Am J Physiol Endocrinol Metab 306: E1406–E1417, 2014. First published April 29, 2014; doi:10.1152/ajpendo.00111.2014.

Running exercise alleviates trabecular bone loss and osteopenia in hemizygous ␤-globin knockout thalassemic mice Kanogwun Thongchote,1,2,3 Saovaros Svasti,4 Jarinthorn Teerapornpuntakit,1,2 Nateetip Krishnamra,1,2 and Narattaphol Charoenphandhu1,2 1

Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand; 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 3Exercise Science Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand; and 4Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand Submitted 5 March 2014; accepted in final form 23 April 2014

Thongchote K, Svasti S, Teerapornpuntakit J, Krishnamra N, Charoenphandhu N. Running exercise alleviates trabecular bone loss and osteopenia in hemizygous ␤-globin knockout thalassemic mice. Am J Physiol Endocrinol Metab 306: E1406 –E1417, 2014. First published April 29, 2014; doi:10.1152/ajpendo.00111.2014.—A marked decrease in ␤-globin production led to ␤-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous ␤-globin knockout thalassemic (BKO) mice and to determine whether endurance running (60 min/day, 5 days/wk for 12 wk in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1–2 mo old) showed growth retardation as indicated by smaller body weight and femoral length than their wild-type littermates. A decrease in BMD was more severe in female than in male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines IL-1␣ and -1␤. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice compared with those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, whereas running exercise appeared to be an effective intervention in alleviating bone microstructural defect in ␤-thalassemia. bone histomorphometry; bone mineral density; endurance exercise; running wheel; osteoporosis

in the erythrocytes—is composed of two ␣-globin chains and two ␤-globin chains. Inadequate production or absence of ␤-globin is found in a hereditary disease known as ␤-thalassemia, which is a common autosomal recessive anemic disorder in several populations, such as Chinese, Southeast Asian, and Mediterranean populations (6, 15, 34, 59). Thalassemic patients and rodents both manifest ineffective erythropoiesis, extramedullary erythropoiesis, splenomegaly, iron overload, growth retardation, HEMOGLOBIN—OXYGEN-CARRYING METALLOPROTEIN

Address for reprint requests and other correspondence: N. Charoenphandhu, Dept. of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand (e-mail: [email protected]). E1406

short stature, and skeletal deformity (16, 28, 31, 38, 43, 56, 62). However, little is known regarding the detailed changes in bone mineral density (BMD) and microstructural defect as well as the cellular mechanism of bone loss, especially at the early stages of the disease in young growing and adolescent mice. Our recent study has provided evidence that defective bone matrix mineralization and trabecular bone resorption did occur in sexually mature (12 wk old) male ␤IVSII-654 knockin thalassemic mice, of which a decrease in ␤-globin production was caused by aberrant splicing of ␤-globin RNA from C¡T mutation at nucleotide 654 of intron 2 (49). Interestingly, the hemizygous ␤-globin knockout thalassemic (BKO) mice also exhibited a marked decrease in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-dependent calcium absorption in the small intestine, which might, in turn, diminish matrix mineralization and bone formation (7). Moreover, the thalassemia-induced intestinal malabsorption of vitamin D and disturbances of calciotropic hormones, such as low serum levels of parathyroid hormone (PTH) and vitamin D, could further aggravate the impairment of bone metabolism (2, 50, 63). Thus, thalassemic patients were vulnerable to osteoporosis, multiple fragility fractures, and deformities after fracture healing. Since physical activities and exercise, particularly endurance impact exercise (e.g., walking, running, and jumping), have been known to increase BMD, bone mass, and bone strength (3, 27, 51), it was hypothesized that running exercise may effectively alleviate osteopenia and microstructural defect in BKO mice. In exercising humans and rodents, mechanical stress on bone during muscle contraction and force impact as well as osteogenic myokines [e.g., insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF)-2, and irisin] played important roles in bone-forming responses (9, 19, 37). Iwamoto et al. (23) reported that 12-wk voluntary wheel running markedly improved trabecular bone density and bone area in rat tibiae and femora. Both impact and nonimpact endurance exercises, such as treadmill running and swimming, respectively, were also capable of enhancing the intestinal calcium absorption, which provided additional calcium for matrix mineralization (48, 60). The main objectives of the present study were, therefore, 1) to determine the age- and sex-dependent changes in BMD, bone mineral content (BMC), and bone microstructural parameters in BKO mice by using dual-energy X-ray absorptiometry (DEXA) and bone histomorphometric analysis and 2) to investigate whether endurance exercise in running wheels could effectively alleviate the thalassemia-induced bone loss and microstructural defect in BKO mice.

