Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.

Fibroblast growth factor 23 (FGF23) induces ventricular arrhythmias and prolongs QTc interval in mice in an FGF receptor 4-dependent manner.

Fibroblast growth factor 23 (FGF23) is a phosphate regulating protein hormone released by osteocytes. FGF23 becomes markedly elevated in chronic kidney disease (CKD), for which the leading cause of death is cardiovascular disease, particularly sudden cardiac death. Previously, we found that FGF23 increases intracellular Ca2+ in cardiomyocytes and alters contractility in mouse ventricles ex vivo via FGF receptor 4 (FGFR4). In the present study, we demonstrate that FGF23 induces cardiac arrhythmias and prolongs QTc interval in mice, and we tested whether these effects are mediated through FGFR4. In isolated Langendorff perfused hearts, FGF23 perfusion increased mechanical arrhythmias in the form of premature ventricular beats (PVBs), and induced runs of ventricular tachycardia in 6 of 11 animals, which were attenuated with pretreatment of an anti-FGFR4 blocking antibody. Ex vivo ECG analysis of isolated intact hearts showed increased ventricular arrhythmias and QTc prolongation after FGF23 infusion compared with vehicle. In vivo, injection of FGF23 into the jugular vein led to the emergence of premature ventricular contractions (PVCs) in 5 out of 11 experiments. FGF23 also produced a significant lengthening effect upon QTc interval in vivo. In vivo FGFR4 blockade ameliorated the arrhythmogenic and QTc prolonging effects of FGF23. Finally, FGF23 increased cardiomyocyte Ca2+ levels in intact left ventricular muscle which was inhibited by FGR4 blockade. We conclude that FGF23/FGFR4 signaling in the heart may contribute to ventricular arrhythmogenesis and repolarization disturbances commonly observed in patients with CKD via Ca2+ overload and may be an important therapeutic target to reduce cardiac mortality in CKD.NEW & NOTEWORTHY Here we provide direct evidence that fibroblast growth factor 23 (FGF23), a phosphaturic hormone elevated in chronic kidney disease, is proarrhythmic. FGF23 acutely triggered ventricular arrhythmias and prolonged corrected QT interval (QTc) in isolated mouse hearts and in vivo. FGF23 also increased Ca2+ levels in ventricular muscle tissue. Blockade of the FGF receptor 4 signaling pathway using a monoclonal antibody ameliorated ventricular arrhythmias, QTc prolongation, and elevated ventricular Ca2+ induced by FGF23, and may represent a potential therapeutic target in chronic kidney disease.

Trimethylamine-N-oxide acutely increases cardiac muscle contractility.

Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.

MCP-1 promotes detrimental cardiac physiology, pulmonary edema, and death in the cpk model of polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by slowly expanding renal cysts that damage the kidney, typically resulting in renal failure by the fifth decade. The most common cause of death in these patients, however, is cardiovascular disease. Expanding cysts in PKD induce chronic kidney injury that is accompanied by immune cell infiltration, including macrophages, which we and others have shown can promote disease progression in PKD mouse models. Here, we show that monocyte chemoattractant protein-1 [MCP-1/chemokine (C-C motif) ligand 2 (CCL2)] is responsible for the majority of monocyte chemoattractant activity produced by renal PKD cells from both mice and humans. To test whether the absence of MCP-1 lowers renal macrophage concentration and slows disease progression, we generated genetic knockout (KO) of MCP-1 in a mouse model of PKD [congenital polycystic kidney (cpk) mice]. Cpk mice are born with rapidly expanding renal cysts, accompanied by a decline in kidney function and death by postnatal day 21. Here, we report that KO of MCP-1 in these mice increased survival, with some mice living past 3 mo. Surprisingly, however, there was no significant difference in renal macrophage concentration, nor was there improvement in cystic disease or kidney function. Examination of mice revealed cardiac hypertrophy in cpk mice, and measurement of cardiac electrical activity via ECG revealed repolarization abnormalities. MCP-1 KO did not affect the number of cardiac macrophages, nor did it alleviate the cardiac aberrancies. However, MCP-1 KO did prevent the development of pulmonary edema, which occurred in cpk mice, and promoted decreased resting heart rate and increased heart rate variability in both cpk and noncystic mice. These data suggest that in this mouse model of PKD, MCP-1 altered cardiac/pulmonary function and promoted death outside of its role as a macrophage chemoattractant.

Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility.

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.

FGF23 directly impairs endothelium-dependent vasorelaxation by increasing superoxide levels and reducing nitric oxide bioavailability.

Fibroblast growth factor 23 (FGF23) is secreted primarily by osteocytes and regulates phosphate and vitamin D metabolism. Elevated levels of FGF23 are clinically associated with endothelial dysfunction and arterial stiffness in chronic kidney disease (CKD) patients; however, the direct effects of FGF23 on endothelial function are unknown. We hypothesized that FGF23 directly impairs endothelial vasorelaxation by hindering nitric oxide (NO) bioavailability. We detected expression of all four subtypes of FGF receptors (Fgfr1-4) in male mouse aortas. Exogenous FGF23 (90-9,000 pg/ml) did not induce contraction of aortic rings and did not relax rings precontracted with PGF2α. However, preincubation with FGF23 (9,000 pg/ml) caused a ∼36% inhibition of endothelium-dependent relaxation elicited by acetylcholine (ACh) in precontracted aortic rings, which was prevented by the FGFR antagonist PD166866 (50 nM). Furthermore, in FGF23-pretreated (9,000 pg/ml) aortic rings, we found reductions in NO levels. We also investigated an animal model of CKD (Col4a3(-/-) mice) that displays highly elevated serum FGF23 levels and found they had impaired endothelium-dependent vascular relaxation and reduced nitrate production compared with age-matched wild types. To elucidate a mechanism for the FGF23-induced impairment, we measured superoxide levels in endothelial cells and aortic rings and found that they were increased following FGF23 treatment. Crucially, treatment with the superoxide scavenger tiron reduced superoxide levels and also restored aortic relaxation to ACh. Therefore, our data suggest that FGF23 increases superoxide, inhibits NO bioavailability, and causes endothelial dysfunction in mouse aorta. Together, these data provide evidence that high levels of FGF23 contribute to cardiovascular dysfunction.

FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertrophy.

Fibroblast growth factor 23 (FGF23) is a hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism. Recent observational studies in humans suggest that circulating FGF23 is independently associated with cardiac hypertrophy and increased mortality, but it is unknown whether FGF23 can directly alter cardiac function. We found that FGF23 significantly increased cardiomyocyte cell size in vitro, the expression of gene markers of cardiac hypertrophy, and total protein content of cardiac muscle. In addition, FGFR1 and FGFR3 mRNA were the most abundantly expressed FGF receptors in cardiomyocytes, and the coreceptor α-klotho was expressed at very low levels. We tested an animal model of chronic kidney disease (Col4a3(-/-) mice) that has elevated serum FGF23. We found elevations in common hypertrophy gene markers in Col4a3(-/-) hearts compared with wild type but did not observe changes in wall thickness or cell size by week 10. However, the Col4a3(-/-) hearts did show reduced fractional shortening (-17%) and ejection fraction (-11%). Acute exposure of primary cardiomyocytes to FGF23 resulted in elevated intracellular Ca(2+) ([Ca(2+)](i); F/F(o) + 86%) which was blocked by verapamil pretreatment. FGF23 also increased ventricular muscle strip contractility (67%), which was inhibited by FGF receptor antagonism. We hypothesize that although FGF23 can acutely increase [Ca(2+)](i), chronically this may lead to decreases in contractile function or stimulate cardiac hypertrophy, as observed with other stress hormones. In conclusion, FGF23 is a novel bone/heart endocrine factor and may be an important mediator of cardiac Ca(2+) regulation and contractile function during chronic kidney disease.

FGF23 production by osteocytes.

Fibroblast growth factor 23 (FGF23), a known regulator of phosphate homeostasis, is produced by cells residing in bone, namely, osteocytes, to target a distant organ, the kidney. Elevated FGF23 levels have recently been found systemically and in osteocytes in patients and animal models of chronic kidney disease. Associations between serum FGF23 level and vascular dysfunction, vascular calcification, and increased risk of cardiovascular disease have also been observed. In this review we discuss FGF23 expression in osteocytes and the potential means to regulate expression and function of this protein at the osteocyte level.

The Role of Zinc in Modulating Acid-Sensing Ion Channel Function.

