Sarcoplasmic Reticulum Proteins Research Articles

Identification of a Calsequestrin-1 Mutation in a Human Vacuolar Myopathy

Calsequestrin-1 (CASQ1) is the major calcium binding protein of the sarcoplasmic reticulum (SR) of skeletal muscle cells. It is mainly localized in the junctional domain of the SR where it is part of a quaternary complex, which includes the ryanodine receptor calcium release channel, junctin and triadin. Calsequestrin-1 can modulate Ca2+ release by either directly bind the ryanodine receptor and/or by binding to junctin and triadin. We recently identified a D244G mutation in CASQ1 in patients with a myopathy characterized by the presence of vacuoles containing aggregates of SR proteins. The mutation affects a conserved aspartic acid located in one of the high-affinity Ca2+ binding sites of CASQ1. We found that muscle fibers from patients carrying the CASQ1 mutation show alterations in the Ca2+ release kinetics, thus suggesting that the D244G mutation may alter the intracellular Ca2+ signaling in the affected fibers. Interestingly, mutations in the CASQ2 protein identified in patients affected by catecholaminergic polymorphic ventricular tachycardia (CPVT) were shown to alter either Ca2+ buffering or Ca2+ release properties of cardiac muscle cells and to reduce the ability of calsequestrin to bind junctin and triadin. In order to understand the cellular mechanisms responsible for alterations of Ca2+ release kinetics in skeletal muscle cells carrying the D244G mutation, interactions between mutated and wild type CASQ1 and the ryanodine receptor type 1, junctin and triadin are being investigated.

Titin and Obscurin: Giants Holding Hands and Discovery of a New Ig Domain Subset

The sarcomere, the fundamental unit of muscle contraction, contains some extraordinarily large polypeptides (0.6–3 MDa). Most of these proteins belong to the titin family, which was initially defined by the sequences of Caenorhabditis elegans twitchin [1,2] and human titin [3,4]. These proteins consist of multiple immunoglobulin (Ig) and fibronectin type III domains (Fn3), one or two protein kinase domains and, in some cases, highly elastic unique regions. As exemplified by studies of titin, the Ig domains serve as binding sites for other proteins and help assemble the sarcomere (reviewed in Ref. [5]). The newest member of the titin family is obscurin (Obs) (~800 kDa), which was initially identified as a ligand of a Z-disk portion of titin [6,7], although it primarily localizes to the sarcomeric M-band in mature muscle [6,8]. Using the power of C. elegans molecular genetics, we described the essential roles of the Obs homolog, UNC-89, in signaling and scaffolding in the M-band 5 years before Obs was discovered [9]. UNC-89 is required for the assembly or stability of the M-band and for thick filament organization [10–13]. The M-band is the portion of the sarcomere where myosin-containing thick filaments are cross-linked by a protein network that, in vertebrate muscle, consists of, at least, the C-terminal portion of titin, myomesin (also a poly-Ig/Fn3 protein) and Obs. The linkage between myosin thick filaments occurs through antiparallel dimers of myomesin that interact via their C-terminal Ig domains and bind to myosin via their N-terminal Ig/Fn3 domains [14,15]. Obs binds myo-mesin and titin but it is also believed to act as a linker between myofibrils and the SR (sarcoplasmic reticulum). This was first suggested by finding that the C-terminus of some isoforms of Obs interacts with the SR membrane proteins, small ankyrin-1 isoform 5 (sAnk1.5) and ankyrin-2 (Ank2) [16,17]. The Obs knockout mouse supports this model: it shows changes in longitudinal SR architecture even though it has normal sarcomeric organization somewhat surprisingly [18]. The role of Obs in linking the sarcomere to the SR is likely conserved in nematode UNC-89. Although ankyrin-like molecules have not yet been reported to interact with UNC-89, there is genetic evidence for this function: UNC-89 is required for the proper organization of the ryandonine receptor and SERCA, as well as for optimal calcium signaling [19]. Moreover, independent studies on nematode UNC-89 [13] and mouse Obs [20] demonstrate an evolutionarily conserved and novel mechanism by which UNC-89/Obs regulates ubiquitin-mediated protein degradation at the M-band, and this involves inhibition of a cullin 3 complex.

