The phospholipase A(2) (PLA(2)) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA(2) types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA(2) enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA(2)/membrane interactions are addressed.

Hydrogen sulfide (H2S) is a biological gasotransmitter that has been employed for the treatment of ischemia-reperfusion injury. Despite its therapeutic value, the implementation of this gaseous molecule for this purpose has required H2S-releasing prodrugs for effective intracellular delivery. The majority of these prodrugs, however, spontaneously release H2S via uncontrolled hydrolysis. Here, we describe a Ru(II)-based H2S-releasing agent that can be activated selectively by red light irradiation. This compound operates in living cells, increasing intracellular H2S concentration only upon irradiation with red light. Furthermore, the red light irradiation of this compound protects H9c2 cardiomyoblasts from an in vitro model of ischemia-reperfusion injury. These results validate the use of red light-activated H2S-releasing agents as valuable tools for studying the biology and therapeutic utility of this gasotransmitter.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), specifically Group VIIA PLA(2), is a member of the phospholipase A(2) superfamily and is found mainly associated with LDL and HDL in human plasma. Lp-PLA(2) is considered as a risk factor, a potential biomarker, a target for therapy in the treatment of cardiovascular disease, and evidence suggests that the level of Lp-PLA(2) in plasma is associated with the risk of future cardiovascular and stroke events. The differential location of the enzyme in LDL/HDL lipoproteins has been suggested to affect Lp-PLA(2) function and/or its physiological role and an abnormal distribution of the enzyme may correlate with diseases. Although a mutagenesis study suggested that a surface helix (residues 362-369) mediates the association between Lp-PLA(2) and HDL, the molecular details and mechanism of association has remained unknown. We have now employed hydrogen deuterium exchange mass spectrometry to characterize the interaction between recombinant human Lp-PLA(2) and human HDL. We have found that specific residues 113-120, 192-204, and 360-368 likely mediate HDL binding. In a previous study, we showed that residues 113-120 are important for Lp-PLA(2)-liposome interactions. We now find that residues 192-204 show a decreased deuteration level when Lp-PLA(2) is exposed to apoA-I, but not apoA-II, the most abundant apoproteins in HDL, and additionally, residues 360-368 are only affected by HDL.The results suggest that apoA-I and phospholipid membranes play crucial roles in Lp-PLA(2) localization to HDL.

Phospholipases represent one of the earliest enzyme activities to be identified and studied, and the phospholipase A2 superfamily traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. Both electrostatic and hydrophobic interactions contribute to the interfacial binding of sPLA2 to anionic phospholipid membranes. The interaction between basic residues on the binding surface with anionic vesicles plays an important role in interfacial binding. The major functions will be summarized below and include the ability to kill Gram-positive and Gram-negative bacteria, thereby affecting host defense against bacterial infections. sPLA2 may be involved in the pathogensis of inflammatory bowel disease including Crohn's disease and ulcerative colitis. GIIA sPLA2 protein and mRNA were detected in Paneth cells of the small intestinal mucosa in the intestine in Crohn's disease patients.

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis, and sepsis. Lp-PLA(2) also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA(2) plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA(2) with dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA₂ binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA₂ and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA₂'s specificity toward oxidized phospholipids.

BACKGROUND: The prognostic significance of left atrial (LA) dysfunction in cardiac amyloidosis is being increasingly acknowledged, but its importance in stage II light-chain cardiac amyloidosis (AL-CA) remains unclear. This study aimed to determine the prognostic value of LA strain in stage II AL-CA. METHODS: Patients with stage II AL-CA who underwent cardiac magnetic resonance at Peking Union Medical College Hospital between January 1, 2015, and October 31, 2021, were consecutively enrolled in this cohort study. Telephone and clinical follow-ups were conducted monthly. The primary endpoints were all-cause mortality, and the secondary endpoints were the combination of all-cause mortality and cardiac progression. Cox regression and Kaplan-Meier survival analyses were performed to identify associations between variables and outcomes. RESULTS: We included 131 patients with stage II AL-CA [61% male; median age 59 years; interquartile range (IQR), 51-65 years]. Among these patients, 37 (28%) reached the primary endpoint (all-cause mortality), and 52 (40%) reached the secondary endpoint (median follow-up 28 months; IQR, 10-50 months). Serum biomarker N-terminal pro-B-type natriuretic peptide and LA strain were significantly associated with the outcomes (all P values <0.05). In the multivariable models, reduced LA booster strain remained associated with a higher risk of all-cause mortality [hazard ratio (HR) 0.64, 95% confidence interval (CI): 0.42-0.96; P=0.031] and the secondary endpoint (HR 0.68, 95% CI: 0.49-0.93; P=0.015). The metrics of cardiac function that showed superior accuracy in predicting the risk of mortality were LA reservoir strain [concordance index (C-index) 0.72; 95% CI: 0.64-0.80] and LA booster strain (C-index 0.71; 95% CI: 0.64-0.80). The addition of LA booster strain to established prognostic predictors improved the discrimination and goodness of fit (P<0.001). CONCLUSIONS: LA booster strain is an independent prognostic indicator in stage II AL-CA and is superior to metrics of LV function. LA strain has potential clinical value as an early prognostic marker and may aid in identifying underdiagnosed patients with poor prognosis.

Aim

To investigate the survival of bone marrow mesenchymal stem cells (BMSCs) and the therapeutic effect for acute myocardial infarction (AMI) after co-transplantation with the functionalized self-assembling peptide nanofiber RAD/PRG or RAD/KLT.

Methods

AMI of balb/c mice was induced. Mice were randomly divided into four groups, and received treatment of phosphate buffered saline (PBS) (Group A), GFP/Fluc-BMSCs (Group B), GFP/Fluc-BMSCs + RAD/PRG (Group C), and GFP/Fluc-BMSCs + RAD/KLT (Group D), respectively. Bioluminescence imaging (BLI) was performed on day 1 (d-1), d-4, d-7, d-10, and d-13 after transplantation. Magnetic resonance imaging (MRI) was performed at baseline (d-4 before transplantation) and d-29 after treatment. Mice were euthanized on d-29 following treatment. Paraffin sections were obtained from the top, mid and bottom part of the infarcted region along the long-axis of the heart. Hematoxylin and eosin (HE) staining and immunohistochemical staining were performed. The infarct ratio micro-vascular density (MVD) was quantified.

Results

There was a significant higher of BLI signal intensity of BMSCs in Group C than that in Group B (d-4, 9713±320 vs. 8164±378, P=0.0008; d-7, 6489±241 vs. 5417±361, P=0.0026; d-10, 3768±255 vs. 0, P < 0.0001). The left ventricular ejection fraction (LVEF) via MRI examination was significantly improved in both Group C and Group D. Infarct ratio and MVD were significantly improved in both Group C and Group D.

Conclusion

Our data highlights BMSCs combining functionalized self-assembling peptide nanofibers RAD/PRG or RAD/KLT as promising therapy for AMI.