Introduction

In an impressive study, Eisenberg et al. showed that supplementation with the naturally occurring polyamine spermidine (Spd) is cardioprotective and prolongs lifespan in mice [1,2]. These authors also demonstrated that high intake of dietary Spd correlates with attenuated blood pressure and reduced incidence of cardiovascular disease in humans [1]. Recently, this group provided data in a human cohort of 829 individuals showing that high intake of Spd is associated with reduced mortality [3]. The possible mechanisms behind Spd-evoked cardioprotection are thought to involve direct cardiac effects such as increased autophagy and mitophagy which, in turn, leads to increased mitochondrial activity, reduced hypertrophy and enhanced titin phosphorylation and thereby improved mechano-elastical properties of the cardiomyocytes (Figure 1). Eisenberg et al. [1] propose that stimulation of autophagy by Spd may activate cardiac stem cells to produce new and functional cardiomyocytes. Spd supplementation also seems to have systemic effects involving lowered blood pressure and attenuated systemic inflammation demonstrated by reduced salt-induced hypertension and lowered levels of circulating TNF-α [1]. Treatment with Spd attenuates salt-induced hypertension in the Dahl salt-sensitive rat through a mechanism which is thought to involve elevated NO production accomplished by increased arginine bioavailability [1]. NO activates soluble guanylyl cyclase and PKG, the latter causing phosphorylation and activation of titin and thereby improved cardiomyocyte mechano-elastical properties. 

Reduced systemic levels of TNF-α is also regarded to increase titin phosphorylation via improved NO bioavailability. The exact molecular mechanism responsible for Spd-induced activation of autophagy in the cardiomyocytes is not known. Eisenberg et al. [1] showed that the cardiac tissue of mice supplemented with Spd is enriched in Spd, but they do not study and characterize uptake of Spd by cardiomyocytes, although it is assumed that cardiomyocytes from Spd-supplemented mice, rats and humans are enriched in Spd and that this polyamine acts intracellularly. Hence, it is of great importance to demonstrate that exogenous Spd is internalized/imported by cardiomyocytes and clarify the underlying molecular mechanism behind Spd import.

Polyamine metabolism

Mammalian polyamines usually comprise putrescine, Spd and spermine (Figure 2). The first step in the biosynthesis of the mammalian polyamines is the conversion of ornithine into putrescine, which is catalyzed by the enzyme ornithine decarboxylase (ODC) [4]. Spd and spermine are produced by the sequential addition of an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine and Spd, catalyzed by the enzymes spermidine synthase (SPDSY) and (SPMSY), respectively. The enzyme S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the synthesis of dcAdoMet from S-adenosylmethionine. The substrate for polyamine production, ornithine, is derived from arginine in a step catalyzed by arginase (ARG1). Both ODC and AdoMetDC are strongly regulated and considered to catalyze rate-limiting steps in the biosynthesis of mammalian polyamines [4]. Difluoromethylornithine (DFMO) is a highly specific irreversible inhibitor of ODC.

 Addition of exogenous polyamines to mammalian cells rapidly down-regulates both ODC and AdoMetDC as part of a feedback control of cellular polyamine homeostasis, which may shift arginine metabolism from polyamine production to NO formation [5]. Spd may also be derived from the interconversion of spermine, which can occur via two different pathways (Figure 2) [4]. In one of the pathways, spermine is first converted into N1 -acetylspermine, a reaction catalyzed by spermidine/spermine N1 -acetyltransferase (SSAT), followed by an oxidative deamination catalyzed by a polyamine oxidase (PAOX) (Figure 2). Spd can similarly be converted into putrescine by the action of SSAT and PAOX. Like ODC and AdoMetDC, SSAT is a strongly regulated enzyme [4]. Spermine can also be converted into Spd in a single step catalyzed by spermine oxidase (SMOX). Unlike PAOX, which is considered to be a constitutive enzyme, SMOX is highly induced by a variety of stimuli [6].

Uptake of polyamines

Besides biosynthesis, polyamines may be provided through cellular uptake. The major extracellular sources for polyamines are polyamines synthesized by the intestinal microbiota and food-derived polyamines [7,8].

 Polyamines are imported from the lumen of the gut to the portal circulation via the intestinal epithelial cells through a process which is thought to be temperature-dependent but Na+ -independent [9,10]. For putrescine, intestinal diamine oxidase represents a barrier between the gut-lumen and the portal blood, but no such barrier seems to exist for Spd [11]. The concentration of Spd in whole blood is estimated to be ∼6 mM, and most of Spd occurs in blood cells while the plasma concentration of Spd is very low [12,13]. Polyamines are protonated at physiological pH which hampers passive transport over the plasma membrane. Instead, active transport of polyamines through polyamine transporters, characterized by energy and temperature dependence as well as saturation at low mM concentrations of polyamines, is suggested [14,15]. Putrescine and Spd import show different pH optimum with putrescine uptake being more sensitive than Spd uptake to acidification ( pH 6.0–7.0) in human breast cancer cells, indicating that putrescine and Spd are imported through separate transporters in these cells [16]. In a study where the authors characterize a cation-Cl-cotransporter (SLC12A8A) in HEK-293 cells, they also showed that polyamine transport is independent of extracellular Na+ , K+ and Cl− [17]. In fact, polyamine transport was long believed to be a co-transport with Na+ using the inward Na+ gradient as the driving force, but later studies strongly argue that polyamine transport is Na+ -independent [15]. Different transporters for polyamines and molecular mechanisms for polyamine import have been proposed but the exact pathway is not understood.

