PROTEIN-KINASE-C ISOFORM EXPRESSION AND REGULATION IN THE DEVELOPING RAT-HEART

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.