Caloric restriction mimetics for the treatment of cardiovascular diseases

Abstract

Caloric restriction mimetics (CRMs) are emerging as potential therapeutic agents for the treatment of cardiovascular diseases. CRMs include natural and synthetic compounds able to inhibit protein acetyltransferases, to interfere with acetyl coenzyme A biosynthesis, or to activate (de)acetyltransferase proteins. These modifications mimic the effects of caloric restriction, which is associated with the activation of autophagy. Previous evidence demonstrated the ability of CRMs to ameliorate cardiac function and reduce cardiac hypertrophy and maladaptive remodelling in animal models of ageing, mechanical overload, chronic myocardial ischaemia, and in genetic and metabolic cardiomyopathies. In addition, CRMs were found to reduce acute ischaemia–reperfusion injury. In many cases, these beneficial effects of CRMs appeared to be mediated by autophagy activation. In the present review, we discuss the relevant literature about the role of different CRMs in animal models of cardiac diseases, emphasizing the molecular mechanisms underlying the beneficial effects of these compounds and their potential future clinical application.

Caloric restriction mimetics, Cardiovascular diseases, Autophagy, Cardiac ageing, Starvation

1. Introduction

Cardiovascular diseases (CVDs) still represent a serious concern for public health, since they continue to be among the most common causes of death in western world, with a huge impact on societies and national health services. In fact, patients affected by overt CVDs face several morbidities that cause reduced quality of life and recurrent hospitalizations before they die.^1,2 The characterization of the molecular mechanisms regulating cardiac and vascular homoeostasis is opening new promising scenarios for the development of novel therapeutic approaches for the treatment of these illnesses.

Previous evidence demonstrated that caloric restriction (CR) exerts beneficial effects in several diseases, including cancer, neurodegeneration, and CVDs.^3,4 CR is defined as the reduction of calorie intake (30–40% of reduction) without malnutrition that would result from reduced intake of essential nutrients.^5,6 Several studies revealed that CR extends lifespan in different organisms, from invertebrates to vertebrates, including non-human primates.^3,4,7 CR delays cardiac ageing and cardiovascular complications related to ageing,^8,9 as it minimizes myocardial injury in response to stress.⁹ At a molecular level, CR reduces oxidative stress, inflammation, apoptosis, telomere shortening, and mitochondrial dysfunction, which represent common features involved in the pathogenesis of CVDs.^10,11 In non-obese individuals, CR also reduces metabolic syndrome and other risk factors associated with CVDs.^12,13

CR represents an effective autophagy inducer.¹⁴ Autophagy is an evolutionary conserved mechanism aimed at the removal of damaged intracellular cargoes, such as dysfunctional organelles and misfolded proteins.^15,16 Autophagy acts as a self-defense process against cellular stress, in particular, energy stress.^15,16 In addition, the physiological activation of autophagy during cardiac stress limits myocardial injury.^17–19 For these reasons, it has been hypothesized that the beneficial effects of CR on the cardiovascular system are prevalently mediated by autophagy activation, although additional mechanistic studies are needed to confirm this hypothesis.^8,17

Despite the promising beneficial effects of CR, its potential application in the clinical setting is severely affected by low patient compliance and potential adverse effects, such as infections. In addition, it is difficult to estimate the duration of a dietary regimen sufficient to obtain a clinically relevant effect.²⁰

In recent years, several compounds that mimic the biochemical and functional properties of CR, have been characterized. These compounds, named caloric restriction mimetics (CRMs), which include dietary supplements and pharmaceutical agents, do not require the patient to reduce food intake.^21–23 CRMs have been shown to exert beneficial effects on the cardiovascular system in the context of several pathological conditions, such as cardiac remodelling, ischaemia reperfusion (I/R) injury, cardiac ageing, and genetic or metabolic cardiomyopathies.^9,24–26 Some of these beneficial effects appeared to be mediated by autophagy activation.

In this review, we summarize the underlying mechanisms involved in the protective role of CRMs in cardiac ageing and in CVDs, as well as their potential clinical applications, with a particular focus on their effects on autophagy.

2. CR: cardiovascular effects and molecular features

CR delays cardiac ageing and cardiovascular complications related to ageing.^8,9 Long-term CR management in aged rats showed improved diastolic dysfunction and reduced cardiac fibrosis that correlated with reduced cardiac lipofuscin and β-galactosidase levels, which are accepted markers of senescence.^27,28 Long-term CR also exerted beneficial effects on cardiac senescence when administered to healthy mice at different ages. Short-term CR was able to rescue cardiac dysfunction in older mice suffering from cardiomyopathy.²⁹ Similarly, short-term CR reversed pre-existing cardiac hypertrophy and diastolic function in aged mice, by restoring the expression of proteins involved in proteome turnover and remodelling.³⁰ The effects of CR on cardiac function have been evaluated in humans, and long-term CR improved diastolic function and attenuated systemic inflammation, hypertension, and fibrosis in healthy individuals.³¹ Multiple studies showed that CR reduces myocardial injury in response to stress. Short-term CR reduced cardiac hypertrophy and dysfunction in response to pressure overload in mice.³² It also reduced infarct size and apoptosis in response to I/R injury.³³ In addition, long-term CR was shown to reduce cardiac remodelling and ameliorate cardiac function in a model of chronic myocardial infarction.³⁴

Alternative approaches to CR have also been introduced and tested in the cardiovascular system. Intermittent fasting (IF) is a diet regimen, which alternates fasting and re-feeding cycles. As shown for CR, IF has been reported to extend lifespan from yeast to human, protects the myocardium against ischaemia–reperfusion injury, and attenuates proteotoxic cardiomyopathy.^35–37 IF stimulates autophagy and improves protein quality control.³⁶ A so-called ‘fasting mimicking diet’ (FMD) represents another approach, consisting in periodic cycles of 3–5 days of low-calorie, low-protein, and high-fat intake. FMD has been demonstrated to improve longevity in mice and also to decrease blood pressure and risk factors associated with age in humans.^38,39

At a molecular level, the beneficial effects of CR depend on: (i) autophagy induction, due to its role in eliminating damaged cytoplasmic organelles and proteins⁴⁰; (ii) a decrease of the insulin/IGF1-like signalling pathway (IIS); and (iii) the modulation of intracellular key nutrient sensors, such as adenosine monophosphate-activated protein kinase (AMPK), histone (de)acetylase Sirtuin1 (SIRT1), and Protein Kinase B (PKB, also known as Akt), that are associated with the pro-healthy and pro-longevity effects of CR.^3,41,42

2.1. Insulin/IGF-1/GH pathway

It is well known that starvation causes large perturbations of metabolome in different organs in vivo^43,44 and reduces the bioavailability of circulating insulin growth factor-1 (IGF-1) levels,⁴⁴ thereby decreasing the pro-growth hormone (GH)/IGF1 axis. Generally, ketone bodies and the insulin-like growth factor-binding protein (IGFBP)-1 increase in the circulation, whereas IGF-1 decreases. Thus, a 72 h of fasting reduced circulating IGF-1 by 70% and increased the level of the IGF-1 inhibitor IGFBP-1 by 11-fold in mice.⁴⁵ Inhibition of insulin/IIS activates the Forkhead (Fox)O1 transcription factor and inhibits the mechanistic (previously called “mammalian") target of rapamycin (mTOR) complex 1 (mTORC1) pathway.^42,46,47 IIS is the most conserved ageing-controlling pathway⁴¹ and its effects on lifespan are evolutionary conserved, from yeast to human.⁴⁸ Mice deficient in growth hormone and IGF1 pathways show enhanced longevity, reduced age-dependent insulin resistance, and reduced cancer formation, as compared to their wild type (WT) counterparts.^49,50 Interestingly, human subjects with mutations in the GH receptor gene (GHR) exhibited prominent resistance to neoplasia and diabetes.⁵¹ Nonetheless, the role of the insulin pathway in promoting lifespan, as well as its role in CVD prevention and protection, are still under debate. Further details about this topic can be found in previous published articles.^52–54

2.2. Energy sensing pathways

The intracellular concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), which together determine the energy charge, influence the activity of AMPK.^14,55 When ATP production is reduced, as in CR conditions, AMP levels increase, leading to AMPK activation and autophagy induction.⁵⁶

The AMPK activity is linked to both the SIRT1 and mTOR pathways. In particular, AMPK can modulate SIRT1 activity, by increasing the levels of nicotinamide dinucleotide (NAD⁺), which represents a key nutrient sensor. This phenomenon triggers the activation of different downstream targets and can lead to the induction of mitochondrial activity, respiratory metabolism, and oxidative stress responses.⁵⁷ Of note, AMPK-independent changes in NAD⁺/NADH levels also modulate the activation of SIRT1. In particular, during CR, Krebs cycle and glycolysis are downregulated, and these events lead to an increase in NAD⁺ and a decrease in NADH levels.⁵⁸ SIRT1 is involved in the CR-mediated lifespan extension and autophagy induction.⁵⁹ Moreover, in mammals, SIRT1 exerts a role in controlling the insulin pathway, fat storage and glucose metabolism, and in regulating the pro-inflammatory regulator nuclear factor-κB (NF-κB), which is involved in obesity and metabolic syndrome.⁶⁰

Another energy sensing pathway modulated by CR is mTOR. When growth factors like GH, insulin or IGF-1 are reduced, or amino acids depleted, as this occurs upon starvation, mTORC1 is inhibited, leading to autophagy induction and cell survival⁶¹ (further details will be addressed in the next section).

CR also dampens mTORC1 activity, thereby reducing ageing in several animal models.^46,62 In support of these observations, abnormal activation of mTORC1 pathway is linked to some age-related disorders, such as obesity, cancer, and CVDs, underlining its role in metabolism and ageing.⁴²

3. Overview on autophagy

Three different forms of autophagy have been characterized. The term macroautophagy (hereafter autophagy) refers to the formation of double-membrane vesicles, called autophagosomes, which engulf cytoplasmic elements that are then delivered to lysosomes.^63–65 In chaperone-mediated autophagy (CMA), proteins containing a specific KFERQ amino acidic sequence are digested in lysosomes, after their binding to the chaperone Hsc70 and their import through a protein complex involving lysosome-associated membrane protein 2A (LAMP2A). In microautophagy, cytoplasmic elements are directly sequestered by lysosomes.^63–65

The autophagic process requires the involvement of several proteins. Autophagy-related proteins (ATGs) regulate all the phases of autophagy: autophagosome formation, maturation, and fusion with lysosomes. Among ATG proteins, Beclin 1 (the mammalian ortholog of the yeast ATG6) is a critical protein acting during the initial phase of autophagy. In its inactive form, Beclin 1 interacts with B-cell lymphoma 2 (BCL2). Once activated by phosphorylation, Beclin 1 binds to vacuolar protein sorting VPS34 and VPS15, resulting in the formation of a complex needed for autophagy initiation.

Non-selective and cargo-specific forms of autophagy have been described. Among cargo-specific forms, mitophagy plays a major role in cells. Mitophagy refers to the selective form of autophagy devoted to the removal of senescent or damaged mitochondria.^66,67 Mitophagy can be sub-classified into Parkin-mediated or Parkin-independent mitophagy. Briefly, in the Parkin-mediated mitophagy, the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) recruits Parkin to the outer mitochondrial membrane, particularly during stress, when mitochondria are damaged. Once activated, Parkin ubiquitinates several proteins, thus facilitating the interaction of ubiquitinated proteins with mitophagy receptors/adaptors, which in turn contribute to autophagosome formation. The Parkin-independent forms of mitophagy are mediated by Bcl2-like protein 13 (BCL2L13), Bcl2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), or FUN14 domain-containing protein 1 (FUNDC1).^66,67

Different mechanisms regulate autophagy in the cardiovascular system.¹⁹ mTORC1 inhibits catabolic mechanisms, such as autophagy. mTORC1-dependent inhibition of autophagy occurs mainly through phosphorylation at serine 757 of unc-51–like autophagy-activating kinase 1 (ULK1), a protein required for the initiation of autophagy.¹⁹ mTOR also inhibits the nuclear translocation of transcription factor EB (TFEB), a positive regulator of autophagy, and lysosomal biogenesis.⁶⁸ Finally, AMPK triggers autophagy directly by activating ULK1 or indirectly by inhibiting mTORC1. The latter occurs through the direct phosphorylation of regulatory-associated protein of mTOR (RAPTOR), a subunit of mTORC1, or by the activation of tuberous sclerosis proteins 1/2 (TSC1/2), which are inhibitors of mTOR signalling.¹⁹

4. Molecular mechanisms underlying the beneficial effects of CRMs

Compounds able to mimic the positive effects of nutrient starvation should be considered as bona fide CRMs (Figure 1). It has been reported that CR is metabolically characterized by (i) autophagy induction, (ii) rapid decrease in the cytosolic acetyl coenzyme A (AcCoA) pool, and (iii) inhibition of acetyltransferase EP300 (E1A binding protein p300).^21,22,69 Based on their mode of action, CRMs can be divided into three different groups: (i) direct inhibitors of protein acetyltransferases, including inhibitors of EP300; (ii) inhibitors of AcCoA biosynthesis that indirectly affect the activity of acetyltransferases; (iii) activators of protein (de)acetylases, such as SIRT1.²² The net result of these three effects is a reduction in the acetylation of cellular proteins, which may occur both in the cytosol (with activation of AMPK and inhibition of mTORC1, the major nutrient sensors) and in the nucleus (with the consequent transcriptional and epigenetic reprogramming), thereby leading to the activation of autophagy³¹ (Table 1).

Figure 1

Schematic representation of the intracellular effects of CRMs. CRMs may be classified in (i) direct inhibitors of protein acetyltransferases, (ii) inhibitors of AcCoA biosynthesis, and (iii) activators of protein (de)acetylases. The net result of these actions leads to nutrient depletion, which triggers autophagy and AMPK and switch off mTORC1 signalling. Arrow-headed lines indicate activation, whereas bar-headed lines indicate inhibition.

Table 1

Effects of CRMs in the promotion of autophagy and extension of lifespan

CRM	Type	Class	Promotion of autophagy	Extension of lifespan	Reference
Curcumin	Natural	1	Yes	NA	70
Epigallocatechin-3-gallate	Natural	1/3	NA	Yes	71,72
4,4’-dimethoxychalcone	Natural	1	Yes	Yes	73
Spermidine	Natural	1	Yes	Yes	74,75
Anacardic acid	Natural	1	Yes	NA	76
C646	Synthetic	1	Yes	NA	77,78
Salicylate	Natural	1	Yes	NA	79
Metformin	Synthetic	1	Yes	NA	80
Hydroxycitrate	Natural	2	Yes	NA	44
Radicicol	Natural	2	NA	NA	81
SB-204990	Synthetic	2	NA	NA	82
Perhexiline maleate	Synthetic	2	NA	NA	83,84
UK5099	Synthetic	2	NA	NA	22
1,2,3-benzene-tricarboxylate	Synthetic	2	NA	NA	22
Resveratrol	Natural	3	Yes	Yes	59,85
Piceatannol	Natural	3	Yes	NA	11
Quercitin	Natural	3	Yes	Yes	86,87
Myricetin	Natural	3	Yes	NA	86
Berberine	Natural	3	Yes	Yes	88
Nicotinamide riboside	Natural	3	NA	NA	89,90
Nicotinamide mononucleotide	Natural	3	NA	NA	91
Vitamin B3	Natural	3	NA	NA	92
SRT1720	Synthetic	3	NA	Yes	93
SRT2104	Synthetic	3	NA	Yes	94

1, inhibitors of protein acetyltransferases; 2, inhibitors of acetyl coenzime A (AcCoA) biosynthesis; 3, activators of protein (de)acetylases; NA, not available.

Notably, based on their major molecular effects, CRMs show significant differences with respect to rapamycin derivatives, the so-called ‘rapalogs’. Rapamycin (sirolimus), produced by Streptomyces hygroscopicus, induces autophagy via the inhibition of mTORC1. Actually, it was first used as an immunosuppressant in the context of organ transplantation.⁹⁵ It was previously shown that rapamycin increases lifespan in Caenorhabditis elegans, Drosophila megalonaster, and mice.⁹⁵ Despite their pro-healthy effects, the ‘rapalogs’ present several side effects, including immunosuppression, limiting their use in clinics.¹⁴ On the contrary, the lack of relevant side effects represents one of the key hallmarks of novel identified CRMs.

4.1 Direct inhibitors of protein acetyltransferases

Several natural compounds were revealed to have an inhibitory effect on different classes of acetyltransferases and to trigger cytoprotective autophagy.¹¹ Such agents may include curcumin (from the South Asian spice Curcuma longa), one of the main ingredient of curry powder,⁹⁶ and epigallocatechin-3-gallate (EGCG), one of the major active compounds in green tea.²² EGCG, mainly through inhibition of several histone acetyltransferases (HATs) and activation of the deacetylase SIRT1,⁹⁷ can improve lifespan of C. elegans⁷¹ and prolong the lifespan of Wistar rats.⁷² ECGC supplementation was also found to reduce body weight in obese rodents, whereas the same effect was not observed in obese human subjects.^98,99 Along the same line of evidence, another polyphenol, the 4,4’-dimethoxychalcone (present in the plant Angelica keiskei, which is used in Japanese folk medicine) was recently described as a new possible CRM for its ability to extend the lifespan of yeast and flies.⁷³ However, it should be noted that these polyphenols were previously described for their antioxidant properties, and more studies are warranted to determine their exact mode of action.¹⁰⁰

Another well-established acetyltransferase inhibitor is spermidine, a natural polyamine present in wheat germs, soybeans, nuts, and mature cheese, that is able to compete with AcCoA for the binding to the catalytic site of EP300, inhibiting its acetyltransferase activity.⁷⁷ Moreover, supplementation of spermidine can extend lifespan from yeast to mice.⁷⁴ This effect is intimately dependent on autophagy and mitophagy that are increased following spermidine administration.⁷⁵ Moreover, epidemiological studies correlated high dietary polyamine uptake with diminished CVDs and cancer-related mortality.⁷⁵ A natural polyphenol, anacardic acid (AA) (also known as 6-pentadecyl-salicylic acid), from the nutshell of the cashew Anacardium occidentale, was described as competitor with AcCoA, for the binding to the active site of EP300, and as an autophagy inducer.^76,77 AA was shown to have anti-inflammatory, anti-oxidative, and antitumor effects.^101,102

Starting from these natural compounds, new molecules have been synthetized as more selective inhibitors of acetyltransferase activity. One example is represented by the chemical compound C646, a specific inhibitor of EP300,⁷⁸ that is able to trigger autophagy in heart, liver, and muscles of mice.

Two molecules already used in clinics, namely aspirin and metformin, were also recently described as potential CRMs.¹¹ Salicylate, the active metabolite of aspirin, found in willow bark and used in folk medicines during years, inhibits EP300 by competing with AcCoA and triggers autophagy.⁷⁹ Metformin, the synthetic molecule derivative of natural guanidines presents in Galena officinalis, was also proposed as a new potential CRM. Metformin is a well-established anti-diabetic drug,¹⁰³ but it is now under investigation for novel applications. Metformin induces autophagy through the direct activation of AMPK. However, metformin also reduces intracellular acetyl coenzyme A levels, presumably by modulating acetyltransferase activity.^104,105 In human patients, metformin seems to be beneficial in cognitive and cardiovascular disorders.¹⁰⁶ Of note, it will be important to test whether the beneficial cardiovascular effects of aspirin and metformin are attenuated in autophagy-deficient mice to understand whether autophagy directly contributes to the beneficial effects of these drugs. In fact, both aspirin and metformin exert several autophagy-independent effects. For example, aspirin reduces platelet aggregation and inhibits the production of pro-inflammatory prostaglandins.^107,108 Similarly, metformin reduces glucose levels, improves insulin sensitivity and lipid metabolism, and influences the composition of the intestinal microbiota.¹⁰⁶

4.2 Inhibitors of AcCoA biosynthesis

The first compound that falls in this class is the natural molecule hydroxycitrate (HC). HC is present in several tropical plants, such as Garcinia cambogia and Hibiscus subdariffa, and it is an inhibitor of ATP citrate lyase (ACLY), the cytosolic enzyme that converts citrate into oxaloacetate and AcCoA, modulating the AcCoA cytosolic pool.^22,58 Additionally, HC triggers autophagy in vitro and in vivo in several mouse organs, such as heart, liver, and muscle.⁴⁴ HC is currently used as an over-the-counter weight loss agent in the USA.²² Interestingly, in mouse models, its ability to decrease weight gain is lost in autophagy-deficient mice, as reported in Atg4b^−/− animals,¹⁰⁹ indicating that autophagy is required for body weight reduction induced by HC. Furthermore, HC improves the efficacy of antitumour chemotherapy, increasing the depletion of regulatory T cells from the tumour bed.⁴⁴ Another natural ACLY inhibitor is radicicol, a molecule first extracted from the fungus Diheterospora chlamydosporia, which shows protective effects against myocardial ischaemia–reperfusion damage in rodent models.¹¹⁰ However, its pro-autophagic effects remain to be elucidated.

