High-intensity interval training using electrical stimulation ameliorates muscle fatigue in chronic kidney disease-related cachexia by restoring mitochondrial respiratory dysfunction.

Background: Exercise, especially high-intensity interval training (HIIT), can increase mitochondrial respiratory capacity and enhance muscular endurance, but its systemic burden makes it difficult to safely and continuously prescribe for patients with chronic kidney disease (CKD)-related cachexia who are in poor general condition. In this study, we examined whether HIIT using electrical stimulation (ES), which does not require whole-body exercise, improves muscle endurance in the skeletal muscle of 5/6 nephrectomized rats, a widely used animal model for CKD-related cachexia. Methods: Male Wistar rats (10 weeks old) were randomly assigned to a group of sham-operated (Sham) rats and a group of 5/6 nephrectomy (Nx) rats. HIIT was performed on plantar flexor muscles in vivo with supramaximal ES every other day for 4 weeks to assess muscle endurance, myosin heavy-chain isoforms, and mitochondrial respiratory function in Nx rats. A single session was also performed to identify upstream signaling pathways altered by HIIT using ES. Results: In the non-trained plantar flexor muscles from Nx rats, the muscle endurance was significantly lower than that in plantar flexor muscles from Sham rats. The proportion of myosin heavy chain IIa/x, mitochondrial content, mitochondrial respiratory capacity, and formation of mitochondrial respiratory supercomplexes in the plantaris muscle were also significantly decreased in the non-trained plantar flexor muscles from Nx rats than compared to those in plantar flexor muscles from Sham rats. Treatment with HIIT using ES for Nx rats significantly improved these molecular and functional changes to the same degrees as those in Sham rats. Furthermore, a single session of HIIT with ES significantly increased the phosphorylation levels of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK), pathways that are essential for mitochondrial activation signaling by exercise, in the plantar muscles of both Nx and Sham rats. Conclusion: The findings suggest that HIIT using ES ameliorates muscle fatigue in Nx rats via restoration of mitochondrial respiratory dysfunction with activation of AMPK and p38 MAPK signaling. Our ES-based HIIT protocol can be performed without placing a burden on the whole body and be a promising intervention that is implemented even in conditions of reduced general performance status such as CKD-related cachexia.

Skeletal muscle endurance declines with impaired mitochondrial respiration and inadequate supply of acetyl-CoA during muscle fatigue in 5/6 nephrectomized rats.

Chronic kidney disease (CKD)-related cachexia increases the risks of reduced physical activity and mortality. However, the physiological phenotype of skeletal muscle fatigue and changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. In the present study, we performed detailed muscle physiological evaluation, analysis of mitochondrial function, and comprehensive analysis of metabolic changes before and after muscle fatigue in a 5/6 nephrectomized rat model of CKD. Wistar rats were randomized to a sham-operation (Sham) group that served as a control group or a 5/6 nephrectomy (Nx) group. Eight weeks after the operation, in situ torque and force measurements in plantar flexor muscles in Nx rats using electrical stimulation revealed a significant decrease in muscle endurance during subacute phase related to mitochondrial function. Muscle mass was reduced without changes in the proportions of fiber type-specific myosin heavy chain isoforms in Nx rats. Pyruvate-malate-driven state 3 respiration in isolated mitochondria was impaired in Nx rats. Protein expression levels of mitochondrial respiratory chain complexes III and V were decreased in Nx rats. Metabolome analysis revealed that the increased supply of acetyl CoA in response to fatigue was blunted in Nx rats. These findings suggest that CKD deteriorates skeletal muscle endurance in association with mitochondrial dysfunction and inadequate supply of acetyl-CoA during muscle fatigue.NEW & NOTEWORTHY Mitochondrial dysfunction is associated with decreased skeletal muscle endurance in chronic kidney disease (CKD), but the muscle physiological phenotype and major changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. By using a 5/6 nephrectomized CKD rat model, the present study revealed that CKD is associated with reduced tetanic force in response to repetitive stimuli in a subacute phase, impaired mitochondrial respiration, and inadequate supply of acetyl-CoA during muscle fatigue.

Role of Erythropoiesis-Stimulating Agents in Cardiovascular Protection in CKD Patients: Reappraisal of Their Impact and Mechanisms.

