Impaired Sarcoplasmic Reticulum Ca2 Research Articles

Intrinsic contractile dysfunction due to impaired sarcoplasmic reticulum Ca2+ release in compensatory hypertrophied muscle fibers following synergist ablation.

Synergist ablation (SA) is an experimental procedure for the induction of hypertrophy. However, SA causes a decrease in specific force (i.e., force per cross-sectional area), likely due to excessive muscle use. Here, we investigated the mechanisms behind the SA-induced intrinsic contractile dysfunction, especially focusing on the excitation-contraction (EC) coupling. Male Wistar rats had unilateral surgical ablation of gastrocnemius and soleus muscles to induce compensatory hypertrophy in the plantaris muscles. Two weeks after SA, plantaris muscle was dissected from each animal and used for later analyses. SA significantly increased the mean fiber cross-sectional area (+18%). On the other hand, the ratio of depolarization-induced force to the maximum Ca2+-activated specific force, an indicator of sarcoplasmic reticulum (SR) Ca2+ release, was markedly reduced in mechanically skinned fibers from the SA group (-51%). These functional defects were accompanied by an extensive fragmentation of the SR Ca2+ release channel, the ryanodine receptor 1 (RyR1), and a decrease in the amount of other triad proteins (i.e., DHPR, STAC3, and junctophilin1). SA treatment also caused activation of calpain-1 and increased the amount of NADPH oxidase 2, endoplasmic reticulum (ER) stress proteins (i.e., Grp78, Grp94, PDI, and Ero1), and lipid peroxidation [i.e., 4-hydroxynonenal (4-HNE)] in SA-treated muscles. Our findings show that SA causes skeletal muscle weakness due to impaired EC coupling. This is likely to be induced by Ca2+-dependent degradation of triad proteins, which may result from Ca2+ leak from fragmented RyR1 triggered by increased oxidative stress.NEW & NOTEWORTHY Synergist ablation (SA) has widely been used to understand the mechanisms behind skeletal muscle hypertrophy. However, compensatory hypertrophied muscles display intrinsic contractile dysfunction, i.e., a hallmark of overuse. Here, we demonstrate that SA-induced compensatory hypertrophy is accompanied by muscle weakness due to impaired sarcoplasmic reticulum Ca2+ release. This dysfunction may be caused by the degradation of triad proteins due to the reciprocal amplification of reactive oxygen species and Ca2+ signaling at the junctional space microdomain.

Abstract 12156: Loss of Thiol on C674 SERCA2 Leads to Ca2 + Mishandling With Hypertrophy in Right Ventricular Exposed to Hypoxia

Introduction: Right ventricular (RV) dysfunction is the strongest predictor of mortality in pulmonary artery hypertension (PAH). Reactive oxygen species in heart failure cause irreversible oxidation of sarcoplasmic reticulum (SR) Ca 2+ -ATPase2 (SERCA2) C674, which results in SERCA2 dysfunction and intracellular Ca 2+ overload. However, the contribution of the loss of the thiol on C674 SERCA2 and RV failure in PAH remains unclear. Hypothesis: The loss of the thiol on C674 SERCA2 contributes to the RV dysfunction in PAH. Methods & Results: We employed the SERCA2 C674S heterozygote knock-in (SKI) mice, in which C674 is replaced by serine, mimics oxidative modification of SERCA2 by loss of the specific thiol. Wild-type mice (WT) and SKI were exposed to either normoxia (Nx) or chronic hypoxia (Hx) for four weeks. Hypoxia elevated RV systolic pressure (WT-Nx: 21.4mmHg vs. WT-Hx: 37.0mmHg, P<0.0001) and represented pulmonary vascular remodeling similarly both in WT and SKI. In histology, SKI-Hx showed the markedly increased Fulton's Index with higher levels of the mRNA expression of atrial natriuretic peptide and B-type natriuretic peptide than WT-Hx. In the exercise test, exercise duration was shorter and peak oxygen consumption was decreased in SKI-Hx than WT Hx. The mRNA levels of SERCA2 were not changed among the four groups. In Ca 2+ transient measurement of isolated myocytes with Fluo-4, SKI prolonged the time to 50% of cytosolic Ca 2+ extrusion (T50) and lower the peak Ca 2+ transient amplitude (F/F0) than WT under Nx. Prolonged T50 and lowered peak F/F0 were observed in WT-Hx, both of which additionally progressed in SKI-Hx. Conclusions: Hypoxia impaired SR Ca 2+ homeostasis in RV. SKI additionally impaired SR Ca 2+ homeostasis with the progression of RV hypertrophy. The loss of the thiol on C674 SERCA2 contributed to the pathogenesis of RV failure in PAH.

