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Myocardial function is intimately dependent on the precise spatiotemporal regulation of membrane-bound proteins and ion channels. Phospholipases play critical roles in the maintenance of membrane structure and function thereby fundamentally integrating dynamic alterations in myocardial performance with membrane composition and dynamics. The major phospholipases in myocardium belong to a family of proteins known as calcium-independent phospholipases (iPLA2s). In addition to their role in maintaining normal membrane structure and function, iPLA2 catalytic activity results in the generation of a variety of lipid 2nd messengers that facilitate cellular signaling. Through its multiple effects on cardiac myocyte bioenergetics, cellular signaling and membrane function, the iPLA2 family of enzymes is of primary importance in modulating the pathologic sequelae of myocardial ischemia, diabetic cardiomyopathy and remodeling during hemodynamic stress. This review will provide a brief overview of myocardial iPLA2s and of their significance in cardiac pathology and physiology.
Myocardium is an electrically active tissue whose function depends upon the precisely orchestrated interplay of transmembrane ion channels and ion pumps. Biologic membranes fulfill critical roles in the electrophysiologic and hemodynamic function of myocardium by integrating the activities of multiple subcellular compartments, each possessing distinct functions through the accrual of specialized lipid and protein constituents. These compartments dynamically interact both chemically and functionally to integrate myocardial metabolism and signaling with hemodynamic function to permit adaptation to a wide variety of physiologic and pathophysiologic conditions. In addition to modulating the activity of membrane-associated proteins, biologic membranes provide a surface for the construction of signaling scaffolds critical to integrated cellular responses to pathophysiologic perturbation. Moreover, myocardial membranes serve as endogenous storage depots for the precursors of lipid 2nd messengers. These biologically latent chemical moieties are activated through hydrolysis by phospholipases to generate a wide variety of biologically active signaling metabolites. Concomitant with hydrolysis of membrane constituents, phospholipases induce alterations in membrane biophysical properties which facilitate cellular signaling. These include changes in stereoelectronic relationships, molecular dynamics and surface charge that modulate the function of membrane-associated proteins. Substantial experimental evidence has now demonstrated the critical role played by phospholipase-mediated alterations in membrane structure and function that contribute to the pathophysiologic sequelae of myocardial ischemia, reperfusion, diabetic cardiomyopathy and congestive heart failure.
To understand the integrated roles of individual members of the iPLA2 family in myocardial bioenergetics and signaling, it is first necessary to understand the chemical diversity and specialized nature of lipids in discrete myocardial subcellular membrane compartments. Detailed structural and kinetic information on alterations in triglyceride and phospholipid content during physiologic metabolic transitions as well as during pathologic processes has been used to assess the functional status of myocardial metabolism, cellular signaling and the bioenergetic efficiency of cardiac myocyte metabolism at different levels of cardiac work. Accordingly, we will first provide a background for understanding the specialized nature of membranes in general and subsequently focus on the unique nature of the phospholipid constituents present in myocardial membranes.
The dominant lipid components of biologic membrane bilayers are glycerophospholipids. Glycerophospholipids are composed of a wide variety of acyl (aliphatic) chains covalently linked to a glycerol molecule at the sn-1 and sn-2 positions and a more limited number of polar head groups covalently bound to the sn-3 position of the glycerol backbone. Glycerophospholipids are categorized into classes based upon the nature of their polar head group. The head group of each glycerophospholipid class has specialized chemical characteristics (e.g., a positively charged quaternary amine, a negatively charged serine, monophosphoinositides, polyphosphoinositides, etc.) which result in a broad diversity of physicochemical properties at the hydrophobic/hydrophilic membrane interface. This repertoire of specialized surface properties can be used to modulate surface charge, ionic interactions, hydrogen bonding and hydrophilic interactions facilitating specific stereoelectronic interactions with proteins and protein complexes at the membrane surface. Thus, the membrane itself serves as a prominent signaling platform by providing a highly specialized chemical foundation for assembly of multiprotein signaling scaffolds.
Additional diversity is provided by variation in the nature of the bond between the sn-1 carbon of the glycerol backbone and its aliphatic chain. This bond can either be a carboxy ester, vinyl-ether, or alkyl-ether as shown in Fig 1. The nature of the linkage at the sn-1 carbon determines the phospholipid subclass. The varying characteristics of the aliphatic chains (chain length and degree of unsaturation) and their regiospecificity (sn-1 versus sn-2) represent an additional level of phospholipid diversification, and dictate glycerophospholipid molecular dynamics, conformational isomers and structural rotomers present in the membrane interior. Varying the dynamic physical and chemical properties of these aliphatic chains can thus influence the activity of transmembrane proteins. Moreover, within membranes there is a heterotopic distribution of lipids, permitting associations of specialized lipids and proteins not only in discrete subcellular organelles, but also into specific membrane domains (e.g., lipid rafts) within an organelle facilitating biologic functions. Mammalian myocardium, including humans, is unique by virtue of its large sarcolemmal content of choline containing plasmalogens (the aliphatic chain is linked to the sn-1 carbon of the glycerol backbone via a vinyl ether linkage with a choline headgroup at the sn-3 position) . The significance of this particular class of glycerophospholipid (i.e., plasmalogen) as a reservoir of arachidonic acid susceptible to the actions of activated myocardial phospholipases will be addressed later in this text.