The synthetic molecule SB-204990 is a high-affinity ACLY inhibitor. It was described as a molecule able to reduce tumour growth in mouse models⁸² and to decrease cholesterol and triglycerides,¹¹¹ exerting putative protective effects against metabolic syndrome. Further synthetic agents can reduce the cytosolic AcCoA pool, thus enhancing autophagy, through different mechanisms of action and hence can be classified as potential CRMs. Perhexiline maleate, a carnitine palmitoyltransferase 1 (CPT1) inhibitor, reduces beta-oxidation by interfering with the transport of fatty acids into mitochondria,^22,83 and promotes autophagy by inhibiting mTORC1.¹¹² Other examples are represented by the synthetic molecules UK5099 and 1,2,3-benzene-tricarboxylate that are, respectively, inhibitors of MPC (mitochondrial pyruvate carrier) and CPT (citrate transport), thereby reducing the cytosolic AcCoA pool.³¹ However, their in vivo effects remain to be elucidated.

4.3 Activators of protein (de)acetylases

The third class of CRMs includes molecules that activate protein deacetylases, which are enzymes that remove acetyl groups from lysine residues in proteins. Sirtuins (as the enzyme SIRT1) are a class of NAD⁺-dependent deacetylase, activated by NAD⁺ precursor agents and CR. Indeed, nutrient starvation is also characterized by a decrease in the concentrations of nicotinamide adenine dinucleotide (NADH); parallel to the decrease of NADH, CR leads to an increase in NAD⁺ levels. Changes in NAD⁺/NADH ratio then activate SIRT1, which represents a bona fide key nutrient-sensing core.¹¹³ The activation of deacetylases contributes to the positive effects and pro-autophagic activity of CR-based strategies, with the final results of increased protein deacetylation and autophagy induction.¹¹³

The natural compound resveratrol (RSV), a polyphenolic phytoalexin present in the skin of grapes and in red wine, is also a potent autophagy inducer in worms, mice, and mammalian cells via the activation of SIRT1^59,85 and AMPK.¹¹⁴ However, recent work showed that RSV is not a direct activator of SIRT1, particularly in vivo.¹¹⁵ This suggests that the beneficial effects of RSV may also be SIRT1-independent. Other direct or indirect targets were also demonstrated to be important mediators of the health-promoting effects of RSV in CVDs. These factors mediate anti-inflammatory and anti-oxidant responses.¹¹⁶ For example, RSV decreases the pro-inflammatory NF-κB activity, whereas it increases nuclear factor (erythroid-derived 2)-like 2 (NRF2), which activates anti-oxidant pathways.^117,118 The reduction of platelet aggregation and LDL oxidation are also important SIRT1-independent effects of RSV.¹¹⁹ Moreover, RSV was shown to prolong longevity in different animal models and exert protective effects in models of metabolic syndrome, cancer, neurodegeneration, and CVDs.¹¹⁴ Of note, anti-ageing and lifespan extending effects of RSV appear to be autophagy-dependent, since depletion of essential ATG proteins attenuated its effects.^59,85

The stilbenoid piceatannol (from Picea abies and red wine), an analogue of RSV, the flavonoid quercetin (a natural molecule from different plants, red wine, and green tea), and myricetin (from black tea, garlic, curcuma, and fruits) also activate SIRT1, decrease protein acetylation, and stimulate autophagy flux.¹¹ Supplementation of quercetin extends life span in worms⁸⁶ and Drosophila.⁸⁷ However, these effects are not observed in mice.¹²⁰ Quercetin also reduces HFD-induced gain of body weight in mice,¹²¹ suggesting that the beneficial effects of this compound may be favoured by weight loss and metabolic improvement. Finally, the alkaloid berberine activates autophagic flux via SIRT1,¹²² extending life span in Drosophila⁸⁸ and protecting mice from neurodegeneration.¹²³ As mentioned above, the NAD⁺ precursor agents represent another class of deacetylase activators. NAD⁺ levels decrease with age in rodents and humans.¹²⁴ For this reason, molecules able to restore NAD⁺ bioavailability have been proposed as a new strategy to protect from CVDs and age-associated disorders. Nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), present in different vegetables, fruits, and meats, can ameliorate vascular ageing,¹²⁴ improve diabetic pathology and reduce stroke occurrence^89,125 in rodents. Intriguingly, nicotinamide (NAM), better known as vitamin B3, is able to restore NAD⁺ levels, promoting health span, but not lifespan, in aged rodents, as well as ameliorating signs of high fat diet (HFD)-associated pathology.⁹² NR and NAM displayed no adverse effects in pre-clinical models.¹²⁶ Intriguingly, NAM has undergone phase III trial testing, revealing its ability to reduce the incidence of non-melanoma skin cancer.¹²⁷

Beside these natural compounds, new synthetic deacetylase activators have been developed. SRT1720, and its related compound SRT2104, two small-molecules that activate SIRT1, extend health and lifespan in mice, ameliorating the signs of metabolic syndrome, decreasing inflammation, and protecting from neurodegeneration.⁹⁴ Of note, SRT2104 underwent Phase I and II clinical trials, showing only minor side effects,⁹⁴ with promising results.

5. The role of CRMs in CVDs

Accumulating lines of evidence indicate that CR may be able to reduce cardiac injury in response to stress. Previous experimental studies showed that CR reduces stress-induced cardiac hypertrophy and dysfunction and limits I/R injury.^128–130 New data suggest that CRMs could represent a valid alternative to CR for the prevention and treatment of CVDs (Table 2). Since autophagy is a fundamental mechanism for maintaining the homoeostasis of the cardiovascular system, autophagy activation may contribute to the beneficial effects of CRMs on heart and blood vessels (Figures 2 and 3).

Figure 2

Effects of CRMs on cardiac ageing and cardiac hypertrophy. List of CRMs exerting their protective effects through autophagy activation. The beneficial effects for some CRMs have not been demonstrated to be directly associated with autophagy modulation. Arrow-headed lines indicate activation, whereas bar-headed lines indicate inhibition. AA, anacardic acid; AMPK, 5' adenosine monophosphate-activated protein kinase; ASP, aspirin; C646, synthetic p300 inhibitor; CAEA, caffeic acid ethanolamide; CUR, curcumin; EGCG, epigallocatechin-3-gallate; FOXO-1, forkhead box O-1; GA, gallic acid; GATA4,6, Transcription Factor GATA-4 and 6; MET, metformin; mTOR, mechanistic target of rapamycin; NAD+, nicotinamide dinucleotide; NMN, nicotinamide mononucleotide; PIC, piceatannol; PTEN, phosphatase and tensin homolog; RAP, rapamycin; RSV, resveratrol; SIRT1,2, sirtuin 1 and 2; SP, spermidine; SRT120, synthetic deacetylase activators; TGF-β, transforming growth factor; TRE, trehalose.

Figure 3

Effects of CRMs in ischaemia/reperfusion (I/R) injury and in genetic and metabolic cardiomyopathy. List of CRMs exerting beneficial effects during I/R and genetic and metabolic cardiomyopathy. Arrow-headed lines indicate activation, whereas bar-headed lines indicate inhibition. AMPK, 5' adenosine monophosphate-activated protein kinase; BER, berberine; CUR, curcumin; CAF, caffeine; DMC, dimethoxychalcone; EGCG, epigallocatechin-3-gallate; FOXO-1, 3a, Forkhead box O-1 and 3a; GATA, Transcription Factor GATA; HC, hydroxycitrate; MET, metformin; mTOR, mechanistic target of rapamycin; mTORC1,2, mechanistic target of rapamycin complex 1 and 2; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; p38 MAPK, mitogen activated protein kinase; PER, Perhexiline maleate PIC, piceatannol; QU, quercitin; RAP, rapamycin; RAD, radicicol; RSV, resveratrol; SIRT1,3, sirtuin 1 and 3; SRT120, synthetic deacetylase activators; STAT-1, signal transducer and activator of transcription-1; TRE, trehalose.

Table 2

Relevant mechanistic studies underlying the protective effects of CRMs in cardiovascular diseases through autophagy activation

CRM	Model	Cardiac condition	Cardiac outcomes	Cardiac protection abolished by autophagy inhibition	Reference
Spermidine	23-month-old mice	Cardiac ageing	↑ Diastolic function ↓ Cardiac hypertrophy	Cardiac-specific ATG5 knockout mice	75
SRT120	Cardiomyocytes isolated from aged mice	Cardiac ageing	↓ Contractile dysfunction	Parkin deletion	131
Resveratrol	Mice undergoing left coronary artery ligation Rats undergoing ischaemia–reperfusion	Myocardial infarction Ischaemia/reperfusion injury	↓ Left ventricular dilatation ↓ Adverse remodelling ↑ Cardiac function ↓ Cardiomyocytes apoptosis	Chloroquine Wortmannin	24 132
Trehalose	Mice subjected to permanent ligation of the left anterior descending artery	Chronic ischaemic remodelling	↓ Left ventricular dilatation ↓ Adverse remodelling ↓ Cardiac fibrosis ↓ Cardiac apoptosis	Beclin 1 heterozigous knockout mice	133
4,4-dimethoxychalcone	Mice subject to ligation of the left anterior descending artery	Myocardial ischaemia	↓ Infarct size	Cardiac-specific ATG7 knockout mice	73
Rapamycin	Mice fed with high fat diet subjected to ischaemia	Metabolic syndrome	↓ Infarct size	Beclin 1 knockout mice	134

5.1 Ageing

As it progressively deteriorates, the ageing heart undergoes histological and morphological modifications and becomes more susceptible to stress. Left ventricular hypertrophy, fibrosis, collagen, and lipofuscin accumulation are considered typical features of the aged heart.^135–138 In addition, diastolic dysfunction and left atrium dilatation occur during ageing.^17,135 Increased inflammation and unbalanced reactive oxygen species (ROS) homoeostasis lead to damage of proteins, lipids, and mitochondrial DNA and can eventually cause mitochondrial dysfunction and cardiomyocyte abnormalities.^8,9,17 In addition, misfolded proteins and dysfunctional mitochondria accumulate in the aged heart.¹³⁹ These derangements may be the consequence of reduced cardiac autophagy, a fundamental mechanism limiting age-associated abnormalities. Autophagy reactivation attenuates age-induced cardiac abnormalities and extends lifespan.¹⁴⁰ Several preclinical studies revealed that CRMs decrease ageing-related cardiac dysfunction.^8,9 Eisenberg et al.⁷⁵ provided compelling evidence demonstrating that spermidine administration in vivo to mice could extend their lifespan and reduce ageing-associated cardiac abnormalities. The authors found that spermidine treatment promotes cardiac autophagy and mitophagy, improves mitochondrial function and reduces inflammation. Of note, the protective effects of spermidine were abolished in cardiac-specific ATG5 knockout mice, in which autophagy is completely disrupted in cardiomyocytes. These data indicate that spermidine treatment attenuates the ageing process through the activation of autophagy. On the other hand, spermidine did not affect body weight in this study. Epidemiological analyses conducted on large human cohorts also demonstrated that a higher amount of dietary intake of spermidine is associated with diminished cardiovascular death and increased longevity.^75,141,142 Similarly, polyphenols, such as EGCG and RSV, were reported to retard cardiac ageing. RSV administration to old mice was shown to mimic the effect of CR, rejuvenating the general cardiac profile of gene expression and improving cardiac function.¹⁴³ On the other hand, RSV did not affect body weight in this study.¹⁴³ Similarly, EGCG attenuated age-induced cardiac hypertrophy, apoptosis, and oxidative stress in rats.¹⁴⁴ However, it remains unclear whether these beneficial effects are mediated by autophagy activation.

Rapamycin was also shown to retard cardiac ageing. A prolonged treatment with this drug for 3 months reduced cardiac dysfunction and increased the expression of anti-hypertrophic and anti-inflammatory genes in old mice.¹⁴⁵ Similarly, rapamycin was observed to reduce cardiac oxidative stress and ubiquitination in elderly mice.³⁰ Rapamycin restored mitochondrial biogenesis and cardiac energy metabolism in old mouse hearts, concomitantly with a transient activation of autophagy.¹⁴⁶ Previous work demonstrated that restoration of NAD⁺ levels during ageing may also be an alternative reliable strategy to reduce vascular and cardiac complications.¹¹⁴ In fact, NMN was able to improve capillary density and increase blood flow in aged organs.¹²⁴ Moreover, NMN ameliorated glucose intolerance and lipid profile of aged mice with type II diabetes.⁸⁹ Similarly, the synthetic SIRT1 inducer SRT120 was reported to rescue contractile dysfunction in cardiomyocytes isolated from aged mice in vitro, likely through deacetylation of FoxO1, an autophagy-related gene. Of note, the anti-ageing effects of SRT120 were abrogated when autophagy was inhibited by insulin, via Akt or by deletion of Parkin.¹³¹ Additional studies are warranted to understand the effects of other CRMs during cardiac ageing.

5.2 Hypertrophy and remodelling

Cardiac hypertrophy results from chronic pressure and volume overload or neuro-hormonal stimuli. It consists in the abnormal augmentation of heart muscle mass due to the increase in cardiomyocyte size and/or extracellular matrix.^147,148 In physiological conditions, such as physical exercise and pregnancy, cardiac hypertrophy acts as a compensatory mechanism. During chronic stress, such as hypertension or valvular diseases, cardiac hypertrophy may become maladaptive, culminating in cardiac dysfunction and heart failure.^147,148 The term cardiac remodelling usually refers to all the events that contribute to change heart morphology after a cardiac injury.¹⁴⁹ Accumulating evidence demonstrates that autophagy plays a pivotal role in the response to hypertrophic signals. Autophagy and mitophagy activation exert protective effects against adverse cardiac remodelling during myocardial infarction and pressure overload.¹⁹

