Cardioprotective Effects of Palmitoleic Acid (C16:1n7) in a Mouse Model of Catecholamine-Induced Cardiac Damage Are Mediated by PPAR Activation

Palmitoleic acid (C16:1n7) has been identified as a regulator of physiological cardiac hypertrophy. In the present study, we aimed to investigate the molecular pathways involved in C16:1n7 responses in primary murine cardiomyocytes (PCM) and a mouse model of isoproterenol (ISO)-induced cardiac damage. PCMs were stimulated with C16:1n7 or a vehicle. Afterwards, RNA sequencing was performed using an Illumina HiSeq sequencer. Confirmatory analysis was performed in PCMs and HL-1 cardiomyocytes. For an in vivo study, 129 sv mice were orally treated with a vehicle or C16:1n7 for 22 days. After 5 days of pre-treatment, the mice were injected with ISO (25 mg/kg/d s. c.) for 4 consecutive days. Cardiac phenotyping was performed using echocardiography. In total, 129 genes were differentially expressed in PCMs stimulated with C16:1n7, including Angiopoietin-like factor 4 (Angptl4) and Pyruvate Dehydrogenase Kinase 4 (Pdk4). Both Angptl4 and Pdk4 are proxisome proliferator-activated receptor α/δ (PPARα/δ) target genes. Our in vivo results indicated cardioprotective and anti-fibrotic effects of C16:1n7 application in mice. This was associated with the C16:1n7-dependent regulation of the cardiac PPAR-specific signaling pathways. In conclusion, our experiments demonstrated that C16:1n7 might have protective effects on cardiac fibrosis and inflammation. Our study may help to develop future lipid-based therapies for catecholamine-induced cardiac damage

Der Einfluss von Palmitoleinsäure (C16:1) auf die PI3K/AKT/FOXO und ERK 1/2 (MAPK) Signalkaskaden im Kontext physiologischer kardialer Hypertrophie

