chronic low-grade inflammation and sustained obesity may all play a role toward compromised cardiac structure and function in the elderly.^11,16,17 However, none of these theories has been fully validated by clinical findings to explain the complex cardiac aging process.

Several theories of aging have been articulated at length elsewhere^18-20 and, thus, will not be emphasized here. Many of these classical theories of aging have also been implicated in cardiac aging.²¹ The “free radical theory of aging” attributes cumulative exposure of biomolecules to oxygen-derived free radical species as the cause of aging.¹² Indeed, the accumulation of lipofuscin pigments in the heart has been considered as the “tell-tale sign” of free radical damage to the heart.¹⁵ This theory was consolidated by the evidence of extended lifespan following overexpression of endogenous antioxidants such as superoxide dismutase, catalase and metallothionein.^5,22-25 Build-up of advanced glycation end products (AGEs) associated with aging forms the basis for the “glycation theory of aging.”²⁶ AGEs may trigger oxidative stress, thus abridging the free radical theory with the glycation theory of aging.^27,28 To maintain physiological contractile function of the heart, cardiomyocytes have to rely on a constant supply of high-energy phosphates from mitochondria.²⁹ The “mitochondrial decline theory” postulates that the aging is associated with decline in mitochondrial function (hence ATP supply), reduction in mitochondrial numbers and the presence of functionally compromised enlarged mitochondria.³⁰ Mitochondria is also the primary source of reactive oxygen species (ROS) produced by the electron transport chain during mitochondrial respiration, which triggers mitochondrial-derived (endogenous) apoptosis, recognized as a leading cause of cell death in cardiomyocytes.³⁰ Recent evidence suggested that mitochondrial expression of Nox4 a member of the NADPH oxidase family, is upregulated in aging hearts, which serves as a major source of oxidative stress to promote cardiac aging.³¹ More evidence has implicated that compromised mitochondrial function in aging hearts may likely result from mitochondrial permeability transition pore (MPTP) opening through a NAD⁺-dependent deacetylase SIRT3-cyclophilin D-dependent manner.³² These observations have caught much attention, as MPTP is associated with release of Ca²⁺ and ROS in cardiomyocytes.³³ Telomere shortening also contributes to cellular senescence in replicating cells, while defective telomere in cardiac stem cells has been recently identified as a useful marker of cardiac aging.^34,35 Genome-wide transcriptome analysis suggests that age-associated alterations in transcripts govern the cardiac aging process.^8,36 The role of epigenetic modifications (such as differential levels of DNA methylation, microRNA) or activation of certain protein kinase cascade has been implicated in cardiac aging.^37-39 Despite this enriched knowledge of the molecular basis of cardiac aging, the precise aging process remains rather complex and cannot be explained in a unifying manner. As a result, novel molecular mechanisms are being explored, and the role of the autophagic process is gaining prominence as a converging point in the biological aging process. This mini-review will summarize some of the recent advances in the understanding of the interplay between autophagy and cardiac aging.

Autophagy is a highly conserved cytoprotective, rather than self-destructive, process involving degradation and recycling of intracellular organelles and proteins.^40,41 A variety of cellular stress conditions such as nutrient or growth factor deprivation, ROS accumulation, aggregation of the long-lived or damaged proteins and organelles and hypoxia usually turn on autophagy. Three distinct forms of autophagy have been identified (1) microautophagy, wherein the lysosomal membrane invaginates to engulf cytosolic components, (2) chaperone-mediated autophagy, wherein chaperones, such as heat-shock proteins, help deliver macromolecules to the lysosomes via direct binding to the lysosomes through specific receptors on the lysosomal membrane and (3) macroautophagy (commonly referred to as autophagy), wherein double membrane vesicles, referred to a autophagosomes, are first formed, which eventually fuse with the lysosomes. The autophagic process is initiated with the formation of a phagophore (from the endoplasmic reticulum, mitochondria or other cellular components), which engulfs cytosolic components and organelles to from the autophagosome (Fig. 1). The autophagy initiation process is mediated by activation of the class III phosphatidylinositol-3-kinase (PI-3K) and beclin-1, which help recruit autophagic proteins. These autophagosomes then undergo an elongation process mediated by several autophagy-related genes (ATG) that are evolutionarily conserved.⁴² The Atg genes are also responsible for the recruitment of microtubule light chain-3 (LC3) to the autophagosome. Completion of autophagosome formation is characterized by the proteolytic cleavage of LC3, which undergoes lipidation to LC3-II, the form that associates with autophagic membrane. At this stage, the outer membrane of the autophagosome fuses with a lysosomes to form autophagolysosome. Lysosomal contents subsequently degrade the cytosolic components inside autophagosomes. In addition to the ATG-genes, the class 3 PI-3K, Beclin-1, Unc-51-like kinase (Ulk), lysosome membrane protein (LAMP-2) and a small GTPase, Rab7, are all involved in various process of autophagy.¹⁷

