Sarcoplasmic Reticulum Function Research Articles

Developmental programming of sarcoplasmic-reticulum function improves cardiac anoxia tolerance in turtles.

Oxygen deprivation during embryonic development can permanently remodel the vertebrate heart, often causing cardiovascular abnormalities in adulthood. While this phenomenon is mostly damaging, recent evidence suggests developmental hypoxia produces stress-tolerant phenotypes in some ectothermic vertebrates. Embryonic common snapping turtles (Chelydra serpentina) subjected to chronic hypoxia display improved cardiac anoxia tolerance after hatching, which is associated with altered Ca2+ homeostasis in heart cells (cardiomyocytes). Here we examined the possibility that changes in Ca2+ cycling, through the sarcoplasmic reticulum (SR), underlie the developmentally programmed cardiac phenotype of snapping turtles. We investigated this hypothesis by isolating cardiomyocytes from juvenile turtles that developed in either normoxia (21% O2; "N21") or chronic hypoxia (10% O2; "H10") and subjected the cells to anoxia/reoxygenation, either in the presence or absence of SR Ca2+-cycling inhibitors. We simultaneously measured cellular shortening, intracellular [Ca2+], and intracellular pH (pHi). Under normoxic conditions, N21 and H10 cardiomyocytes shortened equally, but H10 Ca2+ transients (Δ[Ca2+]i) were twofold smaller than N21 cells, and SR inhibition only decreased N21 shortening and Δ[Ca2+]i. Anoxia subsequently depressed shortening, Δ[Ca2+]i, and pHi in control N21 and H10 cardiomyocytes, yet H10 shortening and Δ[Ca2+]i recovered to pre-anoxic levels, partly due to enhanced myofilament Ca2+ sensitivity. SR blockade abolished the recovery of anoxic H10 cardiomyocytes and potentiated decreases in shortening, Δ[Ca2+]i, and pHi. Our novel results provide the first evidence of developmental programming of SR function and demonstrate that developmental hypoxia confers a long-lasting, superior anoxia-tolerant cardiac phenotype in snapping turtles, by enhancing myofilament Ca2+ sensitivity and modifying SR function.

The Effects of Aging on Sarcoplasmic Reticulum-Related Factors in the Skeletal Muscle of Mice.

The pathogenesis of sarcopenia includes the dysfunction of calcium homeostasis associated with the sarcoplasmic reticulum; however, the localization in sarcoplasmic reticulum-related factors and differences by myofiber type remain unclear. Here, we investigated the effects of aging on sarcoplasmic reticulum-related factors in the soleus (slow-twitch) and gastrocnemius (fast-twitch) muscles of 3- and 24-month-old male C57BL/6J mice. There were no notable differences in the skeletal muscle weight of these 3- and 24-month-old mice. The expression of Atp2a1, Atp2a2, Sln, and Pln increased with age in the gastrocnemius muscles, but not in the soleus muscles. Subsequently, immunohistochemical analysis revealed ectopic sarcoplasmic reticulum calcium ion ATPase (SERCA) 1 and SERCA2a immunoreactivity only in the gastrocnemius muscles of old mice. Histochemical and transmission electron microscope analysis identified tubular aggregate (TA), an aggregation of the sarcoplasmic reticulum, in the gastrocnemius muscles of old mice. Dihydropyridine receptor α1, ryanodine receptor 1, junctophilin (JPH) 1, and JPH2, which contribute to sarcoplasmic reticulum function, were also localized in or around the TA. Furthermore, JPH1 and JPH2 co-localized with matrix metalloproteinase (MMP) 2 around the TA. These results suggest that sarcoplasmic reticulum-related factors are localized in or around TAs that occur in fast-twitch muscle with aging, but some of them might be degraded by MMP2.

