Type-2 diabetic aldehyde dehydrogenase 2 mutant mice (ALDH 2*2) exhibiting heart failure with preserved ejection fraction phenotype can be determined by exercise stress echocardiography

E487K point mutation of aldehyde dehydrogenase (ALDH) 2 (ALDH2*2) in East Asians intrinsically lowers ALDH2 activity. ALDH2*2 is associated with diabetic cardiomyopathy. Diabetic patients exhibit heart failure of preserved ejection fraction (HFpEF) i.e. while the systolic heart function is preserved in them, they may exhibit diastolic dysfunction, implying a jeopardized myocardial health. Currently, it is challenging to detect cardiac functional deterioration in diabetic mice. Stress echocardiography (echo) in the clinical set-up is a procedure used to measure cardiac reserve and impaired cardiac function in coronary artery diseases. Therefore, we hypothesized that high-fat diet fed type-2 diabetic ALDH2*2 mutant mice exhibit HFpEF which can be measured by cardiac echo stress test methodology. We induced type-2 diabetes in 12-week-old male C57BL/6 and ALDH2*2 mice through a high-fat diet. At the end of 4 months of DM induction, we measured the cardiac function in diabetic and control mice of C57BL/6 and ALDH2*2 genotypes by conscious echo. Subsequently, we imposed exercise stress by allowing the mice to run on the treadmill until exhaustion. Post-stress, we measured their cardiac function again. Only after treadmill running, but not at rest, we found a significant decrease in % fractional shortening and % ejection fraction in ALDH2*2 mice with diabetes compared to C57BL/6 diabetic mice as well as non-diabetic (control) ALDH2*2 mice. The diabetic ALDH2*2 mice also exhibited poor maximal running speed and distance. Our data suggest that high-fat fed diabetic ALDH2*2 mice exhibit HFpEF and treadmill exercise stress echo test is able to determine this HFpEF in the diabetic ALDH2*2 mice

The Diminishing Returns of Mechanical Loading and Potential Mechanisms that Desensitize Osteocytes

Adaptation to mechanical loading is critical to maintaining bone mass and offers therapeutic potential to preventing age-related bone loss and osteoporosis. However, increasing the duration of loading is met with diminishing returns as the anabolic response quickly becomes saturated. As a result, the anabolic response to daily activities and repetitive bouts of loading is limited by the underlying mechanisms that desensitize and render bone unresponsive at the cellular level. Osteocytes are the primary cells that respond to skeletal loading and facilitate the overall anabolic response. Although many of osteocytes\u27 signaling mechanisms activated in response to loading are considered anabolic in nature, several of them can also render osteocytes insensitive to further stimuli and thereby creating a negative feedback loop that limits osteocytes\u27 overall response. The purpose of this review is to examine the potential mechanisms that may contribute to the loss of mechanosensitivity. In particular, we examined the inactivation/desensitization of ion channels and signaling molecules along with the potential role of endocytosis and cytoskeletal reorganization. The significance in defining the negative feedback loop is the potential to identify unique targets for enabling osteocytes to maintain their sensitivity. In doing so, we can begin to cultivate new strategies that capitalize on the anabolic nature of daily activities that repeatedly load the skeleton