Acid-sensing ion channels (ASICs) are proton-gated, voltage-independent sodium channels widely expressed throughout the central and peripheral nervous systems. They are involved in synaptic plasticity, learning/memory, fear conditioning and pain. Zinc, an important trace metal in the body, contributes to numerous physiological functions, with neurotransmission being of note. Zinc has been implicated in the modulation of ASICs by binding to specific sites on these channels and exerting either stimulatory or inhibitory effects depending on the ASIC subtype. ASICs have been linked to several neurological and psychological disorders, such as Alzheimer's disease, Parkinson's disease, ischemic stroke, epilepsy and cocaine addiction. Different ASIC isoforms contribute to the persistence of each of these neurological and psychological disorders. It is critical to understand how various zinc concentrations can modulate specific ASIC subtypes and how zinc regulation of ASICs can contribute to neurological and psychological diseases. This review elucidates zinc's structural interactions with ASICs and discusses the potential therapeutic implications zinc may have on neurological and psychological diseases through targeting ASICs.

Acid-Sensing Ion Channels in Glial Cells.

Acid-sensing ion channels (ASICs) are proton-gated cation channels and key mediators of responses to neuronal injury. ASICs exhibit unique patterns of distribution in the brain, with high expression in neurons and low expression in glial cells. While there has been a lot of focus on ASIC in neurons, less is known about the roles of ASICs in glial cells. ASIC1a is expressed in astrocytes and might contribute to synaptic transmission and long-term potentiation. In oligodendrocytes, constitutive activation of ASIC1a participates in demyelinating diseases. ASIC1a, ASIC2a, and ASIC3, found in microglial cells, could mediate the inflammatory response. Under pathological conditions, ASIC dysregulation in glial cells can contribute to disease states. For example, activation of astrocytic ASIC1a may worsen neurodegeneration and glioma staging, activation of microglial ASIC1a and ASIC2a may perpetuate ischemia and inflammation, while oligodendrocytic ASIC1a might be involved in multiple sclerosis. This review concentrates on the unique ASIC components in each of the glial cells and integrates these glial-specific ASICs with their physiological and pathological conditions. Such knowledge provides promising evidence for targeting of ASICs in individual glial cells as a therapeutic strategy for a diverse range of conditions.

Body weight influences musculoskeletal adaptation to long-term voluntary wheel running during aging in female mice.

Frailty is the hallmark of aging that can be delayed with exercise. The present studies were initiated based on the hypothesis that long-term voluntary wheel running (VWR) in female mice from 12 to 18 or 22 months of age would have beneficial effects on the musculoskeletal system. Mice were separated into high (HBW) and low (LBW) body weight based on final body weights upon termination of experiments. Bone marrow fat was significantly higher in HBW than LBW under sedentary conditions, but not with VWR. HBW was more protective for soleus size and function than LBW under sedentary conditions, however VWR increased soleus size and function regardless of body weight. VWR plus HBW was more protective against muscle loss with aging. Similar effects of VWR plus HBW were observed with the extensor digitorum longus, EDL, however, LBW with VWR was beneficial in improving EDL fatigue resistance in 18 mo mice and was more beneficial with regards to muscle production of bone protective factors. VWR plus HBW maintained bone in aged animals. In summary, HBW had a more beneficial effect on muscle and bone with aging especially in combination with exercise. These effects were independent of bone marrow fat, suggesting that intrinsic musculoskeletal adaptions were responsible for these beneficial effects.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) potentiates cardiac contractility via activation of the ryanodine receptor.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction.

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.

Store-operated calcium entry is present in HL-1 cardiomyocytes and contributes to resting calcium.

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Fibroblast growth factor 9 (FGF9) inhibits myogenic differentiation of C2C12 and human muscle cells.

Osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. The biochemical crosstalk between muscle and bone seems to play a role in OS. We have previously shown that osteocytes produce soluble factors with beneficial effects on muscle and vice versa. Recently, enhanced FGF9 production was observed in the OmGFP66 osteogenic cell line. To test its role in myogenic differentiation, C2C12 myoblasts were treated with recombinant FGF9. FGF9 as low as 10 ng/mL inhibited myogenic differentiation, suggesting that FGF9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. FGF9 (10-50 ng/mL) significantly decreased mRNA expression of MyoG and Mhc while increasing the expression of Myostatin. Consistent with the phenotype, RT-qPCR array revealed that FGF9 (10 ng/mL) increased the expression of Icam1 while decreased the expression of Wnt1 and Wnt6 decreased, respectively. FGF9 decreased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and reduced the expression of genes (i.e. Cacna1s, RyR2, Naftc3) directly associated with intracellular Ca2+ homeostasis. Myogenic differentiation in human skeletal muscle cells was similarly inhibited by FGF9 but required higher doses of 200 ng/mL FGF9. FGF9 was also shown to stimulate C2C12 myoblast proliferation. FGF2 and the FGF9 subfamily members FGF16 and FGF20 also inhibited C2C12 myoblast differentiation and enhanced proliferation. Intriguingly, the differentiation inhibition was independent of proliferation enhancement. These findings suggest that FGF9 may modulate myogenesis via a complex signaling mechanism.