Phospholipases are a family of enzymes that catalyze the hydrolysis of phospholipids. These enzymes are broadly classified into four groups, depending on their catalytic activity. Phospholipases A1 and A2 are responsible for catalyzing hydrolysis of the carboxy ester linkages between the glycerol backbone of the phospholipid, and the acyl chains attached at the sn-1 and sn-2 positions, respectively. Phospholipase C catalyzes hydrolysis of the ester linkage between the sn-3 position of the glycerol backbone and the inorganic phosphate moiety of the polar head group and phospholipase D catalyses hydrolysis of the bond between inorganic phosphate and the polar head group. Finally, lysophospholipases catalyze the hydrolysis of the ester bond in lysophospholipids that remains after phospholipase A2 action to produce a free fatty acid and glycerophosphate species. There are presumably thousands of chemically distinct proteins that have phospholipase activity arising from dozens of distinct genes each of which generates potentially many hundreds of splice variants encoding protein products with varied post translational modifications. Collectively, through alterations in transcription, mRNA processing, translation and post-translational modification, these distinct chemical entities contribute to the diversity, function and adaptability of biologic membranes through specific effects on activity, subcellular location and their participation in multiple signaling scaffolds.
The reaction products of phospholipase A2 (PLA2) catalyzed phospholipid hydrolysis are unique among phospholipases in that they simultaneously produce free fatty acids (e.g., arachidonic acid) and lysophospholipids which are both biologically active signaling molecules. Non-esterified arachidonic is clearly present in myocardium and is likely a physiologic effector of multiple signaling pathways. In addition, some arachidonic acid is likely bound to hydrophobic binding sites on carrier proteins or is otherwise compartmentalized. Selective oxidation of arachidonic acid by cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes leads to the production of a plethora of eicosanoids each with highly specific biologic functions . The rate-determining step for the production of these eicosanoids is the release of arachidonic acid. The precise complement of eicosanoids produced is determined by the activity of downstream oxidases that facilitate adaptation to both physiologic and pathophysiologic perturbations. In myocardium, the sarcolemmal phospholipid pool is highly enriched in arachidonic acid as a result of a combination of lipid remodeling , and the differential distribution of phospholipids among subcellular pools from their original sites of synthesis. This arachidonic acid can be released by phospholipase activity to regulate the electrophysiologic properties of cardiac myocytes, integrate conduction in the heart, and modulate cellular signaling. Lysophospholipids are also important signaling molecules, working via multiple pathways including their role as ligands for G-protein coupled receptors [4, 5]. In addition, lysolipids have prominent effects on a number of different ion channels (e.g., the ryanodine receptor) and modulate the molecular dynamics of cell membranes due to their differential polar head group to aliphatic volume ratio [6–10].
There are many types of PLA2 expressed in mammalian tissues. The human genome contains over 25 genes encoding phospholipases A2 that have been broadly classified into types based upon their substrate preference, regiospecificity for hydrolysis, cofactor requirements, dependence on calcium for activity, size, and their catalytic mechanism.
Secretory phospholipases (sPLA2) are relatively low molecular weight entities (13–18 kDa) that require millimolar amounts of calcium ion for catalytic activity and thus are envisioned to act almost exclusively extracellularly. sPLA2 catalysis requires calcium binding to the calcium-binding loop to polarize the sn-2 carbonyl of the substrate through a highly conserved active site geometry. Catalysis is mediated by a catalytic triad composed of histidine, aspartate and water. Nucleophilic attack of the sn-2 carbonyl by the activated water molecule results in non-acyl-chain selective phospholipolysis [11, 12].
Intracellular PLA2s are substantially larger (63–87 kDa) and can be divided into cytosolic (cPLA2; typically calcium-dependent) and calcium-independent phospholipase families (iPLA2s which are all calcium-independent). With the exception of cPLA2g, all known cPLA2s require micromolar concentrations of calcium for membrane association via a C2 domain [13, 14]. Notably, cPLA2g does not possess a C2 domain, but instead is anchored to membranes by a farnesylated and carboxymethylated C-terminus, a C-terminal basic/hydrophobic region, and by long chain fatty acylation. Once membrane associated, cPLA2s catalyze phospholipolysis via an active site serine residue whose primary hydroxyl acts as a nucleophile resulting in the generation of an acyl-enzyme intermediate. Catalysis requires the presence of an adjacent aspartate residue that facilitates proton transfer and decreases the energy of the transition state [16–18].
Calcium-independent phospholipases (iPLA2s), as their name indicates, do not require calcium for membrane association or enzymatic activity . Like cPLA2s, iPLA2s catalyze phospholipolysis via an active site serine nucleophile in concert with an essential active site aspartate to form a similar catalytic dyad [20–23]. Bioinformatic studies have demonstrated elements of a conserved primary structure and preserved stereochemical relationships at the active sites of cPLA2 and iPLA2 family members confirming their common evolutionary origins. Neither family of intracellular phospholipases is exclusively substrate specific, however, and both will also hydrolyze ester bonds at the sn-1 position, in both phospholipid and lysophospholipid substrates [23, 24].
iPLA2s are a group of enzymes that are phylogenetically related to patatin lipases . First isolated from potato tubers, this family of enzymes is characterized by having a consensus GXSXG lipase motif with a conserved Asp residue residing across from the active site Ser, and a GXGXXG ATP (nucleotide) binding motif [21, 25, 26]. Highly homologous sequences and predicted secondary structures surround the active site of all iPLA2s. Outside of the active site region, however, sequence variation is substantial. Recent results have indicated that multiple regulatory modules outside of the lipase and ATP binding regions dictate subcellular localization, modulate activity, and contribute to the chemical properties and biologic roles of these enzymes as shown in Figure 2 [20, 27]. Patatin-like phospholipases catalyze phospholipolysis through initial binding of substrate followed by nucleophilic attack by the primary hydroxyl of the active site serine leading to the formation of an acyl enzyme intermediate and the resultant production of lysophospholipid. The acyl-enzyme is subsequently hydrolyzed resulting in the release of free fatty acid [28–31]. Given the variation in specific activity with both the chemical nature of the bond at the sn-1 position, and the identity of the liberated fatty acid from the sn-2 position, it is likely that the catalytic rate is determined by both the association constant of the enzyme for lipid substrate in the lateral plane of the membrane and the rate of acyl-enzyme intermediate hydrolysis [23, 32–34].
iPLA2s have been implicated in a number of physiologic and pathophysiologic processes. They have been shown to play fundamental roles in mediating electrophysiologic alterations during ischemia , in modulating the function of mitochondria through changes in the molecular species distribution of cardiolipin during the onset of diabetes , and as important effectors of calcium signaling via their role in capacitative Ca2+ entry [37–41]. In addition, prominent roles for various members of the iPLA2 family in mediating cellular signaling through arachidonic acid release, in facilitating adipogenesis and adipocyte lipolysis [42, 43], and in mediating smooth muscle cell proliferation and physiology [38, 40, 44, 45] have been identified. In myocardial cytosol, the dominant PLA2 activity is calcium-independent [19, 33]. Although cPLA2α is expressed in the heart, it contributes relatively little to the total measurable myocardial PLA2 activity. Little is known about the function of cPLA2α in myocardium although K/O (knockout) of cPLA2α results in cardiac hypertrophy . A recent report identifies the possibility that cPLA2α serves to modulate (attenuate) calcium signals via β2 adrenergic receptors based upon pharmacologic evidence. However, the inhibitor (arachidonoyl trifluromethylketone) used to reach these conclusions inhibits both iPLA2 and cPLA2 activities [47, 48].
Although alterations in membrane dynamics modulate the activity of transmembrane proteins in all cell types, cardiac myocytes are particularly susceptible to such changes. In large part, this is due to the prominent role these proteins play in action potential generation and conduction, their importance in thermodynamically efficient respiration to maximize the efficiency of myocardial bioenergetic functions, and their participation in complex membrane scaffolds which facilitate electromechanical coupling. The thermodynamic efficiency of mitochondrial electron transport is intimately dependent on the glycerophospholipid composition of the inner membrane [49–51]. Furthermore, the efficient coupling of electron transport to ATP production depends on the maintenance of an optimized transmembrane electrochemical gradient. To maximize the efficiency of myocardial electromechanical coupling, Ca2+ must be released synchronously during depolarizing impulses which are conducted rapidly from cell to cell throughout the myocardium. This is largely mediated by the spatiotemporal coupling of sarcolemmal membrane depolarization and voltage sensitive calcium channel opening initiating ryanodine receptor mediated calcium-induced calcium release. Alterations in the reuptake of released calcium can also be modulated by membrane microenvironments that affect the activity of SR (sarcoplasmic reticular) and SL (sarcolemmal) ion pumps potentially resulting in diastolic dysfunction from delayed calcium sequestration. In addition to the participation of the sarcoplasmic reticulum, electromechanical coupling requires organized Ca2+ fluctuations mediated by mitochondrial calcium ion release and reuptake. Both of these systems are intimately dependent on membrane structure and the kinetics of transmembrane enzymes. Thus, the maintenance and adaptation of membrane constituents providing optimized biophysical properties is essential for the integrated function of the multiple ion channels and ion pumps necessary for myocardial conduction and effective contraction.
Among the members of the patatin-like phospholipase family, iPLA2β and iPLA2γ have been demonstrated to be of vital importance in regulating myocardial function. This review will therefore focus predominantly on the functions of these two enzymes in myocardial physiology and pathophysiology. The lack of information on the roles played by more recently described members of the iPLA2 family in myocardial physiology does not exclude the possibility that enzymes such as iPLA2ζ (also termed adipose triglyceride lipase or ATGL and PNPLA2), iPLA2ε (adiponutrin or PNPLA3), or iPLA2η (GS2 or PNPLA4) are important in this setting and they will therefore be covered in a cursory manner at the end of this review.
One of the first mammalian intracellular phospholipases to be identified was calcium-independent phospholipase activity in canine myocardial cytosol which was distinguished from previously known activities based upon its unique kinetic characteristics, Ca2+ independence and substrate selectivity . This enzyme is currently termed calcium-independent phospholipase A2β (iPLA2β), phospholipase group VIA, or patatin like phospholipase 9 (PNPLA9 according to HUGO nomenclature). This myocardial cytosolic phospholipase activity was first purified by anion exchange, chromatofocusing, ATP affinity, and high performance hydroxylapatite chromatographies to yield a 40 kDa isoform that hydrolyzed phospholipids with a high specific activity and that was inhibited and covalently modified by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) . It has since been confirmed that this activity is the major measurable phospholipase activity present in myocardium from most mammalian species including human [52–54]. This enzyme catalyzes the hydrolysis of diacyl and vinyl ether containing phospholipids, lysophospholipids, and acyl-CoAs with a two- to three-fold substrate preference for plasmalogens with arachidonic acid at the sn-2 position . Based upon gel filtration chromatography and immunoprecipitation experiments, iPLA2β is proposed to exist in a 350 kDa complex with phosphofructokinase (PFK)  potentially facilitating the integrated regulation of phospholipolysis and glycolysis during metabolic transitions.
After purification, the gene encoding iPLA2β was subsequently cloned from Chinese hamster ovary (CHO) cells revealing a parent protein with a mass of approximately 85 kDa [22, 23]. The recombinant protein as expressed in Sf9 cells possesses a much lower specific activity than the 40 kDa isoform initially identified in canine myocardium, indicating the possibility of an activating post-translational proteolytic modification(s) or the presence of different splice variants in myocardium. Subsequently, iPLA2β has been cloned or purified from hamster, mouse, and rat, and found to yield similar 85 kDa polypeptides [56–58]. Human iPLA2β has been cloned from multiple tissue sources, and found to be highly homologous to that found in rodents. The affinities of both recombinant and cytosolic iPLA2β for regulatory molecules (ATP and calmodulin) have been exploited to create a high yield tandem affinity chromatographic approach for the rapid purification of catalytically active iPLA2β.
The activity of iPLA2β is regulated at multiple levels. First, the iPLA2β gene is transcriptionally regulated and the resultant mRNA is differentially processed in a cell type and tissue specific manner . At present, at least 4 different splice variants have been identified  including a splice variant that does not possess the active site . Once translated, the holoprotein is subject to a multitude of tissue specific proteolytic cleavages that presumably explain the differences in the molecular weight and the specific activity of the naturally occurring originally purified enzyme from myocardium and that of the full-length gene product or one of its splice variants. One well characterized example of iPLA2β proteolysis is Caspase 3 mediated cleavage at Asp 183, which results in the production of a 70 kDa peptide with increased catalytic activity [61–63]. The identities of other myocardial proteases responsible for proteolytic processing of iPLA2β is currently an area of active investigation.
The activity of iPLA2β is further regulated by allosteric inhibition and activation. The catalytic activity of cytosolic iPLA2β is augmented, and the enzyme is stabilized by ATP . ATP increases catalytic activity between 2 and 5-fold as determined by initial rate kinetics. Furthermore, ATP is critical for stabilization of the enzyme, decreasing the rate of thermal denaturation by over three orders of magnitude . Both ATPγS and GTP have similar effects demonstrating the ability of different purine containing nucleotides to modulate activity.
iPLA2β is also regulated by calmodulin. In early experiments, we recognized that although the crude cytosolic phospholipase activity was inhibited by calcium ion, the purified enzyme was not. We purified this calcium dependent inhibitory factor and identified it as calcium-activated calmodulin [66, 67]. These results indicate the possibility that iPLA2β activity may be regulated on a beat-to-beat basis in myocardium by alterations in Ca2+ concentration in specialized subcellular compartments.
