p70S6K via the inhibition of its upstream kinase, the mammalian
target of rapamycin (mTOR) in its hetero-multimeric protein com-
plex called mTOR complex 1 (mTORC1) via a dual mechanism.
AMPK phosphorylates and activates the tuberous sclerosis complex
2 (TSC2) [11] (Fig.1). TSC2 is the GTPase-activating protein of
the small G protein Rheb involved in mTORC1 activation. TSC2
activation results in Rheb inactivation and subsequent mTORC1
inactivation. AMPK also phosphorylates the mTOR binding part-
ner raptor inducing mTORC1 inhibition [12] (Fig.1). Both eEF2
and p70S6K have been proved to be regulated downstream of
AMPK in the heart [13, 14].
In line with its inhibitory action on protein synthesis via eEF2
and mTOR/p70S6K, AMPK has been rapidly proposed to be a
potent inhibitor of cardiac hypertrophy. In agreement, several
studies indicate that AMPK activation blocked cardiomyocyte
hypertrophy and, in the same time, inhibited protein synthesis as
well as its regulators p70S6K and eEF2 [15, 16]. In addition,
AMPK activation also correlated to the inhibition of other impor-
tant players of cardiac hypertrophy including pro-hypertrophic
gene expression pathways involving the transcription factor nuclear
factor of activated T cells (NFAT) and the mitogen-activated pro-
tein kinases ERK1/2 [15, 17, 18] (Fig.1).
We describe in this chapter the main experimental procedures
employed to investigate protein synthesis, cardiomyocyte hypertro-
phy development, and their molecular markers (detailed above) in
line with AMPK activation in a cellular model, the hypertrophied
neonatal rat cardiomyocyte in primary culture [19]. Cardiomyocyte
hypertrophy is induced by treatment with the pro-hypertrophic
stimulus phenylephrine (PE) [20]. AMPK activation is achieved
by using AICAr, which is metabolized into ZMP (an AMP analog),
or the biguanide phenformin, which increases intracellular AMP
concentration by inhibiting mitochondrial complex 1.2 Materials
2.1 Isolation
and Culture
of Neonatal Rat
Ventricular
Cardiomyocytes
for Protein Synthesis
and Signaling Analysis
Trypsin digestion of neonatal hearts- Hank’s balanced salt solution (HBSS) without Ca++and Mg++,
with phenol red (Lonza Formulation: KCl (400 mg/L),
KH 2 PO 4 (60 mg/L), NaCl (8000 mg/L), NaHCO 3
(350 mg/L), NaH 2 PO 4 ·7H 2 O (90 mg/L), glucose
(1000 mg/L)). Store at room temperature. - Trypsin (stored at 4C) (seeNote 1).
- Two petri dishes.
- Cleaned scissors with ethanol.
- Neonatal rats aged from 0- to 3-day-old.
Study of Cardiac Hypertrophy and Protein Synthesis 323