Pre-clinical studies revealed that CRMs also play beneficial effects against maladaptive cardiac hypertrophy and remodelling. In particular, the effects of different natural compounds have been investigated in animal models of cardiac hypertrophy. For instance, curcumin, a HAT inhibitor, was demonstrated to prevent cardiac hypertrophy in both hypertensive rats^150,151 and in rats undergoing surgically induced myocardial infarction.¹⁵² The anti-hypertrophic action of curcumin was associated with reduced acetylation of GATA4, a pro-hypertrophic transcription factor known to be a substrate of P300.^150,151 Consistently, curcumin was shown to decrease apoptosis and cardiac hypertrophy through the inhibition of the P300-induced acetylation of p53 either in vivo and in vitro.¹⁵³ Similarly, AA was reported to reduce cardiac hypertrophy in mice with pressure overload, thereby inhibiting P300.¹⁵⁴ ECGC also reduced cardiac hypertrophy in animal models with angiotensin-II- and pressure overload-induced cardiac injury,¹⁵⁵ preventing telomere shortening and loss of telomere repeat-binding factor 2 (TRF2).¹⁵⁶ Furthermore, caffeic acid ethanolamide (CAEA), a synthetic derivate of caffeic acid, reduced maladaptive cardiac remodelling, and oxidative stress in mice subjected to isoproterenol injection.¹⁵⁷ Piceatannol, an RSV derivative, was able to reduce cardiac hypertrophy induced by β-adrenergic stimuli in vivo and in vitro, in association with the reduction of GATA6-mediated hypertrophic signals.¹⁵⁸ However, these studies did not investigate the direct contribution of autophagy to the anti-hypertrophic effects of the indicated CRMs.

RSV was also shown to exert protective effects against maladaptive cardiac remodelling and hypertrophy. In a murine model of myocardial infarction, RSV attenuated left ventricular dilatation and improved cardiac function. In the same study, RSV-treated mice displayed increased AMPK signalling and activation of autophagy, together with mTOR inhibition. Consistently, cardiac protection was abolished in mice that were co-treated with chloroquine, an inhibitor of autophagic flux.²⁴ This suggests that autophagy may act as an importantly mediator of the health promoting effects of RSV during myocardial infarction. Similarly, RSV was shown to inhibit proteasome digestion of phosphatase and TENsin homologue (PTEN), resulting in mTOR inhibition and AMPK activation in vitro, and in mice undergoing transverse aortic constriction (TAC).¹⁵⁹ The enhancement of AMPK activity was also demonstrated in another study in a model of TAC-induced pressure overload and reduced ejection fraction treated with RSV.¹⁶⁰ It was also found that gallic acid (GA), a natural CRM, is able to attenuate cardiac hypertrophy in hypertensive rats and to reduce cardiac fibrosis in pressure overload mice.^161,162 GA stimulated cardiac autophagy in vivo and in vitro. The anti-hypertrophic effects of GA were abrogated by autophagy inhibition in vitro.¹⁶³ Further studies should evaluate the in vivo effects of GA in conditions of autophagy inhibition.

We recently demonstrated in a mouse model of chronic myocardial infarction that the natural disaccharide trehalose improves cardiac remodelling and function and reduces apoptosis and fibrosis. Mechanistically, we showed that autophagy is the main mediator of the protective effects of trehalose. Heterozygous Beclin 1 knockout mice subjected to myocardial infarction did not show any beneficial effects when treated with trehalose as compared to wild-type control mice.¹³³ In addition, Taneike et al.¹⁶⁴ reported that trehalose is able to rescue cardiac dysfunction in cardiac-specific tuberous sclerosis complex 2 (TSC2) knockout mice, a model that is characterized by increased mTOR activity and cardiac hypertrophy and decreased autophagy. In this study, trehalose administration was associated with increased autophagic flux, together with reduced accumulation of dysfunctional mitochondria.

Previous work also showed that spermidine reduces cardiac hypertrophy and inflammation and improves diastolic function in a model of salt-induced cardiac hypertrophy.⁷⁵ NMN was also shown to rescue NAD⁺ levels and suppress cardiac hypertrophy in mice undergoing TAC by decreasing the acetylation of cardiac mitochondrial proteins and increasing long chain fatty acid oxidation.^165,166 However, the specific role of autophagy in this condition has not been evaluated. Moreover, it was reported that metformin attenuates cardiac hypertrophy in Ang-II treated mice. These effects were abolished in SIRT2 knockout mice, indicating that SIRT2 mediated the antihypertrophic effects of metformin via LKB1-AMPK signalling.¹⁶⁷ On the other hand, rapamycin reduced cardiac hypertrophy via ERK/MAPK signalling and activation of cardiac autophagy.¹⁶⁸

Synthetic CRMs were also tested in pre-clinical models of cardiac hypertrophy. For example, the synthetic EP300 inhibitor C646 was reported to attenuate adverse cardiac remodelling in Ang-II hypertensive mice.¹⁶⁹ Similarly, the Sirt1 activator SRT1720 improved cardiac function and remodelling by inhibition of transforming growth factor (TGF)-β signalling.¹⁷⁰ Salicylate, the active metabolite of aspirin and a novel CRM, promoted mitophagy in cardiomyocytes, resulting in the selective elimination of aged mitochondria and improved cardiac function.⁷⁹ Aspirin reduced cardiac fibrosis and improved cardiac function in isoproterenol-induced and surgically induced animal models of heart injury.¹⁷¹ However, the anti-fibrotic effects of aspirin appeared to be mediated by inhibition of maladaptive autophagy in cardiac fibroblasts in vitro.¹⁷¹ For these reasons, the role of aspirin in the regulation of autophagy requires further investigations, especially in autophagy-deficient models.

5.3 Ischaemia/reperfusion

Cardiac ischaemia/reperfusion (I/R) injury occurs when coronary circulation is restored after a phase of ischaemia. A burst of inflammation and oxidative stress during reperfusion represents the main event contributing to heart damage.^172,173 The role of autophagy during I/R is still under debate, and likely is strictly related to the duration of ischaemia and reperfusion phases. Generally, autophagy is protective during ischaemia, limiting cardiomyocyte death. Similarly, mitophagy is beneficial in response to I/R. On the other hand, a massive and prolonged activation of autophagy in response to severe I/R may be detrimental through the promotion of a type of cell death that is called autosis.^19,174

Several studies investigated the role of different CRMs in the attenuation of cardiac I/R injury. The flavonoid 4,4’-dimethoxychalcone (DMC) was shown to reduce infarct size in a murine model of ischaemia in an autophagy-dependent manner, since the protective role of DMC was lost in ATG7-deficient mice. The effects of DMC were independent of mTOR signalling, whereas they involved specific GATA transcription factors.⁷³

Similarly, in isolated perfused hearts, curcumin improved post-ischaemic cardiac function, and attenuated mitochondrial oxidative damage and inflammation in a SIRT1-dependent manner.^175,176 Curcumin decreased infarct size in rats in response to I/R in vivo.¹⁷⁷ A combination of curcumin with D942, a pharmacological activator of AMPK, was reported to enhance cell survival in cardiomyocytes subjected to oxygen deprivation/reperfusion in vitro, and to enhance autophagy.⁷⁰ In contrast, another study showed that curcumin inhibited autophagy in cardiomyocytes during I/R, but this effect increased cell survival.¹⁷⁸ The effects of RSV in I/R injury were also extensively investigated. This molecule increased cardiomyocyte survival in an I/R model in vitro,¹⁷⁹ and improved cardiac recovery after ischaemia in isolated hearts, also reducing oxidative stress¹⁸⁰ and inflammation in vivo.^181,182 Of note, it was demonstrated that low doses of RSV attenuate cardiac ischaemic damage through the activation of autophagy. In fact, RSV failed to improve cell survival in the presence of autophagy inhibition in isolated hearts, as well as in cardiomyocytes subjected to I/R in vitro. The induction of autophagy by RSV was reported to be mediated by mTORC2 activation.¹³² Radicicol, quercetin (QU), ECGC, caffeine, berberine, metformin, and NMN represent other potential CRMs able to improve I/R recovery. Radicicol increased cardiomyocyte survival in a model of in vitro ischaemic injury, likely through the activation of the heat shock proteins.⁸¹ QU was observed to reduce I/R injury, inflammation, apoptosis, and oxidative stress in rats.¹⁸³ Similarly, EGCG attenuated I/R injury and apoptosis in isolated hearts through signal transducer and activator of transcription-1 (STAT1) inhibition, a transcription factor promoting cardiac apoptosis during I/R.¹⁸⁴ Moreover, long-term administration of caffeine prior to I/R injury improved cardiac function in rats, reducing inflammation, and apoptosis.¹⁸⁵ Berberine also attenuated myocardial I/R injury, inflammation, oxidative stress, and apoptosis in rats by activating SIRT1.¹⁸⁶ However, the specific contribution of autophagy to the beneficial effects of these compounds has not yet been investigated. Metformin administration at the time of reperfusion improved cardiac function by means of AMPK signalling activation, which in turns promoted eNOS and PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) signallaing upregulation. In fact, metformin did not exert beneficial effects in mice harboring a dominant-negative form of AMPK.¹⁸⁷ Similar results were reported for isolated rat hearts subjected to reperfusion injury and in cardiomyocyte cultures in the presence of AMPK inhibition.¹⁸⁸ It was also demonstrated that NMN is able to protect the heart from I/R injury by restoring cardiac NAD⁺ levels in mice. In the same study, the protective effects of NMN were abrogated in cardiac-specific SIRT1 KO mice.⁹¹ Additional investigations are warranted to understand whether autophagy activation contributes to the effects of metformin and NMN during I/R.

Lastly, mTORC1 inhibition by rapalogs was proven to reduce infarct size in animal models of ischaemia without reperfusion. That said, rapamycin effects on I/R models still need to be fully clarified. Rapamycin administration was shown to decrease cardiac function and to increase myocardial necrosis in vivo in pig hearts subjected to I/R, without affecting autophagy.¹⁸⁹ Moreover, rapamycin abrogated the cardiac protection conferred by ischaemic preconditioning.¹⁹⁰ In contrast, other studies reported that rapamycin pretreatment improves cardiac function in response to I/R injury in mice.¹⁹¹ These divergent results are likely due to the dual effects of mTORC1 inhibition/autophagy activation, which are beneficial during ischaemia but may be maladaptive during reperfusion.

5.4 Genetic cardiomyopathies

Autophagy plays an important role in genetic forms of heart diseases, particularly in proteotoxic cardiomyopathies, in which an accumulation of misfolded proteins is observed. Of note, Beclin 1 deletion increases mortality and heart failure in a model of desmin-related cardiomyopathy, whereas autophagy activation improved cardiac function in another cardiomyopathy caused by αB-crystalline mutation.^192,193 Several studies showed that CRMs exert protective effects in different forms of genetic cardiomyopathies. It was demonstrated that either CR or rapamycin can improve the phenotype of a mouse model of genetic cardiomyopathy caused by mutations in the MYBPC3 gene, which encodes the sarcomeric protein myosin-binding protein-C. The protective effects of CR and rapamycin were associated with autophagy reactivation.¹⁹⁴ Rapamycin was also reported to improve cardiac performance in Lamin A/C-deficient mice, a genetic model of dilated cardiomyopathy, in association with a significant improvement of autophagy.^25,195 Rapamycin also palliated a mouse model of LEOPARD syndrome.¹⁹⁶ Of interest, Zhou et al.¹⁹⁷ recently demonstrated that rapamycin rescued autophagy in vivo, in rats with a genetic form of dilated cardiomyopathy due to titin-truncating variants (TTNtv).

Reactivation of sirtuin metabolism by CRMs seems to protect against genetic forms of hypertrophic cardiomyopathies. For example, Martin et al. reported that NMN was able to ameliorate cardiac function in a Friedreich’s ataxia cardiomyopathy mouse model (FXN-KO), together with an improvement of energy metabolism. The authors further demonstrated that NMN supplementation failed to confer cardioprotection to FXN-KO crossed with SIRT3 cardiac-deficient mice. This suggests that SIRT3 mediates the protective effects of NMN, likely by reducing the acetylation of the proteins involved in mitochondrial energetics and antioxidant defense.¹⁹⁸ Similarly, NAD⁺ supplementation through nicotinamide riboside (NR) attenuated heart failure in vivo in a genetic model of dilated cardiomyopathy.¹⁹⁹ Different studies also revealed the potential efficacy of RSV to improve cardiac function in several models of genetic cardiomyopathies, such as dystrophin deficiency, in which RSV improved cardiac function in association with the downregulation of p300.²⁰⁰ In the same model, RSV reactivated cardiac mitophagy and increased SIRT1 and FOXO3a, without affecting mTORC1 activity.²⁰¹ RSV also improved cardiac function in vivo in a genetic model of iron-overload cardiomyopathy.²⁰² Finally, the synthetic acetyl-CoA depleting agent perhexiline maleate ameliorated cardiac function and energy metabolism in individuals affected by hypertrophic cardiomyopathy.⁸³

5.5 Metabolic derangements

Autophagy impairment may contribute to the pathogenesis of cardiomyopathies caused by metabolic derangements, such as diabetes, obesity, and metabolic syndrome.^203,204 CRMs have emerged as potential protective agents for cardiac abnormalities related to metabolic diseases. In many instances, the beneficial effects of CRMs were mediated by restoration of autophagy and mitophagy, which are usually impaired in the context of obesity, diabetes, and metabolic syndrome. For example, HC induced myocardial autophagy and reduced weight in rodents and in obese subjects.¹¹ RSV was found to improve cardiac function in streptozotocin (STZ)-induced diabetic rats, together with restoration of cardiac SIRT1 activity and reduction of apoptosis induced by endoplasmic reticulum stress.²⁰⁵ RSV ameliorated diabetic cardiomyopathy in STZ-induced diabetic mice in a SIRT1-dependent manner and in association with an increase of sarcoplasmic reticulum calcium-ATPase (SERCA2a), which is crucial for maintaining diastolic function.²⁰⁶

Curcumin was shown to ameliorate cardiac function in diabetic mice and in humans with diabetic cardiomyopathy.²⁰⁷ Curcumin administration to diabetic mice improved cardiac function and increased autophagy.²⁶ mTOR resulted inhibited in cardiac cells in response to curcumin treatment, suggesting that the activation of autophagy induced by curcumin may depend on mTOR signalling. ECGC was reported to attenuate myocardial I/R injury in diabetic rats in a SIRT1-dependent manner.²⁰⁸ Similarly, TRE was found to attenuate cardiac damage in Akt2 knockout mice, a model of insulin resistance. This effect was associated with the upregulation of autophagy and with the dephosphorylation of p38 MAPK and FoxO1.²⁰⁹

Metformin was observed to protect diabetic hearts independently of its hypoglycaemic effects. It activated cardiac autophagy and improved cardiac function in diabetic mice through AMPK-dependent mechanisms.²¹⁰

Chronic administration of rapamycin was reported to preserve cardiac function in type 2 diabetic mice (db/db) by inhibiting mTOR signalling, which is overactivated in obesity and metabolic syndrome conditions.²¹¹ Accordingly, in a model of high-fat diet-induced obesity and metabolic syndrome, rapamycin attenuated cardiac ischaemia through autophagy activation.¹³⁴ Thus, rapamycin administration was not effective in autophagy-deficient mice that were haploinsufficient for Beclin 1.¹³⁴ Interestingly, in db/db mice, rapamycin reduced body weight.²¹¹ Therefore, the protective effects of rapamycin on diabetes-induced cardiac dysfunction may also be attributable to both weight loss and mTOR inhibition. It would be interesting to evaluate whether rapamycin-induced weigh reduction is lost in diabetic models in the presence of autophagy inhibition.

Little evidence is available regarding the effects of ECGC, piceatannol, SRT1720, and quercetin on metabolic cardiomyopathy, although these compounds were found to ameliorate metabolic status in obese and diabetic mice.^93,212–216 In these models, piceatannol and SRT1720 showed no effects on body weight, whereas ECGC and quercetin rescued body weight gain induced by high-fat diet.^93,212–216 It will be important to understand whether a reduction of body weight contributes to the cardiac protective effects of different CRMs in metabolic diseases.

6. Perspectives and conclusions

Available data clearly indicate that CRMs are potentially useful therapeutic agents in cardiovascular medicine. Notwithstanding the beneficial effects of both synthetic and natural CRMs highlighted in pre-clinical models of CVDs, some questions still remain to be addressed. First of all, it remains to be demonstrated to which extent the effects of CR and CRMs are mediated by the induction of autophagy or other mechanisms. In fact, a considerable number of the above-mentioned studies reporting the effects of CR or CMRs on autophagy did not mechanistically test whether autophagy directly contributed to the beneficial effects of these molecules. CRMs may have different off-target effects, which are independent of autophagy activation. In order to address this issue, further studies must evaluate whether the beneficial cardiac effects of CR or CRMs are abrogated in autophagy loss-of-function animal models. It will be important to understand whether weight loss or metabolic improvement contributes to the beneficial cardiovascular effects of CMRs. Spermidine and resveratrol were found to exert protective cardiovascular effects independently of weight loss, since no reduction in body weight was observed following the supplementation of these compounds in cardiovascular preclinical studies.^24,75 Conversely, metabolic improvement may contribute to the beneficial cardiovascular effects of quercetin.²¹⁶ Of note, HC was found to reduce body weight through an autophagy-dependent mechanism.¹⁰⁹

In addition, an improved classification and definition of CRMs should be defined. The use of specific biomarkers of AcCoA depletion, autophagy-associated protein deacetylation and sirtuin activity should be standardized in order to facilitate the identification and synthesis of new potential CRMs. Additional studies are warranted to test the effects of all CRMs on different types of cardiac stress in a systematic manner, placing emphasis on those compounds with known beneficial effects in other organs. An interesting approach may be to evaluate whether the combination of CRMs acting on different molecular targets would be able to synergistically boost the effects of single molecules during cardiac stress. It should also be important to assess whether CRMs are able to reverse established heart diseases. Lastly, additional clinical studies testing the effects of CRMs on patients at high risk to develop CVDs are needed to better define the actual potential role for these promising compounds in novel therapeutic strategies. In this regard, it is difficult to interpret epidemiological studies on CR and CRMs in human population studies, since it is difficult to correctly estimate the true intake of calories and natural CRMs ingested with the diet. However, some data were obtained in controlled clinical trials. The CALERIE (Comprehensive Assessment of Long Term Effects of Reducing Caloric Intake) multicentre study investigated the effects of CR on overweight and healthy individuals. The results of this study were consistent with pre-clinical studies, showing a CR-induced decrease of oxidative stress, an improvement of plasma lipid composition and insulin sensitivity, as well as a decrease of cardio-metabolic risk factors.^{203,204,217–219} Meyer et al.³¹ also showed that, in human subjects, CR improved diastolic function and decreased blood pressure levels and systemic inflammation. The protective cardiometabolic effects of CR may be in part the direct consequence of body weight reduction, particularly of fat mass reduction. In fact, body weight loss by CR was found to promote the reduction of cardiovascular risk factors in both obese subjects and in healthy non-obese individuals.^220,221 However, a significant cardiometabolic improvement was shown to persist even after 1 year of CR, although body weight reduction was shown to stop after this time point, with a substantial weight maintenance.^12,219 In addition, body weight was shown to mediate only 11% of the effects of CR on longevity.²²² Overall, this evidence suggests that the protective effects of CR are also independent of body weight reduction. In support of this notion, it should be considered that other weight loss-inducing interventions, such as exercise, did not elicit the same beneficial effects as CR, especially in extending lifespan. In order to better understand to what extent, the beneficial cardiovascular effects of CR are mediated by the reduction of body weight, metabolic and cardiovascular parameters should be evaluated and correlated after prolonged CR intervention in human subjects.

Some clinical trials also investigated the effects of CRMs in CVDs showing divergent results. For example, RSV was reported to improve cardiovascular status in some clinical conditions but not in others.²²³ This is probably attributable to the low bioavailability of RSV.²²⁴ Another important issue regarding the use of CRMs in patients is the establishment of the correct dose and schedule of administration, depending on oral bioavailability, tissue distribution and excretion. In the case of resveratrol, <1% of bioavailability was reported.^119,224 Curcumin also displayed a short half-life and poor availability.²²⁵ Increasing the administered dose to obtain greater effects may lead to toxic effects. A mixture of different CMRs eliciting synergic effects may overcome the problem of excessive doses.

Conflict of interest: G.K. has been holding research contracts with Bayer Healthcare, Biomérieux, Glaxo Smyth Kline, Eleor, Kaleido, Lytix Pharma, PharmaMar, Sotio, and Vasculox/Tioma. G.K. is on the Board of Directors of the Bristol Myers Squibb Foundation France. G.K. is a scientific co-founder of everImmune, Samsara therapeutics, and Therafast bio.

Funding

M.C.M., S.S., F.C., J.S., and G.K. are supported by the Leducq Foundation (15CBD04). G.K. was supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR)—Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085 and GDW20181100051), Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; the LabEx Immuno-Oncology (ANR-18-IDEX-0001); the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM). This work was supported in part by grants from the Italian Ministry of Research (PRIN 2017N8K7S2_002) and from the Pasteur Institute, Cenci-Bolognetti Foundation to S.S.