The catalytic activity of iPLA2β is also regulated by covalent modification of the enzyme. In the presence of low micromolar concentrations of oleoyl-CoA, iPLA2β is acylated and CaM-mediated inhibition of iPLA2β is reversed leading to full reconstitution of catalytic activity . The acylation sites and the mechanism(s) responsible for the reversal of calmodulin-mediated regulation are the topics of active investigation. It should be recognized that acyl-CoA molecular species are acutely increased during myocardial ischemia and chronically elevated during diabetic cardiomyopathy [69, 70]. Thus, the disinhibition of iPLA2β by increased cytosolic acyl-CoA is anticipated to result in acute activation during myocardial ischemia and chronic activation during diabetic cardiomyopathy. Moreover, the catalytic products (i.e., fatty acids converted to their corresponding acyl-CoA derivatives by acyl-CoA synthetases) provide a feed forward amplification of iPLA2β activity thereby increasing the lipid burden presented to ischemic or diabetic myocardium.
iPLA2β is also the target of multiple regulatory protein kinases and kinase cascades. These include protein kinase A and the MAP kinases, both of which have been demonstrated to directly phosphorylate the enzyme, although the sites of phosphorylation and the direct catalytic effects of phosphorylation have not yet been identified [71–73]. Protein kinase C also has been implicated as a regulator of iPLA2β catalytic activity [37, 74, 75], but no direct evidence of phosphorylation has yet been demonstrated in intact cells.
The subcellular localization of individual iPLA2β isoforms has the potential to play a prominent role in its physiologic and pathophysiologic functions. Under normal physiologic conditions, the majority of measurable iPLA2β in the heart is present in the cytosol, although substantial amounts of the enzyme are also present in mitochondria [76, 77]. During myocardial ischemia, the enzyme translocates from the cytosol to the microsomal fraction mediated either by its interaction with PFK [52, 78–80], or facilitated by (auto)acylation as indicated above. In addition, caspase 3-mediated proteolysis has been demonstrated to lead to translocation of iPLA2β to the perinuclear compartment in pancreatic β cells, potentially indicating a role for the enzyme in apoptosis .
Substantial evidence has been presented demonstrating the activation of iPLA2β during ischemia. In various systems including ischemic rabbit myocardium, ischemic C2C12 myotubes, and ischemic human coronary artery endothelial cells, iPLA2β is activated [52, 78, 81, 82]. To study the mechanisms underlying iPLA2β activation during ischemia, we generated mice overexpressing iPLA2β in a cardiac myocyte specific manner. Wild-type mice express only diminutive amounts of iPLA2β in myocardium while the dominant iPLA2 isoform in this species is presumably iPLA2γ. This is in contrast to the isoform profile present in other mammalian species including humans. This fact likely explains the lack of a detectable defect in the myocardial function of iPLA2β K/O mice . Interestingly, as a species, mice are relatively refractory to ischemia-induced cardiac arrhythmias. We thus exploited this unusual “species-specific knockdown” of iPLA2β and its concomitant functional phenotype to examine the role of iPLA2β in myocardium. When transgenic mice overexpressing recombinant iPLA2β in a cardiac myocyte restricted manner were subjected to ischemia, prominent arrhythmias (including multiple episodes of ventricular tachycardia) were induced after only a 5 min ischemic episode. These arrhythmias were completely suppressed by the administration of BEL, a mechanism-based inhibitor of iPLA2, prior to the induction of ischemia. Consistent with an augmentation of iPLA2β activity, a dramatic increase in the release of free fatty acids into the coronary sinus effluent was present in transgenic animals subjected to ischemia in comparison to controls. Moreover, extracts of ischemic myocardium from transgenic animals displayed significant increases in lysophosphatidylcholine compared to non-ischemic transgenic, as well as ischemic and non-ischemic wild-type controls. Notably, at baseline, lipid class and individual molecular species analyses by electrospray ionization mass spectrometry of myocardium from iPLA2β overexpressing mice were not substantially different from wild-type mice demonstrating that no substantial alterations in phospholipid remodeling were present during normal flow conditions . It is important to recognize that in addition to precipitating membrane dysfunction and arrhythmias [84–86], the lysolipid product of iPLA2β can directly activate adenylate cyclase through relieving the tonic inhibition caused by its regulatory domain . Furthermore, using an isolated rabbit heart model, mitochondrial iPLA2β activation was demonstrated to be induced by ischemia, ischemia and reperfusion, as well as ischemic preconditioning. Prior treatment with BEL in this system decreased infarct size . Similar findings have also been demonstrated in rat hearts exposed to ischemia . Collectively, these results suggest that under non-ischemic conditions, iPLA2β is tonically inhibited presumably through its association with Ca2+ activated calmodulin. Calmodulin-mediated inhibition of iPLA2β is then rapidly reversed by lipid products generated during ischemia (e.g., acyl-CoA) leading to activation of iPLA2β and precipitation of lethal ventricular arrhythmias and apoptosis. (Figure 3). Interestingly, iPLA2β retains its acyl-CoA hydrolase activity even when bound to CaM. This property of iPLA2β may allow for “internal editing” of acyl-enzyme intermediate(s), resulting in enzymatic self-regulation of activity through intramolecular transacylation and hydrolysis thereby modulating membrane localization and/or CaM disinhibition of enzymic activity . Thus it is envisaged that acylation relieves the tonic inhibition by calmodulin and that the enzyme continues actively hydrolyzing membrane lipids until it undergoes intramolecular deacylation and recombines with inhibitory calmodulin rendering the complex catalytically inactive. It is likely, however, that other mechanisms including phosphorylation and oxidative stress contribute to the regulation of iPLA2β during myocardial ischemia perhaps in part by altering the relative rates of autoacylation and deacylation in addition to potential direct effects on catalytic efficiency.
Little is currently known regarding the normal physiologic function of iPLA2β in myocardial cells. The presence of ankyrin domains in the N-terminal portion of iPLA2β suggests that the association of iPLA2 with ion channels. One might speculate, given its catalytic activity, subcellular distribution and the effects generated by its overexpression, that iPLA2β plays pleiotropic roles in modulating the function of mitochondrial and sarcolemmal ion channels. This modulation may result from modification of the phospholipid environment surrounding a channel, or from effects generated by protein-protein interactions, likely facilitated via association with the N-terminal ankyrin repeats in iPLA2β. Future research will need to concentrate on elucidating normal protein binding partners for iPLA2β, and on the specific effects it has in the regulation of myocardial ion fluxes, both in intact myocytes, as well as on individual ion channels.
The second dominant iPLA2 present in myocardium is iPLA2γ. The initial identification of iPLA2γ (also called phospholipase group VIB or PNPLA8) was made by recognizing the primary sequence homology of a human gene with the ATP binding and active site motifs characterizing the patatin-like phospholipase family . The iPLA2γ gene is located on chromosome 7, and characterization of the gene product demonstrated that it possessed calcium-independent phospholipase activity with separate and distinct kinetic characteristics in comparison to iPLA2β . As is the case with iPLA2β, iPLA2γ undergoes extremely complex differential mRNA splicing resulting in the presence of at least 20 distinct isoforms, suggesting its importance in the regulation of cellular lipid metabolism, signaling and bioenergetics . When cloned and expressed heterologously in Sf9 cells, recombinant iPLA2γ was localized almost exclusively to the membrane fraction, indicating that unlike iPLA2β, it is likely predominantly membrane bound in vivo. The primary amino acid sequence of the holoprotein revealed dual subcellular localization signals including a mitochondrial import signal at the N-terminus and an SKL Type 1 peroxisomal localization sequence at the C-terminal end of the protein . Northern blot analysis from human mRNA revealed that iPLA2γ is expressed in multiple tissues, and at a particularly high level in myocardium . Like iPLA2β, iPLA2γ is inhibited by racemic BEL, thus presenting a problem in the pharmacologic discrimination of the roles of these two proteins. In an attempt to address this issue, we resolved optical enantiomers of BEL and demonstrated that iPLA2γ is selectively inhibited by (R)-BEL, while iPLA2β is selectively inhibited by (S)-BEL . The discrimination achieved by optical antipodes of BEL for iPLA2b and iPLA2g has proven useful in identifying the unique roles of these enzymes in multiple settings.
The 63kD isoform of iPLA2γ has been characterized in the greatest detail because it is partially soluble and can be purified to near homogeneity. The 63kDa isoform is capable of hydrolyzing the carboxy ester bond of phospholipids at either the sn-1 or sn-2 positions, as well as possessing lysophospholipase activity. Notably, iPLA2γ selectively liberates saturated or monounsaturated fatty acid from the sn-1 position when the sn-2 position is occupied by a polyunsaturated fatty acid (e.g., arachidonic acid), thereby generating polyunsaturated lysolipid products (e.g., 2-arachidonoyl lysophosphatidylcholine (2-AA LPC)). 2-arachidonoyl LPC can be subsequently hydrolyzed to release arachidonic acid and glycerophosphocholine, or alternatively to release phosphocholine and 2-arachidonyl glycerol. It is thus likely that 2-AA LPC represents an important metabolic node for multiple signaling pathways in myocardium (for both eicosanoids as well as endocannabinoids) by either the release of AA by lysophospholipase or production of 2-arachidonoyl glycerol by lysophospholipase C [91, 92]. The physiologic relevance of these in vitro kinetic data is supported by the fact that transgenic overexpression of iPLA2γ in a cardiac myocyte specific manner results in the elevation of 2-AA LPC levels in myocardium .
iPLA2γ is subject to regulation at multiple levels during transcription, mRNA maturation, translation and posttranslational modification (Figure 4). Transcription of iPLA2γ is modulated by a repressor sequence at the 5′ end of the gene encoding the holoprotein to which nuclear repressor proteins bind, as well as by the presence two separate transcriptional start sites. The initial transcript is processed to multiple known differentially spliced mature mRNA species, and the coding sequence contains three separate translation initiation start sites which are each employed in intact tissue. Finally, multiple different post-translational modifications have been identified including proteolytic processing of the translated protein. In myocardium, at least 25 iPLA2γ isoforms generated by differential splicing, translation initiation and post translational processing have already been identified based on 2-D SDS PAGE . The significance and regulation of the transcription of each of these different splice variants is a focus of ongoing research.