Novel OBSCN variants associated with a risk to exercise-intolerance and rhabdomyolysis

Obscurin, encoded by the OBSCN gene, is a muscle protein consisting of three main splice isoforms, obscurin-A, obscurin-B, and obscurin kinase-only protein (also known as KIAA1639 or Obsc-kin). Obscurin is located at the M-band and Z-disks and interacts with titin and myomesin. It plays an important role in the stability and maintenance of the A- and M-bands and the subsarcolemmal organization of the microtubule network. Furthermore, obscurin is involved in Ca2+ regulation and sarcoplasmic reticulum function and is connected to several other muscle proteins. OBSCN gene variants have been reported to be relatively common in inherited cardiomyopathies. Here we reported two young patients with a history of cramps, myalgia, exercise intolerance, rhabdomyolysis, and myoglobinuria without any evidence of concomitant cardiomyopathy in association with novel OBSCN variants (c.24822C>A and c.2653+1G>C). Obscurin-deficient muscle fibers seem to have increased susceptibility to damage triggered by exercise that may lead to rhabdomyolysis. More studies are needed to clarify the diverse clinical phenotypes and the pathophysiology of OBSCN gene variants.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 μg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC–MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

S100A1 expression is increased in spinal cord injury and promotes inflammation, oxidative stress and apoptosis of PC12 cells induced by LPS via ERK signaling.

Spinal cord injury (SCI) is a severe neurological disorder and the molecular mechanisms leading to its poor prognosis remain to be elucidated. S100A1, a mediator of Ca2+ handling of sarcoplasmic reticulum and mitochondrial function, operates as an endogenous danger signal (alarmin) associated with inflammatory response and tissue injury. The aim of the present study was to investigate the expression and biological effects of S100A1 in SCI. A rat model of SCI and a PC12 cell model of lipopolysaccharide (LPS)‑induced inflammation were established to examine S100A1 expression at the mRNA and protein levels. The inflammation level, which was mediated by S100A1, was determined based on inflammatory factor (IL‑1β, IL‑6 and TNF‑α) and anti‑inflammatory factor (IL‑10) expression. The effects of S100A1 on cellular oxidation and anti‑oxidation levels were observed by detecting the levels of reactive oxygen species, superoxide dismutase, catalase activities and nuclear factor erythroid 2‑related factor 2 expression. The protein levels of Bax, Bcl2 and cleaved caspase‑3 were used for the evaluation of the effects of S100A1 on apoptosis. Phosphorylated (p‑)ERK1/2 expression was used to evaluate the effects of S100A1 on ERK signaling. The results revealed that S100A1 expression was significantly upregulated invivo and invitro in the PC12 cell model of LPS‑inflammation. The silencing and overexpression of S100A1 helped alleviate and aggravate LPS‑induced inflammation, oxidative stress and apoptosis levels, respectively. S100A1 was found to regulate the ERK signaling pathway positively. An inhibitor of ERK signaling (MK‑8353) partially abolished the promoting effects of the overexpression of S100A1 on inflammation, oxidative stress damage and apoptosis. In conclusion, S100A1 expression was elevated in model of SCI and in the PC12 cell model of LPS‑induced inflammation. Furthermore, the overexpression/silencing S100A1 aggravated/mitigated the inflammation, oxidative stress damage and the apoptosis of LPS‑stimulated PC12 cells via the ERK signaling pathway. The present study revealed the mechanism of S100A1 in SCI, which provided a new theoretic reference for future research on SCI.

Antenatal Glucocorticoid Administration Promotes Cardiac Structure and Energy Metabolism Maturation in Preterm Fetuses.

Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects on other organs remain controversial. Furthermore, the effects of antenatal GC therapy on the fetal heart show both pros and cons. Translational research in animal models indicates that constant fetal exposure to antenatal GC administration is sufficient for lung maturation. We have established a premature fetal rat model to investigate immature cardiopulmonary functions in the lungs and heart, including the effects of antenatal GC administration. In this review, we explain the mechanisms of antenatal GC actions on the heart in the fetus compared to those in the neonate. Antenatal GCs may contribute to premature heart maturation by accelerating cardiomyocyte proliferation, angiogenesis, energy production, and sarcoplasmic reticulum function. Additionally, this review specifically focuses on fetal heart growth with antenatal GC administration in experimental animal models. Moreover, knowledge regarding antenatal GC administration in experimental animal models can be coupled with that from developmental biology, with the potential for the generation of functional cells and tissues that could be used for regenerative medical purposes in the future.

Impaired fatigue resistance, sarcoplasmic reticulum function, and mitochondrial activity in soleus muscle of db/db mice.

