BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo . METHODS: Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively. RESULTS: The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators. CONCLUSION I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.
Animals ; Mice ; Calcium/metabolism* ; Cysteine Endopeptidases/metabolism* ; Myocardial Reperfusion Injury/metabolism* ; Myocardium/metabolism* ; Myocytes, Cardiac/metabolism* ; Proteins/metabolism* ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics*

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis.
Adipose Tissue, Brown ; Adipose Tissue, White ; Basal Metabolism ; Diabetes Mellitus ; Energy Intake ; Energy Metabolism* ; Hand ; Humans ; Metabolism ; Motor Activity ; Muscle, Skeletal* ; Obesity ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; Thermogenesis* ; Weight Gain

OBJECTIVETo compare the effect of Shen-Fu Injection (SFI) and epinephrine on the expression of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) in a pig model with post-resuscitation myocardial dysfunction.

METHODSVentricular fibrillation (VF) was electrically induced in Wu-zhi-shan miniature pigs. After 8 min of untreated VF and 2 min of cardiopulmonary resuscitation (CPR), all animals were randomly administered a bolus injection of saline placebo (SA group, n=10), SFI (0.8 mg/kg, SFI group, n=10) or epinephrine (20 μg/kg, EPI group, n=10). After 4 min of CPR, a 100-J shock was delivered. If the defibrillation attempt failed to attain restoration of spontaneous circulation (ROSC), manual chest compressions were rapidly resumed for a further 2 min followed by a second defibrillation attempt. Hemodynamic variables were recorded, and plasma concentrations of catecholamines were measured. Adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP) and the expressions of β1-adrenoceptor (AR) and SERCA 2a were determined.

RESULTSCardiac output, left ventricular dp/dtmax and negative dp/dtmax were significantly higher in the SFI group than in the SA and EPI groups at 4 and 6 h after ROSC. The expression of β1-AR and SERCA2a at 24 h after ROSC were significantly higher in the SFI group than in the SA and EPI groups (P<0.05 or P<0.01).

CONCLUSIONSThe administration of epinephrine during CPR decreased the expression of SERCA2a and aggravated postresuscitation myocardial function (P<0.01). SFI attenuated post-resuscitation myocardial dysfunction, and the mechanism might be related to the up-regulation of SERCA2a expression.

Adenylyl Cyclases ; metabolism ; Animals ; Blotting, Western ; Cardiac Output ; drug effects ; Cardiopulmonary Resuscitation ; Cyclic AMP ; metabolism ; Dopamine ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Epinephrine ; blood ; Heart Ventricles ; drug effects ; metabolism ; physiopathology ; Hemodynamics ; drug effects ; Injections ; Male ; Myocardium ; enzymology ; pathology ; Norepinephrine ; blood ; Receptors, Adrenergic, beta-1 ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Swine ; Swine, Miniature ; Up-Regulation ; drug effects

OBJECTIVEThe explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.

METHODSThirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.

RESULTSThe half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.

CONCLUSIONIn the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.

Animals ; Calcium ; metabolism ; Cecum ; Diaphragm ; drug effects ; metabolism ; Endoplasmic Reticulum ; metabolism ; Ligation ; Male ; Muscle Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sepsis

OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on contraction capacity of diaphragm in type 1 diabetic rats.
 METHODS: Thirty-two male SD rats were randomly divided into a normal group (NC), a diabetic group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8). Intraperitoneal injection of streptozotocin was utilized to establish diabetic rat model. After the modeling, the rats in the DM+NaHS and the NaHS groups were intraperitoneally injected with 28 μmol/kg NaHS solution. 8 weeks later, the diaphragm contractility was assessed by isolated draphragm strips perfusion. The peak twitch tension (Pt), maximum tetanic tension (Po) and maximal rates of contraction/relaxation (±dT/dtmax) were determined. The alterations in diaphragm ultrastructure were observed under electron microscopy. The diaphragm weight/body weight (DW/BW) was measured. The activities of succinic dehydrogenase (SDH), lactate dehydrogenase (LDH) and sarcoplasmic reticulum Ca2+ ATPase (SERCA) were analyzed by spectrophotometric method. The mRNA levels of SERCA and prospholamban (PLB) in diaphragm were detected by RT-PCR.
 RESULTS: Compared with the NC group, there was no significant change in all measured index in the NaHS group (P>0.05), while Pt, Po and ±dT/dtmax were significantly decreased in the DM group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the diaphragm. The DW/BW ratio and the activities of SDH, LDH and SERCA were decreased. The SERCA mRNA was decreased, while PLB mRNA was increased. Compared with the DM group, the diaphragm contractility and ultrastructure damage were improved in the DM+NaHS group. The DW/BW ratio and the activities of SDH, LDH and SERCA were increased. The SERCA mRNA was increased, while PLB mRNA was decreased (all P<0.05).
 CONCLUSION H(2)S can enhance the contraction capacity of diaphragm in type 1 diabetic rats, which is involved in regulating the activities of biological enzymes and the gene expressions of calcium regulatory proteins.
Animals ; Body Weight ; Diabetes Mellitus, Experimental ; physiopathology ; Diaphragm ; drug effects ; ultrastructure ; Hydrogen Sulfide ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Male ; Muscle Contraction ; drug effects ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Succinate Dehydrogenase ; metabolism ; Sulfides ; pharmacology

OBJECTIVETo observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats.

METHODSThirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues.

RESULTSCompared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure.