Sarcoplasmic Reticulum Ca 2+ Handling in Gastrocnemius Muscles from 10‐week‐old C57 and D2‐ mdx Mice

Background Duchenne Muscular Dystrophy (DMD) is an X-linked muscle wasting disease caused by an absence of dystrophin. Secondary mechanisms including elevated myoplasmic Ca2+ as a result of dysfunctional sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pumps and leaky ryanodine receptors (RyR) can perpetuate disease pathology. Although the widely used preclinical C57BL/10 (C57) mdx mouse model displays moderate muscle damage and weakness, this model falls short in its ability to recapitulate the more severe human phenotype. Conversely, the DBA/2J (D2) mdx mouse model displays severe and early-onset muscle weakness, damage and histopathology; and therefore, more closely mimics human DMD. To date, there has been no characterization of muscle Ca2+ handling in the D2-mdx mouse. Thus, we examined sarcoplasmic reticulum (SR) Ca2+ handling in both D2-mdx and C57-mdx mice. Methods Eight-week-old male C57-WT, C57-mdx, D2-WT and D2-mdx mice were purchased from Jackson Laboratories. All mice underwent a hangwire test and were temporarily housed (48 hr) in a Promethion Metabolic Cage System to measure energy expenditure and cage activity. At 10 weeks of age, mice were euthanized, and gastrocnemius muscles were collected to measure Ca2+ uptake and leak via an Indo-1 fluorimetric assay. Serum creatine kinase (CK) activity was measured using a commercially available kit. Results Consistent with previous literature, serum CK was elevated in both C57-mdx and D2-mdx mice compared with WT, but to a greater extent in the former (8.9-fold vs 3.3 fold, p = 0.04). While both C57-mdx and D2-mdx mice had lowered hangwire time, these impairments were more severe in the D2-mdx mouse (-30% in C57-mdx vs -50% in D2-mdx, p = 0.09). Similarly, while both C57-mdx (-15%) and D2-mdx (-33%) mice had lowered total cage activity, the D2-mdx mice were most affected (p = 0.03). Interestingly, metabolic cage analyses further revealed that D2-mdx (1.3-fold increase) but not C57-mdx mice, had significantly elevated daily energy expenditure compared to their respective WT groups. When examining SR Ca2+ uptake, we found that the D2-mdx mice had significantly elevated starting Ca2+ levels compared with D2-WT mice (5500 vs 2300 nM). When activated by ATP, there was significantly less removal of Ca2+ in the D2-mdx muscles compared with WT (2.6-fold greater area-under-the-curve). However, none of these effects were observed in the C57-mdx mice. Furthermore, when SERCA was inhibited with cyclopiazonic acid, the rate of SR Ca2+ leak (-50%) and the amount of SR Ca2+ released back into the cytosol (-48%) were significantly lowered in the D2-mdx but not in the C57-mdx mice. This is likely reflective of impaired SR Ca2+ uptake and filling. Conclusions Unlike the C57-mdx, the D2-mdx mice displayed early onset muscle weakness with lowered cage ambulation and hangwire time when compared with their respective WT groups. These findings were associated with impaired SR Ca2+ handling in the D2-mdx mice but not the C57-mdx mice. Together, the extensive muscle damage and impaired SR Ca2+ handling in the D2-mdx mice likely enhances their daily energy expenditure.

Impaired sarcoplasmic reticulum Ca2+ release is the major cause of fatigue-induced force loss in intact single fibres from human intercostal muscle.

