Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase.

OBJECTIVES: We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. MATERIALS AND METHODS: Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. RESULTS: When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength (t-test, p = 0.029) and 92% higher stiffness (t-test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. CONCLUSION: This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression.Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:-97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2.

Regulation of sarcoplasmic reticulum calcium pump gene expression by hindlimb unweighting.

Hindlimb unweighting (HU) causes upregulation of several muscle-specific genes responsible for the slow-to-fast transition in soleus skeletal muscle properties despite the profound muscle atrophy. The purpose of this study was to examine the expression of the fast and slow isoforms of the sarcoplasmic reticulum Ca(2+)-ATPase at the mRNA and protein level in the soleus muscle over a time course of HU and relate them to Ca(2+)-dependent ATPase activity and selected contractile properties. mRNA levels of the acetylcholine receptor (AChR) were measured to compare the signal of unweighting with denervation. Atrophy of the soleus muscles from tail-suspended rats was observed at all time points with muscle mass decreased by 52% at 28 days of HU (P < 0.05). Northern blot analysis showed the relative expression of the fast Ca2+ pump mRNA increased by 0, 250, 910, 1,340, and 4,050% over control levels at 1, 4, 7, 14, and 28 days of HU, respectively, whereas changes in slow mRNA were variable and modest in comparison. For the same time points, Western blot analysis showed relative expression of the fast Ca2+ pump protein increased by 30, 110, 320, 280, and 300% over control levels, whereas the slow-pump protein expression was unchanged except for a 75% decrease at 28 days of HU. Specific Ca(2+)-dependent ATPase activity was increased (P < 0.05) by 170% at 28 days of HU. Contractile properties measured in vitro at 14 and 28 days revealed time to peak tension and one-half relaxation time were shortened (P < 0.05) and a rightward shift in the tension-frequency curves in unloaded soleus muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.

The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats.

Elevated interstitial fluid volume in rat soleus muscles by hindlimb unweighting.

Hindlimb unweighting is a commonly used model to study skeletal muscle atrophy associated with disuse and exposure to microgravity. However, a discrepancy in findings between single fibers and whole muscle has been observed. In unweighted solei, specific tension deficits are greater in whole muscle than in single fibers. Also, metabolic enzyme activity when normalized per gram of mass is depressed in whole muscle but not in single fibers. These observations suggest that soleus muscle interstitial fluid volume may be elevated with atrophy caused by unweighting in rats. The purpose of this study was to determine if soleus muscle atrophy induced by unweighting is accompanied by alterations in muscle interstitial fluid volume and to calculate the effect of any such alterations on the muscle specific tension (N/cm2 muscle cross-sectional area). Nine female Wistar rats (200 g) were hindlimb unweighted (HU) by tail suspension. Soleus muscles were studied after 28 days and compared with those from five age-matched control (C) rats. Interstitial fluid volume ([3H]inulin space) and maximum tetanic tension (Po) were measured in vitro at 25 degrees C. Soleus muscles atrophied 58% because of unweighting (C = 147.8 +/- 2.3 mg; HU = 62.3 +/- 3.6 mg, P less than 0.001). Relative muscle interstitial fluid volume increased 107% in HU rats (35.5 +/- 2.8 microliters/100 mg wet mass) compared with the control value of 17.2 +/- 0.5 microliters/100 mg (P less than 0.001); however, absolute interstitial fluid volume (microliters) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)