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Table 1. Blood parameters and spleen weights of BKO and WT mice 1 Mo Old Parameters 6

RBC (⫻10 cells/␮l) Hb, g/dl Hct, % MCV, fl MCH, pg MCHC, g/dl RDW RET, % Spleen weight, mg

8 Mo Old

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BKO (n ⫽ 7)

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BKO (n ⫽ 7)

8.61 ⫾ 0.18 12.78 ⫾ 0.22 42.65 ⫾ 0.31 49.60 ⫾ 0.73 14.85 ⫾ 0.09 29.98 ⫾ 0.34 16.98 ⫾ 0.47 6.92 ⫾ 1.02 50.00 ⫾ 4.08

7.12 ⫾ 0.15*** 7.50 ⫾ 0.11*** 32.79 ⫾ 0.48*** 46.14 ⫾ 1.08* 10.50 ⫾ 0.14*** 22.83 ⫾ 0.30*** 38.17 ⫾ 0.29*** 37.79 ⫾ 0.50*** 348.60 ⫾ 6.70***

8.49 ⫾ 0.22 12.35 ⫾ 0.35 39.13 ⫾ 0.82 46.40 ⫾ 0.38 14.63 ⫾ 0.10 31.53 ⫾ 0.31 13.45 ⫾ 0.10 3.46 ⫾ 0.13 67.50 ⫾ 2.50

7.75 ⫾ 0.20* 7.29 ⫾ 0.15*** 28.13 ⫾ 0.62*** 36.37 ⫾ 0.80*** 9.43 ⫾ 0.12*** 25.96 ⫾ 0.40*** 34.34 ⫾ 0.56*** 18.22 ⫾ 1.15*** 390.00 ⫾ 19.6***

Values are means ⫾ SD. WT, wild-type; BKO, hemizygous ␤-globin knockout thalassemic. RBC, red blood cell count; Hb, hemoglobin concentration; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; RET, reticulocyte count. *P ⬍ 0.05, ***P ⬍ 0.001 vs. corresponding WT group.

MATERIALS AND METHODS

Animals. Male and female hemizygous ␤-globin knockout (␤⫹/th3; BKO) thalassemic mice and their wild-type C57BL/6 littermates (1, 2, 4, and 8 mo old) were obtained from the Thalassemia Research Center, Institute of Molecular Biosciences (Mahidol University, Bangkok, Thailand). Their genotype was verified from tail DNA as described previously (24). In the present study, only hemizygous mice were used because of the lethality of homozygous ␤-globin knockout. All newborn BKO mice exhibited a phenotype of ␤-thalassemia intermedia (29, 46, 49). Mice were housed in the husbandry unit (polystyrene shoebox cages) with room temperature of 25 ⫾ 2°C and humidity of ⬃55% under a 12:12-h dark-light cycle (lights on at 0600) and were fed regular chow containing 0.9% wt/wt phosphorus, 1.0% wt/wt calcium, and 4,000 IU/kg vitamin D (CP, Bangkok, Thailand) and reverse-osmosis (RO) water ad libitum. Body weight was recorded weekly. Two doses of calcein (10 mg/kg; Sigma, St. Louis, MO) were subcutaneously injected on day 7 and day 1 prior to euthanasia to label bone for dynamic histomorphometric study. This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University. All animals were cared for in accordance with the principles and guidelines of the American Physiological Society’s Guiding Principles in the Care and Use of Animals. Wheel running protocol. In the wheel running experiment, 3-wkold female BKO mice were randomly divided into two groups, i.e., running and sedentary groups. The running group was assigned to run for 3 mo on motorized running wheels (model 80800A, Lafayette Instrument), whereas sedentary mice were kept in stationary (locked) wheels. Running mice were initially trained for a week at 2– 4 m/min for 30 –50 min/day. Running speed was gradually increased to 3–5 m/min for 60 min/day by the end of week 2 and maintained at 4 – 6 m/min until 3 mo. Running frequency was 5 days/wk. The present running protocol was considered a mild- to moderate-intensity endur˙ O2max), as described by Høydal et al. (21). ance exercise (40 – 60% V Sample collection and tissue preparation. Wild-type and BKO mice (1, 2, 4, and 8 mo old) as well as 4-mo-old running and sedentary BKO mice were anesthetized with 70 mg/kg ip pentobarbitone sodium (Ceva Santé Animale, Libourne, France). To determine the characteristics of thalassemic anemia, venous blood was collected from the tail vein for hematological analysis by using an automated analyzer (model ADVIA 120; Bayer, Tarrytown, NY). Arterial blood samples were also collected from the left ventricle using a commercial sterile heparinized syringe (model REF364314; BD Diagnostics, Plymouth, UK) with no exposure to air for blood chemistry or cytokine analyses. In the wheel running experiment, gastrocnemius and soleus muscles of running and sedentary BKO mice were collected for citrate synthase and succinate dehydrogenase activity assays, while the heart was collected for dry heart weight measurement. Spleen weight was also

recorded as an indicator of splenomegaly. Finally, femora, tibiae, and L5– 6 lumbar vertebrae were removed and cleaned of adhering connective tissues. Femoral length was measured by a vernier caliper. Ex vivo bone specimens were analyzed by DEXA, bone histomorphometry, and/or peripheral quantitative computed tomography (pQCT). Since impact exercise usually improves both trabecular and cortical structures (23), we used methods that accurately determined trabecular microstructure (i.e., bone histomorphometry) and cortical thickness (i.e., pQCT) in the exercise study. DEXA. Whole body, femora, and L5– 6 BMD and whole body BMC were obtained from Lunar PIXImus2 system (image area of 100 ⫻ 80 mm; GE Medical Systems, Madison, WI), operated with software version 2.10. The dual-energy supply was 80/35 kVp at 500 ␮A. The DEXA system was calibrated daily with standard material with known BMD of 0.0690 g/cm2. The interassay coefficient of variation was ⬍ 0.3%.

