Chad D. Touchberry

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 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.

Acute heat treatment improves insulin-stimulated glucose uptake in aged skeletal muscle

Aging is associated with insulin resistance and decreased insulin-stimulated glucose uptake into skeletal muscle. Although the mechanisms underlying age-related insulin resistance are not clearly defined, impaired defense against inflammation and tissue oxidative stress are likely causes. Heat shock proteins (HSPs) have been shown to protect tissue from oxidative stress and inhibit the activation of stress kinases such as JNK, known to interfere with the insulin signaling pathway. While the induction of HSPs via chronic heat treatment has been shown to protect skeletal muscle from obesity-related insulin resistance, the ability of heat treatment to improve insulin action in aged skeletal muscle is not known. In the present study, one bout of in vivo heat treatment applied to 24-mo-old Fischer 344 rats improved insulin-stimulated glucose uptake after 24 h in slow-twitch soleus muscles. In vitro heat treatment applied to young (3-mo-old) and aged (24-mo-old) soleus muscles increased expression of HSP72 and inhibited anisomycin-induced activation of JNK. In contrast, heat treatment had no effect on p38 MAPK, a MAPK strongly activated with anisomycin. Prior inhibition of HSP72 transcription with the pharmacological inhibitor KNK437 eliminated the ability of heat treatment to blunt JNK activation. This suggests that the ability of heat treatment to inhibit JNK activation in skeletal muscle is dependent on increased HSP72 expression. In conclusion, an acute bout of heat treatment can increase insulin-stimulated glucose uptake in aged skeletal muscle, with the underlying mechanism likely to be HSP72-mediated JNK inhibition.

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.

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 Ca2+ homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca2+ ([Ca2+]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 [Ca2+]i. The PI(3,5)P2-induced elevation of [Ca2+]i was not present in conditions free of extracellular Ca2+ and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca2+ release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [3H]ryanodine binding and increased the open probability (Po) 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 Ca2+ release and thereby modulates cardiac contractility.

Inhibition of Thromboxane A2-Induced Arrhythmias and Intracellular Calcium Changes in Cardiac Myocytes by Blockade of the Inositol Trisphosphate Pathway

We have recently reported that left atrial injections of the thromboxane A2 (TXA2) mimetic, (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA2 may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA2R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA2R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA2R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP3) formation, gentamicin, or with an inhibitor of IP3 receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA2 can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP3 pathway. In summary, this study provides novel evidence for direct TXA2-induced cardiac arrhythmias and provides a rationale for IP3 as a potential target for the treatment of TXA2-mediated arrhythmias.

Supplemental Carbohydrate Ingestion Does Not Improve Performance of High-intensity Resistance Exercise

Kulik, JR, Touchberry, CD, Kawamori, N, Blumert, PA, Crum, AJ, and Haff, GG. Supplemental carbohydrate ingestion does not improve performance of high-intensity resistance training. J Strength Cond Res 22: 1101-1107, 2008-The effects of supplemental carbohydrate (CHO) ingestion on the performance of squats to exhaustion (STE) were investigated with eight resistance-trained men. Subjects participated in a randomized, counterbalanced, double-blind, placebo-controlled protocol with testing separated by 7 days. Subjects consumed 0.3g·kgCHO·bodymass−1 or a placebo (PLC) of equal volume immediately before exercise and after every other successful set of squats. The STE consisted of sets of five repetitions at an intensity of 85% 1 repetition maximum (1RM). Performance measured as total sets (CHO 3.5 ± 3.2, PLC 3.5 ± 2.7), repetitions (CHO 20.4 ±14.9, PLC 19.7 ± 13.1), volume load (CHO 2928.7 ± 2219.5 kg, PLC 2772.8 ± 1951.4 kg), and total work (CHO 29.9 ± 22.3 kJ, PLC 28.6 ± 19.5 kJ) was not statistically different between the CHO and PLC treatments. The results suggest that CHO supplementation does not enhance performance of squats performed with 85% 1RM to volitional failure.