Changes in intramuscular Ca2+ handling contribute to development of fatigue and disease-related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described. We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca2+ ] at physiological temperature. Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca2+ release in both groups of fibres. Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue-resistant fibres. The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle. Changes in intracellular Ca2+ handling of individual skeletal muscle fibres cause a force depression following physical activity and are also implicated in disease-related loss of function. The relation of intracellular Ca2+ handling with muscle force production and fatigue tolerance is best studied in intact living single fibres that allow continuous measurements of force and myoplasmic free [Ca2+ ] during repeated contractions. To this end, manual dissections of human intercostal muscle biopsies were performed to isolate intact single fibres. Based on the ability to maintain tetanic force at >40% of the initial value during 500 fatiguing contractions, fibres were classified as either fatigue sensitive or fatigue resistant. Following fatigue all fibres demonstrated a marked reduction in sarcoplasmic reticulum Ca2+ release, while myofibrillar Ca2+ sensitivity was either unaltered or increased. In unfatigued fibres, acidosis caused a reduction in myofibrillar Ca2+ sensitivity that was offset by increased tetanic myoplasmic free [Ca2+ ] so that force remained unaffected. Acidification did not affect the fatigue tolerance of fatigue-resistant fibres, whereas uncertainties remain whether or not fatigue-sensitive fibres were affected. Following fatigue, a prolonged force depression at preferentially low-frequency stimulation was evident in fatigue-sensitive fibres and this was caused exclusively by an impaired sarcoplasmic reticulum Ca2+ release. We conclude that impaired sarcoplasmic reticulum Ca2+ release is the predominant mechanism of force depression both in the development of, and recovery from, fatigue in human intercostal muscle.

Peroxynitrite-Induced Intracellular Ca2+ Depression in Cardiac Myocytes: Role of Sarco/Endoplasmic Reticulum Ca2+ Pump

Several studies have shown that peroxynitrite (ONOO–), formed upon the reaction of •NO and O2–, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO–-induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO– donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1–200 μM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO– donor with FeTMPyP (1 μM), an ONOO– decomposition catalyst, inhibited the effects induced by ONOO–. Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO– (SIN-1 200 μM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO– induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO–. Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO– in cardiac tissue.

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.

Central activation, metabolites, and calcium handling during fatigue with repeated maximal isometric contractions in human muscle.

To determine the roles of calcium (Ca2+) handling by sarcoplasmic reticulum (SR) and central activation impairment (i.e., central fatigue) during fatigue with repeated maximal voluntary isometric contractions (MVC) in human muscles. Contractile performance was assessed during 3 min of repeated MVCs (7-s contraction, 3-s rest, n=17). In ten participants, in vitro SR Ca2+-handling, metabolites, and fibre-type composition were quantified in biopsy samples from quadriceps muscle, along with plasma venous [K+]. In 11 participants, central fatigue was compared using tetanic stimulation superimposed on MVC in quadriceps and adductor pollicis muscles. The decline of peak MVC force with fatigue was similar for both muscles. Fatigue resistance correlated directly with % type I fibre area in quadriceps (r=0.77, P=0.009). The maximal rate of ryanodine-induced Ca2+-release and Ca2+-uptake fell by 31±26 and 28±13%, respectively. The tetanic force depression was correlated with the combined reduction of ATP and PCr, and increase of lactate (r=0.77, P=0.009). Plasma venous [K+] increased from 4.0±0.3 to 5.4±0.8mM over 1-3-min exercise. Central fatigue occurred during the early contractions in the quadriceps in 7 out of 17 participants (central activation ratio fell from 0.98±0.05 to 0.86±0.11 at 1min), but dwindled at exercise cessation. Central fatigue was seldom apparent in adductor pollicis. Fatigue with repeated MVC in human limb muscles mainly involves peripheral aspects which include impaired SR Ca2+-handling and we speculate that anaerobic metabolite changes are involved. A faster early force loss in quadriceps muscle with some participants is attributed to central fatigue.

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

Increased Ca2+‐activated Ca2+ influx and impaired Ca2+ buffering in coronary smooth muscle from metabolic syndrome Ossabaw swine (1076.2)

Coronary smooth muscle (CSM) phenotype is affected by intracellular Ca2+ regulation and we have previously correlated changes in CSM Ca2+ signaling from Ossabaw swine with varying stages of coronary artery disease (CAD). Ossabaw swine are a model of metabolic syndrome (MetS) and CAD. Three year old pigs were fed an excess‐calorie, atherogenic or control diet for 6 months. CAD progression, coronary artery function, and CSM Ca2+ signaling were assessed. MetS coronaries had advanced, lipid‐laden atherosclerotic lesions containing calcium deposits, while lean had no CAD. In isometric tension studies diseased coronary rings generated significantly less tension than healthy rings in response to depolarization with 20 mM K+. CSM were enzymatically isolated and loaded with the Ca2+ indicator, fura‐2 AM. Diseased CSM showed less K+‐induced Ca2+ influx compared to healthy cells. Diseased cells recovered less from the cytosolic Ca2+ increase back to baseline, suggesting impaired Ca2+ buffering. In physiologic salt solution (PSS), there was no difference in peak Ca2+ transient elicited by caffeine‐induced intracellular Ca2+ store release. The caffeine‐induced Ca2+ transient in Ca2+‐free PSS, which represents exclusively sarcoplasmic reticulum (SR) Ca2+ release, was decreased in diseased cells. Taken together, these data suggest that lesser SR Ca2+ release elicited greater Ca2+‐activated Ca2+ influx in diseased CSM. After caffeine challenge healthy CSM buffered Ca2+ significantly below basal Ca2+ compared to diseased CSM. Overall, the data suggest impaired CSM SR Ca2+ buffering, decreased SR Ca2+ store, and increased Ca2+‐activated Ca2+ influx in CAD.Grant Funding Source: Supported by NIH HL062552, HL092245, NIH/NCATS CTSI TL1 TR000162

Searching for Sudden Death SNPs in Calcium Handling Genes

Cardiac arrhythmias are a major clinical problem and lead to more than 250 000 sudden cardiac deaths (SCDs) each year in the United States alone.[1][1] Our ability to predict those at risk for sudden death remains poor. The majority of patients who die suddenly are not identified as being at high

GW24-e1015 Nitrate improves postresuscitation myocardial dysfunction and the involvement of nitric oxide and phospholamban protein

ObjectivesThe abnormality of Sarcoplasmic reticulum (SR) Ca2+ handling proteins was shown to be essential in myocardial dysfunction after acute reperfusion injury, which might be attenuated by NO therapy. Cardiac arrest...

Junctophilins and μ‐calpain: partners in excitation–contraction uncoupling

Junctophilins and μ‐calpain: partners in excitation–contraction uncoupling

Phenanthrene exposure causes cardiac arrhythmia in embryonic zebrafish via perturbing calcium handling

Phenanthrene (Phe) is one of the most abundant and ubiquitous polycyclic aromatic hydrocarbons in the aquatic environment. It is known that Phe has cardiotoxic effects, but knowledge concerning the mechanism of cardiac dysfunction caused by Phe is still limited. In this study, zebrafish embryos were exposed to environmentally relevant concentrations of Phe, and an increase of an irregular rhythm was observed in Phe treated embryos. Disordered calcium (Ca2+) handling characterized by impaired sarcoplasmic reticulum Ca2+ uptake, and obvious Ca2+ accumulation in the cytoplasm was observed in rat embryonic cardiac myoblasts (H9C2) exposed to Phe. The mRNA level as well as protein expression of the SERCA2a Ca2+ pump in zebrafish hearts or H9C2 cells was significantly decreased by Phe exposure. The activity of Ca2+-ATPase in H9C2 cells was inhibited by Phe. Both the mRNA and protein levels of TBX5, a direct regulator of SERCA2a, were significantly decreased by Phe exposure. These results suggested that exposure to Phe could lead to arrhythmia in zebrafish embryos via perturbing the calcium handling pathway.

Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Calcium cycling proteins and heart failure: mechanisms and therapeutics

Ca2+-dependent signaling is highly regulated in cardiomyocytes and determines the force of cardiac muscle contraction. Ca2+ cycling refers to the release and reuptake of intracellular Ca2+ that drives muscle contraction and relaxation. In failing hearts, Ca2+ cycling is profoundly altered, resulting in impaired contractility and fatal cardiac arrhythmias. The key defects in Ca2+ cycling occur at the level of the sarcoplasmic reticulum (SR), a Ca2+ storage organelle in muscle. Defects in the regulation of Ca2+ cycling proteins including the ryanodine receptor 2, cardiac (RyR2)/Ca2+ release channel macromolecular complexes and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a)/phospholamban complex contribute to heart failure. RyR2s are oxidized, nitrosylated, and PKA hyperphosphorylated, resulting in "leaky" channels in failing hearts. These leaky RyR2s contribute to depletion of Ca2+ from the SR, and the leaking Ca2+ depolarizes cardiomyocytes and triggers fatal arrhythmias. SERCA2a is downregulated and phospholamban is hypophosphorylated in failing hearts, resulting in impaired SR Ca2+ reuptake that conspires with leaky RyR2 to deplete SR Ca2+. Two new therapeutic strategies for heart failure (HF) are now being tested in clinical trials: (a) fixing the leak in RyR2 channels with a novel class of Ca2+-release channel stabilizers called Rycals and (b) increasing expression of SERCA2a to improve SR Ca2+ reuptake with viral-mediated gene therapy. There are many potential opportunities for additional mechanism-based therapeutics involving the machinery that regulates Ca2+ cycling in the heart.

