ABSTRACT
BACKGROUND: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. METHODS: This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RESULTS: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. CONCLUSIONS: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.
Subject(s)
Calcium/metabolism , Disease Models, Animal , Malignant Hyperthermia/metabolism , TRPC Cation Channels/metabolism , TRPC6 Cation Channel/metabolism , Animals , Female , Homeostasis/drug effects , Homeostasis/physiology , Indans/pharmacology , Male , Malignant Hyperthermia/genetics , Malignant Hyperthermia/pathology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Ryanodine Receptor Calcium Release Channel/genetics , TRPC Cation Channels/antagonists & inhibitors , TRPC Cation Channels/genetics , TRPC6 Cation Channel/antagonists & inhibitors , TRPC6 Cation Channel/geneticsABSTRACT
Mutations in the ryanodine receptor 1 (RYR1) gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive RYR1 myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic versus mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.
Subject(s)
Gene Expression Regulation , Muscle Fibers, Slow-Twitch/metabolism , Muscle Strength , Mutation, Missense , Ryanodine Receptor Calcium Release Channel/biosynthesis , Amino Acid Substitution , Animals , Male , Mice , Mice, Mutant Strains , Ryanodine Receptor Calcium Release Channel/geneticsABSTRACT
AIMS: Atrial remodeling, including structural and electrical remodeling, is considered as the substrate in the development of atrial fibrillation (AF). Structural remodeling mainly involves atrial fibrosis, and electrical remodeling is closely related to the changes of ion channels in atrial myocytes. In this study, we aimed to investigate the changes of ion channels in atrial remodeling induced by CIH in rats, which provide the explication for the mechanisms of AF. MATERIALS AND METHODS: 80 male Sprague-Dawley rats were randomized into two groups: Control and CIH group (n = 40). CIH rats were subjected to CIH 8 h/d for 30 days. Atrial epicardial conduction velocity, conduction inhomogeneity and AF inducibility were examined. Masson's trichrome staining was used to evaluate the extent of atrial fibrosis, and the expression levels of ion channel subunits were measured by RT-qPCR, Western blot, and IHC, respectively. The remaining 40 rats were used for whole-cell patch clamp experiments. Action potential, INa, ICa-L, Ito were recorded and compared between two groups. KEY FINDINGS: CIH rats showed increased AF inducibility, atrial interstitial collagen deposition, APD, expression levels of RyR2, p-RyR2, CaMKII, p-CaMKII, and decreased atrial epicardial conduction velocity, expression levels of Nav1.5, Cav1.2, Kv1.5, Kv4.2, Kv4.3 compared to the Control rats, and the current density of INa, ICa-L, Ito were significantly decreased in CIH group. SIGNIFICANCE: We observed significant atrial remodeling induced by CIH in our rat model, which was characterized by changes in ion channels. These changes may be the mechanisms of CIH promoting AF.
Subject(s)
Atrial Remodeling/physiology , Hypoxia/physiopathology , Ion Channels/physiology , Action Potentials/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/biosynthesis , Chronic Disease , Fibrosis/complications , Fibrosis/pathology , Heart Atria/metabolism , Heart Atria/pathology , Heart Atria/physiopathology , Hypoxia/complications , Hypoxia/metabolism , Ion Channels/biosynthesis , Male , Membrane Potentials/physiology , Rats , Ryanodine Receptor Calcium Release Channel/biosynthesisABSTRACT
The skeletal muscle ryanodine receptor (RyR1), i.e., the Ca2+ channel of the sarco/endoplasmic reticulum (S/ER), and the voltage-dependent calcium channel Cav1.1 are the principal channels involved in excitation-contraction coupling in skeletal muscle. RYR1 gene variants are linked to distinct skeletal muscle disorders, including malignant hyperthermia susceptibility and central core disease (CCD), mainly with autosomal dominant inheritance, and autosomal recessive myopathies with a broad phenotypic and histopathological spectrum. The age at onset of RYR1-related myopathies varies from infancy to adulthood. We report the identification of four RYR1 variants in two Italian families: one with myopathy and variants c.4003C>T (p.R1335C) and c.7035C>A (p.S2345R), and another with CCD and variants c.9293G>T (p.S3098I) and c.14771_14772insTAGACAGGGTGTTGCTCTGTTGCCCTTCTT (p.F4924_V4925insRQGVALLPFF). We demonstrate that, in patient-specific lymphoblastoid cells, the c.4003C>T (p.R1335C) variant is not expressed and the in-frame 30-nucleotide insertion variant is expressed at a low level. Moreover, Ca2+ release in response to the RyR1 agonist 4-chloro-m-cresol and to thapsigargin showed that the c.7035C>A (p.S2345R) variant causes depletion of S/ER Ca2+ stores and that the compound heterozygosity for variant c.9293G>T (p.S3098I) and the 30-nucleotide insertion increases RyR1-dependent Ca2+ release without affecting ER Ca2+ stores. In conclusion, we detected and functionally characterized disease-causing variants of the RyR1 channel in patient-specific lymphoblastoid cells. This paper is dedicated to the memory and contribution of Luigi Del Vecchio.
Subject(s)
Family , Gene Expression Regulation , Genetic Variation , Malignant Hyperthermia , Muscle, Skeletal , Myopathy, Central Core , Ryanodine Receptor Calcium Release Channel , Adult , Child, Preschool , Female , Humans , Italy , Male , Malignant Hyperthermia/genetics , Malignant Hyperthermia/metabolism , Malignant Hyperthermia/pathology , Middle Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myopathy, Central Core/genetics , Myopathy, Central Core/metabolism , Myopathy, Central Core/pathology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Ryanodine Receptor Calcium Release Channel/geneticsABSTRACT
INTRODUCTION: Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. METHODS: We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. RESULTS: Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca2+ ] were significantly decreased in the rat sepsis model compared with controls. DISCUSSION: Decreased intracellular [Ca2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017.
Subject(s)
Calcium Channels, L-Type/biosynthesis , Calcium/metabolism , Diaphragm/metabolism , Intracellular Fluid/metabolism , Ryanodine Receptor Calcium Release Channel/biosynthesis , Sepsis/metabolism , Animals , Calcium Channels, L-Type/genetics , Diaphragm/physiopathology , Gene Expression , Male , Muscle Contraction/physiology , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Ryanodine Receptor Calcium Release Channel/genetics , Sepsis/genetics , Sepsis/physiopathologyABSTRACT
Centronuclear myopathies are early-onset muscle diseases caused by mutations in several genes including MTM1, DNM2, BIN1, RYR1 and TTN. The most severe and often fatal X-linked form of myotubular myopathy (XLMTM) is caused by mutations in the gene encoding the ubiquitous lipid phosphatase myotubularin, an enzyme specifically dephosphorylating phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate. Because XLMTM patients have a predominantly muscle-specific phenotype a number of pathogenic mechanisms have been proposed, including a direct effect of the accumulated lipid on the skeletal muscle calcium channel ryanodine receptor 1, a negative effect on the structure of intracellular organelles and defective autophagy. Animal models knocked out for MTM1 show severe reduction of ryanodine receptor 1 mediated calcium release but, since knocking out genes in animal models does not necessarily replicate the human phenotype, we considered it important to study directly the effect of MTM1 mutations on patient muscle cells. The results of the present study show that at the level of myotubes MTM1 mutations do not dramatically affect calcium homeostasis and calcium release mediated through the ryanodine receptor 1, though they do affect myotube size and nuclear content. On the other hand, mature muscles such as those obtained from patient muscle biopsies exhibit a significant decrease in expression of the ryanodine receptor 1, a decrease in muscle-specific microRNAs and a considerable up-regulation of histone deacetylase-4. We hypothesize that the latter events consequent to the primary genetic mutation, are the cause of the severe decrease in muscle strength that characterizes these patients.