Skeletal Muscle, but not Cardiovascular Function, Is Altered in a Mouse Model of Autosomal Recessive Hypophosphatemic Rickets

Autosomal recessive hypophosphatemic rickets (ARHR) is a heritable disorder characterized by hypophosphatemia, osteomalacia, and poor bone development. ARHR results from inactivating mutations in the DMP1 gene with the human phenotype being recapitulated in the Dmp1 null mouse model which displays elevated plasma fibroblast growth factor 23. While the bone phenotype has been well-characterized, it is not known what effects ARHR may also have on skeletal, cardiac, or vascular smooth muscle function, which is critical to understand in order to treat patients suffering from this condition. In this study, the extensor digitorum longus (EDL-fast-twitch muscle), soleus (SOL–slow-twitch muscle), heart, and aorta were removed from Dmp1 null mice and ex-vivo functional tests were simultaneously performed in collaboration by three different laboratories. Dmp1 null EDL and SOL muscles produced less force than wildtype muscles after normalization for physiological cross sectional area of the muscles. Both EDL and SOL muscles from Dmp1 null mice also produced less force after the addition of caffeine (which releases calcium from the sarcoplasmic reticulum) which may indicate problems in excitation contraction coupling in these mice. While the body weights of the Dmp1 null were smaller than wildtype, the heart weight to body weight ratio was higher. However, there were no differences in pathological hypertrophic gene expression compared to wildtype and maximal force of contraction was not different indicating that there may not be cardiac pathology under the tested conditions. We did observe a decrease in the rate of force development generated by cardiac muscle in the Dmp1 null which may be related to some of the deficits observed in skeletal muscle. There were no differences observed in aortic contractions induced by PGF2α or 5-HT or in endothelium-mediated acetylcholine-induced relaxations or endothelium-independent sodium nitroprusside-induced relaxations. In summary, these results indicate that there are deficiencies in both fast twitch and slow twitch muscle fiber type contractions in this model of ARHR, while there was less of a phenotype observed in cardiac muscle, and no differences observed in aortic function. These results may help explain skeletal muscle weakness reported by some patients with osteomalacia and need to be further investigated.

Effects of an acute bout of resistance exercise on fiber-type specific GLUT4 and IGF-1R expression

The effects of resistance exercise on fiber-type-specific expression of insulin-like growth factor I receptor (IGF-1R) and glucose transporter 4 (GLUT4) was determined in 6 healthy males. The expression of both genes increased in Type I fibers (p < 0.05), but only GLUT4 increased (p < 0.05) in Type II fibers. These data demonstrates that an acute bout of resistance exercise can up-regulate mechanisms of glucose uptake in slow and fast-twitch fibers, but the IGF signaling axis may not be as effective in fast-twitch fibers.

Intramuscular heating through fluidotherapy and heat shock protein response.

CONTEXT Therapeutic modalities that can increase intramuscular temperature commonly are used to treat injuries in the clinical setting. Researchers recently have suggested that the physiologic changes occurring during an increase in temperature also could provide a cytoprotective effect for exercise-induced muscle damage. OBJECTIVE(S) To determine if the Fluidotherapy treatment increases the inducible expression of heat shock protein (HSP), to identify the rate of heating that occurs in the lower extremity with Fluidotherapy treatment, and to evaluate the relationship between the inducible expression of HSP and temperature. DESIGN Controlled laboratory study. SETTING Laboratory. PATIENTS OR OTHER PARTICIPANTS Six male (age = 21.67 ± 1.63 years, height = 180.09 ± 4.83 cm, mass = 87.60 ± 10.51 kg) and 6 female (age = 24.60 ± 4.59 years, height = 151.05 ± 35.76 cm, mass = 55.59 ± 14.58 kg) college-aged students. INTERVENTION(S) One lower extremity was randomly selected to receive the heat treatment, and the other extremity received no treatment. MAIN OUTCOME MEASURE(S) We measured intramuscular temperature every 10 minutes, determining peak intramuscular temperature by 2 identical sequential measurements, and we analyzed the time to peak temperature. We analyzed the amount of HSP70 expression and HSP27P:T (ratio of HSP27 to the total HSP27 expression) in the gastrocnemius and soleus muscles and measured baseline skinfold thickness and estradiol levels. RESULTS Fluidotherapy increased intramuscular temperature by 5.66 ± 0.78°C (t11 = 25.67, P < .001) compared with baseline temperature, with a peak temperature of 39.08°C ± 0.39°C occurring at 84.17 ± 6.69 minutes. We did not find a heat treatment effect for HSP70 or HSP27P:T in the gastrocnemius or soleus muscles (P > .05). Peak temperature and the percentage change of HSP70 were positively correlated for the gastrocnemius and soleus muscles (P < .05). We found no other correlations for skinfold thickness, sex, or estradiol levels (P > .05). No effect of sex for skinfold thickness or estradiol levels at baseline was discovered (P > .05). CONCLUSIONS This Fluidotherapy protocol increased the intramuscular temperature to a therapeutic level; however, it did not stimulate inducible HSP70 or HSP27P:T in the soleus and gastrocnemius muscles regardless of sex or skinfold thickness. These data confirmed that Fluidotherapy is an effective heating modality but suggested it is not an effective method for stimulating an HSP response in the lower limb.