Type 2 diabetes mellitus (T2DM) is characterized by reduced exercise tolerance due to increased fatigability in skeletal muscle. In this study, we investigated muscle fatigue resistance of soleus (SOL) muscle in obese type 2 diabetic model mice (db/db). No differences in muscle volume, absolute force, or specific force in SOL muscle were observed between db/db mice and control mice (db/+), while fatigue resistance evaluated by repeated tetanic contractions was significantly lower in db/db mice (30th tetani, db/+: 63.7 ± 4.7%, db/db: 51.3 ± 4.8%). The protein abundance related to Ca2+ release from the sarcoplasmic reticulum (SR) in SOL muscle was not different between db/db mice and db/+ mice, while SR Ca2+‐ATPase (Ca2+ reuptake to SR) protein was decreased in db/db mice compared to db/+ mice (db/+: 1.00 ± 0.17, db/db: 0.60 ± 0.04, relative units). In addition, mitochondrial oxidative enzyme activity (succinate dehydrogenase) was decreased in the SOL muscle of db/db mice (p < 0.05). These data suggest that fatigue resistance in slow‐twitch dominant muscle is impaired in mice with T2DM. Decreased mitochondrial oxidative enzyme activity and impairment of Ca2+ uptake to SR, or both might be involved in the mechanisms.

Contrasting Effects of Age on Muscle Contractility in Male and Female Rats

This study aimed to describe changes in forelimb grip strength and ex vivo muscle force production in adult (Ad) and aged (Ag, 75% lifespan) male (M) and female (F) F344/BN hybrid rats. We hypothesized that differences in M vs. F would be explained by muscle mass, but that age differences would persist after normalizing to mass in fast, but not slow muscles. Forelimb grip was expressed in absolute terms (Grip) and relative to body mass (NGrip). Ex vivo forces of the extensor digitorum longus (EDL) and Soleus (Sol) were assessed at 3 stimulation frequencies and 2 pulse durations, and expressed as absolute muscle force (AMF) and muscle quality (MQ; force/anatomical cross‐sectional area). We also assessed sarcoplasmic reticulum (SR) Ca2+ release and reuptake from homogenates of both muscles. Body mass was higher in Ag vs. Ad and M vs. F (both p &lt; 0.001), while EDL and Sol mass were smaller in F vs. M (p &lt; 0.001), with no effect of age. Grip showed no effect of age, but was significantly (p &lt; 0.001) lower in F while NGrip was higher in Ad vs. Ag (p &lt; 0.001) and F vs. M (p &lt; 0.001). EDL AMF exhibited a significant age X sex interaction (p = 0.006) across frequencies, as AMF declined with age in M, but increased in F. EDL MQ showed a similar pattern (p = 0.039), but only at the highest frequency (120Hz). In Sol, M showed a trend (p = 0.078) for higher AMF relative to F. In contrast, MQ was significantly higher in F vs. M (p = 0.025). Differences in SR function did not appear to contribute to these differences as the only significant effect observed was for SR Ca2+ uptake to be reduced in F vs. M in both EDL and Sol (p = 0.046 &amp; 0.050, respectively). These findings indicate that grip and isolated muscle contractility are generally higher in M vs. F when expressed in absolute terms, but lower in M vs. F when expressed as relative measures (normalized to body mass or muscle size). Minimal declines in muscle contractility occurred with age in either muscle tested, consistent with what we have observed previously with in situ testing of the Sol, but other aged fast muscles.

Functional, electrophysiology, and molecular dynamics analysis of quercetin-induced contraction of rat vascular musculature

Quercetin, a flavonoid abundantly present in the Mediterranean diet, is considered a vasodilator despite its recognized capability to stimulate vascular CaV1.2 channel current (ICa1.2). The present study was undertaken to assess its possible vasocontractile activity. Functional and electrophysiology experiments were performed in vitro on rat aorta rings and tail artery myocytes along with an in-depth molecular modelling analysis. The CaV1.2 channel stimulator (S)-(−)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (Bay K 8644) was used as reference compound. Quercetin and Bay K 8644 caused a significant leftward shift of KCl concentration-response curve. Neither agent affected basal muscle tone, though in rings pre-treated with thapsigargin or 15 mM KCl they caused a strong, concentration-dependent contraction. Both quercetin and Bay K 8644 potentiated the response to Ca2+ in weakly depolarised rings. At high KCl concentrations, however, quercetin caused vasorelaxation. While Bay K 8644 stimulated ICa1.2, this effect being sustained with time, quercetin-induced stimulation was transient, although the molecule in solution underwent only marginal oxidation. Quercetin transient stimulation was not affected by pre-treatment with isoprenaline, sodium nitroprusside, or dephostatin; however, it converted to a sustained one in myocytes pre-incubated with Gö6976. Classical molecular dynamics simulations revealed that quercetin and Bay K 8644 formed hydrogen bonds with target sensing residues of CaV1.2 channel favouring the inactivated conformation. In conclusion, quercetin-induced stimulation of ICa1.2 promoted vasocontraction when Ca2+ buffering function of sarcoplasmic reticulum was impaired and/or smooth muscle cell membrane was moderately depolarised, as it may occur under certain pathological conditions.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Functional sympatholysis in mouse skeletal muscle involves sarcoplasmic reticulum swelling in arterial smooth muscle cells.

The vasoconstrictive effect of sympathetic activity is attenuated in contracting skeletal muscle (functional sympatholysis), allowing increased blood supply to the working muscle but the underlying mechanisms are incompletely understood. The purpose of this study was to examine α‐adrenergic receptor responsiveness in isolated artery segments from non‐exercised and exercised mice, using wire myography. Isometric tension recordings performed on femoral artery segments from exercised mice showed decreased α‐adrenergic receptor responsiveness compared to non‐exercised mice (logEC50 −5.2 ± 0.04 M vs. −5.7 ± 0.08 M, respectively). In contrast, mesenteric artery segments from exercised mice displayed similar α‐adrenergic receptor responses compared to non‐exercised mice. Responses to the vasoconstrictor serotonin (5‐HT) and vasodilator isoprenaline, were similar in femoral artery segments from non‐exercised and exercised mice. To study sarcoplasmic reticulum (SR) function, we examined arterial contractions induced by caffeine, which depletes SR Ca2+ and thapsigargin, which inhibits SR Ca2+‐ATPase (SERCA) and SR Ca2+ uptake. Arterial contractions to both caffeine and thapsigargin were increased in femoral artery segment from exercised compared to non‐exercised mice. Furthermore, 3D electron microscopy imaging of the arterial wall showed SR volume/length ratio increased 157% in smooth muscle cells of the femoral artery from the exercised mice, whereas there was no difference in SR volume/length ratio in mesenteric artery segments. These results show that in arteries surrounding exercising muscle, the α‐adrenergic receptor constrictions are blunted, which can be attributed to swollen smooth muscle cell SR’s, likely due to increased Ca2+ content that is possibly reducing free intracellular Ca2+ available for contraction. Overall, this study uncovers a previously unknown mechanism underlying functional sympatholysis.

Cardiac contractility of the African sharptooth catfish, Clarias gariepinus: role of extracellular Ca2+, sarcoplasmic reticulum, and β-adrenergic stimulation.

This study investigated the dependence of contraction from extracellular Ca2+, the presence of a functional sarcoplasmic reticulum (SR), and the effects of β-adrenergic stimulation using isometric cardiac muscle preparations. Moreover, the expression of Ca2+-handling proteins such as SR-Ca2+-ATPase (SERCA), phospholamban (PLN), and Na+/Ca2+ exchanger (NCX) were also evaluated in the ventricular tissue of adult African sharptooth catfish, Clarias gariepinus, a facultative air-breathing fish. In summary, we observed that (1) contractility was strongly regulated by extracellular Ca2+; (2) inhibition of SR Ca2+-release by application of ryanodine reduced steady-state force production; (3) ventricular myocardium exhibited clear post-rest decay, even in the presence of ryanodine, indicating a decrease in SR Ca2+ content and NCX as the main pathway for Ca2+ extrusion; (4) a positive force-frequency relationship was observed above 60 bpm (1.0 Hz); (5) ventricular tissue was responsive to β-adrenergic stimulation, which caused significant increases in twitch force, kept a linear force-frequency relationship from 12 to 96 bpm (0.2 to Hz), and improved the cardiac pumping capacity (CPC); and (6) African catfish myocardium exhibited similar expression patterns of NCX, SERCA, and PLN, corroborating our findings that both mechanisms for Ca2+ transport across the SR and sarcolemma contribute to Ca2+ activator. In conclusion, this fish species displays great physiological plasticity of E-C coupling, able to improve the ability to maintain cardiac performance under physiological conditions to ecological and/or adverse environmental conditions, such as hypoxic air-breathing activity.

Role of the glycemic level on in the functional state of the myocardium in patients with coronary heart disease and type 2 diabetes mellitus

Abstract Clinical studies show that hyperglycemia is significant factor that worsens the prognosis and course of the cardiovascular diseases. At the same time, there is evidence that an increased level of glycemia in patients with type 2 diabetes mellitus (DM2) is associated with a better outcome of cardiovascular complications compared to the same patients, but with a normal level of glycemia. Aim To investigate the effect of the glycemia level on the functional state of the myocardium in patients with coronary heart disease (CHD) and DM2. Methods The study included patients with a chronic CHD associated with DM2, who were divided into 2 groups. Group 1 included patients with glycated hemoglobin (HbA1c) &amp;lt;8%, and 2 - patients with HbA1c ≥8%. The function of the heart, according to echocardiography (Echo-KG) and the contractile properties of trabeculae isolated from the right atrial auricle obtained during coronary bypass surgery were studied. To assess the contractile function of the trabeculae, exposure to periods of rest was used [4]. Post-rest inotropic reaction was evaluated. The level of expression of Ca2+-ATPase (SERCA2a) and ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR) was also determined in the patients ' myocardial samples by immunoblotting. Results According to the clinical characteristics, the patients of the groups were comparable, including age and duration of the disease of DM2. According to the Echo-KG data, it was found that in patients of group 1, the values of the end systolic and diastolic volumes, the thickness of the interventricular septum and the mass of the myocardium of the left ventricle (LV) were significantly lower in comparison with the corresponding indicators of patients of group 2. The rate of early LV filling (E) was significantly lower in group 1. At the same time, the rate of rapid LV filling did not have significant differences between groups. The amplitude of post-rest contractions of the trabeculae of patients in both groups was potentiated for short periods of rest, but with an increase in the duration of rest periods, it increased significantly only in group 2. After long periods of rest, the post-rest inotropic response of group 1 patient myocardium was not potentiated. As is known, post-rest potentiation characterizes the function of the SR. So, it is indicates better preservation of SR function in patients with HbA1c ≥8%. This statement is confirmed by the data on the expression of Ca2+ - transporting CP proteins SERCA2a and RyR2. Our studies have shown that the expression level of SERCA2a and RyR2 is significantly higher in the myocardium of patients with HbA1c ≥8% compared to patients with HbA1c &amp;lt;8%. Conclusion In the patients with CHD and DM2, an increased level of HbA1c is associated with better the functional state of the myocardium according to the contractile activity of isolated trabeculae, as well as the whole heart, which associated with higher SERCA2a and RyR2 expression. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Russian Science Foundation

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

Key points Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass.The reasons behind this disproportionate loss of muscle force are still poorly understood.We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest.Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity.Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation–contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading. Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)‐positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C‐terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross‐sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation–contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading.

Contrasting strategies of hypoxic cardiac performance and metabolism in cichlids and armoured catfish.

The heart of tropical fishes is a particularly useful model system in which to investigate mechanisms of hypoxic tolerance. Here we focus on insights gained from two groups of fishes, cichlids and armoured catfishes. Cichlids respond to hypoxia by entering a sustained hypometabolism with decreased heart performance to match whole animal circulatory needs. Heart rate is decreased along with protein turnover to reduce adenosine triphosphate demand. This occurs despite the inherent capacity for high levels of cardiac power development. Although highly hypoxic tolerant at the whole animal level, the heart of cichlids does not have high constitutive activities of glycolytic enzymes compared to other species. Information is conflicting with respect to changes in glycolytic gene expression and enzyme activity following hypoxic exposure with some studies showing increases and others decreases. In contrast to cichlids, species of armoured catfish, that are routinely exposed to water of low oxygen content, do not display hypoxic bradycardia. Under hypoxia there are early changes in glucose trafficking suggestive of activation of glycolysis before lactate accumulation. Thereafter, heart glycogen is mobilized and lactate accumulates in both heart and blood, in some species to very high levels. Heart performance under hypoxia is enhanced by defense of intracellular pH. A functional sarcoplasmic reticulum and binding of hexokinase to the outer mitochondrial membrane may also play a role in cardioprotection. Maintenance of heart performance under hypoxia may relate to a tradeoff between air breathing via a modified stomach and circulatory demands for digestion.