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Fig. 1. Body weight (A) and femoral length (B) of 1-, 2-, 4-, and 8-mo-old male and female wild-type (WT) and hemizygous ␤-globin knockout (BKO) thalassemic mice (Thal). Bone length was measured with a vernier caliper. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched WT mice.

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pQCT. pQCT (model XCT 3000 Research SA⫹; Stratec Medizintechnik, Germany) was used to determine cortical density (mg/cm3), cortical content (mg/mm), cortical thickness (mm), and cortical area (mm2) in the femoral and tibial diaphyses. Cortical content was the amount of mineral present in the defined bone area as normalized by

slice thickness. Each specimen was placed on a supporting platform made of carbon fiber, and was then exposed to X-ray beam (0.5 mm thick) at 50 kVp with a current of 0.307 mA. The voxel size of each bone slide was 0.1 ⫻ 0.1 ⫻ 0.1 mm3, and the slide thickness was 0.05 mm. Scanning speed was 7 mm/s. Femoral and tibial diaphyses were

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C Fig. 2. A and B: whole body bone mineral density (BMD) and bone mineral content (BMC), and BMD of femoral metaphysis (C), femoral diaphysis (D), and L5– 6 lumbar vertebrae (E) of 1-, 2-, 4-, and 8-mo-old male and female WT and BKO Thal mice. BMD was measured by DEXA. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched WT mice.

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scanned at the midshaft (50% bone length) and at both sides of the midpoint (0.2 mm apart). All parameters were analyzed by XCT version 6.20 (Stratec Medizintechnik) (52). Bone histomorphometry. Bone histomorphometric analysis was used to evaluate trabecular bone microstructure as described previously (47, 49). After removal of adhering connective tissue, tibia was dehydrated in 70, 95, and 100% vol/vol ethanol for 3, 3, and 2 days, respectively, and then embedded in methyl methacrylate resin at 42°C for 48 h. The resin-embedded tibia was cut longitudinally with a microtome equipped with a tungsten carbide blade (model RM2255; Leica, Nussloch, Germany) to obtain 5- and 10-␮m-thick sections for stained (static) and unstained (dynamic) histomorphometric techniques, respectively. The sections were mounted on standard slides, deplastinated, dehydrated, and processed for Goldner’s trichrome staining (53). The unstained sections were examined for the double fluorescent lines of calcein labeling under a fluorescent microscope (excitation and emission wavelengths of 495 and 515 nm, respectively). Calcein mainly deposited on mineralizing bone surface; thus, the distance between these two calcein lines was used to calculate the mineral apposition rate and bone formation rate during the 6-day period. The region of interest was within the tibial secondary spongiosa (the trabecular part of tibial metaphysis). Image capture and analysis were performed under a light/fluorescent microscope (model BX51TRF; Olympus, Tokyo, Japan) using a computer-assisted Osteomeasure System version 4.1 (Osteometric, Atlanta, GA). The static parameters obtained in the present study were trabecular bone volume normalized by tissue volume (also known as bone volume fraction; BV/TV, %), marrow volume (Ma.V/TV), trabecular number (Tb.N, mm⫺1), trabecular thickness (Tb.Th, ␮m), trabecular separation (Tb.Sp, ␮m), osteoblast surface normalized by bone surface (Ob.S/ BS, %), osteoid thickness (O.Th, ␮m), osteoid surface normalized by

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BS (OS/BS, %), osteoid volume normalized by TV (OV/TV, %), osteoclast surface normalized by BS (Oc.S/BS, %), and active erosion surface normalized by BS (a.ES/BS, %). The dynamic parameters consisted of single-labeled surface (sLS/BS, %; for calculation of mineralizing surface), double-labeled surface (dLS/BS, %), mineral apposition rate (MAR, ␮m/day), mineralizing surface (MS/BS ⫽ dLS/BS ⫹ 0.5·sLS/BS, %), and bone formation rate (BFR/BS, ␮m3/ ␮m2/day). MAR values were corrected for section obliquity with a conversion factor of ␲/4. The nomenclature, symbols, units, and calculations complied with the Guideline of the American Society for Bone and Mineral Research (12, 14). Blood chemistry and osteoclastogenic cytokine assays. Plasma pH was measured with an automated analyzer (model Ultra C; Nova Biomedical, Waltham, MA). Plasma total calcium concentration (ionized plus complex forms) was determined by modified o-cresolphthalein complexone method using a Dimension RxL analyzer (Dade Behring, Marburg, Germany). Free-ionized calcium concentration was measured by ion-selective electrode (model Stat Profile CCX; Nova Biomedical) under anaerobic condition. Plasma lactate concentration was determined by lactate oxidase technique using Stat Profile CCX system (Nova Biomedical). In some experiments, serum levels of IL-1␣, IL-1␤, and IL-6 were determined in hemizygous ␤IVSII-654 knockin thalassemic mice by use of commercial ELISA kits (catalog nos. MLA00, MLB00C, and M6000B, respectively; R&D Systems, Minneapolis, MN). Measurement of citrate synthase and succinate dehydrogenase activities. Gastrocnemius and soleus of 3-mo-running BKO mice (4 mo old) were collected, immediately dipped in liquid nitrogen, and stored at ⫺80°C until assay. Muscle specimens were homogenized in ice-cold homogenizing buffer (pH 7.4) containing 20 mmol/l 2-[4-(2hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 1 mmol/l

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Fig. 3. A: cortical thicknesses in femoral and tibial metaphyseal cortical envelopes of 4-mo-old female WT and BKO Thal mice, as determined by pQCT. B: trabecular bone volume (BV) normalized by tissue volume (TV), trabecular number (Tb.N; C), trabecular thickness (Tb.Th; D), trabecular separation (Tb.Sp; E), and marrow volume (Ma.V) normalized by TV (F) in tibial metaphyses of 1-, 2-, 4-, and 8-mo-old female WT and BKO Thal mice. Trabecular parameters were determined by bone histomorphometry. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched WT mice. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00111.2014 • www.ajpendo.org

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ethylenediaminetetraacetic acid (EDTA), and 0.25 mmol/l sucrose. Citrate synthase activity was determined as previously described (45, 48). In brief, the homogenate was diluted in 100 mmol/l Tris buffer (pH 8.0) containing 1 mmol/l 5,5=-dithiobis-2-nitrobenzoic acid and 3 mmol/l acetyl-CoA (Sigma). The basal activity in diluted homogenate was determined by reading the optical density change at 412 nm every 30 s for 4 min by using a spectrophotometer (model UV-2550; Shimadzu, Kyoto, Japan). The reaction was initiated by adding oxaloacetate (Sigma) dissolved in 100 mmol/l Tris buffer and 100 mmol/l K2HPO4 (3:1) to obtain a final concentration of 6.5 mmol/l. Succinate dehydrogenase activity was determined by complex II enzyme activity microplate assay kit (catalog no. ab109908; Abcam, Cambridge, MA) according to the manufacturer’s instruction. Statistical analysis. The results are expressed as means ⫾ SE. Two groups of data with normal distribution were compared by unpaired Student’s t-test (i.e., Figs. 3, 7, and 8), whereas data with nonnormal

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distribution were compared by nonparametric Mann-Whitney test (i.e., Figs. 1, 2, 4, 5, 6, and 9). Two-way ANOVA was used to determine how the BMD outcome was affected by sex (male vs. female) and genotype (wild-type vs. BKO). The level of significance for all statistical tests was P ⬍ 0.05. Data were analyzed by GraphPad Prism 6.0 for Mac OS X (GraphPad Software, San Diego, CA). RESULTS

Osteopenia and impaired bone microstructure in BKO mice. Blood parameters of both 1- and 8-mo-old BKO mice revealed severe microcytic anemia as suggested by lower values of red blood cell count, hemoglobin level, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration compared with those of the

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Fig. 4. Bone formation-related parameters. A: osteoblast surface (Ob.S) normalized by bone surface (BS). B: osteoid surface (OS) normalized by BS. C: osteoid thickness (O.Th). D: osteoid volume (OV) normalized by tissue volume (TV). E: mineralizing surface (MS) normalized by BS. F: double-labeled surface (dLS) normalized by BS. G: mineral apposition rate (MAR). H: bone formation rate (BFR) normalized by BS in tibial metaphyses of 1-, 2-, 4-, and 8-mo-old female WT and BKO Thal mice. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched WT mice. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00111.2014 • www.ajpendo.org

EXERCISE ALLEVIATES THALASSEMIA-INDUCED OSTEOPENIA

age-matched wild-type mice (Table 1). Blood smear examination (data not shown) together with the increased red blood cell distribution width and spleen weight (Table 1) confirmed the presence of hypochromia, anisocytosis (red blood cells of varying sizes), poikilocytosis (red blood cells with abnormal shape), and splenomegaly (increased spleen weight), all of which are important characteristics of thalassemia. In young growing (1-mo-old) and adolescent mice (2-mo-old), the body weight (Fig. 1A) and femoral length (Fig. 1B) of wild-type mice were significantly greater than those of the age-matched BKO littermates, suggesting the presence of growth retardation in BKO mice. However, catch-up growth was observed in 4and 8-mo-old BKO mice, since body weight and femoral length of both BKO and wild-type mice were similar. In addition, BKO mice (1-, 2-, 4-, and 8-mo-old) had similar growth plate histology and growth plate height compared with the age-matched wild-type littermates (data not shown). Osteopenia was evident in BKO mice in an age- and sexdependent site-specific manner. As shown in Fig. 2, BMD values (whole body, femoral metaphysis, femoral diaphysis, and L5– 6 vertebrae) as well as whole body BMC (Fig. 2B) were lower in 1- to 2-mo-old male and female BKO mice compared with the age-matched wild-type littermates. At 4 and 8 mo of age, osteopenia of the cortical site (femoral diaphysis) was observed only in female, not in male, BKO mice (Fig. 2D). Two-way ANOVA further revealed that whole body BMD was apparently lower in female than in male BKO mice (P ⬍ 0.001, 8-mo-old male vs. female); therefore, female mice were used in

