Carol S. Okamura

The cranial sartorius muscle undergoes true hypertrophy in dogs with golden retriever muscular dystrophy

The degree of atrophy or hypertrophy of selected pelvic limb muscles was determined in the canine homologue of Duchenne muscular dystrophy. While most muscles were atrophied, the caudal and cranial sartorius were hypertrophied. Cranial sartorius weights were corrected for body weight and endomysial space to determine true muscle weights (g/kg; mean+/-SD) in three golden retriever muscular dystrophy age groups, 4-10 (Group 1; n=15), 13-26 (Group 2; n=4), and 33-66 (Group 3; n=4) months and grouped normal dogs (6-20 months; n=12). Group 1 golden retriever muscular dystrophy weights (2.2063+/-0.6884) were greater than those of normal dogs (1.2699+/-0.1966), indicating that young golden retriever muscular dystrophy dogs have true cranial sartorius muscle hypertrophy. Values of Group 2 (1.3758+/-0.5078) and Group 3 (0.5720+/-0.2423) golden retriever muscular dystrophy dogs were less than those of Group 1, suggesting that the cranial sartorius muscle atrophies over time. Given that cranial sartorius muscle weight correlated with tarsal joint angle in affected dogs (r=-0.817), the hypertrophied muscle may play a role analogous to iliotibial band tightness in Duchenne muscular dystrophy.

Isolation and Characterization of a Novel Promoter for the Bovine Growth Hormone Receptor Gene

The use of alternative promoters represents an important mechanism for the regulation of growth hormone receptor (GHR) gene expression. Two promoters have been isolated previously for the GHR gene: the P1 promoter that drives liver-specific expression, and the P2 promoter that drives ubiquitous expression. In the present study, we isolated a third GHR promoter termed P3. The P3 promoter was GC-rich and TATA-less. The P3 promoter was able to drive the expression of a luciferase reporter gene in cell lines Hep G2, PLC/PRF/5, and BHK-21. In vivo, the P3 promoter initiated transcription from two major sites in exon 1C of the GHR gene in many tissues. In the adult bovine liver, the P3-transcribed GHR mRNA represented only 10% of the total GHR mRNA pool. In non-hepatic tissues such as kidney, skeletal muscle, mammary gland, and uterus, P3-transcribed GHR mRNA represented 30–40% of the total GHR mRNA pool. Within the bovine GHR gene, the P3 promoter was located immediately downstream from the P2 promoter. In transfected cells, the P2 promoter served as an enhancer for the P3 promoter. Existence and co-regulation of two ubiquitous promoters may be a mechanism for achieving a high level of expression of the GHR gene in multiple tissues.

Effects of prednisone in canine muscular dystrophy.

Glucocorticoid use may provide short‐term functional improvement in boys with Duchenne muscular dystrophy (DMD). We report functional and histopathologic changes following a 4‐month course of daily oral prednisone in a canine model of DMD, termed golden retriever muscular dystrophy (GRMD). Muscle extension forces in GRMD dogs treated daily with 1 and 2 mg/kg prednisone measured 2.349 ± 0.92 and 3.486 ± 0.67 N/kg, respectively, compared to 1.927 ± 0.63 N/kg in untreated GRMD controls (p < 0.05 for 2mg/kg group); GRMD muscle flexion forces measured 0.435 ± 0.13 and 0.303 ± 0.08 N/kg, respectively, compared to 0.527 ± 0.01 N/kg in untreated GRMD controls (p < 0.05 for both groups). Although cranial sartorius hypertrophy and tibiotarsal joint angles also tended to improve, myofiber calcification increased and fetal myosin expression decreased following prednisone. Thus, functional data indicate benefit but histopathologic changes following prednisone treatment in GRMD suggest possible deleterious consequences. Muscle Nerve, 2004

Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy

Childers MK, Okamura CS, Bogan DJ, Bogan JR, Sullivan MJ, Kornegay JN: Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy. Am J Phys Med Rehabil 2001;80:175–181. ObjectiveTo test the hypothesis that differential skeletal muscle involvement, previously observed in dogs with a homologue of Duchenne muscular dystrophy, correlates with the histochemical markers of myofiber injury and regeneration. DesignEvidence of injury (cellular penetration by Evans blue dye, immunoglobulin G expression, hematoxylin and eosin staining of necrotic figures), myofiber regeneration (fetal myosin heavy chain isoform expression), and morphologic indices in the cranial sartorius (CS), long digital extensor, and vastus lateralis muscles were examined in five dogs with dystrophy and five normal dogs. ResultsOnly the CS muscle, at 1 mo, demonstrated significant differences in injury when compared with age-matched controls. By 6 mo, the long digital extensor and vastus lateralis also suffered greater than normal injury. Only the dystrophic CS tissue expressed a notable increase in mean myofiber diameter when compared with other muscles at 6 mo. Normal CS muscles revealed a distinct population of small myofibers at this age. ConclusionThe CS seems unique in its selective pathologic involvement. These differences may contribute to the marked regenerative response of this muscle in the dystrophic state. An improved understanding of mechanisms by which some dystrophin-deficient canine muscles remain spared from injury may provide clues to investigate and prevent the degenerative processes in humans.

Uterine Progesterone Receptor Expression, Conceptus Development, and Ovarian Function in Pigs Treated with RU 486 During Early Pregnancy

Establishment of pregnancy in the pig depends on down-regulation of progesterone receptor (PGR) in the uterine luminal and glandular epithelium during the first week after breeding. The present study evaluated the regulation of endometrial PGR by progesterone and the possible role of endometrial tumor necrosis factor (ligand) superfamily member 11 (TNFSF11) and nuclear factor-kappa B (NFKB) activation in PGR expression. Mature, cycling gilts were inseminated (Day 0) and assigned to either untreated control (n = 9) or one of two treatments that employed RU 486 to block progesterone action either before (treatment 1 [T1]) or after (treatment 2 [T2]) the initiation of PGR down-regulation. The T1 gilts were treated with RU 486 (400 mg/day) on Days 3–5 of pregnancy (n = 9), and T2 gilts were treated with RU 486 on Days 6 and 7 of pregnancy (n = 9). Uteri and ovaries were collected on Day 8 or 12 of gestation. The diameter of the conceptuses in T1 gilts was approximately half that in controls by Day 8, and normal conceptuses were not collected from any T1 gilts on Day 12. Endometrial PGR mRNA was more abundant in T1 and T2 gilts compared with control gilts. The PGR-B protein decreased from Day 8 to Day 12 in the luminal epithelium and, to some extent, in superficial glandular epithelium in control and T2 gilts. In T1 gilts, the PGR-B protein remained elevated (i.e., failed to undergo down-regulation) on Day 12. Blocking PGR action early in the cycle (i.e., on or before Day 5), therefore, prevented normal conceptus development, caused elevated PGR mRNA, and prevented the decrease in PGR protein that typically occurs in pigs. We could not confirm a role for NFKB activation in PGR down-regulation, because pigs with extreme differences in PGR and TNFSF11 expression (T1 and controls) had similar NFKB activation on Day 8. Activated NFKB within the luminal epithelium and glandular epithelium (both superficial and deep) was observed in T2 and control pigs on Day 12 when elongating conceptuses (presumably releasing interleukin 1 beta to activate NFKB) were recovered. Gilts treated with RU 486 had greater ovarian follicular growth and greater plasma estradiol concentrations. We conclude that the mechanisms controlling PGR down-regulation are progesterone-dependent and occur between Day 3 and Day 6 of pregnancy. NFKB activation did not appear to have a role in PGR down-regulation within the period that we studied. Blocking progesterone action after Day 6 did not reverse the process of PGR down-regulation, nor did it appear to affect the development of conceptuses collected on Day 12.

TCAP knockdown by RNA interference inhibits myoblast differentiation in cultured skeletal muscle cells

Null mutation of titin-cap (TCAP) causes limb-girdle muscular dystrophy type 2G (LGMD2G). LGMD2G patients develop muscle atrophy, and lose the ability to walk by their third decade. Previous findings suggest that TCAP regulates myostatin, a key regulator of muscle growth. We tested the hypothesis that TCAP knockdown with RNA interference will lead to differential expression of genes involved in muscle proliferation and differentiation, impairing muscle cell growth. mRNA from cultured cells treated with TCAP siRNA duplex constructs was analyzed using Northern blots and real-time RT-PCR. siRNA treatment decreased TCAP mRNA expression in differentiating muscle cells. Significant (p<0.05) decreases in mRNA were observed for myogenic regulatory factors. siRNA treatment also prevented development of the normal phenotype of muscle cells. Our findings suggest that TCAP knockdown with RNA interference alters normal muscle cell differentiation.

Short communication: Growth hormone receptor expression in two dairy breeds during the periparturient period.

The growth hormone receptor (GHR) 1A mRNA decreases after calving in the liver of Holstein dairy cows and may coordinate nutrient partitioning. The hypothesis that the decrease in GHR1A mRNA around the time of calving was characteristic of a second dairy breed was tested by examining Guernsey cows in addition to Holstein cows. Holstein and Guernsey cows were housed together and paired by parity and expected calving date. Liver biopsies and blood samples were collected prepartum (d -20 +/- 1) and postpartum on d 3, and d 14 +/- 1. The amounts of GHR1A, GHR1B, GHR1C, and insulin-like growth factor (IGF)1 mRNA were determined by quantitative reverse transcription polymerase chain reaction. Blood concentrations of growth hormone (GH) and IGF1 were measured by RIA. Both breeds underwent a decrease in GHR1A mRNA, a decrease in liver IGF1 mRNA, a decrease in blood IGF1, and an increase in blood GH after calving. The decrease in liver GHR1A and IGF1 mRNA after calving may be an inherent characteristic of dairy breeds that enables nutrient partitioning for greater milk production. Independent genetic selection in 2 dairy breeds seemingly exploited a similar mechanism, reduced GHR1A expression, to decrease blood IGF1 and increase blood GH, a key hormone involved in nutrient partitioning.