Erythropoiesis-stimulating agents (ESAs) have markedly reduced the need for blood transfusion for renal anemia and are included in standard therapies for patients with chronic kidney disease (CKD). Various protective effects of ESAs on the cardiovascular system have been discovered through basic research, and the effects have received much attention because the rates of cardiovascular events and mortality are high in CKD patients. However, randomized clinical trials did not provide strong evidence that ESAs exert cardioprotection in humans, including CKD patients. It is difficult to assess the cardioprotective effects of ESAs in CKD patients through the clinical data that has been reported to date because the relationship between hemoglobin level rather than ESA dose and cardiovascular event rates was examined in most studies. Interestingly, recent studies using a rat model of CKD showed that the infarct size-limiting effect of an ESA was lost when its dose was increased to a level that normalized blood hemoglobin levels, suggesting that the optimal dose of an ESA for myocardial protection is less than the dose required to normalize hemoglobin levels. Furthermore, animal models of traditional coronary risk factors or comorbidities were resistant to the cardioprotective effects of ESAs because of interruptions in signal-mediated mechanisms downstream of erythropoietin receptors. In this review, we briefly discuss basic and clinical data on the impact of anemia on coronary and systemic circulation, the effects of CKD on the cardiovascular system, and the multiple pharmacological actions of ESAs to examine whether the ESAs that are prescribed for renal anemia exert any cardioprotection in patients with CKD.

Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation.

The initiation of heartbeat is an essential step in cardiogenesis in the heart primordium, but it remains unclear how intracellular metabolism responds to increased energy demands after heartbeat initiation. In this study, embryos in Wistar rats at embryonic day 10, at which heartbeat begins in rats, were divided into two groups by the heart primordium before and after heartbeat initiation and their metabolic characteristics were assessed. Metabolome analysis revealed that increased levels of ATP, a main product of glucose catabolism, and reduced glutathione, a by-product of the pentose phosphate pathway, were the major determinants in the heart primordium after heartbeat initiation. Glycolytic capacity and ATP synthesis-linked mitochondrial respiration were significantly increased, but subunits in complexes of mitochondrial oxidative phosphorylation were not upregulated in the heart primordium after heartbeat initiation. Hypoxia-inducible factor (HIF)-1α was activated and a glucose transporter and rate-limiting enzymes of the glycolytic and pentose phosphate pathways, which are HIF-1α-downstream targets, were upregulated in the heart primordium after heartbeat initiation. These results suggest that the HIF-1α-mediated enhancement of glycolysis with activation of the pentose phosphate pathway, potentially leading to antioxidant defense and nucleotide biosynthesis, covers the increased energy demand in the beating and developing heart primordium.

Requirement of the Ca2+ channel ß2 subunit for sympathetic PKA phosphorylation.

Facilitation of cardiac function in response to signals from the sympathetic nervous system is initiated by the phosphorylation of L-type voltage-dependent Ca2+ channels (VDCCs) by protein kinase A (PKA), which in turn is activated by ß-adrenoceptors. Among the five subunits (α1, ß, α2/δ, and γ) of VDCCs, the α1 subunit and the family of ß subunits are substrates for PKA-catalyzed phosphorylation; however, the subunit responsible for ß-adrenergic augmentation of Ca2+ channel function has yet to be specifically identified. Here we show that the VDCC ß2 subunit is required for PKA phosphorylation upon sympathetic acceleration. In mice with ß2 subunit-null mutations, cardiac muscle contraction in response to isoproterenol was reduced and there was no significant increase in Ca2+ channel currents upon PKA-dependent phosphorylation. These findings indicate that within the sympathetic nervous system the ß2 subunit of VDCCs is required for physiological PKA-dependent channel phosphorylation.

Suppression of Sympathetic Nerve Sprouting by Local Administration of an α-antagonist Around the Dorsal Root Ganglion in a Lumbar Radiculopathy Model.

STUDY DESIGN: Animal experimental study with intervention. OBJECTIVE: The purpose of this study was to elucidate whether local administration of an α-antagonist around the dorsal root ganglion (DRG) suppressed sympathetic nerve sprouting, from the acute to the chronic pain development phase, in a lumbar radiculopathy model using immunohistochemical methods. SUMMARY OF BACKGROUND DATA: The abnormal sympathetic-somatosensory interaction may underlie some forms of neuropathic pain. There were several reports suggesting α-antagonists are effective to treat neuropathic pain. However, its pathophysiological mechanisms remain obscure. METHODS: We used 70 male Sprague-Dawley rats. After root constriction (RC), rats received a series of three local injections of the nonselective α-antagonist phentolamine around the DRG for 3 days. There were three groups of rats: those that were injected from the day of surgery and those injected from day 4 and third group injected from day 11. The control rats were subjected to RC but equal-volume normal saline injections, and the naïve rats were not subjected to any surgical procedures. At the 14th postoperative day, the left L5 DRG was removed, embedded in paraffin, and sectioned. Sections were then immunostained with antibodies to tyrosine hydroxylase (TH). To quantify the extent of the presence of sympathetic nerve fibers, we counted TH-immunoreactive fibers in the DRG using a light microscope equipped with a micrometer graticule. We counted the squares of the graticule, which contained TH-immunoreactive fibers for each of five randomly selected sections of the DRG. RESULTS: In the naïve group, TH-immunoreactive fibers were scarce in the DRG. α-antagonist injections from postoperative day 0 and 4 suppressed sympathetic nerve sprouting compared with the control group. α-antagonist injections from postoperative day 11 had no suppressant effect compared with the control group. CONCLUSION: The α-antagonist administered around the DRG could suppress neural plastic changes in the early phase after nerve injury. LEVEL OF EVIDENCE: N/A.