Calcium-independent phospholipase A2γ is unusual in that it possess dual subcellular localization signals. It has been shown that different splice variants localize to either mitochondria or to peroxisomes, depending on the start site of translation initiation or differential proteolytic processing. The mitochondrial localization signal is present following use of the translation initiation site at the N terminus of the full-length sequence, but can be excluded through use of the two internal translation initiation sites. This dual subcellular localization presents the intriguing potential for iPLA2γ to participate in the sequential metabolic processing of long chain fatty acids in the peroxisome (incomplete oxidation) and subsequent shuttling to the mitochondria for the completion of the oxidative process . Given its subcellular localization, it is likely that previously identified iPLA2 activity associated with the microsomal fraction from various tissues is iPLA2γ, as has been suggested in recent work .
To investigate role of iPLA2γ in myocardial function, we genetically ablated iPLA2γ through homologous recombination . The resultant iPLA2γ knockout (K/O) animals had multiple phenotypes including a deficient myocardial reserve, as demonstrated by their poor survival following transverse aortic constriction, as well as by their extremely poor exercise performance relative to wild-type littermates. Their inefficient myocardial performance may arise due to mitochondrial dysfunction since the ability of Complex IV to dynamically increase its function was demonstrated to be severely compromised in mitochondria isolated from the K/O animals. Moreover, the content and composition of critical mitochondrial phospholipids associated with normal Complex IV function were significantly altered. Specifically, iPLA2γ K/O mice had a significant decrease in overall myocardial cardiolipin content which was most evident in the decrease in the relative abundance of the physiologically important symmetric tetralinoleoyl (tetra 18:2) cardiolipin molecular species. Cardiolipin is tightly bound to cytochrome oxidase (complex IV) and alterations in cardiolipin content are known to precipitate dysfunction of the electron transport chain in the mitochondrial inner membrane. The importance of altered mitochondrial cardiolipin species content in precipitating myocardial dysfunction is highlighted clinically by Barth syndrome. Barth syndrome is characterized by defective cardiolipin remodeling that results from nucleotide substitutions in the tafazzin gene (a mitochondrial lysocardiolipin transacylase) that compromise enzymic activity . Patients suffering from Barth syndrome have a severe cardiomyopathy that leads to death from heart failure in the first few years of life . Thus, alterations in cardiolipin content due to pathologic alterations in cardiolipin deacylation (by iPLA2γ and potentially other phospholipases) and reacylation (by tafazzin and acyl transferases) precipitate mitochondrial dysfunction in humans.
To further investigate the role iPLA2γ plays in myocardial physiology, transgenic mice overexpressing iPLA2γ in a cardiomyocyte-restricted manner were generated . These mice had normal myocardial function and exercise tolerance at baseline, but after overnight fasting developed myocardial dysfunction characterized by decreased fractional shortening, increased left ventricular dimensions, and a paradoxic bradycardia. Lipidomic analysis demonstrated that the hearts of these animals had a marked decrease in myocardial phospholipid mass including both choline and ethanolamine phospholipids accompanied by an increase in lysophospholipids with polyunsaturated acyl chains at the sn-2 position. After overnight fasting, there was a dramatic increase in myocardial triglyceride content in the transgenic mice in comparison to wild-type controls. Investigation of mitochondrial function in iPLA2γ overexpressing mice revealed substantially impaired state 3 respiration. This defect in respiration was accompanied by abnormal mitochondrial morphology as assessed by electron microscopy. Transgenic hearts also were noted to utilize fatty acid at the expense of glucose for baseline metabolic requirements likely resulting from increased fatty acid availability due to increased phospholipase activity.
Taken together, these results indicate that iPLA2γ plays a fundamental role in glycerophospholipid remodeling in the myocardium, and more specifically in the mitochondria to optimize bioenergetic efficiency, through precisely tailoring mitochondrial inner membrane composition to adapt to changing hemodynamic demands, alterations in substrate delivery and changes in cellular signaling status. Dysregulation of iPLA2γ expression level and/or activity provokes mitochondrial dysfunction via alterations in cardiolipin content and maladaptive alterations in cardiolipin species, and effects intracellular signaling pathways by altering the production of 2-AA LPC. iPLA2γ has also been demonstrated to play a role in the mitochondrial permeability transition and in the generation of apoptosis via cytochrome c release, further supporting its fundamental role in mitochondrial biology [98–100].
Database searches for proteins containing the GXGXXG and GXSXG consensus sequences and patatin homology domains identified three novel members of the iPLA2 family. These more recently identified members of the patatin-like phospholipase family of proteins have the potential to play important roles in myocardial physiology. iPLA2ζ (ATGL) was recently demonstrated to be the enzyme primarily responsible for adipose tissue triglyceride lipolysis [101, 102]. iPLA2ζ primarily displays triglyceride lipase activity and lower amounts of acylglycerol transacylase PLA2 activity when measured in vitro. Elegant work from the Zechner group demonstrated that genetic ablation of iPLA2ζ results in accumulation of triglycerides in myocardium precipitating congestive heart failure and death from lipotoxicity at 10–12 weeks of age . These results identify iPLA2ζ as the major triglyceride lipase in myocardium. In addition, animals null for iPLA2ζ, predictably, were increasingly dependent on glucose as an energy source, and had defective thermogenesis. Whether iPLA2ζ behaves simply as the primary triglyceride lipase in the heart, or has other catalytic activities as demonstrated in vitro, resulting in alteration of membrane dynamics and lipid second messenger production is the subject of ongoing research.
