Vo. Rybin et Sf. Steinberg, PROTEIN-KINASE-C ISOFORM EXPRESSION AND REGULATION IN THE DEVELOPING RAT-HEART, Circulation research, 74(2), 1994, pp. 299-309
To determine whether age-dependent differences in cardiac responses to
autonomic agonists could result from developmental changes in protein
kinase C (PKC) isoform expression, we probed extracts from the fetal,
neonatal, and adult heart as well as cultured neonatal and isolated a
dult ventricular myocytes with specific antisera to calcium-dependent
(alpha and beta) and calcium-independent (delta, epsilon, and zeta) is
oforms of the enzyme. Although PKC-beta immunoreactivity could not be
detected in cultured neonatal or isolated adult Ventricular myocytes,
adult and neonatal myocytes expressed multiple other isoforms of PKC.
Our studies revealed an age-dependent decline in the immunoreactivity
for three PKC isoforms. PKC-alpha was detected in extracts from the fe
tal and 2-day-old neonatal heart as well as cultured neonatal rat vent
ricular myocytes. Only faint PKC-alpha immunoreactivity was detected i
n extracts from the adult heart, and PKC-alpha was not detected in ext
racts from isolated adult ventricular myocytes, suggesting that PKC-al
pha resides in nonmyocyte elements in the adult heart. PKC-delta also
was detected in greater abundance in fetal and neonatal than in adult
myocardial extracts. The decline in PKC-alpha and PKC-delta expression
occurred during the first 2 postnatal weeks. PKC-zeta was detected in
greatest abundance in extracts from the fetal heart. PKC-zeta express
ion declined markedly by the second postnatal day, and only faint PKC-
zeta immunoreactivity was detected in extracts from adult myocardium.
Failure to detect PKC-zeta in extracts from isolated adult ventricular
myocytes suggests that PKC-zeta resides primarily in nonmyocyte eleme
nts in the adult heart. PKC-epsilon was detected in all preparations,
but it was detected in greatest abundance in extracts from neonatal he
arts. In vitro sympathetic innervation of previously noninnervated neo
natal ventricular myocytes or in vivo chemical sympathectomy of the ne
onatal heart did not modulate PKC isoform expression, suggesting that
sympathetic innervation does not significantly regulate PKC isoform ex
pression. PKC-alpha partitioned to the soluble fraction of unstimulate
d myocytes and was selectively translocated to the particulate fractio
n by Ca2+. In contrast, a major portion of the novel PKC isoforms part
itioned to the particulate fraction of unstimulated myocytes. The subc
ellular distribution of novel PKC isoforms was not influenced by Ca2+.
12-O-Tetra-decanoylphorbol 13-acetate (TPA, 300 nmol/L) induced trans
location of soluble PKC-alpha, PKC-delta, and PKC-epsilon to the parti
culate fraction at 30 minutes and complete (PKC-alpha and PKC-delta) o
r 80% (PKC-epsilon) downregulation at 24 hours. PKC-zeta was not affec
ted by TPA. We conclude that multiple PKC isoforms, which differ in th
eir subcellular distribution and regulation by Ca2+ and phorbol esters
, are expressed in the heart in an age-dependent fashion. The observat
ion that the developmental decline in FKC-zeta precedes the fall in PK
C-LU and PKC-delta suggests that PKC isoform expression is controlled
by distinct mechanisms that are regulated differently during developme
nt.