PTH(1-34) reduces microdamage in the mouse femur under fatigue loading

INTRODUCTION: Parathyroid hormone (PTH) is a calciotropic hormone that plays a critical role in maintaining bone quality [1]. The PTH(1-34) analog, is commonly used to increase bone mass and reduce fracture risk associated with skeletal disorders, such as osteoporosis. Osteoporotic fractures in particular generally occur under two modes of failure: traumatic and fatigue [1-3]. Microdamage generated under cyclic loading during daily activities contributes to fatigue failure. However, the effect of PTH(1-34) treatment on microdamage has been less studied in the literature. Therefore, the primary objective of this study was to determine how PTH(1-34) influences the fatigue properties of cortical bone. We hypothesize that increased bone formation following PTH(1-34) treatment will increase the resistance to microdamage. METHODS: 16 week old male C57B1/6J mice were weight-matched and divided into two groups: vehicle and PTH (1-34). For 21 consecutive days, subcutaneous injections were given to administer 50 μL of a saline solution (0.9% NaCl) as a vehicle control or 40 μg/kg of hPTH (1-34) (Bachem) in saline solution. On day 22, 5 mice from each group were sacrificed and femurs were removed and stripped of the soft tissue. Micro-Ct scans were taken of the left femur and then subjected to cyclic loading under four-point bending (BOSE, Electroforce 32000 series), with the anterior side under tension. The femur was first loaded for 20 cycles at 5N to determine the initial stiffness and force needed to generate 10,000 microstrain along the anterior side. This estimated force was then applied for 40,000 cycles at 2 Hz under load control. Testing was conducted in calcium chloride buffered saline solution at 37°C. After loading, both left and right (non-loaded control) samples were dehydrated in graded Ethanol with 1% Basic Fuchsin, infiltrated in a liquid methyl methacrylate monomer (Koldmount Cold Mounting Liquid), embedded in polymethylmethacrylate, sectioned and polished to 150μm thickness. Sections were imaged using confocal microscopy (Zeiss 100 Axiovert inverted microscope) with 559 nm laser and UPLSAPO 40X 2 objective lens. Z-stack images were obtained along the anterior side of each sample. In each image, crack density (Cr. Dn = # of cracks / cortical area), diffuse damage (Df.Dm.Dn = diffuse damage area / cortical area), and crack length (Cr.Ln) were quantified using ImageJ. The mean and standard deviations of each metric is reported. Statistical interactions between groups were determined using a student T-test with a p-value of \u3c0.05 considered significant. RESULTS: In vehicle treated mice, crack density was significantly greater following fatigue loading compared to the non-fatigued limb (374.53 ± 115.39 #/mm2 vs. 132.52 ± 59.78 #/mm2), while PTH(1-34) treated mice exhibited no significant difference in crack density between fatigue and non-fatigued limbs (270.21 ± 69.59 #/mm2 vs. 151.69±66.72 #/mm2) (Fig. 1A). The density of diffuse damage in the fatigued limb was significantly greater in vehicle treated mice than the fatigued limb of PTH(1-34) treated mice (0.00703 ± 0.0016 mm2/mm2 vs. 0.0055 ± 0.0015 mm2/mm2) (Fig. 1B). Similarly, crack length in the fatigue limb was also significantly greater in vehicle treated mice than the fatigued limb of PTH(1-34) treated mice (35.067 ± 5.95 μm vs. 26.02±4.062 μm) (Fig. 1C). During fatigue loading, the loss in stiffness was smaller in the PTH(1-34) treated samples compared to vehicle (19.88 ± 6.15 % vs. 24.578 ± 7.55 %). No significant differences were observed between vehicle and PTH(1-34) treated groups for cross-sectional area (0.89 ± 0.06 mm vs. 0.90 ± 0.03) and moment of inertia (0.168 ± 0.03 mm4 vs. 0.173 ± 0.04 mm4) at the femur. DISCUSSION: Daily treatment with PTH(1-34) over a short period of time was sufficient to decrease the presence of microdamage following fatigue loading conditions. This increased resistance to microdamage due to PTH(1-34) treatment corresponded with minimal stiffness loss during fatigue loading. This reduction in microda age and stiffness loss during fatigue loading would also explain the increased fatigue life observed in the bovine femur following similar treatment with teriparatide [3].Contrary to our expectations, PTH(1-34) had minimal effect on cortical area and moment of inertia at the femur, while a significant increase in tibial cortical area and bone formation occurred [4]. Studies using a similar dose of PTH(1-34) required only 4 weeks before changes in cortical area of the tibia occurred [5], while displaying minimal effects at the femur, even after 8 weeks of treatment [6]. Thus, our results demonstrate PTH(1-34) can provide improvements in fracture resistance that do not have to be superceded by an increase in bone mass. (Figure Presented)