Subject(s)
Histone Deacetylases/genetics , Muscle, Skeletal/metabolism , Myopathies, Structural, Congenital/genetics , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Repressor Proteins/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Animals , Biopsy , Calcium/metabolism , Child , Child, Preschool , Female , Gene Expression Regulation , Histone Deacetylases/biosynthesis , Humans , Infant , Infant, Newborn , Male , Mice , Mice, Knockout , MicroRNAs/genetics , Muscle, Skeletal/pathology , Mutation , Myopathies, Structural, Congenital/metabolism , Myopathies, Structural, Congenital/pathology , Repressor Proteins/biosynthesis , Ryanodine Receptor Calcium Release Channel/biosynthesis , ZebrafishABSTRACT
RNA binding protein is identified as an important mediator of aberrant alternative splicing in muscle atrophy. The altered splicing of calcium channels, such as ryanodine receptors (RyRs), plays an important role in impaired excitation-contraction (E-C) coupling in muscle atrophy; however, the regulatory mechanisms of ryanodine receptor 1 (RyR1) alternative splicing leading to skeletal muscle atrophy remains to be investigated. In this study we demonstrated that CUG binding protein 1 (CUG-BP1) was up-regulated and the alternative splicing of RyR1 ASI (exon70) was aberrant during the process of neurogenic muscle atrophy both in human patients and mouse models. The gain and loss of function experiments in vivo demonstrated that altered splicing pattern of RyR1 ASI was directly mediated by an up-regulated CUG-BP1 function. Furthermore, we found that CUG-BP1 affected the calcium release activity in single myofibers and the extent of atrophy was significantly reduced upon gene silencing of CUG-BP1 in atrophic muscle. These findings improve our understanding of calcium signaling related biological function of CUG-BP1 in muscle atrophy. Thus, we provide an intriguing perspective of involvement of mis-regulated RyR1 splicing in muscular disease.
Subject(s)
Alternative Splicing/genetics , CELF1 Protein/genetics , Calcium Signaling/genetics , Muscle Fibers, Skeletal/metabolism , Muscular Atrophy/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Animals , CELF1 Protein/biosynthesis , CELF1 Protein/metabolism , Calcium/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Muscle Fibers, Skeletal/cytology , Muscular Atrophy/pathology , RNA Interference , RNA, Small Interfering/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ryanodine Receptor Calcium Release Channel/biosynthesis , Sarcoplasmic Reticulum/metabolism , Up-Regulation/geneticsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Rhizome and root of Smilax glabra Roxb (Liliaceae family) is a widely used traditional Chinese medicine (TCM) named Tu-fu-ling (TFL) for cardiac disease therapy. The TFL flavonoids (TFLF) has been extracted and proven to possess the anti-cardiac hypertrophy effect in our previous reports. Such effect could be mediated by the modulation of intracellular Ca(2+) flux in myocardial cells, in which junctophilin-2 (JP2) and ryanodine receptor 2 (RyR2) play an important role. However, its mechanism of the anti-cardiac hypertrophy effect remains unclarified. MATERIALS AND METHODS: 2µmol/L Ang II was applied to induce hypertrophy model of rat primary cardiomyocytes. After treatment of TFLF at 0.25, 0.5 and 1.0mg/ml, the cell size was microscopic measured, and the protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were estimated by immunofluorescence imaging, ELISA and real-time PCR assay. RESULTS: Obvious hypertrophy of cardiomyocytes was induced by Ang II but reversed by TFLF from 0.5 to 1.0mg/ml. The protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were also inhibited by Ang II but restored by TFLF at its dose range. Such effect of TFLF was exerted at a dose dependent manner, which was even better than that of verapamil. CONCLUSIONS: Our findings may evidence the correlation between JP2/RyR2 and myocardiac hypertrophy, and indicate the JP2/RyR2-mediated anti-hypertrophy mechanism of TFLF for the first time. It deserves to be developed as a promising TCM candidate of new drug for myocardial hypertrophy treatment.
Subject(s)
Angiotensin II/adverse effects , Flavonoids/therapeutic use , Hypertrophy/drug therapy , Membrane Proteins/biosynthesis , Myocytes, Cardiac/pathology , Phytotherapy , Ryanodine Receptor Calcium Release Channel/biosynthesis , Smilax/chemistry , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Flavonoids/isolation & purification , Flavonoids/pharmacology , Hypertrophy/chemically induced , Myocytes, Cardiac/drug effects , Plant Roots/chemistry , Primary Cell Culture , Rats , Rhizome/chemistryABSTRACT
Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.
Subject(s)
Epigenesis, Genetic , Histone Deacetylases/biosynthesis , Muscle Weakness/metabolism , Myotonia Congenita/metabolism , Ryanodine Receptor Calcium Release Channel/biosynthesis , Animals , Histone Deacetylases/genetics , Mice , Muscle Weakness/genetics , Muscle Weakness/pathology , Mutation , Myotonia Congenita/genetics , Myotonia Congenita/pathology , Ryanodine Receptor Calcium Release Channel/geneticsSubject(s)
Calcium/physiology , Homeostasis/physiology , Myocytes, Cardiac/physiology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Testosterone/pharmacology , Transforming Growth Factor beta/metabolism , Animals , Dose-Response Relationship, Drug , Gene Expression Regulation , Homeostasis/drug effects , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Testosterone/physiologyABSTRACT
Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca(2+)-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285â bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175â bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval-pupal and pupal-adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.
