Abstract-Protein kinase C (PKC) ⑀ and PKC␦ translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38 MAPK cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38 MAPK activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKC⑀ and PKC␦ were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38 MAPK in NRVMs. Adv-caPKC⑀ infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKC⑀ levels in a time-and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKC⑀ induced a time-and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding ␤-galactosidase (Adv-ne␤gal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38MAPK was relatively unaffected. Adv-caPKC␦ infection (1 to 25 MOI, 4 to 48 hours) increased total PKC␦ levels in a similar fashion. Adv-caPKC␦ (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38MAPK 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKC␦, but not Adv-caPKC⑀, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner. here is now substantial evidence to indicate a critical role for protein kinase C (PKC) activation in coordinating specific aspects of cardiomyocyte hypertrophy. 1 Previous studies have also implicated PKCs as potential upstream regulators of the mitogen-activated protein kinases (MAPK), which are involved in both hypertrophic signal transduction, 2 as well as apoptosis. 3 However, cardiomyocytes express several PKC isoenzymes that are differentially activated by various stimuli. For instance, the hypertrophic agonists endothelin-1 (ET) and phenylephrine (PE) caused the membrane translocation of PKC⑀, and to a lesser extent PKC␦, in cultured neonatal rat ventricular myocytes (NRVMs). 4,5 ETinduced PKC⑀ and PKC␦ translocation was accompanied by subsequent activation of all three MAPK cascades. [5][6][7][8][9] In contrast, electrical stimulation of contraction induced a similar degree of cardiomyocyte hypertrophy, but predominantly activated PKC␦ rather than PKC⑀. 10 Electrical pacing was also associated with a rapid increase in JNK 10,11 and p38 MAPK 12 activities, but ERKs were not significantly activated. 10,11 None of these hypertrophic stimuli induced the membrane translocation of PKC␣, the major Ca 2ϩ -dependent, phorbol-ester-sensitive PKC in NRVMs. 4,5,10 Although PKC⑀ has been imp...

Abstract-Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis. Costamere-like structures containing integrins are found in cultured neonatal and adult cardiac myocytes. 2 Cultured cardiomyocytes also form focal adhesions (similar to focal adhesions assembled by adherent nonmuscle cells in culture) containing ␤ 1 -integrins and vinculin. Their organization is highly regulated by peptide growth factors and externally applied or intrinsically generated mechanical load. [3][4][5][6] In addition to their structural role, focal adhesions and costameres are major sites for the localization of cell signaling molecules that are activated during cardiomyocyte hypertrophy. 7-9 Furthermore, disruption of normal cardiomyocyte anchorage during the transition from hypertrophy to early failure leads to apoptosis in an experimental model of ascending aortic coarctation. 10 Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that is localized to focal adhesions and costameres in cardiomyocytes. FAK undergoes autophosphorylation at a specific tyrosine residue (Y397) in response to cell adhesion, integrin clustering, and growth factor stimulation. 11 In addition, a homologous protein, known as FRNK (FAK-related non-kinase), is also a product of the FAK gene but is autonomously expressed under the cont...

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2-30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 micromol/l; 1 h pretreatment). Adv-caPKC epsilon, but not Adv-caPKC delta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKC epsilon inhibited ET-induced FAK activation. Y-27632 (10 micromol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC epsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 micromol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC epsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 micromol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKC epsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.

Muscles in the limbs change with age, but the aging process of urethral muscles is unknown. Therefore, we compared smooth and striated muscle content in the female rat urethra in young (12 month) and old (32 month) animals, using immunochemical techniques. All the striated skeletal fibers at both ages contain slow myosin. Urethral diameter does not change with age (young, 1.44 +/- 0.08 mm; old, 1.46 +/- 0.10 mm, n = 5), nor does the external sphincter width (young, 0.088 +/- 0.016 mm; old, 0.080 +/- 0.017 mm, n = 5). Neither smooth nor skeletal muscle volume in the urethra is changed with age (skeletal: young, 20.72 +/- 2.94%; old, 19.95 +/- 2.35%. Smooth: young, 22.26 +/- 2.98%; old, 26.75 +/- 2.35%, n = 5). The external striated sphincter is separate and distinct from the pubococcygeal region of the levator ani muscle, but is closely apposed to another layer of longitudinally oriented fibers into the vaginal musculature. The morphometric analysis shows no difference in urethral architecture in aging female rats.

The nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) has been implicated in cell signaling pathways involved in left ventricular hypertrophy and heart failure, but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wild-type (WT), and mutant forms of PYK2 were used to determine whether PYK2 overexpression activates MAPKs, and downregulates SERCA2 mRNA levels in neonatal rat ventricular myocytes (NRVM). PYK2 overexpression significantly decreased SERCA2 mRNA (as determined by Northern blot analysis and real-time RT-PCR) to 54 +/- 4% of Adv-GFP-infected cells 48 h after Adv infection. Adv-encoding kinase-deficient (KD) and Y(402)F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y(402)F). Conversely, the PTK inhibitor PP2 (which blocks PYK2 phosphorylation by Src-family PTKs) significantly increased SERCA2 mRNA levels. PYK2 overexpression had no effect on ERK1/2, but increased JNK1/2 and p38(MAPK) phosphorylation from fourfold to eightfold compared with GFP overexpression. Activation of both "stress-activated" protein kinase cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of constitutively active (ca)MKK6 or caMKK7, which activated only p38(MAPK) or JNKs, respectively, was not sufficient, whereas combined infection with both Adv reduced SERCA2 mRNA levels to 45 +/- 12% of control. WTPYK2 overexpression also significantly reduced SERCA2 promoter activity, as determined by transient transfection of a 3.8-kb SERCA2 promoter-luciferase construct. Thus a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca(2+) handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.