Does norepinephrine influence pain behavior mediated by dorsal root ganglia?: a pilot study.

BACKGROUND: Postganglionic neurons in the sympathetic nervous system reportedly are involved in lumbar radicular pain and release norepinephrine (NE), a neurotransmitter. Increased numbers of sympathetic nerve fibers have been found in dorsal root ganglion (DRG) neurons in a root constriction model. Whether this is a reasonable model for pain, however, is unclear QUESTIONS/PURPOSES: We asked whether: (1) painful behaviors occurred in the root constriction model; (2) NE enhanced the excitability of DRG neurons in the root constriction model; and (3) which adrenoceptors were related to the mediation of the NE effects. METHODS: The L5 root was sutured proximal to the DRG as the root constriction model. Behavioral tests were performed until 28 days after surgery. At 10 to 14 days after the root constriction, DRG neurons were quickly excised and digested with collagenase for electrophysiologic studies. Action potentials were recorded from single DRG neurons using a whole-cell patch clamp technique. NE (10 µmol/L) was directly applied to the DRG neurons. The adrenergic sensitivity was examined in combination with antagonists. RESULTS: The rats with root constriction exhibited painful behavior. NE increased the excitability of DRG neurons in the root constriction model. The effects of NE were inhibited by pretreatment with an α-antagonist and α(2)-antagonist but not an α(1)-antagonist. CONCLUSIONS: Our observations suggest NE plays an important role in generating lumbar radicular pain mainly via α(2)-adrenoceptors. CLINICAL RELEVANCE: An α(2)-antagonist may be an appropriate agent for trials to treat lumbar radicular pain.

Modulation of phenotype of human prostatic stromal cells by transforming growth factor-betas.

BACKGROUND: We investigated the effects of transforming growth factor (TGF)-betas on morphological and receptor phenotypes, as well as proliferation of four currently established human prostatic myofibroblast cell lines and one commercially available prostatic stromal cell line. METHODS: The effects of TGF-betas on morphological changes and proliferation of the cells were studied by immunohistochemistry and bromodeoxyuridine assay, respectively. The expression of alpha 1-receptor subtypes was measured by real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the radioligand binding assay for the receptors was also performed. RESULTS: TGF-betas 1, 2, and 3 induced expression of desmin and myosin of cells of the established cell lines, and significantly inhibited their growth. The alpha 1a-receptor was expressed only in the commercially available cell line and alpha 1b and 1d, in all cell lines. TGF-beta 1 suppressed the expression of all three subtypes of the alpha 1-receptor. The binding sites of cells of all the cell lines were reduced by treatment with this growth factor. CONCLUSIONS: TGF-betas may induce human prostatic stromal cells to express the smooth muscle phenotype and inhibited their growth. However, the growth factor reduced the binding sites of the receptor and suppressed mRNA expression of its subtypes, suggesting that morphological and receptor phenotypes may be regulated via more than one pathway by TGF-beta(s).

Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes.

The Ca(2+)-independent, voltage-gated transient outward current (I(to)) displays a marked increase during development of cardiomyocytes. However, the molecular mechanism remained unclear. In rat adult ventricular myocytes, I(to) can be divided into a fast (I(to,f)) and a slow (I(to,s)) component by recovery process from inactivation. Voltage-gated K(+) channel-interacting proteins 2 (KChIP2) has recently been shown to modify membrane expressions and current densities of I(to,f). Here we examined the developmental change of I(to) and the putative molecular correlates of I(to,f) (Kv4.2 and Kv4.3) and KChIP2 in rat ventricular myocytes. Even in rat embryonic day 12 (E12) myocytes, we detected I(to). However, I(to) in E12 was solely composed of I(to,s). In postnatal day 10 (P10), we recorded much increased I(to) composed of two components (I(to,f) and I(to,s)), and I(to,f) was dominant. Thus, the developmental increase of I(to) from E12 to P10 can be explained by the dramatic appearance of I(to,f). Real-time RT-PCR revealed that Kv4.2 and Kv4.3 mRNA levels were slightly changed. By contrast, KChIP2 mRNA level increased from E12 to P10 by 731-fold. Therefore, the huge increase of KChIP2 expression was likely to be the cause of the great increase of I(to,f). In order to confirm that KChIP2 is crucial to induce I(to,f), we used adenoviral gene transfer technique. When KChIP2 was over-expressed in E12 myocytes, a great amplitude of I(to,f) appeared. Immunocytochemical experiments also demonstrated that KChIP2 enhanced the trafficking of Kv4.2 channels to cell surface. These results indicate that KChIP2 plays an important role in the generation of functional I(to,f) channels during development.