Palmitoleinsäure (C16:1) ist eine einfach ungesättigte Fettsäure, die kürzlich durch unsere Arbeitsgruppe als wichtiger Mediator physiologischer kardialer Hypertrophie (PCH) charakterisiert wurde. Da die molekulare Wirkweise jedoch weitgehend ungeklärt ist, war das Ziel dieser Arbeit den intrazellulären Wirkmechanismus von C16:1 zu entschlüsseln. Das Hauptaugenmerk sollte hierbei auf der Phosphoinositid-3-Kinase (PI3K)- Proteinkinase B (AKT)- Forkhead-Box-Protein (FOXO) Signalkaskade liegen. Murine HL-1 Kardiomyozyten wurden entweder mit bovinem Serumalbumin als Vehikel, 40 mM C16:1, 130 mM Palmitinsäure (C16:0) oder 10 nM Insulin like Growth factor-1 (IGF-1) als Positivkontrolle stimuliert. Die Stimulationsintervalle variierten hierbei zwischen 5 min und 48 h. Im Anschluss wurden Gesamt-RNA und Proteine isoliert und mittels qRT-PCR und Western Blot analysiert. Darüber hinaus wurden Immunfluorenz-Assays durchgeführt. Hierzu wurden die immungefärbten Zellen mit einem invertierten Fluoreszenz-Phasenkontrast-Mikroskop vergrößert und mit der BZ-II Analyser Software analysiert. Zuletzt wurde ebenfalls der Effekt von C16:1 auf den Mitogen-activated Protein Kinases (MAPK)- Extracellular-signal regulated Kinases (ERK) Signalweg betrachtet. Die atrophieregulierenden FOXO-Transkriptionsfaktoren 1 und 3a zeigten sich nach einer 30-minütigen C16:1 Stimulation durch Phosphorylierung inaktiviert. Zudem beobachteten wir eine Reduktion der FOXO 1 und 3a Gesamtproteinmenge infolge 18-stündiger C16:1 Stimulation. Mittels Immunfluoreszenzmikroskopie konnte die C16-induzierte zytoplasmatische Translokation von FOXO3a nachgewiesen werden. Das Genexpressionslevel des FOXO3a-Zielgens p21 erwies sich nach 24-48 h C16:1 Stimulation signifikant hochreguliert. Ferner zeigte sich die wachstumsfördernde AKT-Kinase durch C16:1 aktiviert. Die Zellzyklus-regulierende Kinase ERK 1/2 präsentierte eine Inaktivierung mittels Dephosphorylierung infolge der C16:1 Stimulation. Zusammenfassend konnten AKT, FOXO und ERK 1/2 als intrazelluläre Schlüsselmediatoren von C16:1 in Kardiomyozyten identifiziert werden. Es konnte gezeigt werden, dass nicht nur die Aktivierung der wachstumsfördernden AKT-Kinase, sondern auch die Hemmung der atrophieregulierenden FOXO-Proteine bei der PCH eine zentrale Rolle spielt. Darüber hinaus scheint C16:1 durch die Inaktivierung von ERK 1/2, möglicherweise auch die Proliferation von Kardiomyozyten zu regulieren. Somit ist es naheliegend, dass die Effekte von C16:1 auf die kardiale Hypertrophie über die Beeinflussung von AKT/FOXO und ERK 1/2 vermittelt werden. Diese Studie liefert ein Hinweis darauf, dass die Fettsäure C16:1 molekulare kardioprotektive Effekte vermittelt, die zukünftig sowohl potentiell in der Prophylaxe als auch in der Therapie kardiovaskulärer Erkrankungen von Bedeutung sein könnten.We have previously identified palmitoleic acid (C16:1) as a novel regulator of physiological cardiac hypertrophy. Since the underlying mechanisms of the C16:1-induced cardiac effects remain uncertain, we aimed to further investigate the molecular cell signaling pathways behind the C16:1-mediated cardiac responses. The primary focus of this study was set on examining the effects of C16:1 on the PI3K-AKT-FOXO signaling pathway, related to cardiac hypertrophy. The murine HL-1 cardiomyocytes were stimulated with either vehicle, 40 mM C16:1, 130 mM C16:0 as well as 10 nM IGF-1 (positive control) between 5 mins to 48 h. Subsequently total-RNA and proteins were isolated and examined by performing qRT-PCR or Western Blot analysis. In addition, immunofluorescence staining was conducted. Furthermore, the impact of C16:1 on MAPK/ERK signaling was also examined. The FOXO transcription factors 1 and 3a revealed to be phosphorylated and therefore inactivated by a 30 min C16:1 stimulation. Moreover, the cells total abundance of FOXO1 and FOXO3a was significantly diminished upon an 18 h C16:1 treatment. As demonstrated by immunofluorescence staining, C16:1 stimulation furthermore resulted in the cytosolic translocation of FOXO3a. The expression level of the FOXO3a target gene p21 proved to be significantly upregulated upon 24-48 h of C16:1 treatment. The growth-promoting AKT kinase was activated by C16:1 treatment, indicating yet another involvement of the AKT-FOXO signaling pathway in C16:1-induced cardiac responses. In addition, the cell cycle-regulating kinase ERK 1/2 demonstrated to be significantly decreased upon C16:1 stimulation. In conclusion, we were able to identify the C16:1-mediated regulation of AKT, FOXO and ERK 1/2 in cardiomyocytes. As FOXO is known to promote protein degradation and cell atrophy, its inactivation may play a crucial role in mediating physiological cardiac hypertrophy. By activating the growth-promoting kinase AKT, C16:1 ultimately enables pro-hypertrophic cardiac effects. The inactivation of ERK 1/2, being a fundamental regulator of cell cycle progression, indicates that while cell hypertrophy is being promoted, C16:1 may inhibit cell proliferation. This study revealed several molecular targets within the AKT-FOXO as well as the MAPK- signaling pathways that seem to be involved in mediating the previously shown, C16:1-induced beneficial cardiac growth processes. Therefore, it is probable that the effects of C16:1 on cardiac hypertrophy are mediated by affecting the AKT/FOXO as well as the ERK 1/2 signaling pathway. As C16:1 induces physiological cardiac growth, our study may contribute to develop potential future lipid-based therapies for cardiac diseases such as cardiomyopathy or heart failure

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1. Abstract

Identification of vat(E) in Enterococcus faecalis Isolates from Retail Poultry and Its Transferability to Enterococcus faecium

Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium