Calcium Handling In Muscle Research Articles

Multiscale computational modeling of the effects of 2'-deoxy-ATP on cardiac muscle calcium handling.

2'-Deoxy-ATP (dATP), a naturally occurring near analog of ATP, is a well-documented myosin activator that has been shown to increase contractile force, improve pump function, and enhance lusitropy in the heart. Calcium transients in cardiomyocytes with elevated levels of dATP show faster calcium decay compared with cardiomyocytes with basal levels of dATP, but the mechanisms behind this are unknown. Here, we design and utilize a multiscale computational modeling framework to test the hypothesis that dATP acts on the sarcoendoplasmic reticulum calcium-ATPase (SERCA) pump to accelerate calcium re-uptake into the sarcoplasmic reticulum during cardiac relaxation. Gaussian accelerated molecular dynamics simulations of human cardiac SERCA2A in the E1 apo, ATP-bound and dATP-bound states showed that dATP forms more stable contacts in the nucleotide binding pocket of SERCA and leads to increased closure of cytosolic domains. These structural changes ultimately lead to changes in calcium binding, which we assessed using Brownian dynamics simulations. We found that dATP increases calcium association rate constants to SERCA and that dATP binds to apo SERCA more rapidly than ATP. Using a compartmental ordinary differential equation model of human cardiomyocyte excitation-contraction coupling, we found that these increased association rate constants contributed to the accelerated rates of calcium transient decay observed experimentally. This study provides clear mechanistic evidence of enhancements in cardiac SERCA2A pump function due to interactions with dATP.

Exercise, but not statins improve endothelial function in obese rats

IntroductionStatins and exercise training (ET) improve endothelial function. The mechanisms by which statins in combination with ET alter vascular function are largely unknown. We investigated how statins influence the effects of ET on vascular function in obese rats.MethodsEight‐week‐old male Wistar rats (n = 46) received a high‐fat diet for 20 weeks. The rats were randomized after into four groups after eight weeks: sedentary (SED; n =11), exercise (EX; n=11), statin (STAT; n=13) and exercise with statin (EX/STAT; n=11). Simvastatin (10mg/d/kg) was administered in drinking water. The rats exercised for 12 weeks, 5 days/week for 1 h/day at 18 m/min. Endothelium‐dependent (acetylcholine [Ach]) and – independent (sodium‐nitroprusside [SNP]) vascular function was assessed in the abdominal aorta using in vitro myography. Gene expression and protein abundance of nitric‐oxide soluble guanylate cyclase (NO‐sGC) signaling was assessed using q‐PCR and Western blot.ResultsStatin treatment significantly reduced cholesterol levels and did not influence exercise duration or intensity. ET independent of statins improved endothelium dependent vasodilation in EX and EX/STAT compared to SED and STAT, respectively. In untreated rats ET did not alter SNP vasodilation. However, in statin treated animals, ET improved SNP vasodilation. ET and statins increased NO protein abundance by 40% and 80%, respectively, compared to SED. However, in treated rats ET reduced NO to 40% above SED. Protein phosphatase 1 regulatory subunit 14A (CPI17), an inhibitor protein of smooth muscle myosin phosphatase showed a significant statin and exercise interaction at gene expression level (p < .0001). In untreated rats, ET upregulated CPI17 gene expression by 50% (p < .05). In treated rats a downregulation of 35% (p < .05) was observed. The results were similar on the protein level. The myosin phosphatase target subunit 1 (MYPT1) which drives the cGMP independent smooth muscle myosin phosphatase calcium desensitization was increased due to statin treatment (40%) and not affected by ET. Statin reduced sGCα protein content by more than 80%. Protein kinase cGMP‐dependent 1 (PRKG1), which phosphorylates multiple targets implicated in modulating cellular calcium was significantly increased (50%; p < .05) in non‐treated but decreased in treated rats. Protein abundance of the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA), which drives the uptake of calcium into the sarcoplasmic reticulum was reduced by statins (50%) and not rescued by ET.ConclusionET independent of statin treatment improved endothelium‐dependent vasodilation. Interestingly, in untreated animals these improvements were most likely driven by NO, while in animals receiving statins, ET altered vascular smooth muscle calcium handling and desensitization. Overall our results suggests that individuals receiving statins should exercise to maintain vascular health.Support or Funding InformationDZHK (German Centre for Cardiovascular Research)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenine-induced chronic renal failure in rats decreases aortic relaxation rate and alters expression of proteins involved in vascular smooth muscle calcium handling.

Rats with adenine-induced chronic renal failure (A-CRF) develop a reduced rate of relaxation of the thoracic aorta. The aim of this study was to elucidate the mechanisms underlying this abnormality. Male Sprague Dawley rats received either chow containing adenine or were pair-fed with normal chow (controls). After 8-14weeks, arterial function was analysed exvivo using wire myography and the expression of proteins involved in vascular smooth muscle excitation-contraction coupling in the thoracic aorta was analysed. The rate of relaxation following washout of KCl was reduced in A-CRF rats vs. controls in the thoracic aorta (P<0.01), abdominal aorta (P<0.05), and common carotid artery (P<0.05), but not in the common femoral artery. Relaxation rates of thoracic aortas increased (P<0.01), but were not normalized, in response to washout of KCl with Ca2+ -free buffer. Microarray and qRT-PCR analyses of genes involved in excitation-contraction coupling identified 10 genes, which showed significantly altered expression in A-CRF thoracic aortas. At the protein level, the α2 subunit of the Na,K-ATPase (P<0.001) and SERCA2 (P<0.05) was significantly downregulated, whereas stromal interaction molecule 1 and calsequestrin-1 and calsequestrin-2 were significantly upregulated (P<0.05). Rats with A-CRF show a marked alteration in relaxation of larger conduit arteries localized proximal to the common femoral artery. This abnormality may be caused by reduced cytosolic Ca2+ clearance in vascular smooth muscle cells secondary to dysregulation of proteins crucially involved in this process.

One Small Step for Muscle: A New Micropeptide Regulates Performance

Micropeptides represent an emerging class of eukaryotic regulators that are easily missed in conventional genome annotation. Anderson et al. (2015) describe how a new tissue-specific micropeptide, myoregulin (MLN), interacts with the skeletal muscle calcium handling machinery to moderate contractile activity, representing a promising drug target for improving muscle performance.

Resveratrol Attenuates Vasocontractile Responses to Phenylephrine and Potassium Chloride via a Nitric Oxide Synthase– and Endothelium–Independent Mechanism

This study examined the effect of acute pre‐incubation without or with resveratrol (RSV) at increasing in‐bath concentrations (0, 20, 50, or 100 µM) on the dose‐dependent vasocontractile response stimulated by either the α1‐adrenergic receptor agonist phenylephrine (PE; ‐9.0 to ‐5.0 LogM) or potassium chloride (KCl; 0 to 60 mM) in isometrically‐mounted common carotid artery rings isolated from 20‐30 week old Spontaneously Hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). In both SHR and WKY, the contractile dose‐response curve to PE was increasingly blunted by preincubation with 20, 50, and 100 µM RSV, as evidenced by a RSV concentration‐dependent rightward shift of EC50 and decrease in maximum amplitude and area under the curve (p&lt;0.05). The contractile dose‐response curve to KCl was similarly affected by preincubation with increasing concentrations of RSV (p&lt;0.05). Neither nitric oxide synthase (NOS) inhibition nor endothelium removal was found to affect the ability of 50 µM RSV to attenuate either PE‐ or KCl‐stimulated contraction in SHR or WKY. These data indicate that RSV can attenuate vasocontractile activity stimulated by either receptor‐mediated (PE) activation or voltage‐mediated (KCl) activation via a vascular wall cell‐signaling mechanism that is independent of NOS or the endothelium. This suggests that RSV may act to attenuate contraction in the vascular wall by directly affecting central pathways of contractile activation, such as vascular smooth muscle calcium handling and/or sensitivity.Funded by NSERC Canada

Calcium homeostasis in myogenic differentiation factor 1 (MyoD)-transformed, virally-transduced, skin-derived equine myotubes.

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells’ calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.

In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ET(B) and 5-HT(1B) receptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2h MCA occlusion followed by reperfusion for 1 or 24h. Alternatively, MCAs from naïve rats were cultured for 1 or 24h. ET(B) and 5-HT(1B) receptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ET(B) and 5-HT(1B) receptors were strongly upregulated and slightly downregulated, respectively, 24h after experimental stroke or organ culture. ET(B) receptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels.

Nonshivering thermogenesis protects against defective calcium handling in muscle

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca(2+) leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca(2+) release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca(2+) and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.

Effects of calcium on protein turnover of incubated muscles from mdx mice

Mdx mice have a genetic defect similar to that which causes Duchenne muscular dystrophy in humans. The influence of calcium on muscle protein metabolism of mdx and wild type (C57BL/10) mice was examined in vitro. Incubation of mdx muscles in a medium containing calcium at a concentration of 2.0 mM (but not 0.2 mM) resulted in proteolytic rates that were greater than those of C57BL/10 muscles. At 2.0 mM extracellular calcium, mdx muscle proteolysis was attenuated by thiol protease inhibitors but not by the weak base methylamine. Protein synthetic rates were higher in incubated mdx muscles than in incubated C57BL/10 muscles, but no effect of extracellular calcium concentration was observed in either strain. These data suggest that mdx mice have an abnormality of muscle calcium handling, which results in activation of nonlysosomal proteolytic processes but does not exert acute effects on protein synthetic rate.

Leukocyte ionized calcium and sodium content and blood pressure in humans.

The relationship between leukocyte ionized calcium concentration, sodium content, and blood pressure was studied in normotensive subjects with (n = 17) and without (n = 21) a family history of hypertension and in untreated patients with essential hypertension (n = 22). There was a highly significant correlation between intracellular ionized calcium and mean supine blood pressure (measured on the same occasion) in normal subjects with no family history of hypertension (r = +0.73, p less than 0.01). This relationship was lost in normal subjects with a family history of hypertension, and in hypertensive patients there was a nonsignificant negative correlation between intracellular ionized calcium and blood pressure (r = +0.08 and -0.31, respectively). Intracellular ionized calcium was similar in the normotensive groups (both, 126 +/- 7 nmol/L) and slightly but nonsignificantly elevated in hypertensive patients (143 +/- 10 nmol/L; p = 0.09). There was no correlation between intracellular ionized calcium and sodium content in any group (r less than 0.1). These results indicate that while leukocyte ionized calcium in normotensive subjects with no family history of hypertension may reflect smooth muscle contractility resulting in the positive correlation between leukocyte ionized calcium and blood pressure, this relationship is lost in hypertensive patients and subjects predisposed to hypertension. This may be due to an altered relationship between leukocyte and smooth muscle calcium handling in these subjects or to non-calcium-mediated influences on blood pressure.