Figure 1. Stages involved in the formation of autolysosome from phagophore for various cellular components.

Autophagy plays a pivotal role in survival and longevity of cells and organisms. This is particularly true for the permanently differentiated cells, such as cardiomyocytes, which rely on autophagic process to remove and recycle unwanted, damaged or long-lived cellular components or proteins in order to maintain homeostasis. Although autophagy is a mechanism by which the cell destroys itself, paradoxically, it also serves as a protective function and a means of meeting metabolic requirements during conditions of energy/nutrient deficit, such as starvation, ischemia reperfusion and heart failure. Autophagy is therefore considered a “double-edged sword,” in that low baseline levels of regulated autophagy is beneficial to help maintain cardiac structure and function, although uncontrolled or excessive autophagy can cause extensive cell death and pathological state.⁴³ It has been shown that autophagy declines with aging, whereas pharmacological intervention and genetic manipulations to induce longevity are associated with autophagy induction.^44,45 Conversely, suppression of autophagy by knockdown of autophagy related-genes induces cell death and displays the opposite consequence for lifespan. In addition, loss of autophagy was demonstrated to accelerate aging.^46-50 In particular, lessened autophagy with aging may be responsible for aging-induced accumulation of damaged intracellular components, resulting in altered cellular homeostasis and loss of function associated with aging.^44,49,51 Autophagy is upregulated by AMP-dependent protein kinase (AMPK) while being negatively regulated by Akt and mammalian target of rapamycin (mTOR) through phosphorylation of its downstream targets, such as p70s6k.^52,53 mTOR signaling has been considered the main driving force for aging, interruption of which triggers reduced lifespan.^54,55 Nonetheless, the precise mechanism(s) through which autophagy contributes to longevity and improved cardiac function with aging still remains elusive. Here, we are attempting to explore some of the emerging theories and controversies involving the role of the autophagic pathway in cellular aging in general and aging-associated heart disease with a special emphasis on the mTOR signaling pathway, as it is gaining prominence as a key regulator of autophagy.

Autophagy plays a critical role in cardiac homeostasis and pathologies, including myocardial hypertrophy, diabetic and alcoholic cardiomyopathy as well as ischemic heart disease.^56-61 Fatal hypertrophic cardiomyopathy is well recognized as a consequence of lysosomal storage defect, such as the Danon disease, a defect of LAMP-2. Tanaka and coworkers demonstrated that LAMP-2 deficiency increases mortality in mice.⁶² Interestingly, surviving mice with LAMP-2 deficiency exhibit abnormal cardiomyocyte morphology, cardiac hypertrophy, impaired contractility and accumulation of autophagic vacuoles. These findings favored a critical role of autophagy in the maintenance of heart morphology and function, the reduction of which predisposes the heart to pathological changes. The presence of autophagic vacuoles in cardiomyocytes was also demonstrated by Kostin and coworkers in hearts from patients with idiopathic dilated cardiomyopathy.⁶³ In these patients, autophagic vacuoles were present in cardiomyocytes in association with depleted contractile elements and disintegrated nuclei distinct from apoptosis. Cells with autophagic vacuoles exhibited polyubiquitinated protein accumulation, suggesting a role of disrupted ubiquitin/proteasomal pathway with aging. Recent findings from Zheng and coworkers demonstrate that inhibition of the proteasome is sufficient to trigger autophagy in cardiomyocytes.⁶⁴ Vigliano and colleagues examined endomyocardial biopsy specimens from 100 patients with idiopathic dilated cardiomyopathy and advanced heart failure and found a positive correlation between autophagic vacuoles and idiopathic dilated cardiomyopathy.⁶⁵ It is likely that the autophagic process in the heart is upregulated under conditions of pathological stress. To better distinguish the role of autophagy in normal vs. stressed heart, Nakai and coworkers established a cardiac-specific knockout murine model of Atg5 gene essential for autophagosome formation.⁶⁶ In adult mice, conditional knockdown of Atg5 resulted in reduced autophagy in cardiomyocytes followed by cardiac hypertrophy, left ventricular dilation, contractile dysfunction and heart failure. Interestingly, embryonic deletion of Atg5 failed to exhibit any cardiac phenotype and the mice displayed normal survival. However, Atg5-knockout mice were much more susceptible to pressure overload-induced heart failure. It is noteworthy that both the embryonic Atg5-knockout mice and the control mice had similar extent of hypertrophy, indicating that autophagy is not critical for the development of hypertrophy.

The serine threonine kinase mTOR is an evolutionarily conserved protein kinase with a central role in cell growth (hypertrophy), cell survival and autophagy in response to nutrients such as amino acids, fatty acids and growth factors, such as insulin and IGF‑1, and energy.^67-69 mTOR exists as two complexes, as mTORC1 and mTORC2, each with distinct structures and activities. mTORC1 is a complex of mTOR with Raptor, mLST8, PRAS40 and is rapamycin-sensitive, whereas mTORC2 consists of Rictor, mSin1, mLST8 and is rapamycin-insensitive (Fig. 2). Of the two complexes, the rapamycin-sensitive mTORC1 has been shown to be essential for the regulation of translation and autophagy. Recent evidence has depicted that inhibition of the TOR pathway extends lifespan in yeast,⁷⁰ C. elegans⁷¹ and Drosophila.⁷² Harrison and coworkers found that inhibition of mTOR with rapamycin dramatically extends maximal lifespan in mice.⁷³ This study was performed in three different sites with genetically heterogeneous mice, and rapamycin was administered late in life (20 mo of age). In a follow-up study, the authors demonstrated that extension of median and maximal lifespan in mice can be achieved by providing rapamycin from 9 mo of age.⁷⁴ Along the same line, Chen and coworkers found elevated mTOR activity in hematopoietic stem cells from aged mice compared with young mice. Interestingly, activation of mTOR using conditional deletion of TSC1 in young mice mimicked the aging phenotypes (decrease in lymphopoiesis, impaired capacity to reconstitute hematopoietic cells) in young hematopoietic stem cells.⁷⁵ A series of elegant studies from Blagosklonny and coworkers has enriched our understanding of the role of mTOR in cellular senescence. In immortalized, cultured cells (e.g., human retinal pigment cells, human fibrosarcoma cells and rodent fibroblasts), inhibition of mTOR by rapamycin partly reconciled the loss of proliferative potential elicited by ectopic p21 or p16 expression or sodium butyrate-induced p21 expression.⁷⁶ Rapamycin successfully re-induced proliferation, only after removal of cell cycle inhibition, thus transforming “irreversible arrest into a reversible condition” without forcing cells to proliferate if the cell cycle was arrested.⁷⁶ These in vitro studies were extended to in vivo setting, wherein the effect of rapamycin on longevity in cancer-prone mice was tested. Interestingly, rapamycin prolonged lifespan and suppressed carcinogenesis in transgenic HER-2/neu cancer-prone mice, further substantiating the in vitro finding.⁷⁷ Furthermore, these authors showed that intermittent (2 weeks per month), life-long administration of rapamycin extends lifespan and inhibits age-related weight gain.⁷⁸ In a recent review, Menendez and coworkers⁷⁹ elegantly discussed the published evidence to support the hypothesis that mTOR inhibition and subsequently autophagy induction can decelerate cellular senescence by upregulating the reprogramming of somatic cells to induced pluripotent stem cells. These studies have convincingly favored a role of TORC pathway in aging and provided the rationale for mTOR inhibition in lifespan extension. However, as calorie restriction is “inconvenient” and may lead to malnutrition in the elderly population, Blagosklonny proposed the usefulness of mTOR inhibitors as “gerosuppressants” to curb senescence and senescence-related comorbidities.⁸⁰

Figure 2. Signaling pathways regulating the activity of mTORC1. Growth factor (such as insulin and IGF1) stimulation activates Akt which inactivates TSC2 via phosphorylation leading to the conversion of Rheb-GTP to Reb-GD preventing inhibition of mTORC1. On the other hand, low levels of ATP can result in the activation of AMPK which can inhibit mTORC1. Activated mTORC1 can induce translation of mRNA via the phosphorylation of 4E-BP1 and p70S6 kinase. mTORC1 also results in suppression of autophagy.

More recently, data from our group revealed that chronic cardiac-specific Akt activation accentuates aging-induced cardiac geometric and functional changes, possibly through deteriorated autophagy. Akt stimulates mTOR to inhibit autophagic activity. Results from our lab revealed cardiac hypertrophy, fibrosis, decreased myocardial contractility, prolonged diastole along with compromised intracellular Ca²⁺ release and clearance in aged mice compared with young mice, the effects of which were accentuated by chronic Akt activation. Aging enhanced Akt and mTOR phosphorylation while reducing that of PTEN, AMPK and ACC with a more pronounced response in Akt transgenic mice. Levels of beclin-1, Atg5 and LC3-II-to-LC3-I ratio were decreased in aged hearts, the effect of which, with the exception of Atg 5, was exacerbated by Akt over-activation. Levels of the autophagosome adaptor protein p62 were enhanced in aged mice, with a more pronounced rise in Akt mice. In addition, rapamycin reduced aging-induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction, while Akt activation suppressed autophagy in young but not aged cardiomyocytes. These data favored that notion that Akt may accentuate aging-induced cardiac geometric and contractile defects through a loss of autophagic regulation.⁸¹

What makes mTOR an attractive target is the fact that many essential genes involved in lifespan control, including components of insulin/IGF signaling, FOXO transcription factors, AMPK and sitruins all converge at mTOR.⁵⁰ Additionally, extension of lifespan associated with caloric restriction has been shown to be mediated through downregulation of mTOR, supporting the notion of mTORC as a bona fide pharmacological target for lifespan extension. Given the essential role of mTOR in the regulation of lifespan and aging, how does rapamycin or inhibition of mTORC1 extend lifespan? It appears that a number of mediators of TORC1 may affect lifespan. Activated TORC1 mediates protein synthesis by phosphorylating a variety of downstream substrates, including S6Kinase (S6K) and the eukaryotic initiation factor 4E binding protein 1 (4EBP1) eIF4E involved in transcription and translation processes (Fig. 2). It may therefore be argued that mTORC1 inhibition extends lifespan through inhibition of protein synthesis. Interestingly, deletion of S6K1, the downstream effector of mTOR, extends lifespan and resists aging-related pathologies.⁸² Conversely, rapamycin fails to extend lifespan in flies overexpressing S6 kinase.⁸³ Furthermore, inhibition of the translation initiation factor eIF4E may extend lifespan in C. elegans.⁸⁴ These pieces of evidence suggest that protein synthesis regulated by TORC1 signaling seems to play an important role in the aging process, while its inhibition mediates lifespan extension. An alternative explanation would be the autophagic pathway that is inhibited TORC1.^85,86 Recent studies have shown that mTORC1 phosphorylates the serine thyronine kinase unc-51-like kinase 1 (ULK1 or Atg1), an initiator of autophagy (Fig. 1).^87,88 Treatment with rapamycin upregulates Atg1 and promotes autophagy, even in the presence of a surplus of nutrients, suggesting a direct link between mTORC1 and autophagy induction. In addition to early stages of autophagy, mTORC also contributes to late stages of recycling lysosomes.⁸⁶

Caloric restriction without malnutrition has been shown to slow aging, extend lifespan and counter age-related diseases in a variety of species.^89-91 Autophagy helps in recycling intracellular constituents to provide amino acids during nutrient deprivation. Interestingly, recent studies by Wohlgemuth and coworkers failed to observe any alteration of autophagy in aging hearts, whereas age combined with calorie restriction resulted in elevated autophagy, indicating that caloric restriction may be responsible for its beneficial effects through autophagy induction in the heart.⁹² Shinmura and coworkers recently demonstrated that the beneficial effects of caloric restriction on cardiac dysfunction associated with aging may be a consequence of mTOR suppression and autophagy induction.⁹³ Not only can autophagy be an essential mediator for caloric restriction-elicited beneficial cardiac response, it also participates in the neuroendocrine regulation of the satiety hormone, leptin.⁹⁴ Pharmacological autophagy induction by rapamycin, spermidine and resveratrol may reduce serum leptin levels, whereas genetic inactivation of leptin promotes autophagy in peripheral tissues, including skeletal muscle, heart and liver. Paradoxically, administration of recombinant leptin triggered autophagy, the effect of which was mediated through AMPK activation and mTOR inhibition. These findings depict an important role of leptin in the neuroendocrine control of autophagy, possibly en route to leptin-induced regulation of food intake.⁹⁴

Autophagy is also critical for energy regulation and has been demonstrated to play an important role in lipolysis, a pathway termed as macrolipophagy.⁹⁵ Decreased autophagy during aging has been attributed to lipid accumulation in metabolic syndrome and aging.⁴⁴ Similar to the hepatic tissues, autophagic pathway also plays a critical role in the formation of lipid droplets in the heart.⁹⁶ Inhibition of autophagy dramatically increases the build-up of triglycerides and lipid droplets, indicating a potential role of autophagy in pathologies associated with lipid accumulation.⁹⁵ Therefore it is tempting to hypothesize that increased autophagy-mediated lipolysis may change bioenergetics and substrate availability in the heart, which may restore the use of fatty acids as the primary source of energy in the heart under stress conditions.

How do we fit the mTORC-senescence paradigm in the aging heart? Unfortunately, not many reports are currently available to address the role of mTORC in cardiac aging. Shinmura and coworkers examined the effect of long-term caloric restriction on cardiac senescence and found that caloric restriction inhibits age-associated decline in diastolic function in the heart.⁹³ Interestingly, caloric restriction was associated with the suppression of mTOR and elevation of autophagic response the heart, suggesting a pivotal role of mTOR suppression-mediated autophagy induction in caloric restriction-elicited retardation of cardiac senescence. Inuzuka and coworkers found that inhibition of PI-3K preserved cardiac function, enhanced autophagy and inhibited senescence markers in murine hearts.⁹⁷ Furthermore, inhibition of mTOR with rapamycin prevented lipofuscin accumulation in aged murine hearts. Shinde and coworkers recently demonstrated that cardiac-specific ablation of raptor resulted in overt dilated cardiomyopathy and accelerated heart failure in response to increased load following dynamic exercise or aortic constriction without exhibiting adaptive hypertrophy.⁹⁸ Similar to knockdown of raptor, ablation of mTORC1 in the heart resulted in cardiomyopathy and accelerated heart failure along with impaired hypertrophic response.⁶⁰ Adaptive hypertrophy of cardiomyocytes is commonly present with pressure overload due to hypertension or hemodynamic stress associated with aging. In a mouse model of pressure overload-induced cardiac hypertrophy, rapamycin either prevented or reversed cardiac hypertrophy.^99,100 These findings confirmed a role of mTORC in the regulation of cardiac geometry and function under pathological conditions, such as pressure overload and aging (Table 1).

Table 1. Upregulation or downregulation of autophagy in the heart under various pathophysiological conditions

	Autophagy
Ischemia	↑	Protective
Reperfusion	↑↑	Detrimental
Compensatory hypertrophy	↓	Adaptive mechanism
Decompensatory hypertrophy	↑↑	Detrimental (cell death)
Starvation	↑	Protective, survival

Conclusion

In summary, autophagy is a cellular protective process through recycling macromolecules in times of stress or through removal of damaged proteins and organelles, although excessive autophagy causes cell death. The mTOR pathway appears to represent a common pathway at which several well-known cell survival pathways converge, leading to autophagy and cell survival. Clinical trials have depicted that structural analogs of rapamycin, such as everolimus, prevent endomyocardial remodeling following heart transplantation.¹⁰¹ Future strategies may be engaged on mTOR-related autophagy regulation to retard or halt aging-associated cardiac anomalies.

Acknowledgment

This work was supported in part by a grant from NIH P20RR016474.