Subject(s)
Inositol 1,4,5-Trisphosphate Receptors/genetics , Metamorphosis, Biological/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Tribolium/genetics , Animals , Cloning, Molecular , Ecdysone/genetics , Gene Expression Regulation, Developmental , Inositol 1,4,5-Trisphosphate Receptors/biosynthesis , Molecular Sequence Data , Pupa/genetics , RNA Interference , Ryanodine Receptor Calcium Release Channel/biosynthesis , Tribolium/growth & development , Wings, Animal/growth & developmentABSTRACT
Capsaicin, the pungent agent in chili peppers, has been shown to act as a tumor-suppressor in cancer. In our previous study, capsaicin was shown to induce apoptosis in the rat pheochromocytoma cell line (PC12 cells). Thus, the aim of the present study was to determine the potential mechanism by which capsaicin induces apoptosis. We treated PC12 cells with 50, 100 and 500 µM capsaicin and measured the reticular calcium content and expression of the reticular calcium transport systems. These results were correlated with endoplasmic reticulum (ER) stress markers CHOP, ATF4 and X-box binding protein 1 (XBP1), as well as with apoptosis induction. We observed that capsaicin decreased reticular calcium in a concentration-dependent manner. Simultaneously, expression levels of the sarco/endoplasmic reticulum pump and ryanodin receptor of type 2 were modified. These changes were accompanied by increased ER stress, as documented by increased stress markers. Thus, from these results we propose that in PC12 cells capsaicin induces apoptosis through increased ER stress.
Subject(s)
Antipruritics/pharmacology , Apoptosis/physiology , Capsaicin/pharmacology , Endoplasmic Reticulum Stress/physiology , Activating Transcription Factor 4/biosynthesis , Activating Transcription Factor 4/genetics , Animals , Apoptosis/drug effects , Calcium/metabolism , Cell Line, Tumor , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/drug effects , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , PC12 Cells , RNA, Messenger/biosynthesis , Rats , Regulatory Factor X Transcription Factors , Ryanodine Receptor Calcium Release Channel/biosynthesis , Transcription Factor CHOP/biosynthesis , Transcription Factor CHOP/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , X-Box Binding Protein 1ABSTRACT
INTRODUCTION: Sepsis often causes diaphragm contractile dysfunction. Dihydropyridine receptors (DHPRα1s and DHPRα1c) and ryanodine receptors (RyR1, RyR2, and RyR3) are essential for excitation-contraction coupling in striated muscles. However, their expression in diaphragm during sepsis have not been explored. METHODS: Eight rats received endotoxin, and 8 more rats received placebo. After 24 hours, 3) diaphragm isometric contractile force was measured. The mRNA and protein levels of DHPRs and RyRs in diaphragm muscles were determined. RESULTS: Sepsis weakened diaphragm contractile function. The expression levels of DHPRα1s and RyR1 were significantly lower in septic rats than in control rats. The expression levels of DHPRα1c and RyR3 were unaffected by sepsis. RyR2 was undetectable at both mRNA and protein levels in the control and sepsis groups. CONCLUSIONS: Weakened diaphragm contraction in the septic rats was associated with reduced mRNA and protein expression of DHPRα1s and RyR1, the isoforms of skeletal muscles.
Subject(s)
Calcium Channels, L-Type/metabolism , Diaphragm/metabolism , Isometric Contraction/physiology , Muscle, Skeletal/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sepsis/metabolism , Animals , Calcium Channels, L-Type/biosynthesis , Calcium Signaling/drug effects , Calcium Signaling/physiology , Diaphragm/drug effects , Diaphragm/physiopathology , Disease Models, Animal , Down-Regulation/drug effects , Down-Regulation/physiology , Isometric Contraction/drug effects , Male , Muscle, Skeletal/drug effects , Protein Isoforms/drug effects , Protein Isoforms/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , Rats , Ryanodine Receptor Calcium Release Channel/biosynthesis , Sepsis/chemically induced , Sepsis/physiopathology , Shock, Septic/metabolism , Shock, Septic/physiopathologyABSTRACT
Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca(2+) transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca(2+) transients, we investigated Ca(2+) cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca(2+) content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca(2+) sparks and a diminished systolic Ca(2+) release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca(2+) influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca(2+) channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca(2+) influx through LTCC fully compensates for a reduced systolic SR Ca(2+) release to preserve the Ca(2+) transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels.
