Regulation Of Contractile Proteins Research Articles

BIOLOGICAL ROLE OF MAGNESIUM SALTS AND USE IN MEDICINE

The increased interest in the element magnesium is determined by the achievement of fundamental sciences in identifying its role in the physiological regulation of various organs and systems of the body. The great importance of magnesium ions in the regulation of various functions of the body is determined by its cofactor role in the activity of enzyme systems — primarily energy metabolism, synthesis of biologically active substances, ion transport systems, protein synthesis, regulation of contractile proteins, and the immune system. The most significant physiological and pathophysiological role of magnesium ions is manifested in systemic or local deficiency. As a consequence of the achievement of this fundamental knowledge in the practice of public health in our country and abroad, several dozen magnesium preparations are actively used for prophylactic and therapeutic purposes.

Abstract 14981: Altered Gene Expression in Diabetic Heart Failure Patients

Introduction: Diabetes and heart failure are very closely associated. Diabetes patients have an increased risk of developing heart failure and heart failure patients are at higher risk of developing diabetes. Changes occur in myocardial glucose metabolism in end stage heart failure patients and newer therapies for diabetes such as SGLTi improve heart failure outcomes. Our objective was to identify transcriptional changes associated with diabetes in heart failure patients. Methods: Transcriptional profiles were compared in heart tissues obtained at the time of LVAD implant among diabetic heart failure (HF) (n=16), non-diabetic (n=10) HF patients and control (n=3) non failing hearts. RNASeq analysis utilized a custom-targeted panel for 140 genes on the Ion Torrent Personal Genome Machine. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser SuiteTM. The Bejamini-Hochberg method was used to control the false discovery rate to be no more than 0.05. Ingenuity Pathway Analysis (IPA) was also performed to understand the biological pathways involved. Results: Our sample population was 82% male with an average age of 51 and 84% Caucasian, 14% African American and 2% Asian. Average ejection fraction was quite low <25% and average NYHA was 3.5 in the HF populations. Expressions of contractile muscle and ion channels proteins decreased significantly between HF patients and control. Increased expression of chemokine ligand 4 (CCL4), chemokine ligand 2 (CCL2) and collagen type 1 alpha1 (COL1A1) were observed in diabetic HF compared to non-diabetic HF patients. IPA analysis with log fold change between HF and control revealed activation of both methylation genes DNMT3A and B, necrosis of cardiac muscle and death of cardiomyocytes with 2.0 Z score. Conclusions: Diabetic heart failure patients have increased expression of chemokines and extracellular matrix genes, which may contribute to disease progression. Up regulation of CCL4 and CCL2 in diabetic HF patients may cause additional risk of heart diseases through inflammatory pathways and increased expression of collagen may contribute to myocardial fibrosis. Down regulation of contractile proteins, and ion channels were similar in diabetic and non-diabetic HF patients.

The study of fetal rat model of intra-amniotic isoproterenol injection induced heart dysfunction and phenotypic switch of contractile proteins.

To establish a reliable isoproterenol induced heart dysfunction fetal rat model and understand the switches of contractile proteins, 45 pregnant rats were divided into 15 mg/kg-once, 15 mg/kg-twice, sham-operated once, sham-operated twice, and control groups. And 18 adult rats were divided into isoproterenol-treated and control groups. H&E staining, Masson staining, and transmission electron microscope were performed. Apoptotic rate assessed by TUNEL analysis and expressions of ANP, BNP, MMP-2, and CTGF of hearts were measured. Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults. Then echocardiography was performed with M-mode view assessment on E18.5 and 6 weeks later, respectively. Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate. ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF. The isoforms transition of troponin I and myosin heavy chain of fetal heart dysfunction were opposite to adult procedure. The administration of intra-amniotic isoproterenol to fetal rats could induce heart dysfunction and the regulation of contractile proteins of fetuses was different from adult procedure.

Regulation of Contractile Proteins and Protein Translational Signaling in Disused Muscle

Background/Aims: Muscle disuse can lead to muscle atrophy and impaired skeletal muscle function. How skeletal muscle modulates protein translational signaling in response to prolonged muscle disuse is not well understood. Using the hindlimb unloading (HU) model of muscle atrophy we examined how hindlimb unweighting affects protein translational signaling, including the activation of Akt/mTOR/p70S6K/S6 signaling and the inhibitory association of 4EBP1 with translation initiation factor eIF4E. Methods: Male F344BN rats were randomized into baseline control, or subjected to HU for 3, 7 or 14 days. Body weight, gastrocnemius muscle, and individual myofiber cross-sectional area were measured to evaluate the degree of muscle atrophy. The amounts of myosin and related muscle contractile proteins were assessed using SDS-PAGE and immunoblotting. Microarray analysis was used to evaluate changes in the mRNA expression of muscle contractile proteins. Total and phosphorylated proteins of Akt/mTOR/p70S6K/S6 pathway were determined via immunoblotting, while the association of 4EBP1 with eIF4E was detected via co-immunoprecipitation. Results: Unloading for 3 days significantly reduced cytosolic myosin content and was associated with increased binding of 4EBP1 to eIF4E, while prolonged unloading (14 days) was associated with the activation of Akt/mTOR/p70S6K/S6 signaling, decreased binding of 4EBP1 to eIF4E, increased cytosolic myosin and elevations in myofibrillar mRNA levels. Conclusion: Taken together, these data suggest that prolonged muscle disuse induces a biphasic translational signaling response that is associated with diminished and then increased muscle contractile protein expression.

Study of O-Glcnacylation of Contractile Proteins in Cardiac Myofibrils by Enzymatic Labelling

Phosphorylation occurs on serine and threonine residues and plays important roles in the regulation of contractile proteins. In heart failure changes in levels of phosphorylation are reported in a number of cardiac sarcomeric proteins. O-linked-N-acetylglucosamine (O-GlcNAc) modification is another possible posttranslational modification on serine and threonine residues and recent publications reported mapping of O-GlcNAc modification sites in some rat contractile proteins, including myosin heavy chain (MHC), actin, cardiac troponin I and myosin light chains (MLC1 and MLC2). O-GlcNAc modification on normal donor hearts (49yr F and unknown) and hypertrophic obstructive cardiomyopathy myectomy samples (33yr M and 42yr M) were studied. Cardiac myofibrils were isolated and the O-GlcNAc groups labelled using an enzymatic labelling system in the presence of PUGNAc (inhibits O-GlcNAc removal enzyme O-GlcNAcase) and protease inhibitors. This method allows coupling of an azido modified N-acetylgalactosamine (UDP-GalNAz) to O-GlcNAc using the mutant enzyme Y289L β1,4-galactosyltransferase (Y289L GalT). The labelled groups were detected by reacting the azide group with an alkyne bearing the tetramethylrhodamine (TAMRA) fluorescent tag for direct imaging following SDS-PAGE. The gel was post-stained with a total protein stain for analysis with densitometry. The labelling process showed no impact on myofibril protein profiles when native and labelled myofibrils were compared. Preliminary results showed that O-GlcNAcylation profiles vary between samples with a total of 7 proteins identified. Strong TAMRA signals from α-actinin and MLC1 were observed in all the four myofibrils samples. In three of the samples the proteins actin, tropomyosin (Tm) and myosin binding protein-C (MyBP-C) were positively labelled. MHC and desmin O-GlcNAcylation were observed in one of the four subjects. This enzymatic labelling method will be investigated further for possibility of quantification and methods for mapping sites of these modifications with mass spectrometry will be explored.

The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta

We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 +/- 5.1% and 22.3 +/- 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.

Regulation of contractile proteins in diabetic heart.

Diabetes is one of the most prevalent chronic conditions that has a high association with death from cardiovascular disease(s). An impaired cardiac function independent of vascular disease suggests the existence of a primary myocardial defect in diabetes mellitus. We and others have documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins could be responsible for the mechanical defects in streptozotocin (STZ)-diabetic hearts. The major focus of research on contractile proteins in the diabetic state has been on myosin ATPase and its isoenzymes. However, in the contractile protein system, this could be only one of the mechanisms that might be a controlling factor in myofilament contraction in diabetes. To define the role of cardiac contractile as well as regulatory proteins (troponin-tropomyosin) as a whole in the regulation of actomyosin system in diabetic cardiomyopathy, individual proteins of the cardiac system were reconstituted under controlled conditions. Enzymatic data confirmed a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1-->V3) could contribute to the impaired cardiac function in the hearts of chronic diabetic rats. It has also been reported that sarcomeric proteins such as myosin light chain-2 (MLC-2) and troponin I (TnI) could be involved in regulating muscle contraction and in calcium sensitivity. Since phosphorylation of cardiac TnI is associated with altered maximum enzymatic activity and calcium force relationship in isolated muscle preparations. TnI phosphorylation could contribute to depressed myocardial contractility in experimental diabetes. While we have yet to understand the exact function of each component in cardiac muscle and their behavior in concert where all of them act in tandem, we have focussed on the role of contractile proteins and their regulation in diabetes in this review. We have also included a brief discussions on other relevant intracellular components. In summary, there is substantial evidence to suggest that there are independent processes associated with diabetes which effect cardiac performance in experimental animals and in man. The focus of this review has been the explication of a biochemical defect which underlies cardiac contractile dysfunction in experimental models of diabetes.

Inhibitory effects of quercetin and staurosporine on phasic contractions in rat vascular smooth muscle

The aim of this work was to analyze the effects of quercetin and staurosporine on the phasic contractile responses in rat aorta induced by noradrenaline, 5-hydroxytryptamine (5-HT, serotonin) and caffeine in Ca 2+-free media. Both quercetin and staurosporine inhibited the contractions induced by 10 −5 M 5-HT and 20 mM caffeine in Ca 2+-free solution. Phorbol 12-myristate 13-acetate (5 × 10 −8 M) enhanced this transient contraction elicited by noradrenaline, an effect that was abolished by quercetin (5 × 10 −5 M). The relaxant effects of quercetin on 80 mM KCl induced contractions were similar in normal and low Na + solution, e.g. when Ca 2+ efflux through the Na +/Ca 2+ exchanger was inhibited. Furthermore, quercetin or staurosporine had no effect on 45Ca 2+ efflux undeer resting conditions or when stimulated by 10 −5 M noradrenaline. These results suggested that the inhibitory effects of quercetin and staurosporine on phasic contractile responses induced by receptor agonists in Ca 2+-free media do not seem to be related to changes in cellular Ca 2+ regulation but to an inhibitory effect on the regulation of contractile proteins, an effect probably related to the decreased sensitivity of contractile elements to Ca 2+ that apparently resulted from the inhibitory effects of quercetin and staurosporine on protein kinases.

Excitation-contraction coupling and uncoupling in airway smooth muscle.

Excitation-contraction coupling and uncoupling in airway smooth muscle.

Regulation of contractile proteins by phosphorylation.

Regulation of contractile proteins by phosphorylation.