Introduction

Cardiovascular disease has reached epidemic proportions in the elderly and remains the worldwide leading cause of death. Human aging is typically accompanied by cardiac hypertrophic remodeling and a progressive decline of left ventricular (LV) diastolic function1,2. Abnormal diastolic function is present in >20% of the population >65 years of age3. Although less than half of all patients with diastolic dysfunction show clinical signs of congestive heart failure, even patients not meeting the diagnostic criteria are at increased risk to develop heart failure4. No treatment has yet been shown to convincingly target and prevent age-associated diastolic dysfunction or heart failure, likely because our understanding of the fundamental mechanisms underlying progressive deteriorations in the (ultra-)structure and function of the aging heart is incomplete. Recent studies have revealed that autophagy, a major cellular quality control mechanism, may be able to minimize the functional decline of aging cardiomyocytes by degrading and recycling long-lived proteins, which are potentially toxic if damaged, as well as cytoplasmic components and dysfunctional organelles (in particular, damaged mitochondria)5,6. Clearance of dysfunctional mitochondria through a specific type of selective autophagy, termed mitophagy, may be beneficial for cardiac function, because mitochondria can overproduce reactive oxygen species if they are functionally impaired and ignite lethal signalling pathways if they are permeabilized. In view of the established longevity-extending effects of enhanced cytoprotective autophagy in model organisms, it seems plausible that autophagy might also be able to counteract cardiac aging7. 

We previously discovered that the natural polyamine spermidine, a dietary compound, extends lifespan and health span through induction of autophagy in yeast, flies and worms8,9. Dietary supplementation of spermidine delayed age-associated memory impairment in flies10, prevented motor impairment in flies elicited by transgenic expression of human α-synuclein11, and protected mice from TDP-43-associated proteinopathies12, in line with a general neuroprotective action of this polyamine. In several model organisms, the lifespan extending and neuroprotective effects of spermidine were abolished upon inactivation of essential autophagy-related genes8,10. Here, we explored the potential cardioprotective effects of spermidine in rodent models of physiological cardiac aging (mice) and high salt-induced congestive heart failure (rats). We also provide evidence that dietary spermidine intake in humans inversely correlates with cardiovascular disease.

Results

Spermidine extends the lifespan of wild-type C57BL/6 mice

In view of the life prolonging effects of spermidine in model organisms8,9, we tested the long-term survival effects of specific polyamines in C57BL/6J wild-type female mice, which had a life-long (Fig. 1a) access to drinking water supplemented with distinct polyamines. Strikingly, spermidine- or spermine-supplemented mice had a significantly extended median lifespan as compared to control (receiving normal drinking water) or putrescinesupplemented mice (Fig. 1b, c and Supplementary Tables 1 and 2). 

To enhance the translational potential of these findings, we administered spermidine late-in-life, (a regimen more applicable to humans) to pre-aged male and female mice (Fig. 1a). Again, we found that spermidine feeding significantly prolonged median lifespan by ~10% (Fig. 1d and Supplementary Fig. 1). Spermidine-fed animals displayed increased circulating spermidine levels, confirming its systemic bioavailability (Fig. 1e). Food and water consumption, body weight and lean/fat mass composition were similar in spermidine-fed and control groups (Supplementary Fig. 2), excluding the possibility that polyamine supplementation extends lifespan by inducing a calorically-restricted state13.

Dietary spermidine delays cardiac aging by improving diastolic function

Tumor burden and cardiac aging are significant predictors of mortality in C57BL/6 mice and humans14,15. Comprehensive pathological characterization of tissues collected from mice at an advanced age (28 months), as well as from old mice that became moribund and were sacrificed as “end-of-life” animals16, revealed similarly high tumor frequencies in spermidine-treated and control mice (Supplementary Fig. 3 and Supplementary Tables 3, 4). This finding suggests that the potential ability of spermidine to inhibit tumor formation, which has been observed after chemo-induction of tumors17, does not explain its life prolonging effects. Since only minor histopathological abnormalities were observed in cardiac tissue obtained from 28-month-old or from “end-of-life” animals (Supplementary Tables 3, 4), we next subjected aged mice with late-in-life spermidine supplementation to structural and functional cardiac phenotyping.