Subject(s)
Calcium Channels, L-Type/metabolism , Calcium/metabolism , Myocardium/pathology , Action Potentials , Animals , Atrophy/physiopathology , Heart Transplantation , Male , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Rats , Ryanodine Receptor Calcium Release Channel/biosynthesis , Sarcoplasmic Reticulum/metabolism , Transplantation, HeterotopicABSTRACT
BACKGROUND: Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and the authors hypothesized that it has a central role in anesthesia. METHODS: Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca imaging and patch clamp electrophysiology. RESULTS: Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in the mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca flux that is altered in dRyr mutants and correlates with strong hyperpolarization. CONCLUSIONS: In Drosophila, neurally expressed dRyr mediates a substantial proportion of the anesthetic effects of halothane in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. The authors' results provide support for Ryr as an important mediator of immobilization by volatile anesthetics.
Subject(s)
Anesthesia, General , Anesthetics, Inhalation/pharmacology , Halothane/pharmacology , Ryanodine Receptor Calcium Release Channel/physiology , Amino Acid Sequence , Animals , Cell Line , Drosophila melanogaster , Immobilization/methods , Male , Molecular Sequence Data , Point Mutation/drug effects , Point Mutation/physiology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Ryanodine Receptor Calcium Release Channel/geneticsABSTRACT
There are controversies concerning the capacity of Rosuvastatin to attenuate heart failure in end-stage hypertension. The aim of the study was to show whether the Rosuvastatin might be effective or not for the heart failure treatment. Twenty-one spontaneously hypertensive rats (SHRs) aged 52 weeks with heart failure were randomly divided into three groups: two receiving Rosuvastatin at 20 and 40 mg/kg/day, respectively, and the third, placebo for comparison with seven Wistar-Kyoto rats (WKYs) as controls. After an 8-week treatment, the systolic blood pressure (SBP) and echocardiographic features were evaluated; mRNA level of B-type natriuretic peptide (BNP) and plasma NT-proBNP concentration were measured; the heart tissues were observed under electron microscope (EM); myocardial sarcoplasmic reticulum Ca(2+) pump (SERCA-2) activity and mitochondria cytochrome C oxidase (CCO) activity were measured; the expressions of SERCA-2a, phospholamban (PLB), ryanodine receptor2 (RyR2), sodium-calcium exchanger 1 (NCX1), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase inhibitor-1 (PPI-1) were detected by Western blot and RT-qPCR; and the total and phosphorylation of protein kinase Cα/ß (PKCα/ß) were measured. Aged SHRs with heart failure was characterized by significantly decreased left ventricular ejection fraction and left ventricular fraction shortening, enhanced left ventricular end-diastolic diameter and LV Volume, accompanied by increased plasma NT-proBNP and elevated BNP gene expression. Damaged myofibrils, vacuolated mitochondria and swollen sarcoplasmic reticulum were observed by EM. Myocardium mitochondria CCO and SERCA-2 activity decreased. The expressions of PLB and NCX1 increased significantly with up-regulation of PPI-1 and down-regulation of CaMKII, whereas that of RyR2 decreased. Rosuvastatin was found to ameliorate the heart failure in aged SHRs and to improve changes in SERCA-2a, PLB, RyR2, NCX1, CaMKII and PPI-1; PKCα/ß2 signal pathway to be suppressed; the protective effect of Rosuvastatin to be dose dependent. In conclusion, the heart failure of aged SHRs that was developed during the end stage of hypertension could be ameliorated by Rosuvastatin.
