Diabetic cardiomyopathy (DCM) is a medical condition characterized by cardiac remodeling and dysfunction in individuals with diabetes mellitus. Sarcoplasmic reticulum (SR) and mitochondrial Ca²⁺ overload in cardiomyocytes have been recognized as biological hallmarks in DCM; however, the specific factors underlying these abnormalities remain largely unknown. In this study, we aimed to investigate the role of a cardiac-specific long noncoding RNA, D830005E20Rik (Trdn-as), in DCM. Our results revealed the remarkably upregulation of Trdn-as in the hearts of the DCM mice and cardiomyocytes treated with high glucose (HG). Knocking down Trdn-as in cardiac tissues significantly improved cardiac dysfunction and remodeling in the DCM mice. Conversely, Trdn-as overexpression resulted in cardiac damage resembling that observed in the DCM mice. At the cellular level, Trdn-as induced Ca²⁺ overload in the SR and mitochondria, leading to mitochondrial dysfunction. RNA-seq and bioinformatics analyses identified calsequestrin 2 (Casq2), a primary calcium-binding protein in the junctional SR, as a potential target of Trdn-as. Further investigations revealed that Trdn-as facilitated the recruitment of METTL14 to the Casq2 mRNA, thereby enhancing the m⁶A modification of Casq2. This modification increased the stability of Casq2 mRNA and subsequently led to increased protein expression. When Casq2 was knocked down, the promoting effects of Trdn-as on Ca²⁺ overload and mitochondrial damage were mitigated. These findings provide valuable insights into the pathogenesis of DCM and suggest Trdn-as as a potential therapeutic target for this condition.
Animals ; Diabetic Cardiomyopathies/pathology* ; RNA, Long Noncoding/genetics* ; Myocytes, Cardiac/metabolism* ; Mice ; Calsequestrin/genetics* ; Calcium/metabolism* ; Male ; Sarcoplasmic Reticulum/metabolism* ; Methyltransferases/metabolism* ; Mice, Inbred C57BL ; Mitochondria, Heart/metabolism* ; Disease Models, Animal ; Mitochondria/metabolism*

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*

Atrial Ca²⁺ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca²⁺ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca²⁺ signals of atria were collected in isolated perfused hearts by optical mapping. Ca²⁺ transient duration (CaD), alternan, and the recovery properties of Ca²⁺ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca²⁺ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca²⁺-RyR pathway, and thus exerts therapeutic effect on POAF.
Administration, Oral ; Animals ; Atrial Fibrillation/etiology* ; Calcium/metabolism* ; Myocytes, Cardiac/metabolism* ; Pericarditis/pathology* ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel/pharmacology* ; Sarcoplasmic Reticulum/pathology* ; TRPV Cation Channels

This study aimed to explore the positive inotropic effect of phosphodiesterase type 9 (PDE9) inhibitor PF-04449613 in ratsand its cellular and molecular mechanisms. The heart pressure-volume loop (P-V loop) analysis was used to detect the effects of PF-04449613 on rat left ventricular pressure-volume relationship, aortic pressures and peripheral vessel resistance in healthy rats. The Langendorff perfusion of isolated rat heart was used to explore the effects of PF-04449613 on heart contractility. The cardiomyocyte sarcoplasmic reticulum (SR) Ca
Animals ; Calcium/metabolism* ; Myocardial Contraction ; Myocytes, Cardiac/metabolism* ; Phosphodiesterase Inhibitors ; Phosphoric Diester Hydrolases ; Rats ; Ryanodine Receptor Calcium Release Channel ; Sarcoplasmic Reticulum

Cardiovascular Diseases ; Heart Failure ; Humans ; Protein Processing, Post-Translational ; Sarcoplasmic Reticulum Calcium-Transporting ATPases

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).
Darier Disease ; genetics ; Heterozygote ; Humans ; Mutation, Missense ; Pedigree ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.
Active Transport, Cell Nucleus ; Calcium Channels, L-Type ; Cell Membrane ; Chloride Channels ; Humans ; Muscle Development ; Muscle Fibers, Skeletal ; Muscle, Skeletal ; Muscular Diseases ; Muscular Dystrophies ; Muscular Dystrophies, Limb-Girdle ; Myoblasts ; RNA, Small Interfering ; Ryanodine Receptor Calcium Release Channel ; Sarcoplasmic Reticulum

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.
Animals ; Calcium ; Cats* ; Egtazic Acid ; Fura-2 ; Methods ; Microscopy, Fluorescence ; Muscle Contraction ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nimodipine ; Pertussis Toxin ; Phospholipases ; Sarcoplasmic Reticulum ; Thapsigargin ; Type C Phospholipases

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

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca²⁺ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca²⁺ level is mainly determined by Ca²⁺ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca²⁺ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca²⁺ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca²⁺ entryway into skeletal muscle. Herein, recent studies on extracellular Ca²⁺ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca²⁺ entry and their influences on skeletal muscle function and disease.
Contractile Proteins ; Cytosol ; Extracellular Space ; Kinetics ; Muscle, Skeletal* ; Posture ; Relaxation ; Sarcoplasmic Reticulum