 Spermidine reversed age-associated (23 months) echocardiography-detectable hypertrophy, as indicated by a reduction in tibia lengthnormalized left ventricular mass (LV mass/TL) and posterior wall thickness (PW/TL) to values below those observed in middle-aged (18 months) wild-type mice (Fig. 1f, Supplementary Table 5). Hypertrophic remodeling is the most common age-related myocardial abnormality that is associated with diastolic and/or systolic dysfunction, eventually leading to heart failure in humans18. Evaluation of cardiac function by invasive hemodynamic pressure-volume measurements revealed that compared with age-matched control mice, mice fed spermidine late-in-life had significantly enhanced diastolic properties, as reflected by a reduction of LV end-diastolic pressure (EDP; Fig. 1g, h) with a trend towards improved active relaxation (shortened time constant of LV pressure decay τ; Supplementary Table 6), as well as significantly reduced LV passive stiffness, as reflected by decreased myocardial stiffness constant β (Fig. 1i), with a downward shift of the enddiastolic pressure-volume relationship [EDPVR] obtained by transient vena cava occlusion for load-independent cardiac function assessment (Supplementary Fig. 4). The systolic properties of aged hearts were less affected by spermidine. Load-dependent parameters, such as ejection fraction (EF) and dP/dtmax as indicators of LV contractility, were comparable in all tested groups (Fig. 1j, Supplementary Tables 5, 6). 

 Electron microscopy did not reveal changes in the volume fraction or absolute volume of collagen, interstitium, capillaries or cardiomyocytes in the LV (Supplementary Fig. 5a and Supplementary Table 8). However, age-related effects on subcellular cardiomyocyte composition were reversed by spermidine, as reflected by increased relative mitochondrial and myofibrillar volumes and a reduced (mitochondria- and myofibril-free) sarcoplasmic volume (Fig. 2a, b, Supplementary Fig. 5b and Supplementary Table 8). These results suggest that spermidine has cardiomyocyte-intrinsic effect. We hypothesized that the increased myocardial compliance (i.e. myocardial elasticity) induced by spermidine originates from improved contractile apparatus and cardiomyocyte function22. Consistent with this idea, both transcriptome and proteome analyses of cardiac tissue extracts (Supplementary Fig. 6 and Supplementary Tables 9, 10) revealed a rejuvenated molecular phenotype with respect to components of the cytoskeletal apparatus (i.e. myosin heavy chain proteins, ankyrins, integrins, dystonin), inflammatory processes and mitochondrial respiratory chain complex I proteins (i.e. members of the Nduf protein family), all of which are essential for cardiomyocyte mechano-elastical functionality23 and healthy cardiac aging24,25. Accordingly, the respiratory competence of cardiac mitochondria through respiratory chain complex I was increased in mice supplemented with spermidine as compared to control mice (Fig. 2c, Supplementary Fig. 7a, b); thus, spermidine reversed an age-induced decline in mitochondrial respiratory function26. 

Therefore, we next tested whether spermidine supplementation improves autophagic flux in aging cardiomyocytes. To assess basal autophagic flux, we treated ad libitum-fed 13-monthold C57BL/6J wild-type mice supplemented with spermidine for the final four weeks with the vacuolar protease inhibitor leupeptin, which blocks autophagosome turnover, and quantified levels of the autophagosomal marker LC3-II30. Treatment with leupeptin induced a significant increase of LC3-II levels in hearts from spermidine-supplemented mice, whereas age-matched controls showed a reduced (and non-significant) elevation of this marker (Fig. 3a, Supplementary Fig. 9e), indicating that spermidine increases cardiac autophagic flux in vivo. Cellular spermidine content in cardiac tissue was significantly increased in spermidine-supplemented animals as compared to controls (Fig. 3b). The capacity of orally supplemented spermidine to induce autophagic flux in vivo in cardiomyocytes was corroborated by using transgenic cardiomyocyte-specific tandemfluorescent mRFP-GFP-LC3 mice31. These mice serve as an autophagy reporter strain, carrying labeled autophagosomes; both red (mRFP) and green (GFP) fluorescence, as well as labeled autolysosomes; red (mRFP) fluorescence only. In this experiment, chloroquine was used to block autophagosome turnover for assessment of autophagic flux. Spermidine substantially increased the number of autophagosomes and autolysosomes under both vehicle- and chloroquine-treated conditions (Fig. 3c, d). Moreover, spermidine stimulated mitophagy in cardiomyocytes of both young and aged mice, as assessed in mice expressing the mitochondrial-targeted form of the fluorescent biosensor Keima (Mito-Keima). 