Subject(s)
Fluorobenzenes/therapeutic use , Heart Failure/drug therapy , Hypertension/drug therapy , Protein Kinase C beta/metabolism , Protein Kinase C-alpha/metabolism , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Aging , Animals , Blood Pressure/drug effects , Calcium-Binding Proteins/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinase Type 2/biosynthesis , Down-Regulation , Electron Transport Complex IV/metabolism , Male , Mitochondria/metabolism , Mitochondria/pathology , Myofibrils/drug effects , Myofibrils/pathology , Natriuretic Peptide, Brain/blood , Natriuretic Peptide, Brain/genetics , Peptide Fragments/blood , Phosphorylation , Proteins/analysis , RNA, Messenger/biosynthesis , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Rosuvastatin Calcium , Ryanodine Receptor Calcium Release Channel/biosynthesis , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/pathology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Signal Transduction , Sodium-Calcium Exchanger/biosynthesis , Stroke Volume/drug effects , Up-Regulation , Ventricular Function, Left/drug effectsABSTRACT
The ryanodine receptor (RyR) is a large, intracellular calcium (Ca(2+)) channel that is associated with several accessory proteins and is an important component of a cell's ability to respond to changes in the environment. Three isoforms of the RyR exist and are well documented for skeletal and cardiac muscle and the brain, but the isoforms in non-excitable cells are poorly understood. The aggressiveness of breast cancers in women has been positively correlated with the expression of the RyR in breast tumor tissue, but it is unknown if this is limited to specific isoforms. Identification and characterization of RyRs in cancer models is important in understanding the role of the RyR channel complex in cancer and as a potential therapeutic target. The objective of this report was to identify the RyR isoforms expressed in widely used prostate cancer cell lines, DU-145 and LNCaP, and the non-tumorigenic prostate cell line, PWR-1E. Oligonucleotide primers specific for each isoform were used in semi-quantitative and real-time PCR to determine the identification and expression levels of the RyR isoforms. RyR1 was expressed in the highest amount in DU-145 tumor cells, expression was 0.48-fold in the non-tumor cell line PWR-1E compared to DU-145 cells, and no expression was observed in LNCaP tumor cells. DU-145 cells had the lowest expression of RyR2. The expression was 26- and 15-fold higher in LNCaP and PWR-1E cells, respectively. RyR3 expression was not observed in any of the cell lines. All cell types released Ca(2+) in response to caffeine showing they had functional RyRs. Total cellular RyR-associated Ca(2+) release is determined by both the number of activated RyRs and its accessory proteins which modulate the receptor. Our results suggest that the correlation between the expression of the RyR and tumor aggression is not related to specific RyR isoforms, but may be related to the activity and number of receptors.
Subject(s)
Prostate/metabolism , Prostatic Neoplasms/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Calcium/metabolism , Cell Line, Tumor , Humans , Male , Prostatic Neoplasms/pathology , Protein Isoforms/biosynthesis , Protein Isoforms/metabolism , Ryanodine Receptor Calcium Release Channel/biosynthesisABSTRACT
We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.
Subject(s)
Arteries/metabolism , Arterioles/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Myocytes, Smooth Muscle/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Boron Compounds/pharmacology , Calcium/metabolism , Calcium Channels/metabolism , Calcium Signaling/drug effects , Inositol 1,4,5-Trisphosphate Receptors/biosynthesis , Macrocyclic Compounds/pharmacology , Male , Mice , Mice, Inbred C57BL , Muscle Development/physiology , Oxazoles/pharmacology , Potassium Channels, Calcium-Activated/metabolism , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Tetracaine/pharmacology , Type C Phospholipases/metabolism , Vasomotor System/metabolismABSTRACT
Endothelial outgrowth cells (EOCs) derived from blood mononuclear cells can differentiate to an endothelial-like phenotype. There are deficits in understanding of the biology of these cells, particularly detailed characterisation of their Ca(2+) signalling mechanisms. In the current study, it was found that human EOCs express two forms of ryanodine receptor (RyR1 and RyR2) Ca(2+) release channel in their endoplasmic reticulum. Individual EOCs display heterogeneous Ca(2+) responses to physiologically relevant regulators fibrinogen and collagen. Some EOCs showed distinctive, multiphasic Ca(2+) responses to fibrinogen consisting of rapid decreases, transient increases then a gradual return to the resting levels. Transient elevations in Ca(2+) required both L-type voltage gated calcium channels and RyRs. Decreases in Ca(2+) stimulated by fibrinogen depended on plasma membrane Ca(2+) ATPase pumps, but did not require thapsigargin-sensitive Ca(2+) ATPases. These results indicate that EOCs possess sophisticated Ca(2+) signalling mechanisms, capable of generating distinct Ca(2+) waveforms in response to different physiologically relevant cues.
Subject(s)
Calcium Signaling , Calcium/metabolism , Endothelium, Vascular/metabolism , Adult , Calcium Channels, L-Type/biosynthesis , Cells, Cultured , Fibrinogen/metabolism , Humans , Ryanodine Receptor Calcium Release Channel/biosynthesisABSTRACT
Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.