Protein acetylation in cardiac aging

Abstract

Biological aging is attributed to progressive dysfunction in systems governing genetic and metabolic integrity. At the cellular level, aging is evident by accumulated DNA damage and mutation, reactive oxygen species, alternate lipid and protein modifications, alternate gene expression programs, and mitochondrial dysfunction (Sun et al., 2016; Nekhaeva et al., 2002; Kong et al., 2014). These effects sum to drive altered tissue morphology and organ dysfunction (Ballard and Edelberg, 2008; Han and Ren, 2010; Pina and Fitzpatrick, 1996). Protein-acylation has emerged as a critical mediator of age-dependent changes in these processes (Sun et al., 2016; Nekhaeva et al., 2002; Kong et al., 2014). Despite decades of research focus from academia and industry, heart failure remains a leading cause of death in the United States while the 5 year mortality rate for heart failure remains over 40% (Benjamin et al. 2019). Over 90% of heart failure deaths occur in patients over the age of 65 and heart failure is the leading cause of hospitalization in Medicare beneficiaries (Strait and Lakatta, 2012). In 1931, Cole and Koch discovered age-dependent accumulation of phosphates in skeletal muscle (Cole and Koch, 1931). These and similar findings provided supporting evidence for, now well accepted, theories linking metabolism and aging. Nearly two decades later, age-associated alterations in biochemical molecules were described in the heart (Kaufman and Poliakoff, 1950). From these small beginnings, the field has grown substantially in recent years. This growing research focus on cardiac aging has, in part, been driven by advances on multiple public health fronts that allow population level clinical presentation of aging related disorders. It is estimated that by 2030, 25% of the worldwide population will be over the age of 65 (Lakatta, 2002). This review provides an overview of acetylation-dependent regulation of biological processes related to cardiac aging and introduces emerging non-acetyl, acyl-lysine modifications in cardiac function and aging.

1. Cardiac aging

Outside of the congenital patient population, heart failure is clearly a disease associated with age [8]. Decreased myocardial reserve capacity and altered autonomic signaling are early hallmarks of cardiac aging that are well conserved across species [8,11,12]. Left ventricular (LV) hypertrophy, fibrosis and prolonged contraction and relaxation kinetics are also evident. Absent other comorbidities, measurable cardiac dysfunction, though present, is modest until advanced stages. In rodent studies, hypertrophy is typically the first observed remodeling, occurring as early as 12 months of age in mice and as early as 15–18 months of age in rats. Signs of diastolic dysfunction have been observed as early 15–18 months of age in mice and 22–26 months of age in rats with systolic dysfunction manifesting later [13-16]. It is important to note that timing of hypertrophy and dysfunction are strain dependent. Clinically, presentation of aging effects in the heart is heterogeneous, and can resemble either heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF), likely dependent on individual comorbidities. Canonically, HFpEF is more associated with advanced age, hypertension, and chronic inflammation while HFrEF is more associated with ischemic injury [17].

2. Acetyl-lysine protein modifications and general mechanisms

Enzymatic addition of short chain fatty acids to coenzyme A (CoA) and subsequent enzymatic transfer to target lysine residues is the central mechanism of enzyme-regulated protein acylation (see below for non-enzymatic acylation). While a variety of metabolically derived short chain fatty acids can serve as acyl groups in this process, the most widely studied acyl-lysine modification is acetylation. Lysine acetylation is governed by the competing actions of two enzyme families. Histone acetyl transferases (HATs) catalyze acetylation while histone deacetylases (HDACs) catalyze removal. Historically associated with epigenetic remodeling of chromatin and regulation of gene expression [18], the discovery of an increasingly long list of non-histone acetyl-targets including transcription factors, enzymes and the cytoskeleton, extend relevance to direct regulation of metabolism, proliferation, cell excitability, muscle contraction/relaxation, and other cellular processes [19].

As an indication of the complexity of acetylation biology, over 100 proteins either catalyze the addition or removal of acetyl groups to lysine residues or bind acetylated lysine residues. This review highlights, predominantly, the role of deacetylating enzymes in cardiac aging. The literature investigating acetyl-lysine interacting proteins, also referred to as reader proteins, in the context of aging is in its infancy. Also, much of the work investigating acyltransferase enzymes (HATs) in aging has been conducted with compounds notorious for having additional non-HAT targets (e.g. curcumin). Several pivotal studies employing genetic manipulation of HAT enzymes and work with improved inhibitors, such as L002 and C646, clearly implicate HAT activity in cardiac development, remodeling, and dysfunction and have been recently reviewed [20].

Eighteen mammalian HDACs are categorized into four classes (Fig. 1). Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), Class II HDACs (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), and Class IV HDACs (HDAC11) require Zn⁺² for catalytic activity. Class III HDACs, also known as Sirtuins, require NAD⁺ as a reactant in deacetylation reactions.

Fig. 1.

Histone deacetylases. Eighteen histone deacetylases are broken into four classes. Class II HDACs are further divided into Class IIa and Class IIb. Class III HDACs, also known as Sirtuins, require NAD as a reactant in deacetylation reactions. All other HDACs utilize Zn+² as a cofactor in deacetylation reactions.

Though commonly referred to as Zn+² dependent, biochemical studies have shown other divalent cations (e.g. Co⁺² and Fe⁺²) can serve as necessary cofactors in the place of zinc for at least Class I HDACs [21,22]. The Class II HDACs have been divided into sub-class as Class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) and Class IIb HDACs (HDAC6 and HDAC10). Class I HDACs are primarily nuclear while Class IIa HDACs are shuttled between the nucleus and cytoplasm in response to stimulus. Class IIa HDACs are canonically considered cardio-protective and will not be discussed further in this review. Class IIb HDACs are found predominantly in the cytoplasm, though roles in the nucleus have also been reported (e.g. [23]). Importantly, HDAC1 and HDAC3 have recently been reported in the mitochondria [24,25]. Throughout this review, the term pan-HDAC inhibitor is used in reference to compounds that inhibit multiple Classes of zinc-dependent HDACs and does not imply activity against sirtuins.

The Sirtuin family (Class III HDACs) is comprised of 7 proteins (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7), [26] that share a highly conserved catalytic domain, and are distributed in different cellular compartments. In vitro, these proteins display both deacylase and ADP-ribosyltransferase (ADP-ribose derived from NAD) activity. While most in vivo investigation of the Sirtuins has centered on deacetylation/deacylation, Sirt4 has been characterized as having predominantly ADP-ribosyltransferase activity in vivo. Of the Sirtuin family, Sirt6 and Sirt7 are found in the nucleus, Sirt1 and Sirt2 are both nuclear and cytoplasmic (though Sirt1 is primarily nuclear and Sirt2 is primarily cytoplasmic), while Sirt3, Sirt4, and Sirt5 are mitochondrial proteins [27,28].

Like other post-translational modifications, acetylation alters the shape and charge of the lysine residue, which can have a number of biochemical and functional consequences that will be discussed in subsequent sections. These include regulation of enzyme activity (e.g. p300, SDHA, HADHA), protein-protein (e.g. histone:bromodomain and GABPβ1:GABPα) and protein-nucleic acid (e.g. histone:DNA) interactions, protein stability (e.g. E2F1), and sub-cellular localization (e.g. NFAT). Select non-histone deacetylation events relevant to cardiac aging are highlighted in Fig. 2. A classic example of acetylation-regulated protein-nucleic acid interaction is found in the histone-DNA interaction of the nucleosome. Acetylation of histone lysine residues neutralizes lysine's positive charge, causing decreased interaction with negatively charged DNA. More recently, it has become appreciated that histone acetyl-lysine residues serve as docking sites for proteins that regulate gene transcription (e.g. BET proteins [29]). Histone acetylation is a critical mechanism for regulation of chromatin remodeling and transcription [30]. As mentioned above, histone acetylation is finely tuned by the opposing enzymatic activity of HATs and HDACs. In addition to loosening histone-DNA interactions, acetylated lysine residues in core histone proteins facilitate binding of proteins with bromodomains, yeast domains, and double PHD domains [31]. These domains are found in a variety of proteins including HATs, chromatin remodelers, DNA repair enzymes, ubiquitin ligases, and transcriptional coactivators [32-34]. Alterations of histone acetylation and associated transcriptional programs are strongly linked with aging [35,36]. Protein acetylation is broadly increased with age (Fig. 3 and [37].

Fig. 2.

Non-histone deacetylation in cardiac aging. Selected deacetylation events are depicted for the nuclear, sarcomere, cytoplasmic and mitochondrial compartments. Arrows indicate an activating effect of the upstream molecule; blocked lines indicate an inhibitory effect of the upstream molecule.

Fig. 3.

Age dependent changes in protein acylation. Protein homogenates were prepared from aged rat left ventricles at the indicated ages. Lysine acetylation, crotonylation, and β-hydroxybutyrylation were assayed by western blot. GAPDH detection indicates equal protein loading. Black line in acetyl-lysine blot indicates a cropped/removed lane due to a deformed well (uncropped image and method information available in supplemental file). Crotonylation blot was re-exposed for a longer duration to capture crotonyl modification of higher molecular weight proteins that were less abundant than those of lower molecular weight proteins.

The effects of acetylation on non-histone proteins are protein- and residue-specific. For instance, residue specific acetylation of E2F1 protects the protein from degradation, leading to E2F1 accumulation, which helps drive the cell through the G1/S cell cycle checkpoint [36]. This protein stability inducing effect of acetylation can be offset, even within the same protein, by acetylation dependent recruitment of protein poly-ubiquitination complexes, and subsequent proteasome degradation [37,38].

Mechanisms also exist for non-enzymatic protein acylation. Here, cysteine thiol groups (and to a lesser extent other amino acids) accept acyl groups from acyl-CoA. The cysteine-acyl bond can be subject to nucleophilic attack from nearby terminal amino groups on lysine residues, thus creating a route for non-enzymatic lysine acylation. This process is governed by Acyl-CoA:CoA ratios, Acyl-CoA concentration, and pH. Particularly in the mitochondrial compartment when acyl-CoA is abundant but mitochondrial membrane potential is low, conditions are favorable for non-enzymatic protein acylation [39-43]. Consequences of non-enzymatic and non-specific acylation events are not well known in the context of cardiac aging. This is likely due to the difficulty in manipulating these events without also altering known regulators of aging. It is though that these events modulate metabolic enzyme activity and stability and can trigger mitochondrial fission and apoptosis. As mitochondrial protein acylation is negatively regulated by Sirtuins, glutathione and glyoxalase II, it is probable that abundant non-specific acylation events are detrimental in the context of aging.

3. Acetylation in the nucleus

In response to age-associated ROS, inflammatory signals, and systemic dysfunction, cells in the heart engage altered gene expression programs that ultimately lead to heart failure. Mounting evidence points toward potential benefit of inhibiting Class I HDACs in cardiac aging. The protective effects of Class I HDAC inhibition are mediated through a variety of mechanisms including via activation of endogenous anti- ROS and anti-inflammation mechanisms such as increasing the expression of catalase (CAT) and superoxide dismutase-2 (SOD2). Class I HDAC inhibition has been shown to increase expression of these enzymes, decrease ROS, decrease inflammatory cytokine expression, and reduce cardiac fibrosis and hypertrophy. For example, inhibition of Class I HDACs preserves cardiac function in an ischemic reperfusion (IR) injury model [44]. Here, Class I HDAC inhibition with MS-275 led to increased SOD2 and catalase expression. This corresponded with enrichment of nuclear FOXO3a, a regulator of stress inducible, prosurvival and cardio protective genes [45]. In this IR model, neither pan-HDAC inhibition with TSA or Class IIb (HDAC6) inhibition with tubastatin A showed benefit in preserving cardiac function. However others have reported smaller infarct size with both TSA and Scriptaid (another pan-HDAC inhibitor) treatment [46].

In non-IR heart failure models, pan-HDAC inhibition clearly shows cardiac protective effects. Both TSA and Scriptaid prevented hypertrophy and cardiac dysfunction in pressure overload models of heart failure and TSA was able to reverse pre-existing hypertrophy and dysfunction [47-50]. Others have also shown that pan-HDAC inhibition prevents remodeling and preserves cardiac function in the myocardial infarction (MI) model and hypertensive rats [51-54]. Class I HDAC inhibition with Apicidin has also been shown to blunt hypertrophy and fibrosis, and preserve systolic function in the TAC model [55,56] while Mocetinostat (MGCD0103) preserved systolic function in the rat myocardial infarction model [57].

Molecularly, the cardio protective and anti-remodeling effects of HDAC inhibition (Class I and Pan-HDAC) are associated with decreased NF-kB dependent pro-inflammatory gene expression, decreased expression of ROS generating enzymes and increased expression of ROS buffering enzymes (e.g. [50], [53], [54]). In addition to potential genomic effects, HDAC3 has been shown to bind and deacetylate NF-kB/p65 at four lysine residues (K122, 123, 314, 315) causing its activation [58]. Note: this is not the same inhibitory deacetylation mediated by Sirt1 and Sirt2 on NF-kB/p65 which has been shown on K310 [59,60]. The effect of Class I HDAC inhibition mediating activation of ROS buffering enzymes appears to be conserved across multiple organs [61]. Moreover, Class I HDAC inhibition blocked mTOR activity by increasing expression of TSC2, a negative regulator of mTOR. This led to blunted hypertrophy and improved cardiac function in vivo [56]. This is noteworthy as along with caloric restriction, mTOR inhibition is among the most reproducible anti-aging strategies [62]. In addition to NF-kB, several other nuclear proteins including p53 and FOXO1 are deacetylation targets of Class I HDACS. These will be discussed in the Nuclear Sirtuin section below as the role for sirtuin deacetylation of these targets has been more thoroughly characterized.

Inhibition of acetyl-lysine reader proteins in the nucleus, particularly BET proteins, has also shown potent cardio-protective effects. These have been shown in genetic cardiomyopathy, pressure overload, and ischemic heart failure models [63-67]. Counter intuitively, HDAC inhibition and BET inhibition provide similar benefits in many aspects. Likely explanations for this including HDAC regulation of BET suppressors [68] and necessary pruning of acetyl-lysine marks to allow efficient targeting of BET proteins to stress activated genes [69]. Recently, BET inhibition with JQ1 prolonged survival, improved systolic function, and blocked cardiac fibrosis in mice lacking cardiomyocyte expression of Lamin A/C [70], which display accelerated aging phenotypes.

3.1. Nuclear situins

The influence of Sirt1 and other nuclear Sirtuins on cardiovascular aging has been well studied. Targets of Sirt1 deacetylase activity including p53, FOXO1, JNK and eNOS are involved in signaling pathways frequently associated with altered cellular processes in cardiac aging [71-73]. P53 is a critical transcriptional regulator of cardiomyocyte homeostasis, cell cycle regulation, DNA damage responses [74] and can either act as a break on cell growth in response to stress, leading to senescence, or can induce apoptosis [75]. Differential residue-specific acetylation of p53 has been shown to both regulate p53 stability and direct p53 to pro-apoptotic vs growth arrest genes. Following DNA damage, CK2 phosphorylates and activates Sirt1, thus causing deacetylation and inhibition of p53 and preventing both apoptosis and senescence [76]. CK2 kinase activation of Sirt1 is diminished with age, relieving a brake on p53 and leading to senescence [77,78]. Sirt1 is also a positive regulator of autophagy, a cellular homeostatic process of resource scavenging through the lysosomes and is broadly considered and protective mechanism during aging [79]. Cardiac specific transgenic overexpression of Sirt1 attenuates cardiac aging through 18 months of age as indicated by preserved systolic function, and reductions in hypertrophy, fibrosis and senescence marker expression. This group produced three transgenic mouse lines overexpressing Sirt1. It should be noted that in the highest expressing line (>12× protein expression over endogenous), cardiac dysfunction was induced by Sirt1 overexpression while up to 7.5 fold induction of Sirt1 was protective [80].

Sirt6 regulates DNA repair, metabolic gene expression, inflammatory gene expression, and telomere maintenance. Notably, in the heart, Sirt6 was shown to inhibit c-Jun expression and protect against cardiac hypertrophy [81]. Shen et al. also demonstrated that inhibition of hypertrophy mediated by Sirt6 was mediated by the deacetylation, ubiquitination and proteasome degradation of p300. This led to reduced expression of NF-kB target genes associated with hypertrophy [82]. Sirt6 knockout mice develop spontaneous cardiac hypertrophy while over expression of Sirt6 prevents hypertrophy in cultured cardiomyocytes [81] and both hypertrophy and fibrosis in hearts of mice fed high-fat and high-sugar diets [83]. This study indicated that Sirt6 activated NRF2 target gene expression by direct interaction with NRF2 and inhibiting expression of the NRF2 negative regulator, KEAP1. NRF2 is a master regulator of nuclear encoded mitochondrial and antioxidant genes, including Sirt3.

Sirt7 has pan-nuclear and nucleolar functions. In the nucleolus, it interacts with RNA-polymerase I and facilitates ribosomal RNA (rRNA) expression [84]. Sirt7 knockout mice show increased acetylation of cardiac Akt, Ras and p53, resulting in cardiac hypertrophy, inflammation, and cardiomyocyte cell death [85]. Additional mechanistic studies have discovered that Sirt7 deacetylation of GABPβ1 allows heterodimerization of GABPβ1 and GABPα to activate expression of nuclear-encoded mitochondrial genes. This Sirt7/GABPβ1 pathway facilitated beneficial mitochondrial adaptations in response to stress and aging [86]. The cardio-protective role of Sirt7 is supported by recent investigation in cardiomyocytes focused on the transcription factor GATA4, a well known mediator of cardiac hypertrophy [87]. Sirt7 was shown to deacetylate GATA4, blocking pro-hypertrophic gene expression. Interestingly, HDAC2 was shown to deacetylate GATA4 in a developmental study where GATA4 deacetylation led do decreased cardiomyocyte proliferation [88]. It has not been established if HDAC2 and Sirt7 target different acetyl-lysine residues in GATA4.

4. Acetylation in the sarcomere and cytoplasm

Acetyl-lysine residues and acetyl modifying enzymes have also been found in the sarcomere. Several studies implicate these modifications as regulators of cardiac function, though few have been directly tested in aging. Many acetyl-lysine residues have been identified in the sarcomere, including in cardiac troponin I (cTnI). cTnI is critical for regulating cardiac contraction and age-associated decreases in cTnI link with diastolic dysfunction. Using cultured rat cardiomyocytes, acetyl-mimetic mutations in cTnI decreased calcium sensitivity while the speed of relaxation was increased [89]. This work supported a previous study by the same group showing that HDAC inhibition protected rodents from diastolic dysfunction, including in a natural aging model. In the same paper, myofibrils incubated with recombinant HDAC2 showed slower relaxation kinetics and decreased myofibril protein acetylation while incubation with recombinant p300 sped relaxation kinetics and increased myofibril protein acetylation [90]. The premise of protein acetylation in the sarcomere improving diastolic function was confirmed with HDAC inhibition in a feline model of the heart failure with preserved ejection fraction (HFpEF), which is strongly associated with age [91]. Altered mitochondrial protein acetylation following treatment with the pan-HDAC inhibitor, SAHA, were also observed in this study. HDAC3 and HDAC4 have also been shown to regulate acetylation and associated functions of both α and β myosin heavy chain proteins and the sarcomere stretch sensor MLP respectively [92,93].

Acetylation of non-sarcomeric cytoskeleton has also been shown to regulate cardiac health. In a mouse model of cardiomyopathy induced by aggregation of misfolded proteins, HDAC6 (class IIb) activity was shown to accelerate aggregate formation. Genetic or pharmacologic inhibition of HDAC6 induced autophagy and prevented aggregate formation in cultured cardiomyocytes while treatment with a pan-HDAC inhibitor (SAHA) was again cardio-protective in vivo. HDAC6 mediated regulation of autophagy was linked to the acetylation state of tubulin, its most well characterized deacetylation target [94]. Also, HDAC6 knockout mice had preserved systolic function in Angiotensin II and transverse aortic constriction (TAC) models of heart failure, though HDAC6 nulls showed more fibrosis in the Angiotensin II infusion model [95]. Myofibrils from HDAC6 knockouts also had higher maximal force generation and higher resting tension than myofibrils from littermate controls.

Also found in the cytoplasm, Sirt2 is highly expressed in the brain and heart, though its expression is decreased following stress and in advanced age. Mice lacking Sirt2 develop age dependent, spontaneous cardiac hypertrophy, fibrosis and dysfunction, while over expression of Sirt2 prevents hypertrophy in cultured cardiomyocytes. This Sirt2 anti-aging/cardio-protective phenotype has been tied to three specific Sirt2 deacetylation targets. In the absence of Sirt2, NFATc2 protein abundance and transcriptional activity are elevated, leading to pathologic remodeling in the heart. Sirt2 mediated deacetylation of NFATc2 prevents its translocation to the nucleus [96]. Also, Sirt2 activates LKB1, an upstream activator of AMPK, through deacetylation. In the absence of Sirt2, LKB1 is hyper-acetylated and the anti-hypertrophic effects of AMPK signaling are not engaged [97]. Finally, Sirt2 was recently shown to activate GSK3β via deacetylation in cultured rat cardiomyocytes and led to decreased cardiomyocyte size [98]. GSK3β has previously been shown to be anti-hypertrophic [99].

5. Acetylation in the mitochondria

Mitochondrial dysfunction is a well characterized component of the aging process [100]. We encourage readers to also see Herr et al. [101] for a recent review on regulation of mitochondrial enzymes by acetylation.

HDAC1 has recently been reported in the mitochondria of mammalian adult cardiomyocytes. Inhibition of mitochondrial HDAC1 by a Class I HDAC inhibitor designed for mitochondrial accumulation, LL-66, conferred protection against reperfusion injury (I/R). LL-66 treatment reduced ROS accumulation and succinate dehydrogenase (SDHA) metabolic activity, leading to improved myocyte viability and left ventricular function within 1 h. SDHA was shown to be a direct deacetylation target of HDAC1. In contrast, no benefit was observed with the cytoplasmic Class I HDAC inhibitor LL-224 [24]. Supporting this notion, pan-HDAC inhibition with SAHA triggered mitochondrial autophagy and renewal and improved cardiomyocyte survival following IR injury [102]. Though not reported in the heart, a recent publication also showed HDAC3 in the mitochondria of activated macrophages. Here HDAC3 mediated deacetylation of HADHA, an enzyme necessary for fatty acid oxidation, was linked with reduced fatty acid utilization, increased ROS generation, and NLRP3 inflammasome activation [25].

5.1. Mitochondrial sirtuins

The first study targeting Sirt3 in cardiac physiology was published over a decade ago by Sundaresan et al. Here, Sirt3 knockout mice were more susceptible to Angiotensin II induced hypertrophy and fibrosis while Sirt3 transgenic mice were protected [103]. Sirt3 inhibited expression of transcription and translation activators associated with cardiac hypertrophy, blocked ROS accumulation and enhanced production of antioxidant SOD and catalase via Sirt3 activation of FOXO3A. A Sirt3 cardio-protective role was also demonstrated in resistance to ischemia-reperfusion injury in aged mice [104]. Similarly, Hafner et al. demonstrated a protective role of Sirt3 in cardiac tissue against stress and aging, proposing a model where Sirt3 downregulation led to in the accumulation of acetylated CypD. Sirt3 deacetylation of CypD increases mitochondrial membrane integrity by restricting mitochondrial pore complex (mPTP) permeability. In the absence of Sirt3, mitochondrial permeability was dramatically increased in an age dependent manner, leading to reduced NAD⁺ and increased mitochondrial calcium and ROS accumulation [105]. Sirt3 also appears to block the progression of cardiac fibrosis. Sirt3 deacetylation and activation of GSK3β blocks TGF-β1/SMAD signaling and prevents cardiac fibrosis in aged mice[106].