The mechanotransduction of MLO-Y4 cells is disrupted by the senescence-associated secretory phenotype of neighboring cells

Age-related bone loss is attributed to the accumulation of senescent cells and their increasing production of inflammatory cytokines as part of the senescence-associated secretory phenotype (SASP). In otherwise healthy individuals, osteocytes play a key role in maintaining bone mass through their primary function of responding to skeletal loading. Given that osteocytes\u27 response to loading is known to steadily decline with age, we hypothesized that the increasing presence of senescent cells and their SASP inhibit osteocytes\u27 response to loading. To test this hypothesis, we developed two in vitro models of senescent osteocytes and osteoblasts derived from MLO-Y4 and MC3T3 cell lines, respectively. The senescent phenotype was unique to each cell type based on distinct changes in cell cycle inhibitors and SASP profile. The SASP profile of senescent osteocytes was in part dependent on nuclear factor-κB signaling and presents a new potential mechanism to target the SASP in bone. Nonsenescent MLO-Y4 cells cultured with the SASP of each senescent cell type failed to exhibit changes in gene expression as well as ERK phosphorylation and prostaglandin E2 release. The SASP of senescent osteocytes had the largest effect and neutralizing interleukin-6 (IL-6) as part of the SASP restored osteocytes\u27 response to loading. The loss in mechanotransduction due to IL-6 was attributed to a decrease in P2X7 expression and overall sensitivity to purinergic signaling. Altogether, these findings demonstrate that the SASP of senescent cells have a negative effect on the mechanotransduction of osteocytes and that IL-6 is a key SASP component that contributes to the loss in mechanotransduction

Osteocytes\u27 expression of the PTH/PTHrP receptor has differing effects on endocortical and periosteal bone formation during adenine-induced CKD.

Osteocytes play a key role in the pathophysiology of chronic kidney disease (CKD). However, the extent to which osteocytes contribute to abnormalities in bone turnover due to excessive levels of parathyroid hormone (PTH) remains poorly understood. The purpose of this study was to determine the extent to which bone formation and tissue strength during the progression of CKD is modified through osteocytes\u27 response to PTH. Conditional knockout mice targeting osteocytes\u27 expression of the PTH/PTH-related protein type 1 receptor (PPR) were subjected to adenine-induced CKD. After 6-weeks of treatment, adenine-induced CKD was found to reduce bone formation at the periosteal and endocortical surfaces of the tibia. The loss in bone mass corresponded with a significant decrease in structural-level mechanical properties. In knockout mice, the loss of PPR expression in osteocytes further exacerbated the loss in bone formation at the endocortical surface, but inhibited bone loss at the periosteal surface. In general, the effects of adenine-induced CKD were not as extensive in female mice. Collectively, these findings demonstrate that osteocytes\u27 response to PTH under adenine-induced CKD has a unique impact on bone turnover that is specific to the periosteal and endocortical surfaces

Bone adaptation in response to treadmill exercise in young and adult mice.

Exercise is a key determinate of fracture risk and provides a clinical means to promote bone formation. However, the efficacy of exercise to increase bone mass declines with age. The purpose of this study was to identify age-related differences in the anabolic response to exercise at the cellular and tissue level. To this end, young (8-weeks of age) and adult (36-weeks of age) male mice were subjected to a moderate exercise regimen of running on a treadmill. As a result, exercise had a significant effect on PTHrP and SOST gene expression during the first week that was dependent upon age. In particular, young mice displayed an increase in PTHrP expression and decrease in SOST expression, both of which remained unaffected by exercise in the adult mice. After 5-weeks of exercise, a significant decrease in the percentage of osteocytes expressing sclerostin at the protein level was found in young mice, but not adult mice. Mechanical testing of the tibia found exercise to have a significant influence on tissue-level mechanical properties, specifically ultimate-stress and modulus that was dependent on age. Adult mice in particular experienced a significant decrease in modulus despite an increase in cortical area and cortical thickness compared to sedentary controls. Altogether, this study demonstrates a shift in the cellular response to exercise with age, and that gains in bone mass at the adult stage fail to improve bone strength

Osteocytes\u27 response to PTH(1-34) regulates perilacunar tissue composition

INTRODUCTION: The mechanical strength of bone is highly regulated by chemical composition and heterogeneity [1]. Unlike osteoblasts, osteocytes are ideally located throughout cortical bone to augment the intrinsic properties by modifying the perilacunar tissue through \u27osteocytic osteolysis\u27 [2]. Several studies have identified PTH as potential mechanism to modify the intrinsic properties of bone through osteocytes\u27 remodeling their lacuna structure [2-5]; however, the manner in which perilacuar remodeling influences the tissue composition and mechanical properties of cortical bone remain unclear. Therefore, the purpose of this study was to establish how PTH influences perilacunar composition alongside whole bone mechanical properties. We hypothesize that bone strength gained through PTH(1-34) treatment is associated with an increase in perilacunar density. METHODS: 16 week old male C57Bl/6J mice were divided into two weight-matched groups and received daily subcutaneous injections of PTH(1-34) (40μg/kg) or vehicle control (50-l of 0.9% saline) for 21 consecutive days. On day 22, each mouse was sacrificed and the tibea were removed. The left tibia was used for ex-vivo micro-CT scans to measure the cross-sectional geometry and mechanical testing under four-point bending. The right tibia was embedded in PMMA, sectioned at the mid-diaphysis, polished to a thickness of 200 μm, and then imaged using a locally constructed Raman microprobe with a line focused 785-nm diode laser (Invictus, Kaiser Optical Systems) and 40X/0.75DIC objective (Plan Fluor, Nikon Instruments). Within the medial cortex of each tibia, 3 to 4 lacunae were identified and Raman spectroscopic signatures were taken across the perilacunar region (within 0-5 μm from the lacunae wall) and non-perilacunar region (within 5-10 μm from the lacunae wall). The carbonate-to-phosphate ratio (CPR) was calculated based on the carbonate (1071 cm-1) and phosphate (959 cm-1) band intensities. The mineral-to-matrix ratio (MMR) was calculated based on the phosphate and hydroxyproline intensities (851 cm-1 + 873 cm-1). Peak fitting was implemented using GRAMS software. The MMR and CPR for each group were averaged across 7 samples, each sample being the average taken from 3 to 4 imaged lacunae. Two-way ANOVA with a post-hoc Tukey-Kramer test was used to establish interactions between groups. RESULTS SECTION: Based on Raman spectroscopy, 3-weeks of PTH(1-34) treatment caused a significant decrease in the MMR of the perilacunar region compared to the non-perilacuar region (Figure 1A). In contrast, the MMR for vehicle treated controls displayed no significant difference between perilacunar and non-perilacunar regions. The CPR was significantly decreased across both regions following PTH(1-34) treatment compared to vehicle (Figure 1B). The cross-sectional area of the lacunae was also greater following PTH(1-34) treatment compared to vehicle (Figure 1C). Based on a linear regression across both groups, the increase in lacuna area correlated with the decrease in perilacunar MMR (p-value = 0.04). Whole bone mechanics indicated that PTH(1-34) compared to vehicle treatment decreased yield-displacement (237 ± 28 mm vs. 269 ± 40 mm) and yieldstrain (19,072 ± 1,016 vs. 21,910 ± 3,172 ). At the same time, PTH(1-34) compared to vehicle treatment increased stiffness (94 ± 13 N/mm vs. 77 ± 16 N/mm) and modulus (12 ± 0.9 GPa vs. 9.4 ± 1.5 GPa). Based on micro-CT analysis, PTH(1-34) treatment increased cortical area compared to vehicle treatment (0.78 ± 0.06 mm2 vs. 0.73 ± 0.06 mm2), but not the moment of inertia (0.092 ± 0.01 mm4 vs. 0.088 ± 0.01 mm4). DISCUSSION: Osteocytes\u27 response to PTH has a large influence over the mineral composition localized around their lacuna structure. Contrary to expectation, perilacunar remodeling following PTH treatment included a reduction in mineral density localized around their lacuna structure along-side a reduction in carbonate substitution throughout entire tissue. Although perilacunar MMR and carbonate-substitution were expected to ncrease given the corresponding increase in mechanical strength, the reduction in perilacunar MMR can allow bone to absorb more energy under repeatative loading. As a result, we anticipate that the reduction in MMR would explain the increase in fatigue properties reported following PTH(1-34) treatment of bovine samples [6]. The reduction in perilacunar mineral density was also considered a function of osteocytes\u27 enlarging their lacuna space through tissue resorption. Lactation studies have also demonstrated osteocytes\u27 ability to remodel the lacunae structure upon activation of the PTH/PTH-related protein receptor [3]. In addition, osteocytes\u27 response to PTH has shown increase their expression of catabolic factors, such as tartrate resistant acid phosphatase and cathepsin-K [3-5]. (Figure Presented)