Another patatin-like phospholipase family member, iPLA2η was recently identified and characterized . It was found to be a 28 kDa protein with relatively high levels of transacylase activity in comparison to its PLA2 activity. iPLA2η is ubiquitously expressed, including expression in myocardial tissue. Although it has not been specifically studied in this context, one might speculate that it serves an important role in lipid storage and potentially in mediating the shuttling of acyl chains in myocardium through transacylation creating a dynamic flux that is capable of rapidly responding to changes in the nutrient status or hemodynamic requirements of the organism as a whole.
Finally, the discovery of yet another calcium-independent phospholipase, iPLA2ε, may prove to have physiologic relevance in normal myocardial metabolism and function. This 53 kDa protein has TAG lipase activity and transacylase activity similar to that of the other two recently identified members of the patatin-like phospholipase family of enzymes . However, its presence in the heart has yet to be identified. There are 4 additional members of the iPLA2 family in the human genome (PNPLA1-9) whose function in myocardial lipid metabolism, energy homeostasis and cellular signaling have not as yet been defined.
Detailed investigations of the diverse cellular processes in which the iPLA2 family of enzymes is involved will require the development of enzyme-specific pharmacologic inhibitors and/or activators. Although no pharmacologic activators of these enzymes have yet been identified, there are several inhibitors (both specific and non-specific) available. Arachidonyl trifluoromethyl ketone (AACOCF3) and methyl arachidonyl fluorophosphonate (MAFP), originally developed as inhibitors of cPLA2 were subsequently demonstrated to also inhibit iPLA2 family enzymes. [32, 104–106] The poor specificity of these compounds led to a search for mechanism-based inhibitors with greater enzyme selectivity. Early studies in pursuit of this goal concentrated on modifying AACOCF3 and MAFP by attaching acyl chains of differing lengths in substitution for arachidonic acid . This line of investigation was successful in identifying two compounds with a relative specificity for iPLA2 compared to cPLA2. These compounds, however, continued to demonstrate inhibition of cPLA2 at concentrations required to inhibit iPLA2. The most specific inhibitor of iPLA2 enzymes (iPLA2β and iPLA2γ) is the previously identified bromoenol lactone (BEL) which demonstrates a 100-fold selectivity for this family of enzymes . BEL is a mechanism-based suicide substrate for iPLA2 enzymes. Hydrolysis of the lactone ring by the active site nucleophilic serine results in the formation of a bromo-keto-acid. This hydrolysis product then alkylates either the active site serine or a nearby reduced cysteine residue resulting in the formation of a covalent complex and irreversible inhibition [108, 109]. In addition, the enantiomers of BEL, when resolved using reverse phase chromatography, have been demonstrated to have selectivity for different iPLA2 isoforms . (S)-BEL has been demonstrated to specifically inhibit iPLA2β while (R)-BEL specifically inhibits iPLA2γ with an approximate 10-fold selectivity. The specificity of BEL inhibition for iPLA2 catalytic activity, however, has been called into question by the demonstration that it also inhibits a macrophage phosphatidate phosphohydrolase . Thus, in spite of the promise that pharmacologic modulation of iPLA2 family enzymes holds for advancing research and potential future medical therapy, the development of specific inhibitors for each enzyme is a challenging but soluble problem given the diversity of the different members of the iPLA2 family. The development of future enzymatic modulators might focus on molecules that exploit the calmodulin binding site for inhibition of the enzyme, or the ATP binding site for activation. The unique nature of these binding sites and of the surrounding protein milieu hold the great promise as targets for drug development.
Calcium-independent phospholipases play fundamental roles in myocardial physiology (Figure 5). Their catalytic activity results in the generation of a variety of lipid 2nd messengers whose role is to facilitate adaptation to physiologic and pathophysiologic perturbations. Appropriate iPLA2 regulation is also essential for maintaining membrane integrity, efficient integral membrane protein and ion channel function and bioenergetic efficiency. Through their pleiotropic effects on cardiac myocyte bioenergetics, cellular signaling and membrane function, the iPLA2 family of enzymes plays a prominent role in the pathologic sequelae of myocardial ischemia, diabetic cardiomyopathy and remodeling during hemodynamic stress. Elucidation of the multiple mechanisms through which discrete iPLA2 gene products modulate cardiovascular function holds great promise for the development of targeted pharmacotherapy to attenuate the deleterious sequelae of ischemic heart disease and congestive heart failure in the future.
This work was supported by National Institutes of Health Grant 2PO1HL057278-11A1.