Characterization of a novel murine Sost ERT2 Cre model targeting osteocytes.

Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene (Sost ERT2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or 'leaky' with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ERT2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.

Technique for quantitative RT-PCR analysis directly from single muscle fibers.

The use of single-cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study, we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and that gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of five male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer-probes for beta(2)-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, insulin-like growth factor I receptor, and glucose transporter subtype 4. The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used one-quarter of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was <8% (cycle threshold value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4). We observed a 13.9-fold change in expression after resistance exercise, which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers.

Sex difference in the association of the angiotensin converting enzyme I/D polymorphism and body mass index.

BACKGROUND: Angiotensin converting enzyme (ACE) catayzes the formation of angiotensin I to angiotensin II. A polymorphism has been identified in intron 16 in which a 287 base-pair alu sequence was found to be present (insertion or I) or absent (deletion or D) in the population. ACE and the components of the renin-angiotensin system are expressed in adipose tissue and therefore the I/D polymorphism within ACE may be associated with obesity. MATERIAL/METHODS: This study involved genotyping two groups with differing body mass indexes (BMI or=30) that were composed primarily of middle-age Caucasian subjects (n=421). RESULTS: The male groups differed significantly in allele frequency at the ACE locus with the I allele more frequent in the BMI >or=30 group (p<0.05). While the female BMI >or=30 group also had a higher I allele frequency than the BMI

FGF23/FGFR4-mediated left ventricular hypertrophy is reversible.

Fibroblast growth factor (FGF) 23 is a phosphaturic hormone that directly targets cardiac myocytes via FGF receptor (FGFR) 4 thereby inducing hypertrophic myocyte growth and the development of left ventricular hypertrophy (LVH) in rodents. Serum FGF23 levels are highly elevated in patients with chronic kidney disease (CKD), and it is likely that FGF23 directly contributes to the high rates of LVH and cardiac death in CKD. It is currently unknown if the cardiac effects of FGF23 are solely pathological, or if they potentially can be reversed. Here, we report that FGF23-induced cardiac hypertrophy is reversible in vitro and in vivo upon removal of the hypertrophic stimulus. Specific blockade of FGFR4 attenuates established LVH in the 5/6 nephrectomy rat model of CKD. Since CKD mimics a form of accelerated cardiovascular aging, we also studied age-related cardiac remodeling. We show that aging mice lacking FGFR4 are protected from LVH. Finally, FGF23 increases cardiac contractility via FGFR4, while known effects of FGF23 on aortic relaxation do not require FGFR4. Taken together, our data highlight a role of FGF23/FGFR4 signaling in the regulation of cardiac remodeling and function, and indicate that pharmacological interference with cardiac FGF23/FGFR4 signaling might protect from CKD- and age-related LVH.

Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle.

The purpose of this investigation was to examine the expression of three commonly used housekeeping genes -- glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta(2)-microglobulin (beta(2)M), and RNA polymerase 2a (polR2a) -- in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 +/- 3.4 yrs) and 11 elderly subjects (73.0 +/- 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, beta(2)M, and polR2a was obtained as the value of cycle threshold (C(T)). An independent t-test with a level of significance at p < or = 0.05 was used to determine differences between groups. There was no difference in average C(T) of GAPDH between groups (p=0.869) (Y = 16.92 +/- 2.25 vs. E = 17.08 +/- 2.09) and polR2a (p = 0.089) (Y = 28.00 +/- 0.89 vs. E = 26.73 +/- 1.91). However, there was a significant difference (p < or = 0.05) in the average C(T) of beta(2)M (Y =21.79 +/- 0.44 vs. E = 21.05 +/- 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.