studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 533A
651
Skeletal muscle mitochondrial fragmentation results in
functional abnormalities during hyperammonemia of
cirrhosis
Gangarao Davuluri 2 , Avinash Kumar 2 , Samjhana Thapaliya 2 ,
Allawy Allawy 2,5 , Dharmvir Singh 2 , Julie H. Rennison 2 , Rafaella
Nascimento e Silva 2 , David R. Van Wagoner 3 , Sathyamangla V.
Naga Prasad 3 , Xin Qi 4 , Hoppel Charles 4 , Takhar Kasumov 2 , Srinivasan
Dasarathy 1 ; 1 Department Of Gastroenterology and Hepatology,
Cleveland Clinic, Cleveland, OH; 2 Pathobiology, Cleveland
Clinic, Cleveland, OH; 3 Molecular Cardiology, Cleveland Clinic,
Cleveland, OH; 4 Pharmacology and Medicine, Case Western
Reserve University, Cleveland, OH; 5 Department Of Internal Medicine,
Cleveland Clinic, Cleveland, OH
Background. Hyperammonemia is a consistent abnormality
in cirrhosis and contributes to sarcopenia. Skeletal muscle is
a major alternate organ for ammonia disposal by the initial
reaction of conversion to glutamate in the mitochondria. We
have observed skeletal muscle mitochondrial functional abnormalities
in cirrhosis and hyperammonemia. It is not known if
the functional abnormalities are due to structural alterations
in the skeletal muscle mitochondria. Methods. Mitochondrial
mass was measured by immunoblots for mitochondrial proteins
citrate synthase, voltage dependent anion channels and cyclooxygenase
IV during hyperammonemia in C2C12 myotubes.
Additional methods to quantify mitochondrial mass included
electron microscopy and immunofluorescence images using
mitochondrial dye, MitoTracker Red. Mitochondrial fragmentation
was quantified using electron microscopy and immunofluorescence
images with Mitotracker red. Membrane polarization
generated by proton pumping by components of the electron
transport chain (ETC) was quantified using the dye TMRE and
uncoupler, FCCP was used as a positive control. The mechanism
of altered mitochondrial dynamics during hyperammonemia
was determined by quantifying genes regulating mitochondrial
fission and fusion by real time PCR and immunoblots of DRP1
protein expression and polymerization polymerization. Finally,
siRNA to knockdown of DRP1 and P110 peptide to block
DRP1 function in myotubes were used to show that ammonia
promoted DRP1 polymerization that resulted in mitochondrial
fragmentation and dysfunction. Results. Despite unaltered mitochondrial
mass with hyperammonemia, increased mitochondrial
fragmentation was observed on both immunofluorescence
and electron microscopy. These changes were accompanied
by consistent alterations in expression of genes regulating mitochondrial
dynamics, Fis 1, OPA1, MFN1, and MFN2. We
also observed increased polymerization of dynamin related
protein-1 (DRP1) during hyperammonemia in the myotubes.
Genetic and pharmacologic inhibition of DRP1 in restored
ATP content and decreased reactive oxygen species in myotubes
regardless of hyperammonemia. These changes were
accompanied by loss of membrane potential during hyperammonemia.
Conclusions. Increased mitochondrial fragmentation
was observed with unaltered mitochondrial mass and was due
to polymerization of dynamin related protein-1 (DRP1). Since
blocking mitochondrial fragmentation reversed the low ATP
and elevated ROS during hyperammonemia, our observations
provide a novel approach to reverse skeletal muscle bioenergetic
dysfunction and sarcopenia of cirrhosis by targeting
mitochondrial fragmentation using anti DRP1 therapies.
Disclosures:
The following authors have nothing to disclose: Gangarao Davuluri, Avinash
Kumar, Samjhana Thapaliya, Allawy Allawy, Dharmvir Singh, Julie H. Rennison,
Rafaella Nascimento e Silva, David R. Van Wagoner, Sathyamangla V. Naga
Prasad, Xin Qi, Hoppel Charles, Takhar Kasumov, Srinivasan Dasarathy
652
Thr505 RelA phosphorylation controls liver proliferative
response and supresses NF-κB tumor-promoting activities
Anna Moles 1 , Jacqueline Butterworth 2 , Jill E. Hunter 2 , Ana M.
Sanchez 2 , Dina Tiniakos 1 , Derek Mann 1 , Fiona Oakley 1 , Neil
D. Perkins 2 ; 1 Institute of Cellular Medicine, Newcastle University,
Newcastle Upon Tyne, United Kingdom; 2 Institute for Cell and
Molecular Biosciences, Newcastle University, Newcastle Upon
Tyne, United Kingdom
Background and aims: NF-κB is a family of transcription factors
that are key regulators of the immune and inflammatory
responses. NF-κB regulation is complex and occurs at many
levels, including direct post-translational modifications (PTMs)
of the subunits. Phosphorylation of RelA (p65) at Thr505 regulates
proliferation, migration and autophagy in in vitro cellular
systems; however its role and relevance in vivo still remains
unclear. Thus, the aim of this study was to analyse the role
of RelA Thr505 phosphorylation in vivo by mutating this site
through creating a knock in mouse. Methods: Knock in mutant
mice where generated where Thr505 was substituted by Alanine,
thus preventing phosphorylation. Partial hepatectomy
(PhX), acute CCl 4
administration and 15 days DEN injection
was performed in WT and knock in mice. Animals were culled
at 36, 72 hours and 5 days for PhX; at 24, 48 and 72 hours
for acute CCl 4
and 30 weeks post-DEN. Paraffin staining for
BrdU and PCNA were used as markers of proliferation and
γH2AX for DNA damage. Western blot was used to determined
p53 and p-JNK expression. Gene set enrichment analysis
(GSEA) was performed in WT and RelA T505 livers after 36
hours of PhX. Tumor assessment was performed by an expert
pathologist. Results: Thr505 mutation of RelA resulted in an
increased liver to body weight ratio after PhX at 36, 72 hours
and 5 days. Hepatocyte proliferation was increased after both
injuries, PhX and acute CCl 4,
at all the time points in RelA
T505 mice versus WT, as assessed by PCNA and/or BrdU
staining. Furthermore, mitotic body counts were also higher
in RelA T505 mice after both injuries. Of note, proliferation
after PhX was sustained for longer in RelA T505 mice, pointing
towards an inefficient resolution of the proliferative response.
Increased DNA damage, as determined by γH2AX staining,
was seen in both WT and RelA T505 after 36 hours of PhX.
However, the DNA damage was significantly higher in RelA
T505 mice in comparison with WT. Western blot analysis of
these samples revealed increased expression of the p53 tumour
suppressor and active JNK in RelA T505 mice. Finally, chronic
DEN administration resulted in a significant increased tumor
number, mostly HCC, in RelA T505 versus WT. Conclusions
RelA Thr505 phosphorlyation provides an important and novel
regulatory mechanism to control the liver proliferative response
and is a critical pathway suppressing NF-κB tumour-promoting
activities of RelA.
Disclosures:
The following authors have nothing to disclose: Anna Moles, Jacqueline Butterworth,
Jill E. Hunter, Ana M. Sanchez, Dina Tiniakos, Derek Mann, Fiona
Oakley, Neil D. Perkins