Impact of caloric restriction on myocardial ischaemia/reperfusion injury and new therapeutic options to mimic its effects

Abstract

Caloric restriction (CR) is the most reliable intervention to extend lifespan and prevent age-related disorders in various species from yeast to rodents. Short- and long-term CR confers cardio protection against ischaemia/reperfusion injury in young and even in aged rodents. A few human trials suggest that CR has the potential to mediate improvement of cardiac or vascular function and induce retardation of cardiac senescence also in humans. The underlying mechanisms are diverse and have not yet been clearly defined. Among the known mediators for the benefits of CR are NO, the AMP-activated PK, sirtuins and adiponectin. Mitochondria, which play a central role in such complex processes within the cell as apoptosis, ATP-production or oxidative stress, are centrally involved in many aspects of CR-induced protection against ischaemic injury. Here, we discuss the relevant literature regarding the protection against myocardial ischaemia/reperfusion injury conferred by CR. Furthermore, we will discuss drug targets to mimic CR and the possible role of calorie restriction in preserving cardiovascular function in humans.

Caloric restriction (CR): an introduction

The level of oxidative stress increases with age. Subsequently, oxidative damage to DNA, proteins or lipids accumulates with age, and as a consequence, an impairment of cellular functions may occur. Mitochondria are a major source of reactive oxygen species (ROS) as a by-product of the normal functioning of the respiratory chain. In cardiomyocytes, which possess a high energy demand, but a relatively poor repair capacity, a chronic state of oxidative stress exists. Life-long CR has long been known to attenuate this age-associated increase in mitochondrial ROS production (Sohal et al., 1994; Barja, 2002), in lipid peroxidation (Matsuo et al., 1993), in protein oxidation (Leeuwenburgh et al., 1997), and in oxidative damage of mitochondrial and nuclear DNA (Gredilla et al., 2001). All these ROS-induced alterations, sensitive to modulation by CR, are considered as manifestations of the free radical theory of ageing (Harman, 1956).

In 1935, Clive McCay and colleagues reported for the first time that decreasing food intake extends the lifespan of rats (McCay et al., 1935). By now, long-term CR is known to retard the ageing process in a number of organisms from yeast to smaller, short-lived mammals, but the detailed mechanisms of its efficacy remain unclear and its actions in different organs are remarkably heterogeneous (Masoro, 2000). The so-called Hormesis Hypothesis (Masoro, 1998) tries to unify various theories of ageing and longevity as merely specific examples of hormetic processes. Hormesis refers to a beneficial action resulting from the response of an organism to a low-intensity stressor otherwise detrimental when administered at higher concentrations or intensities (Masoro, 1998; Sinclair, 2005; Rattan, 2008; Calabrese et al., 2012). The hormesis hypothesis of CR predicts that reduction in calorie intake is a mild stress that provokes a survival response resulting in increased cellular defences, partial repair of age-associated damage, attenuation of stress-induced cell death and altered metabolism.

Beneficial effects of CR were reported using various regimens. The key factor for the efficacy of CR seems to be an overall reduction in energy intake (Masoro, 1988), although a restriction of dietary methionine replicates some of the effects of CR, such as an increase in lifespan in rats (Orentreich et al., 1993) and mice (Miller et al., 2005) or a decrease in mitochondrial ROS production and damage (Sanz et al., 2006). A common CR approach is to measure the ad libitum food intake of an individual animal and then reduce the food by a certain percentage. This approach works well in adult animals, but not in young or senescent animals as food intake is not constant across the lifespan. However, CR animals fed a certain percentage of their own baseline intake will not only experience reduced levels of calories but also a reduction of all micronutrients. Thus, this type of approach should be called dietary restriction instead of CR (Masoro, 2009).

Common, commercially available CR diets provide reduced levels of calories but micronutrients adjusted to the levels of ad libitum fed controls and hence do not lead to malnutrition. The magnitude of CR applied in most rodent studies varies between severe restriction with a reduction in calories of 40–50%, a moderate restriction with a reduction in calories of 20–25%, and a mild restriction with a reduction in calories of 5–10%. The latter protocol is sometimes also used for control groups by restricting food intake in order to prevent obesity as some mouse or rat strains overeat and become quite obese when given free access to food (ad libitum feeding). The maximum lifespan-extending effect of CR is achieved with a 40–45% CR, while a stronger CR reduces lifespan (review in (Speakman and Mitchell, 2011). Another diet regimen is alternate day fasting (also called ‘every-other-day feeding’), in which animals alternate between days where they are fed ad libitum and days of fasting (Goodrick et al., 1990; Anson et al., 2003). Interestingly, these animals also experience enhanced longevity (Goodrick et al., 1990).

Other factors known to influence CR efficacy are the total duration of CR and the time of initiation. Besides life-long CR, short-term CR (3 months) is commonly applied. Some of the metabolic effects of CR have even been reproduced using overnight starvation or prolonged starvation (48 h). Depending on the scientific focus, CR is initiated at birth, in adulthood or late in life. Although there are many beneficial effects on age-related diseases in various organs when CR is initiated late in life (Rae, 2004), the greatest gains are achieved when a strong CR is initiated relatively early in life and sustained to late age. Indeed, there is a linear relationship between the extent of CR and the extent to which lifespan is increased (Weindruch, 1996). CR started late in life results in a lesser extension of lifespan compared with the effect observed when CR is started at weaning (Weindruch and Walford, 1982). Similarly, alternate day fasting results in an increased lifespan only when initiated at the age of 1–6 months in mice, but not when introduced at 10 months of age (Goodrick et al., 1990). However, a recent meta-analysis evaluating laboratory experiments that have investigated life-extending effects of CR in rats and mice suggests significant differences in CR efficacy and even failure to increase lifespan in some species and strains (Swindell, 2012).

To address the question whether CR is also beneficial in long-lived animal species, two independent studies using rhesus monkeys were initiated in the late 1980s. The first report published in 2009 demonstrated that CR extends lifespan in rhesus monkeys (Colman et al., 2009). However, most recently, such beneficial effect of CR in rhesus monkeys was questioned again as CR had no effect on longevity (Mattison et al., 2012). Differences in study design, husbandry and diet composition were alleged to have caused this divergent effect on the life extension by CR. Controlled trials on the effects of long-term CR on longevity and/or cardiac function in humans are lacking for obvious reasons, including unresolved safety issues or difficulties in lifelong observation of participants. One small-scale study described protective effects on diastolic dysfunction in humans practising self-imposed CR for 3–15 years (n = 25; Meyer et al., 2006). The few studies suggesting that CR affects ageing in humans are controversial (Dirks and Leeuwenburgh, 2006; Shanley and Kirkwood, 2006; Morley et al., 2010). However, many human studies have been conducted in obese individuals over short durations and are therefore not comparable with long-term CR performed in normal-weight animals. The Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy (CALERIE) trials systematically investigate the effects of CR in healthy, non-obese human beings (Rochon et al., 2011). Phase 1 of CALERIE used short-term CR (6–12 months), while phase 2 of CALERIE is a randomized, multicentre study that uses dietary and behavioural interventions to achieve 25% CR for 2 years (Rochon et al., 2011). Similar to the data obtained from animal studies, biomarkers of longevity such as fasting insulin level or body temperature are decreased (Heilbronn et al., 2006), and major risk factors for coronary heart disease are substantially improved in humans after 6–12 months of CR (Fontana et al., 2007). The results of phase 2 are expected to be released in late 2013.

Mitochondria in cardiac physiology and pathophysiology: focus on effects during ischaemia/reperfusion

Energetic and ionic homeostasis

Under physiological conditions, mitochondria consume large amounts of oxygen to produce ATP at complex V of the respiratory chain. When the heart becomes hypoperfused and oxygen is lacking, the electron flow along the respiratory chain complexes is inhibited and mitochondrial oxygen consumption as well as ATP production decreases (Lesnefsky et al., 1997; Paradies et al., 2004; Boengler et al., 2007). During ischaemia, glycolysis becomes the major source of ATP production and AMPK activation promotes glycolysis by increasing glucose uptake (Russell et al., 2004) and phosphorylation of phosphofructokinase-2 (Marsin et al., 2000). Mitochondria, instead of producing myocardial ATP, now consume ATP to maintain their inner membrane potential and inhibition of such reversed mode of F₁Fo-ATPase protects cardiomyocytes from irreversible injury. During reperfusion, AMPK activation can stimulate fatty acid oxidation (FAO) by inhibiting acetyl-CoA carboxylase and reducing the production of malonyl-CoA, an inhibitor of the mitochondrial transporter of long-chain fatty acids carnitine palmitoyltransferase-1. Restoration of ATP production contributes to the re-establishment of cellular ion homeostasis, but at the same time may paradoxically contribute to irreversible reperfusion injury. During ischaemia, cardiomyocytes become calcium overloaded, which at reperfusion, when ATP is available, is rapidly taken up into the sarcoplasmic reticulum (SR). However, once the SR is calcium overloaded, it again releases calcium resulting in high cytosolic calcium concentrations. The repetition of this process at early reperfusion has been termed ‘calcium oscillation’. High cytosolic calcium concentrations in the presence of ATP result in hypercontracture of cardiomyocytes, membrane disruption and subsequent necrosis (for review, see Piper et al., 2006). Mitochondria also take up calcium in the presence of high cytosolic calcium concentrations and thus become calcium overloaded (Maack and O'Rourke, 2007). Here, the tight intracellular communication between the SR and mitochondria seems to be involved. Specific domains for direct interaction with mitochondria known as the mitochondria-associated membrane exist (Raturi and Simmen, 2013). As has been recently reviewed, the mitochondria-associated membrane tethers the SR/ER (endoplasmic reticulum) to mitochondria and is enriched with proteins relevant to calcium and lipid metabolism, such as mitofusins (Mfn, especially Mfn 2) (de Brito and Scorrano, 2008; Ruiz-Meana et al., 2010).

ROS formation and mitochondrial permeability transition pore (MPTP) opening

Apart from ATP, mitochondria also generate ROS (Droge, 2002; Balaban et al., 2005; Murphy, 2009). ROS within mitochondria originate from different sources, one of them is the respiratory chain, mainly complexes I and III. Some cytosolic proteins that are important under physiological (like connexin 43) or pathophysiological (like the signal transducer and activator of transcription 3, STAT-3) conditions interact either directly or indirectly with the respiratory chain to modify respiration and/or ROS formation (Boengler et al., 2011; 2012; 2013). ROS are also produced by MAOs located at the outer mitochondrial membrane, which transfer electrons from amine compounds to oxygen leading to hydrogen peroxide formation. Especially under pathophysiological conditions MAO-induced ROS formation contributes to heart failure development secondary to pressure overload or ischaemia/reperfusion injury (see later) (Kaludercic et al., 2010; 2011; 2014). Furthermore, the cytosolic protein p66^Shc becomes phosphorylated under stress conditions and shuttles into the mitochondrial intermembrane space, where it oxidizes reduced cytochrome c resulting in peroxide formation (Giorgio et al., 2005; Carpi et al., 2009) (for further review also see Di Lisa et al., 2009). During hypoperfusion, ROS formation increases, and a further burst of ROS occurs in early reperfusion (Vanden Hoek et al., 1997; Becker et al., 1999; Paradies et al., 2004). Increased ROS formation during reperfusion aggravates cell death (Turrens, 2003; Adlam et al., 2005), and accordingly, hearts from p66^Shc knockout (KO) mice are protected from ischaemia/reperfusion injury in vitro, and inhibition of MAO reduces infarct size following ischaemia/reperfusion injury in vivo (Bianchi et al., 2005; Carpi et al., 2009; Di Lisa et al., 2009).

High concentrations of mitochondrial ROS – together with other factors – facilitate opening of the MPTP, a large conductance pore in the inner mitochondrial membrane, most likely build up by dimerization of the mitochondrial ATP synthase (Giorgio et al., 2013). MPTP opening at reperfusion enhances the inner mitochondrial membrane permeability to solutes with molecular weights up to 1.5 kDa and therefore leads to mitochondrial depolarization and subsequently to ATP depletion. Mitochondrial matrix volume increases and induces rupture of the outer mitochondrial membrane, inhibition of electron flow along the electron transport chain (ETC) and initiation of apoptotic cell death (Zou et al., 1999; Di Lisa et al., 2011; Griffiths, 2012). While prolonged opening of MPTP is detrimental, flickering of MPTP appears to be essential for putting the heart into a protected state and controlling mitochondrial matrix calcium concentration. Indeed, long-term blockade of MPTP opening facilitates the development of heart failure in mice upon increased stress (Hausenloy et al., 2004; Saotome et al., 2009; Elrod et al., 2010; Korge et al., 2011; Wong et al., 2012).

There are two subpopulations of mitochondria located in different regions of the cell, the subsarcolemmal mitochondria (SSM) and the interfibrillar mitochondria (IFM), which differ in their respiratory and calcium retention capacity (Fannin et al., 1999; Judge et al., 2005; Bugger et al., 2006; Hofer et al., 2009). Age-related declines in oxidative phosphorylation rates in rat heart mitochondria occur mainly in the IFM (Fannin et al., 1999; Judge et al., 2005), while the increase in ROS-production during ischaemia and reperfusion (I/R) is comparable in SSM and IFM from rat hearts (Chen et al., 2008). The depletion of cardiolipin and the accompanying cytochrome c loss in SSM but not in IFM during ischaemia results in an amplified ROS production and contributes substantially to the mitochondrial damage and the myocardial I/R injury (Chen and Lesnefsky, 2006; Chen et al., 2010b). Ischaemia was also reported to cause a decreased recovery of citrate synthase in isolates of SSM but not of IFM (Bugger et al., 2006). Besides, loss of mitochondrial connexin 43, which is located at the inner membrane of SSM but not of IFM, abolishes the cardioprotection by ischaemic preconditioning (IPC), suggesting that SSM and IFM also differ in their function and signal transduction during endogenous cardioprotection (Boengler et al., 2009b).

Mitochondrial fusion and fission

Many cardiovascular diseases affect mitochondrial function, but even more affect mitochondrial morphology and the inter-mitochondrial network, that is modify fusion and fission of mitochondria. Mitochondrial fusion proteins include the outer membrane proteins Mfn 1 and 2 and the inner membrane protein optic atrophy protein 1 (Opa1). Mitochondrial fission in mammalian cells is mediated by a large GTPase, the dynamin-related protein 1 (Drp1), along with other proteins such as the outer mitochondrial fission protein 1 (Fis1) and the mainly cytoplasmic endophilin B1. Mitochondrial fusion and fission are constant ongoing processes in many cell types and are essential for the maintenance of normal mitochondrial function. However, in the adult heart where mitochondrial movements are restricted by their tightly packed distribution along myofibrils or beneath the subsarcolemma, the relevance of mitochondrial dynamics is less obvious (Ong et al., 2013). Mitochondrial fusion serves as a pro-survival mechanism and content/protein exchange might help to overcome local functional deficiencies such as mitochondrial DNA (mtDNA) mutations within the mitochondrial network (Nakada et al., 2001; Ono et al., 2001; Chen et al., 2010a). Accordingly, inhibition of fusion results in an accumulation of mtDNA mutations triggering mitochondrial dysfunction, the loss of the mitochondrial genome and finally organ dysfunction (Chen et al., 2010a).

Pathophysiological conditions also affect mitochondrial fusion and fission. Ischaemia reduces Opa1 protein levels in H9c2 cells and increases apoptosis and fragmentation of the mitochondria. Also studies in heart failure suggested a possible reduction in mitochondrial fusion (Chen and Knowlton, 2011). In turn, Opa1 overexpression preserves mitochondrial morphology and protects cells against apoptosis (Chen et al., 2009b). Similarly, overexpression of Mfn-1 or Mfn-2 decreases MPTP sensitivity and protects from I/R injury in the cardiac muscle cell line HL-1 (Ong et al., 2010). However, Mfn-2 deletion was also demonstrated to induce a better cardiac recovery following I/R (Papanicolaou et al., 2011). Cells lacking human Fis1 undergo senescence-associated phenotypic changes associated with extensive mitochondrial elongation and increased ROS production (Lee et al., 2007). Mutation of dynamin-1-like protein, which is also critical for mitochondrial fission, induces mitochondrial dysfunction and cardiomyopathy in mice (Ashrafian et al., 2010). Decreasing mitochondrial fission is cardioprotective, as a Drp1 inhibitor reduces cardiomyocyte death following I/R both in vitro and in vivo (Ong et al., 2010). There are also rare and generally overlooked examples of cardiomyopathies linked either to naturally occurring mutations or to experimentally induced mutagenesis of mitochondrial fusion/fission genes (Dorn, 2013).

Mitophagy

Accumulation of damaged and dysfunctional mitochondria can lead to a cellular energy deficit, increased ROS production and the release of pro-apoptotic factors (Gottlieb et al., 2009). Interestingly, cells have developed a defence mechanism against aberrant mitochondria that can cause harm, in that selective sequestration and subsequent degradation of the dysfunctional mitochondria are initiated before they cause activation of the cell death machinery (Kubli and Gustafsson, 2012). Components required for this organelle-specific type of autophagy include the mitochondrial kinase PTEN-induced novel kinase 1 (PINK1), the E3 ubiquitin ligase Parkin, the atypical B-cell lymphoma 2 homology domain 3-only (BH3-only) proteins BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) and Nip-like protein X, and several autophagy-related genes (Kanki et al., 2011). The elimination of damaged mitochondria by mitophagy is cardioprotective (Gottlieb and Carreira, 2010; Gottlieb and Mentzer, 2010; Sheng et al., 2010) and endogenous cardioprotective interventions such as preconditioning rely on autophagy/mitophagy (Gottlieb and Gustafsson, 2011). Preconditioning results in an up-regulation of autophagy in association with the pro-survival molecule BCL2-associated athanogene 1 protein, while inhibition of autophagy abolishes the cardioprotective effects of preconditioning (Gurusamy et al., 2009). Furthermore, the E3 ubiquitin ligase Parkin mediates cardioprotection by IPC through increased mitophagy (Huang et al., 2011). Bnip3 is activated following I/R and induces fragmentation of the mitochondrial network and increased mitophagy (Hamacher-Brady et al., 2006b; 2007), which was suggested to function as a cytoprotective pathway to oppose I/R-related apoptosis. Bnip3 also functions as a redox sensor during I/R (Kubli et al., 2008). Homodimerization and activation of Bnip3 is observed after I/R (Kubli et al., 2008). Bnip3 induces mitochondrial dysfunction through permeabilization of the mitochondrial membranes, disassembly of Opa1 mitochondrial fusion complexes and their release from the mitochondria (Quinsay et al., 2010). The Bnip3-triggered autophagy and mitochondrial turnover appears to be independent of ROS generation or MPTP opening (Quinsay et al., 2010). The p53-dependent up-regulation of TP53-induced glycolysis and apoptosis regulator (TIGAR) reduces Bnip3 activation and mitophagy in cardiomyocytes, resulting in an accumulation of damaged mitochondria and subsequent apoptosis in ischaemic myocardium (Hoshino et al., 2012). Enhancement of the autophagic flux following I/R through overexpression of Beclin 1 significantly reduces pro-apoptotic activation (Hamacher-Brady et al., 2006a).

Mitobiogenesis

If depletion of damaged mitochondria as described in the previous paragraph is not followed by an appropriate induction of mitochondrial biogenesis, this will have detrimental consequences for the heart as well. Mitochondrial biogenesis requires the coordination of the nuclear and the mitochondrial genome and involves changes in the expression of more than 1000 genes (review in Lopez-Lluch et al., 2008). Transcription factors involved in this process include the nuclear respiratory factors (NRF-1/NRF-2), the mitochondrial transcription factor A (Tfam), the oestrogen-related receptors ERα and ERγ (for nomenclature see Alexander et al., 2013a), ubiquitous transcription factors and the transcriptional coactivators PPARγ coactivator-1α (PGC-1α)or PGC-1-related co-activator (PRC; Goffart and Wiesner, 2003). Many of the pathways regulating mitochondrial biogenesis seem to converge at the transcriptional co-activator PGC-1α,which has been shown to directly dock on some of these transcription factors and modulate their activity (Schreiber et al., 2004; Gleyzer et al., 2005; Zhu et al., 2010; Safdar et al., 2011). Overexpression of PGC-1α in cultured myoblasts increases mitochondrial biogenesis and oxidative respiration (Wu et al., 1999), but PGC-1α also plays a key role in coordinating metabolic flux (Rodgers et al., 2005; Dominy et al., 2010) and regulates processes such as gluconeogenesis or FAO (Handschin and Spiegelman, 2006). The hearts of PGC-1α KO mice (Arany et al., 2005; Leone et al., 2005) demonstrate reduced oxidative capacity and mitochondrial gene expression, but normal mitochondrial volume density, suggesting additional mechanisms controlling cardiac mitochondrial biogenesis. Although cardiac dysfunction under basal conditions is moderate in PGC-1α KO mice (Arany et al., 2005; Leone et al., 2005), aortic constriction leads to accelerated heart failure (Arany et al., 2006). In contrast, PGC-1α overexpression causes uncontrolled mitochondrial proliferation and loss of sarcomeric structure, finally leading to dilated cardiomyopathy and premature death (Lehman et al., 2000). Thus, a well-balanced and tightly controlled change in the expression of PGC-1α appears to be necessary to maintain optimal mitochondrial and cardiac function. With ageing or under pathophysiological conditions (obesity, diabetes mellitus, I/R) major disturbances in mitochondrial biogenesis and function occur in various tissues including the heart (Semple et al., 2004; Sparks et al., 2005; Zahn et al., 2006; Linford et al., 2007; Niemann et al., 2011).

Protection against myocardial ischaemia/reperfusion injury conferred by CR

Ischaemia/reperfusion injury

Ischaemic tolerance decreases with age (Frolkis et al., 1991; Ataka et al., 1992; Tani et al., 1997) and many cardioprotective interventions including IPC and postconditioning are less effective in aged individuals (Abete et al., 1996; Schulman et al., 2001; Lee et al., 2002; Boengler et al., 2008; 2009a). Short- and long-term CR improves ischaemic tolerance in young and in old animals (Shinmura et al., 2005; 2008; Edwards et al., 2010), and IPC reduces post-ischaemic dysfunction in isolated, perfused hearts from food-restricted senescent rats, but not in hearts from ad libitum-fed senescent rats (Abete et al., 2002). Combining CR with exercise training completely restores the protection afforded by IPC in senescent hearts when compared with young hearts (Abete et al., 2005). Apart from ageing, cardioprotective interventions are affected by a number of co-morbidities (for a review, see: Ferdinandy et al., 2007; Ovize et al., 2010; Hausenloy et al., 2013). Indeed, ischaemic postconditioning is less effective in obese or diabetic animals (Bouhidel et al., 2008) (for a review, see Przyklenk, 2011; Oosterlinck et al., 2013).

Only a few data exist on the influence of CR on the protective effects of ischaemic postconditioning. Ischaemic postconditioning improves contractile recovery and cell viability in fed, but attenuates them in fasted hearts. Furthermore, ATP content and ATP synthesis increases while oxidative stress decreases in hearts of fasted rats following ischaemic postconditioning (Marina Prendes et al., 2011). The same group also reported improved functional recovery and increased resistance to MPTP opening after ischaemic postconditioning (Hermann et al., 2012).

Energetic homeostasis

The increased resistance to severe ischaemia and better functional recovery of hearts after CR is strongly related to changes in mitochondrial respiration (Broderick et al., 2002). Mitochondria isolated from CR hearts after I/R demonstrate an increased state 3 respiration, increased respiratory control ratios as a sign of well-coupled mitochondria and a higher efficiency of mitochondrial energy production (Broderick et al., 2002), indicating the involvement of metabolic changes in the mitochondrial compartment as a basis for improved left ventricular (LV) function. A cause and effect relationship between cardiac functional improvement and increased mitochondrial metabolism was, however, not determined in that study (Broderick et al., 2002). In addition to these mitochondrial changes, activation of glucose uptake and glycolysis, improved insulin sensitivity and increased antioxidative defence appear also to be involved in CR-induced protection during I/R (Russell et al., 2004; Mitchell et al., 2010; Wan et al., 2010; Yamagishi et al., 2010).

ROS formation

ROS balance is lost at the extremes of reduction or oxidation of the redox couples involved in electron transport or ROS scavenging. Isolated mitochondria display increased oxidative stress at high reduction potentials, while intact cardiac cells experience oxidative stress when mitochondria are uncoupled or maximally reduced as in ischaemia (Aon et al., 2010). The ‘uncoupling to survive’ hypothesis states that the attenuation of ROS by partial dissipation of the mitochondrial membrane potential while maintaining sufficient ATP production is a potential mechanism for delaying cellular senescence (Papa and Skulachev, 1997; Brand, 2000). Indeed, mild uncoupling has been shown to be associated with longevity in various experimental models (Barros et al., 2004; Speakman et al., 2004; Padalko, 2005; Caldeira da Silva et al., 2008; Lemire et al., 2009), suggesting that the beneficial effects of CR on mitochondrial respiration and lifespan can be mimicked by uncoupling agents. The induction of a mitochondrial biogenic response by uncoupling agents has been suggested to be involved in these beneficial effects as well. Accordingly, the authors of a comparative analysis in mice reported recently that systemic mild uncoupling by dinitrophenol and 40% CR for 6 months similarly increased activities of Akt and the Akt downstream target endothelial NOS (eNOS), leading to mitochondrial biogenesis in adipose tissue and skeletal muscle (Cerqueira et al., 2011).

Mitochondrial fusion and fission

CR increases Mfn-2 protein expression in various organs, suggesting a role for CR in the control of mitochondrial morphology and dynamics (Cerqueira et al., 2011). During nutrient deprivation, PKA activation phosphorylates the pro-fission protein Drp1, which is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion (Gomes et al., 2011). This mitochondrial hyperfusion is required to sustain cellular ATP levels and allow survival of starving cells (Gomes et al., 2011). Blocking fusion and thus elongation results in dysfunctional mitochondria, reduced membrane potential and starvation-induced cell death in vitro (Gomes et al., 2011). How these mechanisms operate in vivo during CR has not yet been investigated.

Mitophagy and mitobiogenesis

Starvation or reduced insulin signalling are strong inducers of autophagy (review in Levine and Kroemer, 2008), and inhibition of autophagy prevents the beneficial effects of CR in all species investigated so far (Rubinsztein et al., 2011). However, the efficiency of this process declines with age (Brunk and Terman, 2002), but this age-related impairment of autophagy was also attenuated by mild, life-long CR in the skeletal muscle of rats (Wohlgemuth et al., 2010).

In vitro starvation results in mitochondrial depletion (Carreira et al., 2010). This mitochondrial depletion may contribute to the observed myocardial dysfunction after food restriction (Pinotti et al., 2010). If depletion of mitochondria is not followed by an induction of mitochondrial biogenesis (Hancock et al., 2011), this will be detrimental for the heart. However in many studies, CR increases the number of functional mitochondria and promotes changes in mitochondrial dynamics (Lopez-Lluch et al., 2006; Civitarese et al., 2007), favouring tighter coupling between beta-oxidation and the tricarboxylic acid (TCA) cycle, and may concomitantly improve insulin sensitivity (Lopez-Lluch et al., 2006).

Mechanisms and mediators of cardioprotection conferred by CR

Mitochondrial quality control

Over their lifespan, cardiomyocytes experience repetitive exposure to ROS resulting in damage to macromolecules and cell organelles, which cannot be diluted via cell division. Stressful conditions such as I/R induce an increase in mitochondrial damage requiring an adequate removal by mitophagy, followed by an increase in cardiac mitochondrial biogenesis to meet the high energetic demand of the heart. Interestingly, CR has a major impact on most processes of mitochondrial quality control from mitochondrial biogenesis to mitochondrial fusion and fission or removal of damaged mitochondria by mitophagy.

Various endogenous and exogenous factors regulate the activity of the transcriptional coregulator of mitochondrial biogenesis PGC-1α, including NO (Leary and Shoubridge, 2003; Nisoli et al., 2003), cAMP response element-binding protein (CREB; Shaw et al., 2005; Than et al., 2011), AMPK (Jager et al., 2007; Canto et al., 2009), Akt or p38 MAPK (Puigserver et al., 2001; Fan et al., 2004), many of which have also been shown to be regulated in response to CR. CR increases the expression of eNOS, accompanied by increased mitochondrial biogenesis, mitochondrial respiration and ATP production (Nisoli et al., 2005; Nisoli and Carruba, 2006). The eNOS-induced mitochondrial biogenic response involves an Akt-dependent phosphorylation of eNOS (Dimmeler et al., 1999; Nisoli et al., 2003) and can be mimicked with serum from CR rats, an effect putatively mediated by adiponectin (Cerqueira et al., 2012a). However, Akt2 also phosphorylates PGC-1α at Ser⁵⁷⁰ preventing its recruitment to the promoter of PGC-1α target genes (Li et al., 2007b), possibly reducing mitochondrial biogenesis. The reversible acetylation and inhibition of PGC-1α by the histone acetyltransferase generally controls non-repressed protein 5 (acetyltransferase), but deacetylation and activation by sirtuin 1 (SIRT1) is another key way to alter PGC-1α activity. PGC-1α contains multiple distinct acetylation sites and PGC-1α deacetylation has been demonstrated to occur via SIRT1 in vitro as well as in vivo during fasting (Rodgers et al., 2005; Gerhart-Hines et al., 2007) and can be mimicked by resveratrol (Lagouge et al., 2006). Recently, a reduced PGC-1α acetylation and increased mitochondrial biogenesis in skeletal muscle was demonstrated following exercise (Canto et al., 2009; Li et al., 2011).

In a screen for upstream regulators of PGC-1α gene transcription, transducer of regulated cAMP-regulated element-binding protein 1 (TORC1) was identified as the most potent activator of PGC-1α gene transcription and thus mitochondrial biogenesis (Wu et al., 2006). Furthermore, it has been shown that phosphorylation of CREB at Ser¹³³ activates the promoter of PGC-1α, increases PGC-1α expression and induces mitochondrial biogenesis (Chowanadisai et al., 2010). Accordingly, CREB-deficient mice are only poorly responsive to CR because of a reduction of the CREB-target genes PGC-1α and neuronal NOS in the brain (Fusco et al., 2012). Indirect evidence also suggests a role for p38 MAPK-mediated changes in PGC-1α activity in response to diet. The p38 MAPK phosphorylates PGC-1α at three residues (Thr²⁶²; Ser²⁶⁵; Thr²⁹⁸), resulting in a more active and stable protein (Puigserver et al., 2001). Insulin has been demonstrated to inhibit the p38 MAPK-mediated increase in PGC-1α expression (Hong et al., 2011) and prolonged exposure to insulin decreases mitochondrial mass, cellular ATP content, and oxygen consumption in hepatocytes (Liu et al., 2009). Under physiological conditions, insulin is increased in the blood in the fed state but falls during CR (Niemann et al., 2008; 2010). Reduced insulin levels in CR-treated animals may thus contribute to increased PGC-1α expression via altered p38 MAPK activation.

Reduced insulin signalling is also a strong inducer of autophagy (review in Levine and Kroemer, 2008) and autophagy seems to have positive effects on longevity. Life-long CR increases the occurrence of autophagy in the hearts of rats (Wohlgemuth et al., 2007) and extends lifespan, at least in part, by increasing expression or activity of the NAD-dependent deacetylase SIRT1 in various species including mammals (Chen et al., 2005; Bordone et al., 2007; Haigis and Sinclair, 2010). Indeed, SIRT1 appears to be necessary for the induction of starvation-induced autophagy (Lee et al., 2008; Morselli et al., 2010) and the protective effects of CR. Lack of SIRT1 inhibits autophagy in vivo, leads to an elevated acetylation of proteins required for autophagy, accumulation of damaged mitochondria and impaired metabolism (Lee et al., 2008). Down-regulation of the nicotinamide phosphoribosyltransferase (Nampt), which is the rate-limiting enzyme in the NAD⁺ salvage pathway and thus influences the SIRT activity, impairs autophagic flux, suggesting that endogenous Nampt positively regulates autophagy (Hsu et al., 2009).

Other pathways involved in CR-mediated effects on autophagy include established modulators of mitochondrial biogenesis such as Akt or AMPK. Signalling pathways converging at the activation of Akt inhibit the negative regulator of mammalian target of rapamycin kinase (mTOR) mTOR (see Alexander et al., 2013b) inhibiting proteins (TSC1/TSC2). The resulting activation of mTOR inhibits autophagy, while the opposite occurs when nutrients are depleted or Akt activation is reduced. Likewise, inhibition of phosphatidylinositide 3-kinase (PI3K), which generates lipid second messengers essential for the translocation and subsequent activation of Akt, preserves cardiac function in aged mice, attenuates cardiac signs of senescence and enhances autophagy (Inuzuka et al., 2009). Accordingly, long-term CR results in a decreased cardiac Akt phosphorylation and increased autophagy associated with preserved cardiac contractile function in mice (Han et al., 2012). In addition, CR also induces autophagy via AMPK activation (Egan et al., 2011; Kim et al., 2011) or through the inhibition of insulin/insulin-like growth factor (IGF) signalling (Kenyon, 2010). However, the fact that nutritional stress induced by serum/glucose deprivation in vitro or starvation in vivo strongly induces autophagy and cell death, which can be inhibited by IGF-1 (Troncoso et al., 2012), also points to the importance of IGF-1 in maintaining cardiac adaptation to nutritional insults. Furthermore, it also suggests that autophagy has the potential to induce harmful effects in response to nutritional challenges.

Role of micro RNAs (miRs)

A new emerging field is now unravelling the role of miRs in cardiac pathophysiology and their regulation in response to diet. Hypercholesterolaemia induces a down-regulation of cardiac miR-25, resulting in increased NADPH oxidase (NOX) 4 expression and consequently increased oxidative stress in the heart (Varga et al., 2013). Recently, it was shown that ageing is associated with a down-regulation of the miR processing enzyme Dicer and multiple miRs in adipose tissue in mice and humans (Mori et al., 2012). Interestingly, these effects in murine adipose tissue are largely prevented by CR in vivo and by nutrient deprivation, AMPK activation or mTOR inhibition in vitro. Oxidative stress, in contrast, induces a prematurely aged phenotype with low Dicer expression in vitro (Mori et al., 2012). Age-associated changes in miR expression in the skeletal muscle of rhesus monkeys and the brain of mice have also been reported to be partially modified by CR (Khanna et al., 2011; Mercken et al., 2013). Recently, increasing levels of circulating miRs, sensitive to modulation by CR, have been implicated in the ageing process in addition to tissue miRs and may thus convey cell–cell communications or influence the function of distant organs (Dhahbi et al., 2013). MiR-80 deletion in Caenorhabditis elegans results in extended lifespan and positive effects on the quality of cardiac-like muscle ageing, effects involving the transcription factor daf-16/FOXO, an important modulator of longevity through insulin signalling, and the CREB-binding protein CBP-1 (Vora et al., 2013). Interestingly, deletion of miR-80, which is highly expressed under ad libitum feeding but low under CR, exhibits these beneficial CR effects regardless of food availability. MiR-80 may thus represent a core regulator of metabolism and provide a novel point of application for CR mimetics (CRM) under normal calorie intake (Vora et al., 2013).

In addition to their role in response to ageing and diet, miRs have been shown to be involved in mitochondrial biology. So far, miR-499, miR-484 and the miR-30 family have been implicated in targeting the mitochondrial fusion or fission machinery in cardiomyocytes. miR-499 expression is reduced in the area at risk following myocardial ischaemia and in myocytes after anoxia (Wang et al., 2011). miR-499 inhibits cardiomyocyte apoptosis induced by anoxia. miR-499 transgenic mice show less apoptosis, reduced infarct size and improved LV function following I/R, while knockdown of miR-499 aggravates cardiac injury (Wang et al., 2011). The catalytic subunit of the serine and threonine protein phosphatase calcineurin was identified as a target of miR-499. Calcineurin dephosphorylates the mitochondrial fission protein Drp1 at Ser⁶⁵⁶ and promotes its translocation to mitochondria, which initiates the fission programme in cardiomyocytes. miR-499 mediates its anti-apoptotic effects through the suppression of calcineurin-mediated dephosphorylation of Drp1 and inhibition of mitochondrial fission (Wang et al., 2011). The promoter region of miR-499 contains p53 binding sites and p53 knockdown results in increased miR-499 levels and attenuation of mitochondrial fission and cell death, an effect that can also be mimicked in vivo (Wang et al., 2011). Recently, it was also reported that FOXO3a-mediated activation of miR-484 suppresses the translation of Fis1, resulting in an inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes. Accordingly, FOXO3a KO mice exhibit low levels of miR-484 and an enhanced mitochondrial fission, apoptosis and myocardial infarction (Wang et al., 2012). Finally, members of the miR-30 family (miR-30a, miR-30b, miR-30d) are able to inhibit mitochondrial fission and apoptosis (Li et al., 2010). Low levels of miR-30 result in a p53-induced transcriptional activation of the mitochondrial fission protein Drp1 in cardiomyocytes in response to oxidative stress and subsequently to apoptosis (Li et al., 2010). Although we are just beginning to unravel the role of miRs in mitochondrial fusion or fission, the modulation of miRs may present a future therapeutic approach to treat apoptosis-related cardiac diseases including ischaemic heart diseases.

Role of SIRTs

SIRTs are a highly conserved group of NAD⁺-dependent class III histone deacetylases and/or ADP-ribosyltransferases, which are localized in different cellular compartments and deacetylate histones and a number of non-histone proteins. The non-histone targets of SIRTs include p53, the FOXO family of transcription factors, PGC-1α, hypoxia-inducible factor 1α (HIF-1α), NF-κB, the apoptosis inhibitor survivin, liver kinase B1 (LKB1), β-catenin, eNOS, Tfam, the mitochondrial acetyl CoA synthetase 2, glutamate dehydrogenase, isocitrate dehydrogenase 2 (IDH2), and others. Among the deacetylated proteins in CR hearts are components of complex I (NDUFS1) and complex III (Rieske subunit) of the respiratory chain. Deacetylation of these ETC components and reduced ROS production can be mimicked in cardiomyocytes treated with the SIRT activator resveratrol and attenuated by SIRT inhibition with nicotinamide (Shinmura et al., 2011b), suggesting that deacetylation of these complex I and complex III subunits is closely associated with reduced mitochondrial ROS production during I/R in CR hearts (Shinmura et al., 2011b). At least seven silent information regulator 2 (Sir2) homologues, SIRTs 1 to 7, have been identified in mammals. Three SIRTs, SIRT3, SIRT4 and SIRT5, localize to mitochondria. Data obtained from yeast, worms and rodents suggest that some SIRTs are conserved mediators of many benefits of CR. The dependence on NAD⁺ availability couples their activation to the cellular energy status. Nampt catalyses the conversion of nicotinamide to nicotinamide mononucleotide and is the rate-limiting enzyme in the NAD⁺ salvage pathway (Revollo et al., 2004; Garten et al., 2009). While intracellular Nampt is part of the NAD salvage pathway, extracellular Nampt acts as a pro-inflammatory cytokine and induces cardiac hypertrophy or adverse ventricular remodelling (Pillai et al., 2013). Intracellular Nampt is up-regulated by fasting (Yang et al., 2007b) or in vitro glucose restriction (Fulco et al., 2008), but reduced in the heart by I/R or pressure overload (Hsu et al., 2009). Cardiac-specific overexpression of Nampt reduces the infarct size after I/R, while down-regulation of Nampt significantly decreases NAD⁺ and ATP levels, increases apoptotic activation and reduces the autophagic flux in cardiomyocytes (Hsu et al., 2009). Thus, the molecular mechanisms by which Nampt protects the heart from ischaemic injury seem to involve a variety of signalling pathways in addition to SIRTs.

SIRT1

CR activates Sir2 by decreasing NADH levels in yeast, resulting in an increased lifespan (Lin et al., 2004). Similar results have been obtained in rodents undergoing CR, where higher NAD⁺ levels increased expression and activity of SIRT1, the mammalian Sir2 homologue (Rodgers et al., 2005). Increased expression of Sir2 in yeast extends lifespan, while Sir2 deletion shortens lifespan (Kaeberlein et al., 1999; Lin et al., 2000). Interestingly, CR does not extend lifespan of SIRT1 KO mice (Boily et al., 2008). Loss of SIRT1 activity leads to dilated cardiomyopathy in adult hearts associated with altered acetylation of different isoforms of myocyte enhancer factor 2 transcription factors and with major mitochondrial morphological and functional disturbances, where the latter may actually occur before the onset of dilated cardiomyopathy (Planavila et al., 2012). Furthermore, SIRT1 is necessary for the induction of starvation-induced autophagy (Lee et al., 2008; Morselli et al., 2010) and required for the lifespan-prolonging effects of CR and resveratrol, a pharmacological activator of SIRT1 (Morselli et al., 2010). SIRT1 is also involved in the CR-mediated increase in mitochondrial biogenesis, as PGC-1α deacetylation occurs via SIRT1 in vitro as well as in vivo during fasting (Rodgers et al., 2005; Gerhart-Hines et al., 2007).

Shinmura et al. (2008) provided evidence that the increase in nuclear SIRT1 is critically involved in the cardioprotection against ex vivo I/R injury conferred by 6 months of CR in middle-aged rats. The nuclear SIRT1 increase is NO-dependent and chronic NOS inhibition prevents not only the SIRT1 translocation but also the CR-induced cardioprotection (Shinmura et al., 2008). Accordingly, myocyte-specific SIRT1 overexpression inhibits I/R injury while cardiac loss of SIRT1 enhances I/R injury (Hsu et al., 2010). The authors suggested that cardioprotection against I/R injury occurs mainly via long-term transcriptional effects on cardioprotective or apoptotic genes. Mild to moderate cardiac overexpression of SIRT1 (2.2–7.5-fold) attenuates age-dependent increases in hypertrophy, apoptosis/fibrosis, cardiac dysfunction, retards cardiac ageing and protects against oxidative stress through FOXO-dependent catalase expression, elevated cellular ATP levels, and increased mitochondrial citrate synthase activity (Alcendor et al., 2007). However, high levels of SIRT1 (12.5-fold overexpression) result in the development of cardiomyopathy (Alcendor et al., 2007). Another group also generated mice with different levels of cardiac SIRT1 overexpression (Kawashima et al., 2011). They reported that moderate, 6.8-fold, overexpression of SIRT1 results in impaired diastolic function, but preserved systolic function in young mice. Interestingly, major metabolic changes were observed in these hearts with reduced fatty acid uptake, but increased glucose uptake, degenerated mitochondria, reduced mitochondrial respiration, mitochondrial gene expression and reduced mitochondrial ROS production (Kawashima et al., 2011). However, even mild overexpression of SIRT1 (3.2-fold) caused early cardiac dysfunction after pressure overload. All the observed cardiac changes demonstrate a clear dependency on the gene dose of SIRT1. Furthermore, it appears that modulation of SIRT1 activity in heart is context-dependent. An increase in lysine deacetylation has been observed following in vitro and in vivo IPC in cellular models and in mouse hearts, which occurred concurrent with an increase in SIRT1 activity (Nadtochiy et al., 2011a). Direct inhibition of SIRT1 as well as the reduction of NAD⁺ levels reverse lysine deacetylation and inhibit IPC-induced cardioprotection. SIRT1(+/−) hearts cannot be preconditioned and exhibit higher cytosolic lysine acetylation (Nadtochiy et al., 2011b). However, infusion of the SIRT1 activator SRT1720 alone does not elicit cardioprotection, suggesting that SIRT1 is necessary, but not sufficient for the cardioprotective effects of IPC (Nadtochiy et al., 2011a). Pharmacological inhibition or down-regulation of SIRT1 in cardiomyocytes/.cardiomyoblasts induces apoptosis because of overactivation of p53 (Alcendor et al., 2004; Passariello et al., 2011). Furthermore, a signalling cascade involving p53, the farnesoid X receptor and miR34a participates in the regulation of SIRT1 in age- and obesity-related diseases (Lee and Kemper, 2010).

SIRT3

CR increases the expression of SIRT3 (Shi et al., 2005; Palacios et al., 2009) and mitochondrial NAD⁺ levels (Nakagawa et al., 2009), it decreases the level of acetylation of mitochondrial proteins and increases mitochondrial SIRT activity, associated with an increased post-ischaemic state 3 respiration and reduced post-ischaemic ROS production (Shinmura et al., 2011b). Interestingly, the protective effects of CR on oxidative damage are diminished in SIRT3 KO mice, an effect involving deacetylation of critical lysine residues on manganese superoxide dismutase (MnSOD) as shown by two independent groups (Qiu et al., 2010; Tao et al., 2010). Therefore, SIRT3 up-regulation during CR (Shi et al., 2005; Palacios et al., 2009) or through interventions leading to a higher SIRT3 expression and activity may promote this cardioprotective potential. Indeed, among the different SIRTs, SIRT3 is the only protein whose increased expression has been directly linked to an increased lifespan in humans. Increased expression of SIRT3 due to a polymorphism in the promoter is associated with longevity in humans, suggesting that SIRT3 is involved in genetic control of lifespan (Bellizzi et al., 2005). However, a more recent larger study did not show a strong association between SIRT3 polymorphism and longevity (Lescai et al., 2009).

SIRT3 expression in the heart, which utilizes more ATP than many other organs, is high and the hearts of SIRT3-deficient mice show a strong reduction in ATP levels, suggesting that SIRT3 is an important regulator of cellular ATP content (Ahn et al., 2008). KO of SIRT3 results in elevated levels of acetylation of mitochondrial proteins in various tissues (Lombard et al., 2007; Ahn et al., 2008). SIRT3 influences a variety of proteins involved in oxidative phosphorylation. In particular, components of complex I of the ETC demonstrate increased acetylation in SIRT3 KO mice and SIRT3 physically interacts with the complex I subunit NDUFA9, increasing its activity in vitro (Ahn et al., 2008). The complex II subunit SdhA (succinate dehydrogenase complex, subunit A) is also a substrate for SIRT3 and deacetylation results in increased complex II activity (Cimen et al., 2010; Finley et al., 2011). Finally, SIRT3 may influence respiratory chain activity via deacetylation of cyclophilin D (CypD) at lysine 145. Subsequently, the reduced peptidyl-prolyl cis-trans isomerase activity of CypD induces dissociation from ANT1 (adenine nucleotide translocator) and thereafter promotes the dissociation of hexokinase II from the mitochondria, resulting in an increase in oxidative phosphorylation (Shulga et al., 2010). SIRT3 also reduces oxidative damage by modulating the activity of the mitochondrial IDH2, which acts as a source of electrons for cellular antioxidants, through deacetylation (Someya et al., 2010). This IDH2 deacetylation occurs in response to CR in various organs, but cardiac tissue or mitochondria were not investigated so far (Someya et al., 2010). SIRT3 also acts as an endogenous negative regulator of cardiac hypertrophy by activating MnSOD and catalase, thereby supressing cellular ROS levels and downstream signals involved in cardiac hypertrophic response (Sundaresan et al., 2009).

SIRT3 deacetylates CypD also on lysine 166, adjacent to the binding site of the CypD inhibitor cyclosporine A, thus reducing mPTP opening and flux through the pore (Hafner et al., 2010). SIRT3 KO mice exhibit an age-dependent increase in mitochondrial swelling, because of increased mPTP opening, and signs of accelerated cardiac ageing (Hafner et al., 2010). Interestingly, SIRT3 does not seem to influence the structurally related cyclophilins A and B, which is an important secondary finding and may be valuable for any treatment of cardiac diseases involving SIRT3 without affecting immune functions. Furthermore, SIRT3 was demonstrated to deacetylate and activate LKB1, resulting in an AMPK-meditated inhibition of cardiac hypertrophy in vitro as well as in vivo (Pillai et al., 2010). The authors also show that pathological cardiac hypertrophy is characterized by low cellular NAD⁺ levels. Exogenous supplementation of NAD⁺ normalizes these levels and blocks pathological hypertrophy through activation of SIRT3 but not SIRT1 (Pillai et al., 2010). SIRT3 is also directly involved in the regulation of apoptotic cell death in cardiomyocytes. The pro-apoptotic protein Bcl2-associated X protein (Bax) translocates from the cytosol to the mitochondrial membranes during the early phase of apoptosis, a process prevented through complex formation of Bax with Ku70. Acetylation of Ku70 results in the release of Bax, thus enabling mitochondrial translocation. In cardiomyocytes, SIRT3 effectively deacetylates Ku70, resulting in anti-apoptotic effects (Sundaresan et al., 2008). Many of these studies clearly suggest that SIRT3 represents a survival factor for cardiomyocytes under stress conditions.

SIRT2, 4, 5, 6, 7

Much less data are available on the role of other SIRTs in the heart as well as in response to CR. A CR-induced up-regulation of SIRT2 in adipose tissue and kidney has been demonstrated in mice (Wang et al., 2007). SIRT2 reduces the acetylation of FOXO3a and increases the expression of FOXO target genes such as MnSOD, enabling a better ROS defence in vitro (Wang et al., 2007). In contrast to SIRT3, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial SIRTs, SIRT4 and SIRT5 (Lombard et al., 2007). SIRT4 has no identified substrate so far and may only show ADP-ribosyltransferase activity. SIRT4 binds ANT (Ahuja et al., 2007) and SIRT5 can deacetylate cytochrome c (Schlicker et al., 2008), although the biological significance of these interactions is as yet not known. In contrast to SIRT1, which stimulates the resveratrol-induced, glucose-stimulated insulin secretion (Vetterli et al., 2011), SIRT4 represses insulin secretion (Haigis et al., 2006; Ahuja et al., 2007). Knockdown of SIRT4 results in increased FAO, expression of genes involved in FAO and cellular respiration (Nasrin et al., 2010). SIRT4 may thus counteract some of the SIRT1 or SIRT3 metabolic effects and putatively also oppose some of the metabolic effects of CR.

SIRT6, which is highly expressed in the heart and brain (Mostoslavsky et al., 2006) and reduced in human failing hearts (Sundaresan et al., 2012), was first shown to be a nuclear, chromatin-associated protein that is important for DNA repair, genomic instability and chromatin silencing at telomeres (Mostoslavsky et al., 2006; Tennen et al., 2011). Although there are no data on the effects of CR on cardiac SIRT6 expression or function, the expression of SIRT6 increases in the ovary after moderate CR, which may have an impact on the reserve of germ cells (Luo et al., 2012). In the CVS, SIRT6 delays vascular ageing through protection from telomere and genomic DNA damage (Cardus et al., 2013) and to inhibit angiotensin II-induced hypertrophy in cardiomyocytes (Cai et al., 2012; Yu et al., 2013). Loss of SIRT6 leads to accelerated ageing, ageing-associated degenerative abnormalities and premature death in mice (Mostoslavsky et al., 2006). Furthermore, SIRT6-deficient mice more rapidly develop heart failure, whereas SIRT6 transgenic mice are protected from hypertrophic stimuli through attenuation of the IGF-Akt signalling pathway (Sundaresan et al., 2012). Recently, it was shown that SIRT6 overexpression extends lifespan only in male transgenic mice (Kanfi et al., 2012).

SIRT7 KO mice demonstrate reduced lifespan and develop cardiac hypertrophy with increased inflammation, apoptosis, lipofuscin accumulation and fibrosis, characteristics also known for the ageing heart (Vakhrusheva et al., 2008). Interestingly, cardiac SIRT7 levels indeed decrease with age (Vakhrusheva et al., 2008). SIRT7 deacetylates p53 in vitro (Vakhrusheva et al., 2008). Accordingly, myocytes from SIRT7 KO mice show a hyperacetylation of p53 and increased rate of apoptosis (Vakhrusheva et al., 2008). SIRT7 myocytes are more susceptible to oxidative stress (Vakhrusheva et al., 2008), which is suggestive for a role of this SIRT also in I/R, although this has not yet been investigated. There seem to be overlapping functions of SIRT1 and SIRT7 in the heart. Both, SIRT7 and SIRT1, protect cardiomyocytes from apoptosis through p53 deacetylation (Alcendor et al., 2004; Vakhrusheva et al., 2008) and higher levels of acetylated p53 have been shown to occur in diseased human myocardium together with reduced activity of SIRT7 and SIRT1 (Alcendor et al., 2004; Pillai et al., 2005; Vakhrusheva et al., 2008).

Role of NO

CR for either 3 or 12 months induces NOS expression and cGMP formation in various tissues of male mice (Nisoli et al., 2005; Cerqueira et al., 2011; 2012b). This is accompanied by mitochondrial biogenesis, with increased oxygen consumption and ATP production, and an enhanced expression of SIRT1 (Nisoli et al., 2005). SIRT1 in turn plays a fundamental role in regulating endothelial NO and endothelium-dependent vascular tone by deacetylating eNOS and thus increasing NOS activity (Mattagajasingh et al., 2007). CR reverses obesity-induced endothelial dysfunction and reduces uncoupling of eNOS and thereby oxidative stress (Ketonen et al., 2010). Short-term CR also reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NOX-mediated superoxide production and stimulation of anti-oxidant enzyme activity) (Rippe et al., 2010), and normalizing the inducible NOS (iNOS)/eNOS ratio (Zanetti et al., 2010). Short-term CR alters BP in spontaneously hypertensive rat (SHR) via stimulation of an adiponectin/AMPK/eNOS signalling axis and may serve as an effective non-pharmacological treatment of hypertension (Dolinsky et al., 2010). Similarly, CR improves endothelial function in obese, hypertensive patients and enhances the response of forearm blood flow to ACh. The i.a. infusion of N^G-monomethyl-L-arginine (L-NAME, 8 mmol·min⁻¹), a NOS inhibitor, decreases the enhanced ACh-induced blood flow response induced by CR, again pointing to the involvement of NOS (Sasaki et al., 2002).

In addition to direct vascular effects, NO might also contribute to angiogenesis. In wild-type mice CR improves revascularization of ischaemic limbs and stimulates the phosphorylation of eNOS in the ischaemic limbs. Administration of the AMPK inhibitor compound C abolishes the CR-induced increase in limb perfusion and eNOS phosphorylation in wild-type mice (Kondo et al., 2009). CR also reduces infarct size following I/R, an effect completely blocked by NOS inhibition using L-NAME (Shinmura et al., 2008). Similar to CR, the adipocytokine adiponectin protects from myocardial contractile dysfunction and limits infarct size following I/R by a mechanism involving activation of AMPK and production of NO (Tao et al., 2007; Gonon et al., 2008).

Role of AMP-activated PK

AMPK is a heterotrimeric complex (see Alexander et al., 2013c), which consists of the catalytic α-subunit with a phosphorylation site at Thr¹⁷² and the two regulatory subunits β and γ. Upstream kinases, which can phosphorylate and thus activate AMPK, include LKB1, calcium/calmodulin-dependent PK kinase (CAMKKβ) and possibly also TGF-β-activating kinase 1 (TAK1). In mammalian cells, the activity of AMPK depends on the ratio between AMP and ATP and a very small rise in AMP can induce an allosteric activation of AMPK (Hardie, 2004). AMPK senses any circumstances that cause energy deficiency including starvation, hypoxia, ischaemia, glucose deprivation, metabolic poisons, oxidative and hyperosmotic stress. Whenever AMPK is activated, ATP-generating catabolic pathways such as FAO and glycolysis are switched on, while ATP-consuming anabolic pathways such as fatty acid synthesis, protein synthesis and cholesterol synthesis are turned off.

It is conceivable that adaptive responses to CR can be triggered by mechanisms that are able to sense and respond to changes in nutrient availability. AMPK acts as such a key nutrient sensor and is therefore thought to be a major regulator of the beneficial effects of CR (Hardie, 2004). The most conclusive genetic evidence supporting an important role for AMPK activation in the effects of CR on longevity is derived from studies performed in C. elegans (Apfeld et al., 2004; Greer et al., 2007a). Among the potential mediators of the protective effects of AMPK and thus CR are SIRT1, the FOXO transcription factors, PGC-1α and mTOR. AMPK activation promotes an increase in cellular NAD⁺ levels, thus increasing the activity of SIRT1 (Fulco et al., 2008; Canto et al., 2009). Furthermore, AMPK phosphorylates PGC-1α, increasing its activity and enabling deacetylation by SIRT1 (Canto et al., 2009). The CR-induced, AMPK-mediated phosphorylation of FOXO3a and consecutively increased transcriptional activity appears to be important for the regulation of genes involved in the control of energy balance and stress resistance (Greer et al., 2007b). Genetic reduction of the nutrient-responsive mTOR signalling results in increased lifespan, resistance to age-associated pathologies and gene expression patterns observed after CR or pharmacological AMPK activation (Selman et al., 2009). In addition, the complex network of AMPK, SIRT1, FOXO and mTOR is also involved in the control of autophagy/mitophagy (Lee et al., 2008; Sengupta et al., 2009; Canto et al., 2010; Hariharan et al., 2010; Egan et al., 2011; Kim et al., 2011) as described earlier. Previous studies suggest a strong activation of AMPK in the heart after long-term CR (Edwards et al., 2010; Niemann et al., 2010) and after short-term CR in mice and rats (Shinmura et al., 2005; 2008), while obesity or high nutrient supply induce reduced AMPK activation and downstream signalling (Niemann et al., 2011; 2013). Short-term CR results in a chronic AMPK activation before ischaemia and in reduced infarct size and improved LV function following ex vivo I/R. These protective effects are abolished by AMPK inhibition in aged hearts (Edwards et al., 2010). However, others have also reported that 24 h of fasting or 4 months of CR did not result in a significant increase in AMPK activation in various mouse organs including the heart (Gonzalez et al., 2004). Similarly, while Edwards et al. reported that AMPK plays a major role in protecting the aged heart from I/R injury after long-term CR (Edwards et al., 2010), others (Shinmura et al., 2008) provided evidence that long-term CR-induced cardioprotection occurs without changes in AMPK activation (Shinmura et al., 2008).

Whether or not cardiac AMPK activation during I/R is indeed beneficial has been explored in different mouse models of AMPK deficiency. Studies in isolated hearts have demonstrated that AMPK deficiency results in a more severe injury after I/R (Xing et al., 2003; Russell et al., 2004; Carvajal et al., 2007) and experimental data even suggest that a stronger AMPK activation in acute ischaemia results in a stronger reduction in infarct size (Nishino et al., 2004). Transgenic mice expressing a kinase dead (KD) form of AMPKα2 fail to augment glucose uptake and glycolysis during ischaemia and FAO during reperfusion. Furthermore, these hearts demonstrate significantly impaired contractile recovery during reperfusion and increased cardiac injury compared with wild-type hearts (Russell et al., 2004). AMPK was shown to be responsible for activation of glucose uptake and glycolysis and to limit cardiac damage by reducing apoptotic activity during low-flow ischaemia and reperfusion in these experiments (Russell et al., 2004). Another study using mice overexpressing a dominant negative AMPKα2 found no impairment in energetic status or cardiac recovery during reperfusion in these hearts in the presence of fatty acids (Folmes et al., 2009). Furthermore, in the presence of insulin, which inhibits AMPK activation and FAO, these hearts demonstrate a better functional recovery than wild-type mice, suggesting that AMPK inhibition is actually beneficial (Folmes et al., 2009). However, the KD mutation almost completely abolishes AMPKα2 activity and also substantially blunts AMPKα1 activity even under baseline conditions (Russell et al., 2004), while the dominant negative AMPKα2 mice demonstrate a more moderate reduction in AMPK activity (Folmes et al., 2009). In contrast, AMPKα2 KO mice are characterized by a higher sensitivity to ischaemic contracture, but their post-ischaemic contractility is similar to wild-type mice in the absence of fatty acids although delayed in the presence of oleate (Carvajal et al., 2007). The latter conditions may represent a setting more closely resembling the in vivo situation, which is also in agreement with deleterious effect of free fatty acids during reperfusion (Lopaschuk et al., 1993; Liu et al., 2002). The higher susceptibility to development of ischaemic contracture in the AMPKα2 KO hearts is most likely related to their low glycogen stores and decreased glucose uptake (Carvajal et al., 2007). In general, these studies using three different mouse models of AMPK deficiency suggest that the role of AMPK during I/R is also dependent on substrate availability and the possible opposing effects of AMPK on fatty acid and glucose metabolism.

Ischaemic tolerance decreases with age (Frolkis et al., 1991; Ataka et al., 1992; Tani et al., 1997), and many cardioprotective interventions including ischaemic pre- and postconditioning are less effective in aged animals and in elderly patients (Abete et al., 1996; Schulman et al., 2001; Lee et al., 2002; Boengler et al., 2008; 2009a). Interestingly, short- and long-term CR improves ischaemic tolerance even in aged animals (Shinmura et al., 2005; 2008; Edwards et al., 2010). In general, ischaemic AMPK activation is significantly lower in aged myocardium (Ma et al., 2010). AMPK activation by IPC not only contributes to reduced infarct size, but also to a better metabolic adaptation of the post-ischaemic myocardium, possibly via increased glucose uptake during reperfusion (Baldwin et al., 2002; Kristiansen et al., 2009), and mechanisms involving PKC-dependent translocation of the glucose transporter type 4 to the plasma membrane (Nishino et al., 2004). A recent study shows that CR significantly improves cardiac recovery following ischaemia, which is associated with a higher rate of glucose oxidation during reperfusion compared with controls (Sung et al., 2011). This improved energy supply also results in higher ATP content and lower AMPK activation at the end of reperfusion. While AMPK activation is decreased, CR results in a higher phosphorylation of Akt and ERK1/2 at the end of reperfusion, suggesting an involvement of the reperfusion injury salvage kinase (RISK) pathway (Sung et al., 2011). Both signalling pathways have previously been shown to be necessary in preconditioning-induced cardioprotection (Hausenloy et al., 2005). A concise summary on mechanisms and mediators of cardioprotection conferred by CR is presented in Figure 1.

Figure 1.

Mechanisms and mediators of cardioprotection conferred by CR because of altered expression (E) or phosphorylation (P) resulting in inhibition () or activation (→) of downstream targets.

Role of adiponectin and the adiponectin paralogues

Many investigations try to address the question about physiological AMPK activators during I/R and the cardioprotection conferred by CR. Among the candidates are adipocytokines such as adiponectin, which has repeatedly been shown to be increased after CR (Shinmura et al., 2007; Zhu et al., 2007; Niemann et al., 2008; 2010; Wan et al., 2010; Ding et al., 2012). The protective effects of short-term CR appear to be mediated by a marked increase in circulating high molecular weight complexes of adiponectin. Accordingly, recombinant adiponectin is able to restore the CR-induced improvement of myocardial ischaemic tolerance in adiponectin-deficient mice (Shinmura et al., 2007). However, inhibition of AMPK completely prevents the CR-induced cardioprotection as deduced from the recovery of LV-developed pressure and reduced infarct size in wild-type mice (Shinmura et al., 2007). While IPC reduces infarct size and results in higher adiponectin plasma levels, also after 6 months of CR, myocardial AMPK phosphorylation is not altered (Shinmura et al., 2008). However, this does not exclude the possibility that an initial AMPK activation is necessary for long-term cardioprotective CR effects. Three months of intermittent fasting, which has been suggested to be as effective as continuous, steady CR, also result in increased circulating adiponectin and cardioprotection against ischaemic injury in rats (Wan et al., 2010).

Adiponectin activates AMPK, resulting in a stimulation of glucose utilization and FAO (Yamauchi et al., 2002). Activation of the AMPK pathway by adiponectin mediates antihypertrophic effects in cardiomyocytes (Chan et al., 2004; Shibata et al., 2004a) and also plays an important role in the improved functional LV recovery and reduction of infarct size after I/R in mice (Shibata et al., 2005; Shinmura et al., 2007). Adiponectin-deficient mice, in contrast, show a more pronounced I/R injury, while exogenous adiponectin results in cardioprotection (Shibata et al., 2005; Tao et al., 2007). Adiponectin deficiency increases TNF-α production, iNOS expression, ROS release by NOX and formation of peroxynitrite in the I/R myocardium (Tao et al., 2007). Adiponectin-mediated activation of AMPK results mainly in an inhibition of apoptosis while the inhibitory effects on TNF-α production appeared to be AMPK-independent in one of these studies (Shibata et al., 2005). The protective effects of adiponectin during I/R do not only involve the myocardium and cardiomyocytes, but also the vasculature and endothelial cells. Angiogenic repair of ischaemic hind limbs is severely impaired in adiponectin-KO mice (Shibata et al., 2004b). Adiponectin promotes angiogenesis mainly through AMPK-dependent mechanisms (Shibata et al., 2004b; 2005). Adiponectin deficiency accelerates the transition from cardiac hypertrophy to heart failure following pressure overload, a mechanism induced through inhibition of AMPK-dependent VEGF induction and microvessel formation (Shimano et al., 2010). Furthermore, the CR-induced stimulation of revascularization in response to ischaemia critically involves the adiponectin-induced, AMPK-mediated activation of eNOS as shown in adiponectin- and eNOS-deficient mice (Kondo et al., 2009). Short-term CR also improves flow-mediated vasodilatation and increases eNOS activity and NO bioavailability in spontaneously hypertensive rats (Dolinsky et al., 2010). These effects are associated with elevated levels of circulating adiponectin and enhanced AMPK activity in mesenteric arteries, suggesting an involvement of the adiponectin/AMPK/eNOS signalling axis in the vasoprotective effects of CR (Dolinsky et al., 2010). Accordingly, administration of a NOS inhibitor abolishes the protective effects of adiponectin on infarct size and LV function following I/R (Gonon et al., 2008). Mice with cardiomyocyte-specific overexpression of a dominant negative AMPKα2 demonstrate larger infarcts, reduced LV function associated with a higher superoxide formation and increased apoptosis compared with wild-type mice after ischaemia/reperfusion (Wang et al., 2009). Exogenous adiponectin reduces infarct size, apoptotic activation and ROS production in both groups of mice similarly, suggesting AMPK-independent protective effects of adiponectin (Wang et al., 2009).

Recently, a family of structural and functional adiponectin paralogues, comprising 15 members so far, was discovered and designated as C1q/TNF-α-related proteins (CTRPs; Wong et al., 2004; 2008; 2009; Peterson et al., 2009; 2010; Wei et al., 2011; 2012a,b; Seldin et al., 2012). CTRPs are widely expressed and may exert their biological effects in a paracrine or autocrine fashion (Wong et al., 2004). Although adiponectin was described to be secreted by cardiomyocytes and to protect against I/R injury (Pineiro et al., 2005; Wang et al., 2010), these locally produced and secreted CTRPs may emerge as major mediators of AMPK-mediated cardioprotection in the heart in addition to the fat cell-derived high amounts of adiponectin. The CTRPs are predicted to be secreted proteins and may form heteromultimers with adiponectin in vivo, but they do not circulate in as high concentrations in blood as adiponectin (Wong et al., 2004). Ageing is associated with a reduction in plasma adiponectin and this reduction can only partially be overcome by a higher degree and duration of CR (Niemann et al., 2008). The adiponectin paralogues CTRP2 and CTRP7, which were among the first described CTRPs, cannot sufficiently compensate the loss of adiponectin in aged animals (Rohrbach et al., 2007). Putatively, other CTRPs could locally compensate in situations of adiponectin deficiency such as obesity or ageing. One of the most relevant CTRPs in terms of cardioprotective effects appears to be CTRP9. Cardiac expression of CTRP9 exceeds adiponectin by more than 100-fold (Su et al., 2013). CTRP9, which is most probably released by the cardiomyocyte itself, reduces myocardial infarct size and cardiomyocyte apoptosis following I/R. These effects appear to be mediated via CTRP9-induced activation of the adiponectin receptor-1 (Adipo1 receptor)-AMPK-signalling pathway (Kambara et al., 2012). Furthermore, CTRP9 administration significantly attenuates NOX expression and superoxide generation, reduces infarct size, and improves cardiac function in diabetic mice (Su et al., 2013). Both conditions, high-fat diet-induced diabetes and I/R injury, result in a strongly reduced CTRP9 expression in vivo (Kambara et al., 2012; Su et al., 2013). Recently, the adipose-derived CTRP3 was also demonstrated to act as an anti-apoptotic, pro-angiogenic and cardioprotective adipokine in a mouse model of myocardial infarction (Yi et al., 2012). CTRP3 improves survival and restores cardiac function after myocardial infarction and attenuates post-ischaemic pathological remodelling, which involves Akt-HIF-1α-VEGF signalling (Yi et al., 2012). The influence of CR on CTRP9 expression in the heart or CTRP3 expression in adipose tissue has not been studied so far.

Drug targets to mimic CR

It is unlikely that CR will be widely adopted because of the difficulty in maintaining long-term CR in modern society and because of potential side effects such as hypotension, infertility, bone thinning and osteoporosis, cold sensitivity, loss of strength and sarcopoenia, slower wound healing, depression, and emotional deadening. Therefore, efforts have been made to develop pharmacological agents to mimic CR as summarized in Figure 2. Such agents, which have been termed CRM, could provide the beneficial metabolic, hormonal and physiological effects of CR without altering dietary intake (Ingram et al., 2006).

Figure 2.

Possible drug targets to mimic the cardioprotective effects of CR and their mechanisms of action.

2-Deoxyglucose (2-DG)

2-DG was the very first agent that was formally investigated as a putative CRM (Lane et al., 1998) via inhibition of the glycolytic pathway. 2-DG is a synthetic analogue of glucose in which a hydroxyl group at position C2 is replaced with a hydrogen atom. It enters the normal glycolysis pathway, however, inhibits the activity of phosphoglucose isomerase, thereby, blocks glycolysis at the second step. With this property, 2-DG could induce the CR-like physiological phenotype in animal models. Feeding rats with low to moderate doses (0.2–0.4%) of 2-DG for up to 7 days lowers the serum insulin and glucose levels, lowers heart rate, basal BP and circulating free fatty acids without affecting the daily food intake. Long-term administration of 2-DG every second day decreases resting BP and heart rate and markedly enhances cardiovascular adaptation to stress (Wan et al., 2004). Likewise, 24 h incubation of neonatal rat cardiomyocytes with 2-DG protects the myocytes against doxorubicin-induced toxicity (Chen et al., 2011). However, feeding of rats with 0.4% 2-DG every second day for 6 months results in premature death (Wan et al., 2003), most likely by vacuolization of the heart, impaired cardiac function, atrial thrombosis and lung and liver congestion (Minor et al., 2010). The toxicity of 2-DG has been related to increased ER stress and is independent of metabolic inhibition. A striking difference between CR and 2-DG ingestion is that CR reduces the expression of the ER stress marker glucose regulated protein 78 while 2-DG ingestion induces it, thereby leading to autophagy – a main cause of its cardiac toxicity (Minor et al., 2010). Although increasing data show that long-term use of 2-DG as a CRM results in cardiac toxicity, a short-term use is protective against apoptosis in both endothelial cells and cardiomyocytes. Clearly, more studies are required to determine whether or not short-term use of 2-DG can be used to pharmacologically precondition the heart.

Metformin

Metformin is a member of biguanides and is the drug of choice for the treatment of type-2 diabetes. Metformin reduces blood glucose levels by decreasing hepatic glucose production and increasing insulin sensitivity in muscular tissue at least in part via activation of LKB1/AMPK signalling (Zhou et al., 2001; Shaw et al., 2005). In addition to its use in diabetes, recent animal studies and clinical trials suggest that the drug has therapeutic potential in other clinical disorders including cardiovascular diseases. Acute administration of metformin before ischaemia or at the onset of reperfusion reduced infarct size in isolated rat hearts (Legtenberg et al., 2002; Bhamra et al., 2008; Calvert et al., 2008). Although administration of a single dose of metformin reduces infarct size after 24 h, this dose does not prevent development of severe cardiomyopathies after 4 weeks. However, cardiomyopathy is prevented when metformin is administered daily for 4 weeks, showing its efficacy in post-infarction cardiac remodelling (Gundewar et al., 2009). Similarly, administration of metformin in murine models of permanent left coronary artery ligation and acute or chronic I/R improves cardiac and mitochondrial function, effects ablated in mice lacking functional AMPK (Calvert et al., 2008; Gundewar et al., 2009). Chronic intake of metformin for 30 days reduces infarct size and elicits cardioprotection both in diabetic and non-diabetic rats (Whittington et al., 2013) and preserves LV ejection fraction. Similarly, metformin improves cardiac function in a ventricular pacing model of heart failure in dogs (Sasaki et al., 2009), and it reduces acute graft rejection and apoptosis in cardiac allografts in a murine model of heart transplantation, when given daily for 4 weeks post-surgery (Chin et al., 2011). It must be noted, however, that metformin does not provide cardioprotection in all models of cardiomyopathy, that is, metformin does not preserve cardiac structure and function in a volume overload-induced model of heart failure (Benes et al., 2011). Beyond its specific cardioprotective effect, metformin improves endothelial function via activation of eNOS (Davis et al., 2006), inhibits platelet aggregation (Gin et al., 1989) and ROS production in endothelial cells (Ouslimani et al., 2005).

Metformin directly interacts with complex I of the mitochondrial respiratory chain and inhibits its activity, thus shutting off the ATP generation along with reduction in mitochondrial ROS production (El-Mir et al., 2000; Owen et al., 2000). This lowers the ATP/AMP ratio, thus activating the LKB1/AMPK pathway. Additionally, metformin inhibits the opening of MPTP and protects mitochondria against swelling, effects involved in cardioprotection against reperfusion injury (Bhamra et al., 2008). This effect was suggested to be mediated via PI3K activation (Bhamra et al., 2008). The metformin-induced cardioprotective effect is dependent at least in part on AMPK activation (Calvert et al., 2008; Gundewar et al., 2009), and part of its protective effect is lost in AMPKα2-dominant negative transgenic mice. Similarly, low-dose metformin was able to activate AMPK in isolated rat hearts and co-perfusion with the AMPK inhibitor compound C attenuates the protective effects of metformin (Paiva et al., 2010). Recently, long-term treatment with low-dose metformin (0.1% w w⁻¹ in diet) was shown to extend lifespan in male mice, while a higher dose (1% w w⁻¹ in diet) was toxic (Martin-Montalvo et al., 2013). Chronic metformin treatment increases AMPK activity and antioxidant protection (Martin-Montalvo et al., 2013). Metformin also induces eNOS phosphorylation and its cardioprotective effect is lost in eNOS KO mice. In contrast to the studies mentioned earlier, Bhamra et al. (2008) showed that acute administration of metformin protects rat hearts against reperfusion injury without activating AMPK, but instead Akt phosphorylation is increased following metformin administration; protection of rat hearts is lost following PI3K inhibition (Solskov et al., 2008). Finally, metformin induces the release of adenosine from cardiac tissue and a non-specific adenosine receptor antagonist attenuated metformin-mediated cardioprotection (Paiva et al., 2009). Thus, metformin exerts its beneficial effects via multiple molecular targets, potentially depending on the animal model used. Other drugs or xenobiotics that have been described to activate AMPK, and may thus also mimic some of the CR effects are the nucleoside aminoimidazole carboxamide ribonucleotide (AICAR), thiazolidindiones, resveratrol, capsaicin, berberine and quercetin (Hardie, 2011). Some of these pharmacological AMPK activators have been tested with regard to their efficiency in limiting the I/R injury. Ex vivo models have shown that AMPK activation by AICAR results in a reduction in infarct size in young hearts, an effect attenuated by AMPK inhibition (Paiva et al., 2010). However, AMPK activation by AICAR failed to reduce infarct size in rabbits (Dorion et al., 1992). Ischaemia/reperfusion injury is an important cause of morbidity and mortality after coronary artery bypass graft (CABG) surgery. In a meta-analysis of five studies, using the adenosine-regulating agent and AMPK activator acadesine, the use of acadesine correlated with significant reductions in the occurrence of perioperative myocardial infarction, cardiac death and reduction in a composite of cardiac death, stroke and myocardial infarction (Mangano, 1997). However, in a randomized, double-blind, placebo-controlled trial (RED-CABG study: Reduction in Cardiovascular Events by Acadesine in Patients Undergoing CABG) acadesine did not reduce all-cause mortality, non-fatal stroke, or need for mechanical support in intermediate to high-risk patients undergoing CABG surgery (Newman et al., 2012).

Resveratrol

The polyphenol resveratrol is a potent activator of SIRT1, possibly through increasing its binding affinity to NAD⁺ and the acetylated substrate (Howitz et al., 2003). Resveratrol is a plant phenolic compound found in the skin of red grapes and other fruits, which has been shown to expand lifespan in yeast, worms and flies (Howitz et al., 2003; Wood et al., 2004). Notably, the life-expanding benefits of resveratrol have been reproduced (Bauer et al., 2004; Jarolim et al., 2004; Viswanathan et al., 2005; Yang et al., 2007a; Greer and Brunet, 2009) in all three organisms, although some negative studies exist as well (Kaeberlein et al., 2005; Bass et al., 2007). Supporting the latter finding, resveratrol does not extend lifespan in mice (Barger et al., 2008; Pearson et al., 2008), but was shown to improve insulin sensitivity and reduce mortality in obese mice (Baur et al., 2006). The ability of resveratrol to directly activate SIRT1 in vitro has recently been questioned (Borra et al., 2005; Kaeberlein et al., 2005; Beher et al., 2009; Pacholec et al., 2010), suggesting that resveratrol modulates SIRT1 activity by regulating other signalling cascades. Furthermore, although many effects of resveratrol are mediated via SIRT1-dependent pathways, there are also a number of SIRT1-independent mechanisms. Many pharmacological compounds that interfere with the mitochondrial respiratory chain are known activators of AMPK and have been used to mimic some of the CR effects. Resveratrol for example has an impact on mitochondrial respiratory chain by inhibiting ATP synthase (Zheng and Ramirez, 2000). Many metabolic effects of resveratrol, including increases in insulin sensitivity, mitochondrial biogenesis, physical endurance and elevation of the NAD/NADH ratio appear to be AMPK-dependent (Um et al., 2010). AMPK may thus serve as a convergent point for many CRM compounds including resveratrol and metformin.

Resveratrol also prevents cardiac hypertrophy in SHR both by reducing haemodynamic load (Dolinsky and Dyck, 2011) and by direct action on cardiomyocytes (Chan et al., 2008) and has been proven beneficial by reducing myocardial infarct size and improving post-ischaemic functional recovery (Ray et al., 1999; Kaga et al., 2005; Shen et al., 2006; Goh et al., 2007). The cardioprotective effects of resveratrol have been attributed to its capacity to induce adenosine release, enhance NO production (Bradamante et al., 2003), increase neovascularization via VEGF signalling (Kaga et al., 2005), its antioxidant activity (Hung et al., 2002; Shen et al., 2006) and anti-inflammatory effects (Shigematsu et al., 2003). Furthermore, resveratrol activates SIRT1 thereby inducing mitochondrial biogenesis (Price et al., 2012), it inhibits hypoxia-induced apoptosis via the SIRT1-FOXO1 pathway in H9C2 cardiomyoblasts (Chen et al., 2009a) and protects the heart from I/R injury by inducing autophagy (Gurusamy et al., 2010). Resveratrol provides significant cardioprotection during ex vivo I/R as evidenced by better post-ischaemic ventricular recovery, reduced infarct size, concentration-dependent protective actions on the vasculature and is associated with enhanced NO-signalling, Akt and p38 MAPK activation (Das et al., 2006; Goh et al., 2007). The survivor activating factor enhancement (SAFE) pathway, which requires the activation of STAT-3 and significantly reduces cardiomyocyte death at the time of reperfusion, is activated by resveratrol in isolated cardiomyocytes (Palomer et al., 2013) and mouse hearts during I/R (Lamont et al., 2011). The cardioprotective effects of resveratrol, at concentrations found in red wine, seem to depend on TNF-α and STAT-3, as resveratrol significantly reduces infarct size in wild-type mice, but fails to protect in TNFRSF1B (also know as TNF receptor 2; see Alexander et al., 2013b)-KO or STAT-3-KO mice after I/R (Lamont et al., 2011).

The expanding body of preclinical evidence for beneficial effects has encouraged the translational researchers to analyse resveratrol for potential clinical use. Several clinical trials are being carried out for potential use of resveratrol in different diseases (http://clinicaltrials.gov). Most of the available data are about the pharmacokinetic and safety profile of resveratrol (review in Patel et al., 2011). The major challenge is the bioavailability of resveratrol after oral administration. A phase I clinical study showed that after a single oral dose of 5 g, the peak plasma concentration reached approximately 0.53 μg·mL⁻¹ within 1.5 h (Boocock et al., 2007). Upon an oral dose, resveratrol is almost completely conjugated to its glucuronide and sulfate metabolites that show several fold higher plasma concentration compared with free resveratrol (Boocock et al., 2007; Brown et al., 2010). Whether these metabolites are therapeutically active, however, remains unknown. The bioavailability of resveratrol could be improved using micronized forms (SRT501, GlaxoSmithKline, Middlesex, UK), reaching plasma concentrations up to 2.2 μg·mL⁻¹ after a single 5 g daily dose (Howells et al., 2011). These high doses of resveratrol are generally well tolerated and show only mild adverse reaction such as diarrhoea, nausea and vomiting (Patel et al., 2011). The most severe adverse effects are observed with the micronized form of resveratrol (SRT501) in a phase II clinical study where the drug was administered to multiple myeloma patients (Popat et al., 2013). The study reported that 38 and 21% of the patients developed anaaemia and acute renal failure, and finally the study was halted following two deaths because of nephrotoxicity. In contrast to the high-dose strategy, recently, two studies reported cardiovascular protective effects with long-term use of very low dosages of resveratrol, although they did not analyse the plasma concentration of the drug. Administration of 10 mg resveratrol day⁻¹ to post-myocardial infarction, patients for 3 months reduces plasma low-density lipoprotein levels and platelet aggregation, and improves endothelial function (Magyar et al., 2012). In another 1 year follow-up of a study in patients undergoing primary prevention of cardiovascular disease, administration of 8 mg daily dose of resveratrol was reported to reduce plasma CRP and TNF-α concentrations and increase the adiponectin levels (Tome-Carneiro et al., 2012). Similarly, administration of 150 mg day⁻¹ for 30 days to healthy volunteers reduces metabolic rate, activates AMPK, induces the expression of SIRT1 and PGC-1α, enhances intramyocellular and reduces intrahepatic lipid levels (Timmers et al., 2011). Finally, resveratrol improves flow-mediated vasodilatation in patients with coronary heart disease (Timmers et al., 2011).

Other SIRT activators

A number of synthetic SIRT1-activating compounds (STACs), that are 1000 times more effective than resveratrol in vitro (Milne et al., 2007), such as SRT1720, SRT2104, SRT2183 and SRT2379, have been developed (Sirtis). Currently, several STACs have already entered clinical trials (see the ClinicalTrials.gov website; http://clinicaltrials.gov). SRT1720, one of the first synthetic SIRT1 activators developed, induces mitochondrial biogenesis via SIRT1-dependent activation of PGC-1α and increases the health span of obese mice on high-fat diet (Feige et al., 2008; Minor et al., 2011). However, its efficacy in metabolic disorders is still controversial (Huber et al., 2010; Pacholec et al., 2010). Although acute perfusion of SRT1720 reduces infarct size in aged as well as SIRT1^+/− mice following I/R (Tong et al., 2013), more work is needed to evaluate and confirm the beneficial effects of SRT1720 in vitro as well as in vivo. Although pharmacological strategies to influence SIRT3 activity are not yet on the market, there are good reasons for considering SIRT3 and its biological effects, as described earlier, as a promising new target for future therapeutic strategies. SIRT3 activation seems to provide beneficial effects in ageing, metabolic and cardiac diseases as well as neurological disorders. However, increasing SIRT3 activity might turn out to be a double-edged sword. Fasting-induced, SIRT3-mediated deacetylation of mitochondrial aldehyde dehydrogenase 2 was reported to be involved in exacerbated hepatotoxicity observed after paracetamol (Lu et al., 2011). However, SIRT3 KO mice are protected from paracetamol-induced hepatotoxicity, recommending at least caution regarding therapeutic strategies aimed at increasing SIRT3 activity (Lu et al., 2011).

Rapamycin and mTOR

Rapamycin (sirolimus) is a natural macrolide antibiotic obtained from Streptomyces hygroscopicus and commonly used as an immunosuppressant. The kinase mTOR is an important mediator of insulin- and growth factor-mediated signalling in multiple organs including the heart. Structurally, mTOR exists with other molecular components in two enzyme complexes, that is rapamycin-sensitive complex I (mTORC1) and rapamycin-insensitive complex 2 (mTORC2). Rapamycin is the best-characterized pharmacological autophagy inducer and inhibits mTOR by directly binding mTORC1.

Pharmacological or genetic inhibition of mTOR extends the lifespan in yeast, nematodes, flies and different mouse strains (Vellai et al., 2003; Jia et al., 2004; Kapahi et al., 2004; Harrison et al., 2009). Moreover, deletion of mTORC1 substrate S6K1 (ribosomal S6 kinase) is sufficient to confer increased lifespan in mice (Selman et al., 2009) and mice lacking S6K1 are protected from obesity and metabolic disorders (Um et al., 2004). Although the role of mTORC1-S6K1 inhibition in longevity is consistently demonstrated in animal models, little information is available about the role of mTORC1-S6K1 in age-associated cardiovascular diseases (Ming et al., 2012).

The most data in relation to cardiovascular disorders are available on pathological cardiac hypertrophy. In cultured cardiomyocytes, treatment with rapamycin attenuates angiotensin- and phenylephrine-induced hypertrophic response (Sadoshima and Izumo, 1995; Boluyt et al., 1997). A decrease in protein degradation because of inhibition of autophagy and an increase in protein synthesis upon stress stimulation by mTORC1-S6K1 signalling have been proposed as underlying mechanisms (Hands et al., 2009). In mice with pressure overload-induced cardiac hypertrophy, acute administration of rapamycin reduces cardiac growth without compromising cardiac function (Shioi et al., 2003), and it even reverses established hypertrophy (McMullen et al., 2004). Likewise, rapamycin treatment also attenuates thyroid hormone-induced cardiomyopathy (Kuzman et al., 2007). Similarly, partial genetic deletion of TOR in a heterozygous zebrafish model protects the animals against doxorubicin-induced cardiac toxicity and remodelling (Ding et al., 2011). In isolated mice hearts and in cultured cardiomyocytes, rapamycin protects against I/R-induced irreversible injury (Khan et al., 2006) and attenuates LV remodelling following myocardial infarction in rats (Buss et al., 2009). Rapamycin was suggested to confer cardioprotection against myocardial infarction through potent preconditioning-like effects involving the PI3K pathway, and opening of mitochondrial K_ATP channels (Khan et al., 2006), but appears to be independent of rapamycin-induced autophagy (Yang et al., 2010).

In contrast to rapamycin, which directly inhibits mTOR, AMPK activates the mTOR inhibitors TSC1/TSC2, thus indirectly inhibiting mTOR-S6K1 signalling (Shaw, 2009). In mammals, CR, which is known to activate AMPK, also suppresses mTOR signalling (Stanfel et al., 2009; Dogan et al., 2011; Shinmura et al., 2011a). The AMPK activators AICAR and metformin also attenuate pressure overload-induced cardiac hypertrophy (Li et al., 2007a; Fu et al., 2011). Conversely, reduced AMPK activity either by genetic deletion of AMPKα2 (Zhang et al., 2008) or lack of adiponectin (Shibata et al., 2004a) increases mTOR-S6K1 signalling and exacerbates cardiac hypertrophy in response to pressure overload. In contrast to the studies mentioned earlier demonstrating a consistent cardioprotection of mTOR inhibition, genetic ablation of mTORC1 signalling either in cardiac-specific KD mTOR transgenic mice or by deletion of raptor, a component of mTORC1 complex, induces dilated cardiomyopathy with enhanced autophagy, apoptosis and mitochondrial structural changes (Zhang et al., 2010; Shende et al., 2011). Interestingly, another group using the same transgenic mouse model (Shen et al., 2008) was unable to observe any protective or destructive effect of mTOR ablation on cardiac morphology or structure in response to exercise-induced hypertrophy and rapamycin again protected against hypertrophic growth (Shioi et al., 2002). Finally, cardiac-specific overexpression of mTOR in mice attenuates pressure overload-induced cardiac hypertrophy (Song et al., 2010) and protects the heart against irreversible injury following I/R and reduces mortality (Aoyagi et al., 2012). Thus, genetic and pharmacological studies differ in their results, and more basic research is needed to understand the underlying causes.

In the clinical arena, pharmacological inhibition of mTOR was proven beneficial. In a retrospective study in cardiac transplant patients receiving rapamycin, cardiac function was shown to be improved and LV mass was reduced compared with patients who were treated with a calcineurin inhibitor (CNI) over 1 year (Kushwaha et al., 2008). In a similar study in kidney transplant patients when CNI was replaced by rapamycin, a marked regression in LV hypertrophy was observed (Paoletti et al., 2008). A similar beneficial effect of CNI to rapamycin switching was also observed in patients who underwent cardiac transplantation (Raichlin et al., 2007). Overall, the current animal and clinical data are consistent in demonstrating some cardioprotective effects by pharmacological mTOR signalling inhibition. However, several challenges, particularly related to the adverse effects need to be addressed in clinical studies. A major adverse effect of chronic rapamycin therapy is glucose intolerance and insulin resistance (Houde et al., 2010; Deblon et al., 2012), causing a condition known as ‘starvation-like diabetes’ (Lamming et al., 2012). Thus, further analyses of the mTOR signalling pathway are required to potentially develop novel therapeutic approaches for the treatment of cardiomyopathies of different aetiologies.

Conclusions

CR has proven to be a valuable experimental tool for studying the process of ageing and its interaction with chronic diseases. Furthermore, results obtained from CR animals have also stimulated the search for drugs that mimic the beneficial metabolic, hormonal and physiological effects of CR without the potential side effects and without altering dietary intake. Evidence from animal studies and a limited number of human trials indicates that CR has the potential to both delay cardiac ageing and help prevent cardiovascular diseases. The mechanisms involved in this cardioprotection are diverse, but many of them involve mitochondrial adaptations. Data obtained from experimental models also suggest that pre-interventional, short-term CR or the pre-interventional application of CRMs may be a promising strategy in cardiac surgery and cardiology to improve tolerance to ischaemia and post-ischaemic cardiac recovery in their patient population. However, it is unlikely that CR will be widely adopted because of the difficulty in maintaining long-term CR in modern society and because of potentially serious side effects. Therefore, several challenges related to the adverse effects of CR and CRMs, and their long-term applicability for older adults and for those with significant comorbidities including cardiovascular diseases need to be addressed in clinical studies. Therefore, currently no general recommendation to implement CR in the general population for extending maximal lifespan in humans and improving cardiovascular diseases can be made.

Glossary

2-DG

2-deoxyglucose

Adipo1 receptor

adiponectin receptor 1

AICAR

aminoimidazole carboxamide ribonucleotide

AMPK

AMP-activated PK

ANT

adenine nucleotide translocator

Bax

BCL2-associated X protein

Bnip3

BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

CABG

coronary artery bypass graft

CALERIE

Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy

CAMKK

calcium/calmodulin-dependent PK kinase

CNI

calcineurin inhibitor

CR

caloric restriction

CREB

cAMP response element-binding protein

CRM

CR mimetic

CTRP

C1Q/TNF-related protein

CypD

cyclophilin D

Drp1

dynamin related protein 1

eNOS

endothelial NOS

ER

endoplasmic reticulum

ETC

electron transport chain

FAO

fatty acid oxidation

Fis1

mitochondrial fission protein 1

FOXO3a

forkhead box O3

HIF-1α

hypoxia-inducible factor 1α

I/R

ischaemia and reperfusion

IDH2

isocitrate dehydrogenase 2

IFM

interfibrillar mitochondria

IGF

insulin-like growth factor

iNOS

inducible NOS

IPC

ischaemic preconditioning

KD

kinase dead

KO

knockout

LKB1

liver kinase B1

LV

left ventricle/left ventricular

Mfn

mitofusin

miR

micro RNA

MnSOD

manganese superoxide dismutase

MPTP

mitochondrial permeability transition pore

mtDNA

mitochondrial DNA

mTOR

mammalian target of rapamycin kinase

mTORC1

mTOR complex 1

Nampt

nicotinamide phosphoribosyltransferase

NOX

NADPH oxidase

NRF

nuclear respiratory factor

Opa1

optic atrophy protein 1

PGC-1α

PPARγ coactivator-1α

PI3K

phosphatidylinositide 3-kinase

PINK1

PTEN-induced novel kinase 1

PRC

PGC-1-related coactivator

ROS

reactive oxygen species

S6K1

ribosomal S6 kinase

Sdh

succinate dehydrogenase

SHR

spontaneously hypertensive rat

SIRT

sirtuin

SR

sarcoplasmic reticulum

SSM

subsarcolemmal mitochondria

STACs

sirtuin-activating compounds

TAK1

transforming growth factor-β-activating kinase 1

TCA

tricarboxylic acid

Tfam

mitochondrial transcription factor A

TIGAR

TP53-induced glycolysis and apoptosis regulator

TORC1

transducer of regulated cAMP-regulated element-binding protein 1

TSC1 and 2

mTOR inhibiting proteins

Conflict of interest

No conflict of interests.