Both histone-acetylations and histone deacetylases have been shown to play a key role in cardiac remodeling. Recently, it has become abundantly clear that many non-histone proteins are modified by post-translational lysine acetylations and that these acetylations regulate protein activity, conformation, and binding. In the present study, non-histone acetylated proteins associated with heart failure were identified. Global screening for lysine acetylated proteins was performed using 2-dimensional gel electrophoresis coupled with immunoblotting with a primary monoclonal anti-acetyl-lysine antibody. Lysine acetylated proteins were compared in two rodent models of hypertensive heart failure, the Dahl salt-sensitive (SS) and spontaneously hypertensive heart failure prone (SHHF) rats with those in corresponding controls, i.e., the Dahl salt-resistant (SR) and W (W) rat strains, respectively. Forty-one and 66 acetylated proteins were detected in SS and SHHF failing hearts, respectively, but either not detected or detected with less abundance in corresponding control hearts. Twelve of these acetylated proteins were common to both models of heart failure. These were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry followed by Mascot Analysis and included mitochondrial enzymes: ATP synthase, long-chain acyl-CoA dehydrogenase, creatine kinase, malate dehydrogenase, and pyruvate dehydrogenase. The abundance of NAD-dependent deacetylase sirtuin-3 (Sirt3), a mitochondrial deacetylase was reduced in SS and SHHF failing hearts. This is the first description of non-histone protein acetylations associated with heart failure and raises the prospect that acetylations of mitochondrial proteins linked to reduced Sirt3 mediate, in part, metabolic changes in heart failure.1

The objective of this study was to determine the role of A-Kinase Anchoring Protein (AKAP)-Lbc in the development of heart failure, by investigating AKAP-Lbc-protein kinase D1 (PKD1) signaling in vivo in cardiac hypertrophy. Using a gene-trap mouse expressing a truncated version of AKAP-Lbc (due to disruption of the endogenous AKAP-Lbc gene), that abolishes PKD1 interaction with AKAP-Lbc (AKAPLbc-ΔPKD), we studied two mouse models of pathological hypertrophy: i) angiotensin (AT-II) and phenylephrine (PE) infusion and ii) transverse aortic constriction (TAC)-induced pressure overload. Our results indicate that AKAP-Lbc-ΔPKD mice exhibit an accelerated progression to cardiac dysfunction in response to AT-II/PE treatment and TAC. AKAP-Lbc-ΔPKD mice display attenuated compensatory cardiac hypertrophy, increased collagen deposition and apoptosis, compared to wild-type (WT) control littermates. Mechanistically, reduced levels of PKD1 activation are observed in AKAP-Lbc-ΔPKD mice compared to WT mice, resulting in diminished phosphorylation of histone deacetylase 5 (HDAC5) and decreased hypertrophic gene expression. This is consistent with a reduced compensatory hypertrophy phenotype leading to progression of heart failure in AKAP-Lbc-ΔPKD mice. Overall, our data demonstrates a critical in vivo role for AKAP-Lbc-PKD1 signaling in the development of compensatory hypertrophy to enhance cardiac performance in response to TAC-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment.

There is over-whelming evidence that protein phosphorylations regulate cardiac function and remodeling. A wide variety of protein kinases, e.g., phosphoinositide 3-kinase (PI3K), Akt, GSK-3, TGFβ, and PKA, MAPKs, PKC, Erks, and Jaks, as well as phosphatases, e.g., phosphatase I (PP1) and calcineurin, control cardiomyocyte growth and contractility. In the present work, we used global phosphoprotein profiling to identify phosphorylated proteins associated with pressure overload (PO) cardiac hypertrophy and heart failure. Phosphoproteins from hypertrophic and systolic failing hearts from male hypertensive Dahl salt-sensitive rats, trans-aortic banded (TAC), and spontaneously hypertensive heart failure (SHHF) rats were analyzed. Profiling was performed by 2-dimensional difference in gel electrophoresis (2D-DIGE) on phospho-enriched proteins. A total of 25 common phosphoproteins with differences in abundance in (1) the 3 hypertrophic and/or (2) the 2 systolic failure heart models were identified (CI>99%) by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Mascot analysis. Among these were (1) myofilament proteins, including alpha-tropomyosin and myosin regulatory light chain 2, cap Z interacting protein (cap ZIP), and tubulin β5; (2) mitochondrial proteins, including pyruvate dehydrogenase α, branch chain ketoacid dehydrogenase E1, and mitochondrial creatine kinase; (3) phosphatases, including protein phosphatase 2A and protein phosphatase 1 regulatory subunit; and (4) other proteins including proteosome subunits α type 3 and β type 7, and eukaryotic translation initiation factor 1A (eIF1A). The results include previously described and novel phosphoproteins in cardiac hypertrophy and systolic failure.

Natriuretic peptides (NPs) are evolutionarily conserved hormones that affect blood pressure and fluid volume through membrane-bound guanylate cyclase (GC)-linked natriuretic peptide receptors-A and -B (NPR-A and NPR-B, respectively) in a variety of vascular, renal, and other tissues. The principal physiological stimulus for cardiac NPs in fish is somewhat debated between two prominent theories: regulation of salt balance (osmoregulatory hypothesis) or prevention of volume expansion (cardioprotective hypothesis). In the present study, we examined atrial and ventricular expression of trout NPs, atrial (ANP), brain (BNP), and ventricular (VNP) using Northern (mRNA), Western (NP pro-hormone), and qPCR (GC-NPR-A and -B mRNA) blot analysis following independent manipulation of plasma salt and volume levels after chronic exposure to freshwater (FW; volume loaded, salt depleted), saltwater (SW; volume depleted, salt loaded), or freshwater trout fed a high-salt diet (FW-HSD; volume and salt loaded). We also measured NP transcriptional response to acute (2 h) volume expansion with dialyzed plasma (VE; 80% blood vol) or volume depletion by hemorrhage (VD, 20% blood volume every 30 min for 2 h) with real-time PCR. In essentially all instances, increased expression of the NP system was associated with FW-HSD or plasma expansion. There were no differences in NP expression between chronically adapted FW and SW fish, and hemorrhage decreased atrial ANP and VNP mRNA. These results indicate that rainbow trout cardiac NPs and cardiovascular GC-NPRs respond principally to volume, not salt overload, and this suggests that the primary function of trout cardiac NP system is to protect the heart.

Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic β-adrenergic receptor (β-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3′, 5′-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-Kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic β-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity.

Although cystic fibrosis (CF) is attributed to dysfunction of a single gene, the relationships between the abnormal gene product and the development of inflammation and progression of lung disease are not fully understood, which limits our ability to predict an individual patient’s clinical course and treatment response. To better understand CF progression, we characterized the molecular signatures of CF disease status with plasma-based functional genomics. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with plasma samples from CF patients ( n = 103) and unrelated, healthy controls ( n = 31). Gene expression levels were measured with an Affymetrix microarray (GeneChip Human Genome U133 Plus 2.0). Peripheral blood samples from a subset of the CF patients ( n = 40) were immunophenotyped by flow cytometry, and the data were compared with historical data for age-matched healthy controls ( n = 351). Plasma samples from another subset of CF patients ( n = 56) and healthy controls ( n = 16) were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA) for numerous cytokines and chemokines. Principal component analysis and hierarchical clustering of induced transcriptional data revealed disease-specific plasma-induced PBMC profiles. Among 1,094 differentially expressed probe sets, 51 genes were associated with pancreatic sufficient status, and 224 genes were associated with infection with Pseudomonas aeruginosa. The flow cytometry and ELISA data confirmed that various immune modulators are relevant contributors to the CF molecular signature. This study provides strong evidence for distinct molecular signatures among CF patients. An understanding of these molecular signatures may lead to unique molecular markers that will enable more personalized prognoses, individualized treatment plans, and rapid monitoring of treatment response.

No abstract

BackgroundA-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport.ResultsShown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction) corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels) that promote the production of AKAP7γ instead of AKAP7δ.ConclusionsThis AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.