Distinct Transcriptomic and Proteomic Profile Specifies Patients Who Have Heart Failure With Potential of Myocardial Recovery on Mechanical Unloading and Circulatory Support.

BACKGROUND: Extensive evidence from single-center studies indicates that a subset of patients with chronic advanced heart failure (HF) undergoing left ventricular assist device (LVAD) support show significantly improved heart function and reverse structural remodeling (ie, termed "responders"). Furthermore, we recently published a multicenter prospective study, RESTAGE-HF (Remission from Stage D Heart Failure), demonstrating that LVAD support combined with standard HF medications induced remarkable cardiac structural and functional improvement, leading to high rates of LVAD weaning and excellent long-term outcomes. This intriguing phenomenon provides great translational and clinical promise, although the underlying molecular mechanisms driving this recovery are largely unknown. METHODS: To identify changes in signaling pathways operative in the normal and failing human heart and to molecularly characterize patients who respond favorably to LVAD unloading, we performed global RNA sequencing and phosphopeptide profiling of left ventricular tissue from 93 patients with HF undergoing LVAD implantation (25 responders and 68 nonresponders) and 12 nonfailing donor hearts. Patients were prospectively monitored through echocardiography to characterize their myocardial structure and function and identify responders and nonresponders. RESULTS: These analyses identified 1341 transcripts and 288 phosphopeptides that are differentially regulated in cardiac tissue from nonfailing control samples and patients with HF. In addition, these unbiased molecular profiles identified a unique signature of 29 transcripts and 93 phosphopeptides in patients with HF that distinguished responders after LVAD unloading. Further analyses of these macromolecules highlighted differential regulation in 2 key pathways: cell cycle regulation and extracellular matrix/focal adhesions. CONCLUSIONS: This is the first study to characterize changes in the nonfailing and failing human heart by integrating multiple -omics platforms to identify molecular indices defining patients capable of myocardial recovery. These findings may guide patient selection for advanced HF therapies and identify new HF therapeutic targets.

Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Protein Arginine Methyltransferase†1-Dependent Labeling and Isolation of Histone H4 through N-Mustard Analogues of S-Adenosyl-l-methionine.

Histones, the fundamental building blocks of nucleosomes, undergo post-translational modifications and play a major role in the regulation of transcriptional processes. Although the significance of these modifications, including methylation, is widely recognized, little is known about the mechanisms connecting such events. To improve our understanding of how protein methylation is intricately linked, we have developed novel N-mustard analogues of S-adenosyl-l-methionine (SAM) functionalized with azides and alkynes to serve as probes of biological methylation. Here, we demonstrate their ability to serve as effective cofactor mimics of SAM and to be enzymatically transferred by protein arginine methyltransferase 1 (PRMT1) to histone H4 with high site selectively for its target Arg3 on the histone tail. Further incorporation of biotin through copper-catalyzed click chemistry permitted visualization and isolation of the analogue-modified histone H4 from a complex mixture. This work validates the future utility of N-mustard analogues as probes of protein methylation events beyond PRMT1.

Cysteine-Directed Bioconjugation of a Platinum(II)-Acridine Anticancer Agent.

Classical maleimide Michael addition chemistry in conjunction with copper-free click chemistry was investigated as a synthetic strategy to attach cytotoxic platinum-acridine hybrid agents to carrier proteins. The structural integrity and selectivity of the model payloads, which were validated in human serum albumin (HSA) using mass spectrometric analysis and heteronuclear 2D 1H-15N HSQC NMR experiments, may have broad utility for the targeted delivery of highly cytotoxic platinum acridines and other nonclassical platinum containing anticancer agents.

GFI1 functions in transcriptional control and cell fate determination require SNAG domain methylation to recruit LSD1.

Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.

Programmed cell death protein 5 interacts with the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) to regulate ß-tubulin folding.

Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and ß-tubulin, a key CCT-folding substrate, and specifically inhibited ß-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of ß-tubulin folding. PDCD5 bound the apical domain of the CCTß subunit, projecting above the folding cavity without entering it. Like PDCD5, ß-tubulin also interacts with the CCTß apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and ß-tubulin suggest that PDCD5 sterically interferes with ß-tubulin binding to the CCTß apical domain and inhibits ß-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of ß-tubulin folding.

Chaperone-mediated assembly of G protein complexes.

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into functional complexes. Formation of the G protein ßγ (Gßγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gß and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and its co-chaperone phosducin-like protein 1 (PhLP1). CCT assists Gß in achieving its ß-propeller structure, while PhLP1 releases Gß from CCT and facilitates its interaction with Gγ. Once Gßγ is formed, PhLP1 remains bound until it is displaced by the Gα subunit and the G protein heterotrimer is brought together. Another obligate dimer is the complex between the G protein ß(5) subunit and a regulator of G protein signaling protein (Gß(5)-RGS). Gß(5)-RGS also requires CCT for Gß(5) folding, but PhLP1 plays a different role. It stabilizes the interaction between Gß(5) and CCT, perhaps to increase folding efficiency. After Gß(5) folding PhLP1 must subsequently release, allowing the RGS protein to bind and form the Gß(5)-RGS dimer directly on CCT. Gß(5)-RGS is then freed from CCT to interact with its membrane anchoring protein and form a stable complex that turns off the G protein signal by catalyzing GTP hydrolysis on Gα.

Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

G protein ß subunits (Gß) play essential roles in phototransduction as part of G protein ßγ (Gßγ) and regulator of G protein signaling 9 (RGS9)-Gß5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gß5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gßγ and RGS9-Gß5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gß complex formation in G protein signaling.

Ultrasonic gene and drug delivery using eLiposomes.

eLiposomes are liposomes encapsulating emulsions and therapeutics for targeted delivery. By applying ultrasound to eLiposomes, emulsion droplets can transform from liquid to gas and rupture the lipid bilayer of the eLiposome to release a drug or plasmid. In this study, perfluoropentane (PFC5) emulsions were encapsulated inside folated eLiposomes carrying a model drug (calcein) or a model GFP plasmid to examine the effects of a folate ligand, PFC5 emulsion and various ultrasonic acoustic parameters in drug delivery and gene transfection into HeLa cells. Confocal microscopy was used to quantify drug delivery and the level of plasmid transfection into HeLa cells. The results showed that drug delivery or transfection was minimal without incorporation of internal PFC5 emulsions and folate ligand on the eLiposome surface. It was also shown that application of ultrasound greatly enhanced the drug delivery and plasmid transfection. Delivery of these therapeutics appears to be to the cytosol, indicating that the expansion of the emulsion droplets disrupted both the eLiposomes and the endosomes.

Potential energy surfaces for small alcohol dimers. II. Propanol, isopropanol, t-butanol, and sec-butanol.

Potential energy landscapes for homogeneous dimers of propanol, isopropanol, tert-butanol, and sec-butanol were obtained using 735 counterpoise-corrected energies at the MP2/6-311+G(2df,2pd) level. The landscapes were sampled at 15 dimer separation distances for different relative monomer geometries, or routes, given in terms of the yaw, pitch, and roll of one monomer relative to the other and the spherical angles between the two monomer centers (taken as the C atom attached to the O). The resultant individual energy surfaces and their complex topographies were also regressed using a site-site pair potential model using a modified Morse potential that provides a mathematically simple representation of the landscapes suitable for use in molecular simulations. Generalized Morse parameters were also obtained for this model from a composite regression of these energy landscapes and those previously reported for methanol and ethanol. The quality of fit for all these energy landscapes suggests that these site parameters have transferability for possible use on other alcohols.

Potential energy surfaces for small alcohol dimers I: methanol and ethanol.

Potential energy landscapes for homogeneous dimers of methanol and ethanol were calculated using counterpoise (CP) corrected energies at the MP26-311+G(2df,2pd) level. The landscapes were sampled at approximately 15 dimer separation distances for different relative monomer geometries, or routes, given in terms of a relative monomer yaw, pitch, and roll and the spherical angles between the monomer centers (taken as the C atom attached to the O). The 19 different routes studied for methanol and the 22 routes examined for ethanol include 607 CP corrected energies. Both landscapes can be adequately represented by site-site, pairwise-additive models, suitable for use in molecular simulations. A modified Morse potential is used for the individual pair interactions either with or without point charges to represent the monomer charge distribution. A slightly better representation of the methanol landscape is obtained using point charges, while the potential energy landscape of ethanol is slightly better without point charges. This latter representation may be computationally advantageous for molecular simulations because it avoids difficulties associated with long-range effects of point-charge-type models.