Preventing Ryanodine Receptor 1 calcium Leak Improves Age-Dependent Muscle Dysfunction

A hallmark of mammalian aging is the progressive decline in muscle function, referred to as sarcopenia. It is commonly found that the force-generating capacity of aged muscle is reduced when normalized to the muscle cross-sectional area (specific force). This indicates defective excitation-contraction coupling. In this process, sarcoplasmic reticulum (SR) Ca2+ release via the ryanodine receptor 1 (RyR1) is a pivotal step that grades muscle contractile force. It has previously been shown that impaired contractility and SR Ca2+ release in muscular dystrophy can be caused by excessive RyR1-cysteine nitrosylation and reduced binding of the stabilizing protein FK506 binding protein 12 (FKBP12 or calstabin1) to RyR1. We hypothesized that maladaptations in the RyR1-SR Ca2+ release system could underlie impaired muscle function also in aging. Using immunoprecipitation and immuonblotting, we found that RyR1 from aged (24-26 month) mouse muscle were oxidized, cysteine-nitrosylated, and depleted of FKBP12, compared to RyR1 from younger (3-6 months) adult mice. This remodeling of the RyR1 resulted in “leaky” channels, which displayed an increased open probability and Ca2+ spark frequency.Moreover, tetanic Ca2+ transients and muscle specific forcewere reduced in 24-month-old mice. Treating aged mice with the RyR-stabilizing compound, S107, restored RyR1-FKBP12 interaction, and improved tetanic Ca2+ release, muscle specific force and exercise capacity.Together, these findings highlight the role of impaired SR Ca2+ release in age-dependent muscle weakness and introduce a novel therapeutic target for sarcopenia.

Adverse Bioenergetic Consequences of Na + -Ca 2+ Exchanger–Mediated Ca 2+ Influx in Cardiac Myocytes

In heart failure, the Na+-Ca²+ exchanger (NCX) is upregulated and mediates Ca²+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca²+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca²+ influx is rather slow and mitochondrial Ca²+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca²+ gradients in a "mitochondrial Ca²+ microdomain," we speculated that NCX-mediated Ca²+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca²+ ([Ca²+](c) and [Ca²+](m), respectively) was determined within the same cell after varying Ca²+ influx via L-type Ca²+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca²+ uptake, indexed by the slope of plotting [Ca²+](m) against [Ca²+](c) during each Ca²+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca²+ release. Depletion of sarcoplasmic reticulum Ca²+ load and increased contribution of the NCX to cytosolic Ca²+ influx independently reduced the efficiency of mitochondrial Ca²+ uptake. The upstroke velocity of cytosolic Ca²+ transients closely correlated with the efficiency of mitochondrial Ca²+ uptake. Despite comparable [Ca²+](c), sarcoplasmic reticulum Ca²+ release, but not NCX-mediated Ca²+ influx, led to stimulation of Ca²+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca²+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca²+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.

Ageing, but not yet senescent, rats exhibit reduced muscle quality and sarcoplasmic reticulum function

Reduced muscle force greater than expected from loss of muscle mass has been reported in ageing muscles. Impaired sarcoplasmic reticulum (SR) Ca(2+) release has been implicated as a possible mechanism, and attributed to several factors, including loss of ryanodine receptor (RYR) expression and protein binding. The aim of this study was to evaluate muscle quality and SR Ca(2+) release in ageing rats that were not so old that major atrophy had occurred. We collected in situ force data from the plantarflexor muscle group and muscle mass from the constituent muscles to determine muscle quality (force/mass) in adult (6-8 months) and ageing (24 months) rats (n=8/group). We evaluated SR Ca(2+) uptake and release, and determined expression of key proteins associated with Ca(2+) release [RYR and FK506 binding protein (FKBP)] and uptake (SERCA, parvalbumin, calsequestrin). Plantarflexor force and muscle quality were reduced with ageing (approx. 28 and 34%, respectively), but atrophy was limited, and significant only in the medial gastrocnemius (approx. 15%). The fast phase of SR Ca(2+) release was reduced with ageing in both gastrocnemii, as was FKBP expression and FKBP-RYR binding, but RYR expression was not affected. Similar, but non-significant changes were present in the plantaris, but the soleus muscle generally showed no ageing-related changes. These data suggest a possible role for impaired SR Ca(2+) release in ageing-related loss of muscle quality, although not through loss of RYR expression.

Ca 2+ Cycling and New Therapeutic Approaches for Heart Failure

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …