Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes.

Protein kinase C (PKC) epsilon and PKCdelta 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 PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

Calcium not strain regulates localization of alpha-myosin heavy chain mRNA in oriented cardiac myocytes.

Our aim was to test a hypothesis that localization of the alpha-myosin heavy chain (alpha-MyHC) mRNA in oriented neonatal rat cardiomyocytes is regulated either by calcium, or by mechanical strain, or by both. Myocytes, grown on collagen aligned on stretchable silicone membranes, were elongated and had an increased length to width ratio (L/W) compared with randomly oriented myocytes grown on conventional substrata. Oriented cells were stretched by 10% in the longitudinal direction, in the transverse direction or passively unloaded for 6 h. As expected, shape changes followed these mechanical deformations. In situ hybridization was used to determine the localization of alpha-MyHC mRNA by quantitative analysis of optical density under various mechanical perturbations in myocytes that were either spontaneously beating or treated with verapamil (10 mM) to block influx of calcium. Unstretched, longitudinally stretched, and cells stretched transversely all had mRNA dispersed to their extremities. Verapamil treatment resulted in a perinuclear pattern of mRNA under all three mechanical perturbations. Additionally, mRNA distribution was examined in myocytes that were passively unloaded in the presence and absence of verapamil. Unloading myocytes with intact calcium cycling does not result in a perinuclear accumulation of mRNA. These data suggest that calcium is essential for alpha-MyHC mRNA distribution throughout the cell whereas stretch and alignment affect myocyte shape but have little effect on mRNA localization.

Mechanical activity in heart regulates translation of alpha-myosin heavy chain mRNA but not its localization.

Mechanical inactivity depresses protein expression in cardiac muscle tissue and results in atrophy. We explore the mechanical transduction mechanism in spontaneously beating neonatal rat cardiomyocytes expressing the alpha-myosin heavy chain (alpha-MyHC) isoform by interfering with cross-bridge function [2,3-butanedione monoxime (BDM), 7.5 mM] without affecting cell calcium. The polysome content and alpha-MyHC mRNA levels in fractions from a sucrose gradient were analyzed. BDM treatment blocked translation at initiation (162 +/- 12% in the nonpolysomal RNA fraction and 43 +/- 6% in the polysomal fraction, relative to control as 100%; P < 0.05). There was an increase in alpha-MyHC mRNA from the nonpolysomal fraction (120.5 +/- 7.7%; P < 0.05 compared with control) with no significant change in the heavy polysomes. In situ hybridization of alpha-MyHC mRNA was used to estimate message abundance as a function of the distance from the nucleus. The mRNA was dispersed through the cytoplasm in spontaneously beating cells as well as in BDM-treated cells (no significant difference). We conclude that direct inhibition of contractile machinery, but not calcium, regulates initiation of alpha-MyHC mRNA translation. However, calcium, not pure mechanical signals, appears to be important for message localization.

Histologic analysis of needle biopsy of urethral sphincter from women with normal and stress incontinence with comparison of electromyographic findings.

OBJECTIVE: Our goal was to compare urethral sphincter biopsy and needle electromyography between women who had genuine stress incontinence and those who did not. STUDY DESIGN: Seventeen continent women and 10 women with stress incontinence had urethral sphincter needle electromyography and urethral biopsy specimens blindly processed for light and electron microscopy. RESULTS: The continent group had greater skeletal muscle content and percentage in each muscle fascicle and each urethral sphincter. The group with genuine stress incontinence had higher connective tissue content. All urethral skeletal muscle was type 1. The smooth muscle was "multiunit" type and was morphologically indistinguishable between the 2 groups. On electromyography, patients with genuine stress incontinence had significantly more fibrillation potentials, fewer motor unit action potentials, a higher percentage of polyphasia, and less maximum voluntary electrical activity than control subjects. CONCLUSIONS: Women with stress incontinence differ from continent women in skeletal muscle volume, amount of fibrosis, and electromyographic parameters; these differences support a neurogenic contribution to genuine stress incontinence. Urethral sphincter has only type 1 skeletal muscle and "multiunit" type smooth muscle.

Pudendal denervation affects the structure and function of the striated, urethral sphincter in female rats.

Our aim was to examine the effects of denervation on urethral anatomy and urine voiding pattern. Rats usually void at one end of their cage, which gives a behavioral index of continence. The voiding preference for denervated rats was decreased to 88.8 + 4.7%, n = 32, P < 0.001, compared to improvements with time for unoperated (117 +/- 10%, n = 16) or sham-operated rats (105 +/- 8%, n = 5). The volume of urine or the frequency of voidings between denervated, unoperated or sham-operated rats did not differ significantly. Urethral sections were analyzed immunochemically and quantified morphometrically. Smooth muscle volume remained constant but skeletal muscle volume decreased after denervation, from 43 +/- 2% to 36 +/- 3% (P < 0.05, n = 5). Fiber diameter decreased from 14.3 +/- 1.4 microm to 8.5 +/- 0.7 microm (P < 0.005). We concluded that pudendal nerve transection in female rats causes behavioral alterations in voiding and muscular atrophy of the striated sphincter.

Morphometry of the aging female rat urethra.

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.