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the subsequent experiments, including the exercise study in which they should gain more bone mass than male mice. In femora and tibiae, cortical thicknesses appeared thinner in BKO mice than in wild-type mice, as determined by pQCT (Fig. 3A). Furthermore, bone histomorphometric analysis showed that female BKO mice had lower trabecular bone volume fraction (Fig. 3B), trabecular number (Fig. 3C), and trabecular thickness (Fig. 3D), whereas trabecular separation (Fig. 3E) and marrow volume (Fig. 3F) in the tibial metaphysis were higher compared with the age-matched wild-type controls. Such trabecular bone loss probably resulted from a consistent decrease in osteoblast-mediated bone formation, as indicated by marked decreases in osteoblast surface (Fig. 4A), osteoid surface (Fig. 4B), osteoid thickness (Fig. 4C), osteoid volume (Fig. 4D), mineralizing surface (Fig. 4E), doublelabeled surface (Fig. 4F), mineral apposition rate (Fig. 4G), and bone formation rate (Fig. 4H) in BKO mice compared with the age-matched wild-type mice. Higher values of osteoclast surface (Fig. 5A) and active erosion surface (Fig. 5B) also suggested an enhanced osteoclast-mediated bone resorption, consistent with the elevated levels of osteoclastogenic cytokines IL-1␣ and -1␤ (Fig. 5, C and D), both of which might be secreted from hematopoietic cells in response to aberrant erythropoiesis (57). However, circulating IL-6 levels were similar in thalassemic and wild-type mice (Fig. 5E). Our results, therefore, elucidated widespread osteopenia as well as impaired trabecular bone microstructure in BKO thalassemic mice.

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Fig. 5. Bone resorption-related parameters. A: osteoclast surface (Oc.S) normalized by bone surface (BS). B: active erosion surface (a.ES) normalized by BS in tibial metaphyses of 1- 2-, 4-, and 8-mo-old female WT and BKO Thal mice. Serum concentrations of IL-1␣ (C), IL-1␤ (D), and IL-6 (E) of 2- and/or 4-mo-old female WT and ␤⫹/IVSII654 knockin thalassemic mice (Thal). Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01 vs. age-matched WT mice.

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Mitigation of ␤-thalassemia-induced bone loss by running exercise. Prior to determination of the effect of running exercise in BKO mice (protocol shown in Fig. 6A), the effectiveness of endurance running exercise was indicated by muscle enzyme activities. Running markedly enhanced the activities of citrate synthase in soleus and gastrocnemius (Fig. 6, B and C) as well as succinate dehydrogenase in gastrocnemius (Fig. 6D) of running BKO mice compared with those of sedentary BKO mice. An increase in dry heart weight (cardiac hypertrophy) with no changes in the blood lactate levels also confirmed that the present running protocol was endurance aerobic exercise without induction of lactic acidosis (Fig. 6, E–G). Plasma total calcium and free-ionized calcium levels remained unaltered in running BKO mice compared with sedentary BKO mice (Fig. 6, H and I). After 3-mo exercise, the 4-mo-old BKO mice exhibited increases in cortical mineral density (Fig. 7A), cortical mineral

content (Fig. 7B), cortical area (Fig. 7C), and cortical thickness (Fig. 7D) in both femoral and tibial diaphyses—representatives of cortical sites—as determined by pQCT (Fig. 7). At the trabecular site, i.e., proximal tibial metaphysis, running exercise markedly increased bone volume fraction (Fig. 8A), bone surface (Fig. 8B), trabecular number (Fig. 8C), and trabecular thickness (Fig. 8D) while reducing trabecular separation (Fig. 8E) and marrow volume (Fig. 8F). Running-induced alleviation of the microstructural defect of BKO bone was probably due to increased bone formation, as indicated by increases in osteoblast surface (Fig. 9A) and several dynamic histomorphometric parameters (i.e., mineralizing surface, double-labeled surface, mineral apposition rate, and bone formation rate; Fig. 9, E–H). In addition, the osteoid surface was reduced (Fig. 9B), whereas the osteoid thickness was greater in running BKO mice than in the sedentary controls (Fig. 9C). Thus, running exercise did not alter osteoid volume in BKO mice (Fig. 9D).

