Cataldi, Amelia

di Giacomo, Viviana

Di Valerio, Valentina

Rapino, Monica

Zara, Susi

Firenze University Press: E-Journals

IJAE Vol. 115, n. 1/2 (Supplement), 2010
179
Morphological and molecular events during H9c2 
differentiation: role for pPKCδ/SC35 interaction
Susi Zara1, Viviana di Giacomo2 Monica Rapino3, Valentina Di Valerio2, Amelia Cataldi2
1 Department of Drug Sciences “G. d’Annunzio” University, Chieti-Pescara, Italy
2 Department of Medicine and Ageing Sciences, “G. d’Annunzio” University, Chieti-Pescara, Italy
3 Institute of Molecular Genetics CNR, Unity of Chieti, Italy
H9c2 cells are rat embryonic myoblasts with skeletal muscle properties which reach 
differentiation when cultured in HS low concentration (1%). Their terminal differentia-
tion consists of fusing and forming multinucleated myotubes. At ultrastructural level 
the organization of differentiated cells undergoes modifications in terms of morphologi-
cal features: they appear thin and elongated, often fused to form multinucleated syncy-
tia (myotubes) and well organized actin filaments are recognizable and detected by im-
munogold labeling. The decreased expression of cyclin A, required cell cycle regulator 
for the onset of DNA replication, and the increased expression of myogenin, marker of 
skeletal muscle differentiation, accompany such morphological modifications.
Among the molecules involved in a cascade of reactions controlling the functioning 
of nuclear complexes and leading to cell differentiation, a role has been assigned to Pro-
tein Kinases C. In our experimental model a ten fold greater signal of pPKCδ in 1%HS 
cells, with respect to 10%FBS cells, is shown. In order to investigate a potential activa-
tion of SC35 splicing factor by pPKCδ, pPKCδ-SC35 co-immunoprecipitation has been 
performed and revealed only in differentiated cells, supporting a possible interaction 
between the two molecules. Immunogold co-localization of SC35 factor with pPKCδ, 
carried out by FEI-SEM and TEM, evidences in 1% HS cells an increased level of pPKCδ 
and its accumulation in the nucleus, which represents reasonable evidence of protein 
activation along with an increased expression of SC35. In order to check the specificity 
of PKCδ involvement in H9c2 differentiation, PKCδ silencing has been performed and 
both PKCδ and myogenin expression have been detected by immunofluorescence anal-
ysis, evidencing a decrease of myogenin expression in 1% HS PKCδ siRNA- transfected 
H9c2 cells in parallel to the reduced percentage of PKCδ positive cells. 
Thus these results suggest that pPKCδ/SC35 interaction represents a crucial event 
resulting in downstream changes in transcription of components which determine the 
morphological modifications related to cardiomyocyte differentiated phenotype.


Morphological and molecular events during H9c2 differentiation: role for pPKCδ/SC35 interaction

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Morphological and molecular events during H9c2 differentiation: role for pPKCδ/SC35 interaction

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