Abstract 15509: SYNE2 Expression Regulates Calcium Cycling and Mitochondria Function in Cardiomyocytes: Implications for Association With Atrial Fibrillation

Introduction: SYNE2 encodes a nuclear membrane protein that connects the nucleus with the cytoskeleton. rs1152591 is a common SNP in the SYNE2 gene that is associated with atrial fibrillation (AF) and with reduced expression of SYNE2α1 , a short isoform, in human left atrial appendage tissue. We previously found that SYNE2α1 over expression (OE) acts as a dominant negative for the nuclear phenotype, similar to the SYNE2 knockdown (KD), leading to enlarged nuclear size and decreased nuclear stiffness. Objectives: To determine if GFP- SYNE2α1 (OE) and KD of all SYNE2 isoforms show similar effects on gene expression and cell physiology in human induced pluripotent stem cell-derived cardiomyocytes (iCMs). Methods and Results: RNAseq after SYNE2α1 OE or SYNE2 KD revealed both congruent changes in expression of specific genes, supporting the dominant negative role of SYNE2α1 , but also divergent changes in expression of some genes, showing specific effects of the SYNE2α1 short isoform. We identified both mitochondrial function and sarcoplasmic reticulum (SR) function as differentially expressed pathways comparing OE vs. KD iCMs. Fura-2 photometry was used to study Ca 2+ cycling in beating iCMs, and revealed delayed calcium reuptake in the SYNE2 KD cells (15.9% increase in reuptake time as % of each contraction, p&lt;0.0001), but not in the GFP- SYNE2α1 OE cells (not significant). Flow cytometry showed significantly lower SERCA2 expression in the SYNE2 KD cells but not in the SYNE2α1 OE cells (12% decrease, p=0.029). Immunofluorescence microscopy revealed that GFP-SYNE2α1 not only localized to the nuclear membrane but also to the SR, stained with anti-SERCA2. Flow cytometry after MitoTracker Orange staining, to monitor the cellular volume of functional mitochondria, was significantly increased in both the SYNE2α1 OE and SYNE2 KD iCMs vs. their respective controls. Thus, despite differential expression of mitochondrial pathway genes, SYNE2α1 mimics the KD of all SYNE2 isoforms in regard to mitochondrial function. Conclusions: SYNE2α1 OE, unlike KD of all SYNE2 isoforms, preserves SR Ca 2+ reuptake activity, and this may contribute to the mechanism by which the AF risk allele in the SYNE2 gene, associated with decreased expression of SYNE2α1, predisposes carriers to AF.

Abnormal Sarcoplasmic Reticulum/Mitochondria Ca2+ Microdomains Impairs Mitochondrial Ca2+ in Cardiomyocytes in Aging

Mitochondrial (Mito) metabolism is regulated by free calcium concentration ([Ca2+]m) in the mitochondrial matrix. Cardiac function is impaired in aging and is associated with Mito disfunction. [Ca2+]m homeostasis depends on the Mito calcium uniporter complex and functional sarcoplasmic reticulum (SR)/Mito calcium microdomains. We hypothesized that Mito dysfunction could be the result of aging‐induced maladaptive changes in protein levels of key proteins of the SR/Mito microdomain resulting in deficient Mito Ca2+ uptake. Therefore, the objective of this work was to determine the protein levels of key members of the SR/Mito microdomain and analyze Mito Ca2+ handling as well as spatial SR‐Mito interaction using the proximity ligation assay. We measured protein levels by Western blot of MFN2, GRP75 and SERCA2a, in hearts of young (YH; 2–3 months) and old (OH; 19–22 months) mice. In addition, we analyzed Mito Ca2+ uptake, release and [Ca2+]m using Mitycam and Mito‐pericam. Our results showed that the SR‐Mito Ca2+ microdomain tethering proteins MFN2 and GRP75 were decreased in the OH by 55% and 60% respectively. SERCA2a, the key mediator of SR Ca2+ loading was also decreased by 41% in the OH. SR‐Mito interaction was dramatically diminished in OH. Consequently, Mito Ca2+ uptake and release as well as [Ca2+]m were significantly decreased. In vivo cardiac AAV‐based expression MFN2 improved SR‐Mito interaction. Adenoviral MFN2 expression in cardiomyocytes increased [Ca2+]m.In conclusion, SR/Mito interaction is impaired in the cardiomyocyte in aging. This results in abnormal Mito Ca2+ homeostasis and can be improved by expression of MFN2.Support or Funding InformationAuthors acknowledge funding from a Merit Grant from Veterans Affairs system I01BX003429. This work was supported by the National Institute on Aging, Grant T35 AG26757 (PI: Dilip V. Jeste, MD), and the Stein Institute for Research on Aging and the Center for Healthy Aging at the University of California, San Diego. Angelica Suarez was a 2018 UC San Diego MSTAR Student.

Caffeine as a tool to investigate sarcoplasmic reticulum and intracellular calcium dynamics in human skeletal muscles.

Caffeine is worldwide used for its power to increase cognitive and physical performance. The ergogenic effects of caffeine, however, do not depend on a direct action on muscles. Actually, the actions of caffeine on skeletal muscles, take place at millimolar concentrations which are far above the micromolar level reached after a regular consumption of coffee or similar drinks, and close to a lethal concentration. At millimolar concentrations caffeine exerts a powerful effect on sarcoplasmic reticulum (SR) activating the release of calcium via ryanodine receptors and, possibly, inhibiting calcium reuptake. For this reason caffeine has become a valuable tool for studying SR function and for diagnostics of SR related muscle disorders. This review aims to briefly describe the effects and the mechanism of action of caffeine on sarcoplasmic reticulum and to focus on its use to study intracellular calcium dynamics in human muscle fibers in physiological and pathological conditions.

Intracellular calcium current disorder and disease phenotype in OBSCN mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy.

Obscurin participates in the development of striated muscles and maintenance of the functional sarcoplasmic reticulum. However, the role of obscurin in arrhythmogenic right ventricular cardiomyopathy (ARVC) is not well understood. We aimed to study the novel obscurin mutations in the pathogenesis of ARVC and the underlying mechanisms.Methods: We generated induced pluripotent stem cells (iPSC) through retroviral reprogramming of peripheral blood mononuclear cells isolated from a 46-year-old female diagnosed with ARVC, carrying a mutation in OBSCN. The cells differentiated into functional iPSC-based cardiomyocytes (iPSC-CMs), whose phenotype was determined by transmission electron microscopy, electrophysiological description, immunofluorescence staining, and Oil Red O staining. Molecular characterization was performed by bioinformatic analyses, and identification by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.Results: ARVC-iPSC-CMs mutation in OBSCN showed significant accumulation of lipids, increased pleomorphism, irregular Z-bands, and increased L type calcium currents. Functional enrichment analysis identified pathways involved in focal adhesion and structure formation; the adipocytokines and PPAR signaling pathways were also activated in the ARVC group. Moreover, our results from ultra-high-resolution microscopy, qRT-PCR and Western blotting confirmed that the mutant OBSCN protein and its anchor protein, Ank1.5, showed structural disorder and decreased expression, but there was increased expression of junctional protein N-Cadherin. Further analysis revealed the gene expression of other desmosomal proteins in ARVC-iPSC-CMs was also decreased but some adipogenesis pathway-related proteins (PPARγ, C/EBPα, and FABP4) were increased.Conclusion: A novel frameshift mutation in OBSCN caused phenotypic alteration accompanied by disrupted localization and decreased expression of its anchoring protein Ank1.5. Furthermore, there was an accumulation of lipids with an increase in fatty fibrosis area and myocardial structural disorder, possibly leading to dysrhythmia in calcium channel-related myocardial contraction. These observations suggested the possibility of attenuating ARVC progression by therapeutic modulation of OBSCN expression.

SERCA2a: a key protein in the Ca2+ cycle of the heart failure.

Calcium ion (Ca2+) cycle plays a crucial role in the contraction and relaxation of cardiomyocytes. The sarcoplasmic reticulum (SR) acts as an organelle for storing Ca2+, which mediated the release and re-uptake of Ca2+ during contraction and relaxation. Disorders of SR function lead to the dysfunction of Ca2+ cycle and myocardial cell function. The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) acts as a subtype of SERCA expressed in the heart, which mediates the contraction of cardiomyocytes and Ca2+ in the cytoplasm to re-enter into the SR. The rate of uptake of Ca2+ by the SR determines the rate of myocardial relaxation. The regulation of SERCA2a activity controls the contractility and relaxation of the heart, affecting cardiac function. The expression and activity of SERCA2a are reduced in failing hearts. Gene therapy by increasing the expression of SERCA2a in the heart has been proven effective. In addition, SERCA2a is regulated by a variety of factors, including transmembrane micropeptides, protein kinases, and post-translational modifications (PTMs). In this review, we discuss the regulatory factors of SERCA2a and provide new insights into future treatments and the direction of heart failure research. In addition, gene therapy for SERCA2a has recently emerged as therapeutic option and hence will be discussed in this review.