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Fig. 6. A: timeline shows exercise protocol for female BKO thalassemic mice. Mice were trained to run in running wheels for 1 wk (1 mouse/wheel), followed by a 3-mo running period. Citrate synthase activity in soleus and gastrocnemius (B and C), succinate dehydrogenase activity in gastrocnemius (D), dry heart weight (E), plasma pH (F), plasma lactate (G), total plasma calcium (H), and plasma ionized calcium levels (I) of sedentary (Sed) and exercise-trained (Ex) female BKO mice. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched sedentary BKO mice.

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Fig. 7. Cortical density (A), cortical content (B), cortical area (C), and cortical thickness (D) in femoral and tibial diaphyses of sedentary (Sed) and running (Ex) female BKO mice. All parameters were determined by pQCT. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched sedentary BKO mice.

Moreover, osteoclast surface (Fig. 9I) and active erosion surface (Fig. 9J), which are indicative of osteoclast-mediated bone resorption, appeared lower in running BKO mice than in sedentary BKO mice. These findings thus suggested that endurance running was effective in mitigating osteopenia and bone microstructural defect in BKO thalassemic mice. DISCUSSION

Since ␤-thalassemia is a hereditary disease, it was hypothesized that bone growth and microstructure would be impaired from infancy. In humans, a reduction in ␤-globin

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production profoundly affected body function, especially when the fetal hemoglobin (e.g., hemoglobin F; ␣2␥2) was switched to hemoglobin A (␣2␤2) at ⬃2–3 years of age. On the other hand, ␤-thalassemia in BKO mice was present from birth because there was no ␥- to ␤-globin switching. Clinical manifestations commenced after precipitation of ␣-globin tetramer and accumulation of free iron in the cytoplasm of erythroid precursor cells, causing oxidative damage of the erythrocytes and ineffective erythropoiesis (6, 39, 40). Anemia in thalassemic patients and rodents thus triggered a number of compensatory responses, such as expansion of the medullary cavity and aberrant release of hematopoietic cytokines, some of which (e.g., IL-1) not only promoted proliferation of erythroid precursor cells but also induced osteoclastogenesis (25, 57). In the present study, both male and female BKO mice exhibited growth retardation, particularly during the growing period (1–2 mo of age), as indicated by lower body weight and shorter femoral length compared with their age-matched wild-type littermates. Female mice were found to have more severe osteopenia than male mice. Defect in bone microstructure was a result of increased osteoclast-mediated bone resorption and decreased osteoblast-mediated bone formation. Indeed, growth retardation, delayed growth, and short stature were common clinical manifestations in young ␤-thalassemic patients (8, 28), presumably due to the impairment of hepatic IGF-I production (44). Iron overload in ␤-thalassemia also damaged the pituitary somatotropic cells and growth hormone release, thereby aggravating growth retardation (42). Interestingly, despite showing signs of growth retardation in the young, adult male and female BKO mice (4 mo old) finally caught up with their wild-type littermates in body weight and femoral length. The underlying mechanism of catch-up growth is unknown. Since both wild-type and BKO mice had similar growth plate histology and growth plate height, we speculated that ␤-thalassemia did not directly impair growth plate chondrocyte function, and thus, in the presence of appropriate stimuli (e.g., sex hormones, growth plate-derived IGF-I, increased body weight, and physical activity), endochondral bone growth and bone elongation could be rapidly recovered, leading to catch-up growth of BKO mice. However, osteopenia—as indicated by low BMD in both primarily trabecular (L5– 6 lumbar vertebrae and femoral metaphysis) and/or cortical sites (femoral diaphysis)—was evident throughout the 8-mo period, with greater severity in female BKO mice. The salient characteristics of trabecular bone loss in female BKO mice were poor trabecular microarchitecture and decreased trabecular connectivity, as shown by reduction in mineralized bone volume (trabecular bone volume), trabecular number, and trabecular thickness as well as an increase in the trabecular separation with expansion of bone marrow volume compared with the age-matched wild-type littermates. Static and dynamic histomorphometric analyses further revealed that this ␤-thalassemia-induced microstructural defect was associated with conspicuous signs of impaired osteoblast function (decreased osteoblast surface), extracellular matrix production (decreases in osteoid surface, thickness, and volume), and bone calcium accretion (decreased mineralizing surface and mineral apposition rate), all of which eventually contributed to a decrease in the rate of bone formation. Similar histomorphometric findings were also observed in the transiliac bone

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Fig. 8. A: bone volume (BV) normalized by tissue volume (TV). B: bone surface (BS) normalized by TV. C: trabecular number (Tb.N). D: trabecular thickness (Tb.Th). E: trabecular separation (Tb.Sp). F: marrow volume (Ma.V) normalized by TV in tibial metaphyses of sedentary (Sed) and running (Ex) female BKO mice, as determined by bone histomorphometry. Numbers in parentheses are numbers of animals. ***P ⬍ 0.001 vs. age-matched sedentary BKO mice.

D

biopsies of thalassemic patients (31). Nevertheless, little is known regarding the cellular and molecular mechanisms of ␤-thalassemia-induced osteoblast dysfunction. The impaired intestinal calcium absorption could partly explain a decrease in bone calcium accretion in BKO mice. We recently showed that daily subcutaneous injection of 1 ␮g/kg 1,25(OH)2D3 for 3 days did not enhance the duodenal calcium absorption in BKO mice, probably due to a decrease in the duodenal 1,25(OH)2D3 responsiveness (7), thereby reducing calcium supply for bone mineralization. Other investigators also reported the presence of hypoparathyroidism and decreased 1,25(OH)2D3 production in thalassemia (30, 43, 50), which might, in turn, aggravate calcium malabsorption and might also diminish osteoblast function (5, 54). A reduction in the circulating levels of certain endocrine factors, particularly IGF-I (13), could also contribute to the impaired bone formation since IGF-I was an important regulator of both osteoblastogenesis and intestinal calcium absorption (4, 20, 61). Furthermore, iron overload and oxidative stress from iron toxicity commonly found in ␤-thalassemia could suppress osteoblast function (11, 17). Iron markedly suppressed alkaline phosphatase activity in osteoblast-like cell lines as well as the expression of several osteoblast-derived bone formation markers, such as runt-related transcription factor 2, osteocalcin, and osteopontin (58). Excessive production of reactive oxygen species after iron overload might also activate the nuclear factor-␬B signaling pathway, leading to a reduction in osteoblast activity or even apoptosis of osteoblasts (1). The enhanced osteoclast-mediated bone resorption as indicated by increases in osteoclast surface and active erosion surface in BKO mice was probably due to excessive production of osteoclastogenic cytokines IL-1␣ and -1␤. Since oxidative stress-related chronic inflammation and compensatory increase in erythropoiesis were accompanied by intramedullary hematopoietic cytokine release (26, 41), osteoclast precursor cells that were also derived from hematopoietic stem cells could

B

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proliferate concurrently (18), thus leading to an exaggerated number of active osteoclasts. IL-1 was capable of activating the transcription of osteoclast-specific genes, such as tartrateresistant acid phosphatase (TRAP) and osteoclast maturation (32). In addition, ineffective erythropoiesis also induced bone marrow expansion that directly eroded bone trabeculae, as suggested by an increase in marrow volume (Fig. 3F). Since endurance exercise not only benefited cardiopulmonary function but also promoted bone gain and prevented osteoporosis (22), BKO mice were allowed wheel running to demonstrate that this intervention was effective for alleviating ␤-thalassemia-induced osteopenia. The present exercise program for BKO mice was an endurance type that efficiently induced cardiac hypertrophy and increased activities of the oxidative muscle enzymes, i.e., citrate synthase and succinate dehydrogenase, in both fast- and slow-twitch muscles without changing plasma lactate levels (an indicator of anaerobic respiration). The positive effects of 3-mo running in BKO mice were clearly observed at the cortical and trabecular sites of long bone, consistent with a previous report in normal mice with tibial bone loading (10). In the diaphysis of BKO mice, pQCT revealed increases in the cortical density, mineral content, and thickness at the end of 3-mo exercise session. Furthermore, endurance running significantly improved the trabecular bone microstructure as indicated by increases in trabecular bone volume, trabecular bone surface, trabecular number, and trabecular thickness with decreases in trabecular separation and marrow volume. These positive effects of exercise on thalassemic bone were likely to have resulted from enhanced osteoblast-mediated bone formation rather than suppression of osteoclast-mediated bone resorption. However, the running-induced decrease in active erosion surface suggested that wheel running indeed suppressed bone resorption, presumably by reducing osteoclast activity. Generally, exercise could enhance bone gain by a number of mechanisms, such as stimulation of intestinal calcium absorp-

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Fig. 9. A: osteoblast surface (Ob.S) normalized by bone surface (BS). B: osteoid surface (OS) normalized by BS. C: osteoid thickness (O.Th). D: osteoid volume (OV) normalized by tissue volume (TV). E: mineralizing surface (MS) normalized by BS. F: double labeled surface (dLS) normalized by BS. G: mineral apposition rate (MAR). H: bone formation rate (BFR) normalized by BS. I: osteoclast surface (Oc.S) normalized by BS. J: active erosion surface (a.ES) normalized by BS in tibial metaphyses of sedentary (Sed) and running (Ex) female BKO mice, as determined by bone histomorphometry. Numbers in parentheses are numbers of animals. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. age-matched sedentary BKO mice.

tion, release of skeletal muscle-derived osteogenic myokines, and induction of mechanotransduction in bone (36). Our group recently demonstrated that mild to moderate endurance swimming markedly stimulated the transepithelial calcium transport in rat duodenum, in part by upregulating the expression of genes related to calcium absorption, namely transient receptor potential vanilloid calcium channel (TRPV)-5, TRPV6, calbindin-D9k, and Na⫹/Ca2⫹-exchanger 1 (48). Yeh and coworkers also reported that female rats subjected to flat-bed treadmill running had a higher rate of duodenal calcium absorption than sedentary rats (48, 60). This enhanced intestinal calcium absorption and the resultant extra calcium could be responsible for bone mineralization, consistent with increases in mineralizing surface and mineral apposition rate in running BKO mice. A decreased osteoid surface (Fig. 9B) in running BKO mice could also be explained by the enhanced calcium supply, which gradually abolished unmineralized spots. However, since ␤-thalassemia severely impaired calcium absorption (7), the exercise-enhanced calcium supply might have remained

inadequate to normalize mineralization rate. An increase in thickness of osteoid, which was probably produced at high level by osteoblasts after exercise, was thus evident in running BKO mice (Fig. 9C). Several contraction-induced factors or myokines, such as IGF-I, FGF-2, and irisin, released during exercise have been reported to exert osteogenic effects on bone (37). For example, it was evident that myoblast-derived irisin could upregulate the expression of alkaline phosphatase and type I collagen in osteoblasts in vitro (9), and FGF-2 was a determinant of bone mass and bone formation (33, 35). Meanwhile, force due to muscle contraction and mechanical loading during exercise induced physical signals (e.g., canalicular fluid flow), which could be sensed by mechanosensitive cells, particularly osteocytes (36). An in vitro study showed that osteocytes subjected to pulsating fluid flow stimulated osteoblast proliferation and differentiation (55). Taken together, impact exercise could increase both osteoblast number and activities, the latter of which was consistent with increases in osteoblast surface and

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osteoid thickness in the tibial metaphysis of BKO mice. Nevertheless, more investigation is required to demonstrate the detailed cellular and molecular mechanisms of the exerciseinduced osteoblast function in these mice. In conclusion, we elaborated herein that hemizygous ␤-globin knockout was associated with growth retardation and impaired longitudinal bone growth, which gradually disappeared after 2 mo of age. Moreover, compared with wild-type mice, the BKO mice manifested lower BMD and impaired bone microstructure, the latter of which evidently resulted from suppression of osteoblast function and mineral accretion as well as the enhancement of osteoclast-mediated bone resorption. The ␤-thalassemia-induced osteopenia was apparently more severe in female than in male mice, as analyzed by two-way ANOVA. Interestingly, endurance wheel running effectively increased bone density and improved bone microstructure in BKO mice. It seems that physical activity or exercise with appropriate intensity could help alleviate osteopenia and reduce fracture risk in ␤-thalassemic patients. ACKNOWLEDGMENTS We thank Prof. Suchinda Malaivijitnond, Department of Biology, Faculty of Science, Chulalongkorn University, for providing the pQCT, and Panan Suntornsaratoon for histological technique and critical comments on the manuscript. GRANTS This research was supported by grants from the Mahidol University (to N. Charoenphandhu), the Thailand Research Fund-Mahidol University through the Royal Golden Jubilee Ph.D. Program (PHD/0352/2550 to K. Thongchote), the Faculty of Science, Mahidol University (to N. Charoenphandhu), and Research Chair Grant, National Science and Technology Development Agency, Thailand. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: K.T. and N.C. performed experiments; K.T., S.S., J.T., N.K., and N.C. analyzed data; K.T., S.S., J.T., N.K., and N.C. interpreted results of experiments; K.T., J.T., and N.C. prepared figures; K.T., S.S., J.T., N.K., and N.C. approved final version of manuscript; J.T., N.K., and N.C. drafted manuscript; J.T., N.K., and N.C. edited and revised manuscript; N.C. conception and design of research. REFERENCES 1. Almeida M, Han L, Ambrogini E, Bartell SM, Manolagas SC. Oxidative stress stimulates apoptosis and activates NF-␬B in osteoblastic cells via a PKC␤/p66shc signaling cascade: counter regulation by estrogens or androgens. Mol Endocrinol 24: 2030 –2037, 2010. 2. Aloia JF, Ostuni JA, Yeh JK, Zaino EC. Combined vitamin D parathyroid defect in thalassemia major. Arch Intern Med 142: 831–832, 1982. 3. Bassey EJ, Ramsdale SJ. Increase in femoral bone density in young women following high-impact exercise. Osteoporos Int 4: 72–75, 1994. 4. Bikle DD, Sakata T, Leary C, Elalieh H, Ginzinger D, Rosen CJ, Beamer W, Majumdar S, Halloran BP. Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res 17: 1570 –1578, 2002. 5. Bronner F. Role of intestinal calcium absorption in plasma calcium regulation of the rat. Am J Physiol Regul Integr Comp Physiol 246: R680 –R683, 1984. 6. Cao A, Galanello R. Beta-thalassemia. Genet Med 12: 61–76, 2010. 7. Charoenphandhu N, Kraidith K, Teerapornpuntakit J, Thongchote K, Khuituan P, Svasti S, Krishnamra N. 1,25-Dihydroxyvitamin D3induced intestinal calcium transport is impaired in ␤-globin knockout thalassemic mice. Cell Biochem Funct 31: 685–691, 2013.

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