 In Table 1, we summarize plausible polyamine transporters and mechanisms for polyamine uptake in mammalian cells as suggested in the literature. Polyamines have been suggested to be imported via caveolae which are Ω-shaped cholesterol-rich invaginations of the plasma membrane. Caveolin-1 has a critical role for both the structure and function of caveolae, and moreover, caveolin-1 seems to be involved in cell proliferation [18]. In intestinal epithelial cells, polyamine uptake has been shown to be regulated via a caveolin-1-dependent import via the solute carrier transporter SLC3A2 (DAX) and in colon cancer cells uptake of polyamines is mediated via caveolin-1-regulated endocytosis, suggesting that caveolin-1 regulates polyamine uptake via different mechanisms [19,20]. Vascular smooth muscle cells lacking the caveolin-1 gene show higher uptake of polyamines than wild-type cells, suggesting that caveolin-1 negatively regulates polyamine uptake in these cells [21]. The enhanced uptake of polyamines in caveolin-1-deficient vascular smooth muscle cells is associated with increased expression of SLC7A1 and SLC43A1. Polyamine uptake has also been suggested to be dependent on extracellular binding to the heparan sulfate proteoglycan glypican-1 and uptake via endocytosis [22–24]. However, uptake of polyamines is also observed in receptor-mediated endocytosis deficient CHO cell mutants, as demonstrated using fluorescence-labeled Spd, arguing that import of Spd is at least partly dependent on non-endocytosis-dependent mechanisms as well [25]. The identity of mammalian polyamine transporters is not known although members of the solute carrier transporter family (SLC gene family) have been suggested to facilitate polyamine uptake and represent polyamine transporters [15,26]. 

 Another argument speaking for polyamine transport through channels is that polyamines are protonated at physiological pH and thus they are attracted by negatively charged molecules in the intracellular compartment. Intracellular polyamines have been shown to block inwardly rectifying K+ channels which are crucial for setting normal electrical activity, implying that polyamines may be involved in modulation of cardiac excitability through this mechanism [33–35]. Furthermore, exogenous polyamines reduce inward Ca2+ currents through voltage-sensitive Ca2+ channels, attenuate intracellular Ca2+ concentration and cause relaxation of intestinal smooth muscle, suggesting that polyamines may act as plasma membrane Ca2+ channel blockers [36,37]. Down-regulation of intestinal smooth muscle cell Ca2+ channel activity is observed both when polyamines are applied from the outside and inside of the plasma membrane [37]. Polyamine-induced inward rectification and inhibition of Ca2+ channel activity will presumably influence both cardiac excitability and systemic blood pressure. Thus, supplementation with dietary Spd, causing elevated levels of this polyamine in cardiac tissue as reported by Eisenberg et al. [1], may modulate the electrical activity and excitability of cardiovascular cells through these mechanisms. Polyamine transport inhibitors (PTIs) have recently been synthesized showing low toxicity and high potency in inhibiting Spd uptake by DFMO-treated L3.6pl human pancreatic cancer cells [38]. In mouse aortic and human coronary vascular smooth muscle cells, these PTIs antagonize both basal and DFMO-stimulated polyamine uptake [39].

 The resting membrane potential in lymphocytes is −60 mV, whereas it is considerably more negative (−85 to −90 mV) in electrically excitable cells such as cardiomyocytes and skeletal muscle cells [41–43]. Hence, it may be argued that import of polyamines via a mechanism driven by the level of negativity of the membrane potential is more important in cardiac cells and skeletal muscle cells when compared with lymphocytes. Inhibition of polyamine uptake (both putrescine and Spd) was observed in human breast cancer cells subjected to high extracellular concentration of K+ providing further evidence with the membrane potential drives polyamine import [31]. Strong evidence that the membrane potential and electrogenic mechanisms are involved in polyamine uptake was provided by Busch et al. [28] in Xenopus oocytes expressing the putative rat polyamine transporter SLC22A1 (OCT1). They demonstrated that this transporter uses both Spd and spermine as substrate and that the uptake of polyamines is electrogenic and shows reduced activity upon depolarization. Polyamine uptake was characterized in cerebellar astrocytes by Dot et al. [32]. These authors showed that polyamines are imported via a single polyamine transport system which is saturable for putrescine (KM = 3.2 mM), Spd (KM = 1.8 mM) and spermine (Km = 1.2 mM) and not mediated via a co-transport with Na+ . Importantly, polyamine uptake by the astrocytes is antagonized by both the Na+ ionophore gramicidin and the Ca2+ ionophore ionomycin implying that membrane potential is critical for polyamine import in these cells as well [32].

 However, Spd is probably imported globally by all cells of the mouse, but perhaps uptake is more pronounced in cardiomyocytes than in other cells. Spd is most likely internalized/diffusing to the cytosol of cardiomyocytes of mice supplemented with Spd, but Eisenberg et al. [1] do not characterize the import of Spd. High-resolution microscopy of fluorescence-labeled Spd may reveal the intracellular distribution and trafficking of Spd imported by the cardiomyocytes. Interestingly, it has been reported that heart tissue of both humans and rats contains low levels of Spd and spermine compared with, for example, pancreas, liver and spleen [45,46]. Skeletal muscle tissue, a tissue harboring electrically excitable cells similar to the heart, also shows low levels of polyamines compared with other tissues [45,46]. It is well recognized that polyamine contents are higher in rapidly growing tissues with high cell turnover than in tissues with cells showing no or low activity of cell proliferation [47]. Interestingly, it has been reported that polyamine formation, assessed by measuring ODC activity, is higher in the immature heart and decrease with age in rats [48]. Although these studies provide no information about the proportion of intracellular bound and free concentrations of polyamines, respectively, it is reasonable to suggest that mature cardiac and skeletal muscle tissues have relatively low intracellular concentrations of polyamines, and thus there may be a reasonable chance to build a concentration gradient for polyamines from outside to inside establishing a driving force for Spd from the extracellular to the intracellular compartment acting together with the inward electrical gradient for Spd.