Indeed, spermidine increased arginine bioavailability, as determined by an elevated global arginine bioavailability ratio (GABR, defined as arginine/[ornithine +citrulline]) (Fig. 4c), and increased the arginine/ornithine ratio, while also decreasing the cumulative level of ornithine and citrulline (Supplementary Fig. 12c, d). These findings suggest the ability of spermidine to improve NO production/bioavailability. Elevation of the GABR and the arginine/ornithine ratio, as well as decreased levels of ornithine plus citrulline (indicative of diminished arginine catabolism) have been associated with reduced cardiovascular risk38,39. To explore whether spermidine attenuates hypertension-induced hypertrophic remodeling and the progression to heart failure in this model, we assessed cardiac dimensions and function. Spermidine treatment reduced tibia length-normalized LV mass, posterior wall thickness and heart weight, indicating that it attenuated the increase in cardiac hypertrophy observed in controls (Fig. 4d and Supplementary Table 14, 15). Furthermore, spermidine enhanced diastolic function, as reflected by a reduction in the E/E’ ratio, a parameter that strongly correlates with mean LV filling pressure40 (Fig. 4e). Indeed, LV-EDP was reduced (Fig. 4f, g) along with a reduction in LV stiffness, as reflected by a decreased myocardial stiffness constant for indexed volumes βi (Fig. 4h, Supplementary Table 16) with a downward shift of the EDPVR (Supplementary Fig. 13) as well as an increase in the levels of total and S4080 phosphorylation of the N2B titin isoform (Supplementary Fig. 14a, b).

 Comparable to our findings in aging mice, enhanced diastolic function in rats was accompanied by a significant reduction of circulating TNFα levels (Supplementary Fig. 14c), a pro-inflammatory marker with increased levels in heart failure patients41. In control animals fed a high-salt diet, relative lung and liver weights (normalized to tibia length) increased from 7 weeks of age to 14 or 19 weeks of age (Fig. 4i). Spermidine treatment significantly delayed the increases in relative lung and liver weights (Fig. 4i and Supplementary Table 15), suggesting that spermidine reduces pulmonary and systemic fluid accumulation, respectively, which are characteristic of heart failure. Ejection fraction was preserved (>70%) in all groups (Fig. 4j), implying that spermidine delays the progression from hypertension-induced hypertrophy to a phenotype that resembles heart failure with preserved ejection fraction (HFpEF). Control animals fed a high-salt diet showed higher arterial elastance (i.e. arterial stiffness) for indexed volumes (Eai ), a surrogate of arterial load42, at 14 or 19 weeks of age, compared to 7 weeks of age. These animals appeared to compensate for this increased arterial elastance by increasing LV contractility, as indicated by an increase in end-systolic elastance for indexed volumes (Eesi ; Supplementary Fig. 13b and Supplementary Table 16), leading to comparable VVC values in the control groups of different ages (Fig. 4k and Supplementary Table 16). Notably, spermidine administration decreased arterial stiffness (Supplementary Table 16), resulting in a significantly improved VVC (Fig. 4k and Supplementary Table 16), similar to the effects we observed in both young and old Atg5-competent mice treated with spermidine. 

In a prospective, population-based cohort (Bruneck Study47), dietary intake of spermidine (as assessed by food questionnaires) was inversely associated with the risk of both fatal heart failure (a ~40% reduction in risk in the high compared to low spermidine intake groups) and clinically overt heart failure; both risks were more pronounced in men (Fig. 5a, b). Intake of spermidine was also inversely related to the risk of other cardiovascular diseases, as assessed by a composite of acute coronary artery disease, stroke and death due to vascular disease (Fig. 5c), and to systolic and diastolic blood pressures (Fig. 5d), which were significantly lower in the high compared to low spermidine intake groups. High intake of spermine or of spermine and spermidine combined showed similar associationsas high intake of spermidine (Supplementary Fig. 16). In contrast, putrescine intake did not show these associations (Fig. 5a-c) and tended to be associated with an increase in blood pressure (Fig. 5d). Notably, spermidine intake showed a significant inverse association with plasma levels of soluble Nterminal pro-B type natriuretic peptide (NT-proBNP), the key clinically-used biomarker for heart failure (r = -0.115, p=0.001). Moreover, in an exploratory approach, we tested whether spermidine intake correlated with the levels of 131 plasma proteins (data not shown).