NAD+ is a necessary cofactor for several cellular processes in addition to Sirt activity, including the mitochondrial electron transport chain [43]. NAD+ concentration has been proposed as an endogenous regulator of Sirt activity in homeostatic signaling. NAD+ concentration is low in senescent cells and aged tissues. Normalizing NAD+ provides cellular protection against aging. CD38, another NAD+ consuming enzyme, is upregulated in cardiac and cerebral tissues of aged animals.[107]. First demonstrated in liver, brain, spleen and skeletal muscle[108], recent work in H9C2 cells suggest that CD38 can regulate Sirt activity by consuming NAD+, causing senescence in a variety of tissues and may contribute to cardiac aging [109]. This appears to be a drugable aging pathway as the CD38 inhibitor, thiazoloquin(az)olin(on)e 78c, increased health span (including cardiac systolic function) and lifespan of aged mice. CD38 inhibition has been associated with a dramatic reduction in protein acylation (acetyl-, succinyl- and malonyl-lysine modifications) and activation of Nrf2 and Foxo1 dependent antioxidant gene expression [110]. The NAD+ sparing Sirt activating effect of CD38 inhibition does not appear to be limited to the mitochondrial or cytoplasmic compartments as acyl-modifications were also reduced on nuclear proteins.

Sirt5 also appears to have cardio-protective and anti-aging properties and is highly expressed in the heart. Sirt5 knockout animals have been shown to be more susceptible to TAC pressure overload induced heart failure and death [111]. Sirt5 has potent deacylase activity for multiple acyl species. As expected, a number of proteins were found to have higher acyl-lysine modifications in the Sirt5 knockout. Specifically succinyl-lysine and malonyl-lysine were enriched in the knockouts, causing decreases in both fatty acid oxidation and glucose oxidation and reducing the NAD/NADH ratio. However, cardiomyocyte specific inducible disruption of Sirt5 failed to show any cardiac functional or survival differences compared to controls when again subjected to TAC [112]. Though protein succinylation was clearly upregulated in these mice, results suggest the importance of either multi-organ involvement or mitochondrial function during early development influencing adult adaptations to cardiac stress. Another group aged Sirt5 knockout mice and discovered spontaneous hypertrophy and systolic dysfunction at 39 weeks of age. Proteomics investigation revealed that in the Sirt5 knockouts, hyper-succinylation of ECHA, a protein involved in fatty acid oxidation, dampened fatty acid oxidation and reduced ATP levels in the heart [113].

However, not all mitochondrial Sirtuins have anti-aging activity. Sirt4 has been recently associated with pathological mechanisms that lead to cardiac hypertrophy, fibrosis and cardiac dysfunction. In an Angiotensin II infusion model of heart failure, Sirt4 knockout mice displayed reduced hypertrophy and fibrosis and had preserved systolic function. Conversely, Sirt4 overexpressing mice showed increased hypertrophy and fibrosis and had worse systolic function. It was shown that Sirt4 blocked the ability of Sirt3 to physically interact with and deacetylate/activate, the antioxidant enzyme mnSOD [114].

5.2. Emerging acyl-modifications

Multiple acyl-lysine modifications (e.g. succinylation, malonylation, glutarylation) have been known in the mitochondria for several years and some effects on mitochondrial enzyme function have been characterized [115-117], for instance regulation of ECHA activity noted above in Sirt5 discussion. Sirt5 desuccinylase activity has been associated with amino acid metabolism in the liver [118], and is a positive regulator of the fatty acid metabolic oxidation as demonstrated in both the liver and heart [113]. Sirt5 is also the only mitochondrial sirtuin with malonylation and glutarylation transferase activity, although the effects these modification and their regulation remain largely unknown [119,120]. Broadly, these modifications likely serve as adaptive regulators of homeostasis amid changing metabolic conditions. Though chronic hyper-acylation brought about by metabolic inflexibility or persistent insult (e.g. diabetes or obesity) are likely to be pathologic.

Non-acetyl, acyl-lysine modifications outside of the mitochondria are of particular interest to many labs. Given the variety in acyl group chemical structures, these modifications are likely to influence a host molecular mechanisms in epigenetics and cardiac physiology differently than acetylation. Many questions remain regarding physiological significance and basic molecular mechanisms of these modifications including sources of extra-mitochondrial acyl-CoA, acyl-CoA transport/organelle membrane permeability, as well as specificity of readers, writers and erasers for these lysine modifications.

Protein succinylation is not limited to the mitochondria. Mass spectrometry analysis of myofibrils from human heart show that 307 amino acid residues on 108 proteins are differentially succinylated in failing vs control samples. Nine of these proteins are located in the sarcomere [121]. Functional implications of these modifications are yet to be determined.

Crotonylation is another acyl-lysine modification in histone and non-histone proteins [122]. When found in histones, the effect of lysine crotonylation appears to be similar to acetylation. Recently, crotonylation was found at 557 lysine residues in 218 proteins in zebrafish. Twenty eight of the crotonylated proteins were in the myofilament, suggesting a potential role for crotonylation in regulation of contractile function [123]. Crotonylation is positively regulated by p300/CBP [124] while Sirt3 and Class I HDACs show decrotonylase activity [125-127]. Our data indicate that unlike acetylation, which is increased in aged rat hearts, crotonylation appears to be reduced with age (Fig. 3).

Several dozen sites of lysine β-hydroxybutyrylation (β-OHB) have been found on histone proteins and were associated with active promoters of genes responsive to changes in metabolism. Recently, it was reported that p53 was modified with β-OHB at three key lysine residues, which was associated with decreased p53 target gene expression [128]. As more investigation of these non-acetyl, acyl-lysine modifications are conducted, a more complete picture of their physiological relevance and associated molecular mechanisms will undoubtedly emerge, but this field is clearly in its infancy.

6. Conclusions and future prospective

As the worldwide population continues to live longer, age-associated cardiac dysfunction and heart failure will become even more prominent. A clear pattern has emerged indicating that Class I/pan-HDAC inhibiting and Sirtuin activating interventions provide cardiac protection in aging. Potential hurdles do exist related to therapeutic intervention with Zn⁺² dependent HDAC inhibitors for age-associated heart failure. Pan-HDAC inhibition with TSA was shown to accelerate atherosclerosis in LDL receptor deficient mice [129]. However, SAHA, another pan-HDAC inhibitor, slowed progression of atherosclerosis in APOE deficient mice fed a high fat diet [130]. Also, beneficial effects of HDAC inhibition can appear counter-intuitive given that lysine acetylation is increased with age (Fig. 3 and [37]). This fact potentially highlights the dominance of anti-inflammatory and anti-NF-KB effects of Class I HDAC inhibition. In reality, we are just beginning to uncover and are far from understanding the interactions between metabolism and epigenetics. Remarkably little is known regarding the emerging non-acetyl acyl-modifications, functional consequences of these modifications, or their role in pathophysiology of aging.

We expect that novel therapies focused on deacetylase activity will soon enter the clinic for cardiac indications. Clinicaltrials.gov lists 787 completed, active, or planned HDAC clinical trials. The only trial including cardiac indications is a limited imaging trial to assess Class I HDAC abundance/distribution in heart failure related diagnoses (e.g. aortic stenosis, diabetes, and LV hypertrophy; NCT03549559). Given the number of trials underway or completed for other indications, it is likely that combined post hoc analysis of cardiac events in these studies would shed light on human cardiac benefits of HDAC inhibition. Far fewer trials can be found related to Sirtuins and the majority of these are focused on observing Sirtuin expression following lifestyle modification or anti-diabetes therapies. Additional opportunities to intervene in age-associated cardiac dysfunction may be found in targeting the reader proteins of acyl-lysine residues and in the emerging field of non-acetyl acyl-modifications. Both of these areas are virtually untapped in relation to cardiac aging, though a recent publication showed that mice haploinsufficient for the BET protein BRD2, were protected from cancer and kidney damage in advanced age and lived 23% longer than control mice [131].