CONCLUSIONThese results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Animals ; Calcium-Binding Proteins ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; Diabetic Cardiomyopathies ; metabolism ; physiopathology ; Disease Progression ; Heart ; physiopathology ; Male ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Streptozocin

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca(2+)-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca(2+) ([Ca(2+)]i) via Ca(2+) influx, Ca(2+) mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca(2+) via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca(2+)]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca(2+) concentration in the ER, a compensatory up-regulation of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and a reduction in depolarization-evoked Ca(2+) influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.
Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Membrane Permeability ; drug effects ; Cells, Cultured ; Down-Regulation ; drug effects ; Endoplasmic Reticulum ; metabolism ; Glucose ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Potassium Chloride ; pharmacology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Thapsigargin ; pharmacology ; Up-Regulation ; drug effects

OBJECTIVETo study the protection of stress protein calreticulin (CRT) in rat skeletal muscle during the adaptation mechanism of calmodulin in the course of heat treatment.

METHODSIncreased heat treatment program would be applied, 40 SD rats were randomly divided into the quiet control group C (n = 8) and heat-treated group H (n = 32), then the heat treatment group would be divided into immediately group (H1), 24-hour post-heat treatment group (H2), 48 -hour post-heat treatment group (H3) and six days post-heat treatment group (H4) (n = 8).

RESULTSAfter heat treatment, the Ca(2+)-ATP activity in rat skeletal muscle sarcoplasmic reticulum in H2 group reached the highest value compared with that in the quiet control group C (P < 0.01), and the value in H1 group showed significant differences compared with control group C (P < 0.05); The Ca(2+)-ATP activity in mitochondrial had the highest value in H1 group, compared with the quiet control group C (P < 0.05), while the Ca2+ concentration in rat skeletal muscle sarcoplasmic reticulum had the highest in group H2, followed by H1 group, both showing significant difference compared with the quiet control group (P < 0.05); The Ca2+ concentration in mitochondrial was high in H1 and H2 group than that of the quiet control group C, and the value in H3 and H4 group was lower than that of the quiet control group C, which had no difference; After heat treatment, the expression of stress proteins of CRT from H1, H2 and H3 group in rat skeletal muscle increased significantly compared with quiet group C.

CONCLUSIONIn the process of increased heat treatment, calreticulin played the regulatory role on the imbalance of calcium homeostasis in skeletal muscle cells, and the adaptation protection from the thermal stimulation could have the very good effect on muscle.

Adaptation, Physiological ; Animals ; Calcium ; metabolism ; Calreticulin ; physiology ; Heat Stress Disorders ; metabolism ; physiopathology ; Male ; Mitochondria ; metabolism ; Muscle, Skeletal ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

OBJECTIVETo investigate the molecular mechanisms of diaphragm injury in rats with liver cirrhosis.

METHODSThirty adult male Sprague-Dawley rats were randomized into control group (n=10) and carbon tetrachloride-induced liver cirrhosis group (LC group, n=20). In the 9th week, the rat body weight and diaphragm to body weight ratio were measured, and the parameters of diaphragm contractility including peak twitch tension (Pt), maximum tetanic tension (Po), time to peak contraction (CT), half relaxation time (1/2RT), and force-frequency curve were assessed using a Medlab-U/4C biological signal collecting system. The activities of superoxide dismutase (SOD), succinic dehydrogenase (SDH) and myeloperoxidase (MPO) and malondiadehyde (MDA) content in the diaphragm were detected. The mRNA expression levels of sarcoplasmic reticulum calcium ATPase (SERCA) and cytoskeletal proteins (titin and nebulin) in the diaphragm were detected by RT-PCR, and the diaphragm ultrastructure was examined with electron microscopy.

RESULTSCompared with those in the control group, body weight, diaphragm to body weight ratio, Pt, Po, and tetanic force under the stimulus frequency of 10, 20, 40, 60, 100 Hz were all significantly decreased (P<0.01), while CT and 1/2RT were significantly prolonged in LC group (P<0.01). SOD and SDH activities were significantly lowered (P<0.01) while the contents of MDA and MPO activity were significantly increased in LC group (P<0.01) with significantly decreased SERCA, titin and nebulin mRNA expressions in the diaphragm (P<0.01). Electron microscopy of the diaphragm in LC group revealed myofibrillar degeneration, absence of the Z line, and mitochondria swelling and edema.

CONCLUSIONLiver cirrhosis increases free radicals and aggravates inflammatory response and lipid peroxidation in the diaphragm, thus leading to mitochondrial damages and decreased expressions of cytoskeletal proteins and SERCA to cause diaphragmatic dysfunction.

Animals ; Body Weight ; Carbon Tetrachloride ; Connectin ; metabolism ; Diaphragm ; metabolism ; Lipid Peroxidation ; Liver ; enzymology ; pathology ; Liver Cirrhosis ; metabolism ; Male ; Muscle Contraction ; Muscle Proteins ; metabolism ; Oxidation-Reduction ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high-frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus (EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase (nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2+ concentration ([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K+ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain (MHC) isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.
Animals ; Calcium Signaling ; Calpain ; metabolism ; Desmin ; metabolism ; Muscle Contraction ; Muscle Fatigue ; Muscle, Skeletal ; physiopathology ; Muscular Atrophy ; physiopathology ; Myosin Heavy Chains ; metabolism ; Rats ; Sarcoplasmic Reticulum ; pathology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Weightlessness Simulation