PTH signaling mediates perilacunar remodeling during exercise.

Mechanical loading and release of endogenous parathyroid hormone (PTH) during exercise facilitate the adaptation of bone. However, it remains unclear how exercise and PTH influence the composition of bone and how exercise and PTH-mediated compositional changes influence the mechanical properties of bone. Thus, the primary purpose of this study was to establish compositional changes within osteocytes\u27 perilacunar region of cortical bone following exercise, and evaluate the influence of endogenous PTH signaling on this perilacunar adaptation. Raman spectroscopy, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS) were used to evaluate tissue composition surrounding individual lacuna within the tibia of 19week old male mice exposed to treadmill running for 3weeks. As a result of exercise, tissue within the perilacunar region (within 0-5μm of the lacuna wall) had a lower mineral-to-matrix ratio (MMR) compared to sedentary controls. In addition, exercise also increased the carbonate-to-phosphate ratio (CPR) across both perilacunar and non-perilacunar regions (5-10μm and 10-15μm from the lacuna walls). Tibial post-yield work had a significant negative correlation with perilacunar MMR. Inhibition of PTH activity with PTH(7-34) demonstrated that perilacunar remodeling during exercise was dependent on the cellular response to endogenous PTH. The osteocytes\u27 response to endogenous PTH during exercise was characterized by a significant reduction in SOST expression and significant increase in FGF-23 expression. The potential reduction in phosphate levels due to FGF-23 expression may explain the increase in carbonate substitution. Overall, this is the first study to demonstrate that adaptation in tissue composition is localized around individual osteocytes, may contribute to the changes in whole bone mechanics during exercise, and that PTH signaling during exercise contributes to these adaptations

Periosteal Bone Formation Varies with Age in Periostin Null Mice

Periostin, also known as osteoblast-specific factor 2, is a matricellular protein predominantly expressed at the periosteum of bone. During growth and development, periostin contributes to periosteal expansion by facilitating osteoblast differentiation and mineralization. Later in life, periosteal expansion provides an adaptive strategy to increase tissue strength without requiring substantial increase in bone mass. However, the function of periostin past skeletal maturity and during advanced aging is relatively unknown. The objective of this study was to examine the function of periostin in maintaining bone mass and tissue strength across different ages. In periostin null mice (Postn-/-), periosteal bone formation was significantly reduced in young (3 months) and adult mice (9 months). The lack of bone formation resulted in reduced bone mass and ultimate strength. Conversely, periosteal bone formation increased at advanced ages in 18-month-old Postn-/- mice. The increase in periosteal mineralization at advanced ages coincides with increased expression of vitronectin and osteopontin. Periosteal progenitors from Postn-/- mice displayed an increased capacity to mineralize when cultured on vitronectin, but not type-1 collagen. Altogether, these findings demonstrate the unique role of periostin in regulating periosteal bone formation at different ages and the potential for vitronectin to compensate in the absence of periostin

Taraxacum marklundii Palmgr. (BR0000011792970)

Belgium Herbarium image of Meise Botanic Garden