Unfolded Protein Response and Triad Formation in Skeletal Muscles of Catecholaminergic Polymorphic Ventricular Tachycardia Mouse / Odgovor Razvijenog Proteina I Formiranje Trijada U Skeletnim Mišićima Miševa Sa Kateholaminergičkom Polimorfnom Ventrikularnom Tahikardijom

Summary Isoform 2 of calsequestrin (CASQ2) is the main calcium-binding protein of sarcoplasmic reticulum (SR), expressed both in cardiac and in skeletal muscles. CASQ2 acts as an SR calcium (Ca2+) sensor and regulates SR Ca2+ release via interactions with triadin, junctin, and the ryanodine receptor. Various mutations of the csq2 gene lead to altered Ca2+ release and contractile dysfunction contributing to the development of arrhythmias and sudden cardiac death in young individuals affected by catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, a transgenic mouse carrying one of the identified CASQ2 point-mutations (R33Q) associated to CPVT was developed and a drastic reduction of the mutated protein was observed. Following a biomolecular approach, several analysis were performed using different antibody treatments in order to identify when the reduction of CASQ2 begins in skeletal muscles, unveil the mechanism involved in the reduction of CASQ2 in slow-twitch and fast twitch muscles and verify if other proteins are affected by the presence of the mutated protein. Mutated CASQ2 decreased soon after birth. Up-regulation of proteins associated to the unfolded protein response (UPR) was also observed. Important proteins in skeletal muscle triads formation were analyzed and increased protein levels were observed in adult knock-in CASQ2-R33Q/R33Q mice. Probably, R33Q mutation induced the decrease of CASQ2 by activation of the UPR and subsequently degradation through proteasome.

Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Unfolded Protein Response Is Activated in the Hearts of Catecholaminergic Polymorphic Ventricular Tachycardia (Cpvt) Mice

Abstract Isoform 2 of calsequestrin (CSQ2) is the main calciumbinding protein of the sarcoplasmic reticulum (SR) and is expressed in both cardiac and skeletal muscle. CSQ2 acts as an SR calcium (Ca2+) sensor and regulates SR Ca2+ release via interactions with triadin, junctin, and the ryanodine receptor. Various mutations of the csq2 gene lead to altered Ca2+ release and contractile dysfunction and contribute to the development of arrhythmias and sudden cardiac death in young individuals affected by CPVT . Transgenic mice carrying one of the identified CSQ2 point mutations (R33Q) associated with CPVT were developed, and a drastic reduction in the mutated protein was observed. Following a biomolecular approach, several analyses were performed using different antibody treatments to identify when the reduction of CSQ2 begins, to unveil the mechanism involved in the reduction of CSQ2 and to verify whether other proteins are affected by the presence of the mutated protein. Th e results of this study showed that mutated CSQ2 levels decreased soon after birth, in conjunction with decreased levels of other important junctional SR proteins, including triadin (TD). Th e up-regulation of proteins associated with the unfolded protein response (UPR) was also observed, and the ATF6- dependent pathway was activated by the UPR. The presence of the R33Q mutation induced the decrease of CSQ2 via UPR activation and subsequent proteasomal degradation.

A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.

G.P.159: Cylindrical spirals originate from sarcoplasmic reticulum of muscles in two Chinese siblings

Cylindrical spirals are morphological abnormalities, reported in fewer than 15 cases so far and presenting with a wide variety of clinical phenotypes. To clarify the nature of cylindrical spirals, we characterized the proteins in muscle specimens from two Chinese siblings presented clinically with muscle weakness and ultrastructurally with cylindrical spirals using a series of antibodies to sarcoplasmic reticulum and its related structures. On electron microscope, the cylindrical spirals are consisted of 10–20 lamellae wrapped around a central core containing glycogen granules. The immunohistochemical stains showed that all of these key calcium-regulatory proteins of the skeletal muscle sarcoplasmic reticulum, including SERCA1 (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum) were expressed in cylindrical spirals. The T-tubule special resident DHPR and nuclear membrane protein emerin were also present in cylindrical spirals, while the Golgi apparatus marker GM130 and the myofilament protein myosin as well as the cellular skeleton protein desmin were not found in cylindrical spirals. Moreover, antibodies to LC3 and Lamp2 relevant to autophagosome and lysosome were negative. Taken together, these results suggested that cylindrical spirals could originate from the whole sarcoplasmic reticulum containing the junctional, longitudinal and cisternae components of the SR and consequently affected the calcium homeostasis.

Nanoscale Changes in the Organisation of Junctional Proteins in JPH2 Transgenic Mice

In cardiomyocytes, transverse tubule (t-tubule) and sarcoplasmic reticulum (SR) membranes form close associations, known as diadic junctions, which are critical for excitation-contraction (E-C) coupling. Ryanodine receptors (RyRs), which are the SR calcium release channels, are concentrated in junctions where they play a key role for calcium release. In human and animal models of heart failure junctions and transverse (t-) tubules often become disorganised but the underlying mechanisms remain unclear. The SR membrane protein junctophilin 2 (JPH2) has been implicated in junction formation and maintenance. In this study, transgenic mouse models were used to investigate the influence JPH2 expression on RyR organisation. Cardiomyocytes from adult mice in which JPH2 had been acutely knocked down or over-expressed and control cells labeled for RyR and JPH2. Confocal laser scanning and optical super-resolution microscopy were used to investigate junctional protein organisation and t-tubule architecture.As previously observed in response to JPH2 knockdown t-tubule organisation was disrupted. Regular t-tubules in line with the z-discs were predominant in the control cardiomyocytes, but greatly reduced and irregular in JPH2 knockdown cells. In JPH2 overexpressing myocytes the t-tubules and longitudinal tubules were more abundant.RyR organisation was also altered in response to changes in JPH2 expression. Large RyR superclusters (>200nm diameter) were present in control cells and more abundant in JPH2 over-expressing cardiomyocytes. Such superclusters were mostly absent from JPH2 knockdown cells in which smaller clusters were observed. These changes only became apparent with the resolution provided by optical super-resolution but were difficult to detect with conventional confocal imaging. Our results indicate that cardiomyocyte organisation is severely affected by altered expression of JPH2 and raise questions on the functional role of RyR superclusters and their effect on local and global Ca2+ release.

Effects of the Inflammatory Cytokines TNF-α and IL-13 on Stromal Interaction Molecule–1 Aggregation in Human Airway Smooth Muscle Intracellular Ca2+ Regulation

Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

Parameter Sensitivity Analysis of Stochastic Models Provides Insights into Cardiac Calcium Sparks

We present a parameter sensitivity analysis method that is appropriate for stochastic models, and we demonstrate how this analysis generates experimentally testable predictions about the factors that influence local Ca2+ release in heart cells. The method involves randomly varying all parameters, running a single simulation with each set of parameters, running simulations with hundreds of model variants, then statistically relating the parameters to the simulation results using regression methods. We tested this method on a stochastic model, containing 18 parameters, of the cardiac Ca2+ spark. Results show that multivariable linear regression can successfully relate parameters to continuous model outputs such as Ca2+ spark amplitude and duration, and multivariable logistic regression can provide insight into how parameters affect Ca2+ spark triggering (a probabilistic process that is all-or-none in a single simulation). Benchmark studies demonstrate that this method is less computationally intensive than standard methods by a factor of 16. Importantly, predictions were tested experimentally by measuring Ca2+ sparks in mice with knockout of the sarcoplasmic reticulum protein triadin. These mice exhibit multiple changes in Ca2+ release unit structures, and the regression model both accurately predicts changes in Ca2+ spark amplitude (30% decrease in model, 29% decrease in experiments) and provides an intuitive and quantitative understanding of how much each alteration contributes to the result. This approach is therefore an effective, efficient, and predictive method for analyzing stochastic mathematical models to gain biological insight.

Proteomic Analysis of Purified Dog Cardiac Sacroplasmic Reticulum Membrane Subcompartments

Ca2+ homeostasis primarily involves two major morphological subdivisions of the sarcoplasmic reticulum (SR): junctional (jSR) cisterna that appose sarcolemma, and free (fSR) tubules surrounding myofilaments. Biochemical isolation of jSR and fSR membranes involves selective increases in density of SR membrane vesicles using the SR Ca2+ pump (SERCA2a) (Jones and Cala, J. Biol. Chem. 1981). The purpose of the current study was to comprehensively characterize cardiac SR subproteomes. Purified SR membranes were solubilized, trypsinized and analyzed by LC-MS/MS on a LTQ-XL mass spectrometer. Abundant species were fragmented with collision-induced dissociation (CID). Data analysis was performed using Proteome Discoverer 1.1 (Thermo) which incorporated the Mascot algorithm (Matrix Science). In replicate experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MSMS spectra with protein false discovery rate (FDR) at 0.8% and peptide FDR at 0.0%. SERCA2a was the most abundant protein in both fractions, as expected, and virtually all of the known cardiac SR proteins were also identified. In jSR, the major 4 components of the Ca2+-release complex were found exclusively, including calsequestrin-2, ryanodine receptor 2 and 3, triadin, and junctin. Another 57 proteins were also identified as specific to this subcompartment. In fSR, 53 proteins were exclusively identified. Relative enrichments in one of the SR subcompartments were found for an additional 86 proteins, and 52 additional proteins were identified in the cardiac SR, without obvious subcompartment origin. SR proteins that showed less specificity were mitochondrial proteins (more enriched in the lower-density jSR), ER chaperones (roughly evenly distributed), lipid-metabolizing enzymes, and filamentous proteins. Proteomic analyses of classical cardiac SR subfractions extends our understanding of the cardiac secretory compartments, and serves as foundation for future exploration and understanding of cardiac cell biology.

The Intraluminal Domain of Junctin Contains Junctional Sarcoplasmic Reticulum Targeting Sequences

Junctin is an integral membrane protein of the sarcoplasmic reticulum (SR) encoded by the Aspartyl beta-hydroxylase (AβH) gene. The AβH gene can undergo different alternative splicing events, generating at least three distinct proteins, the Aspartyl beta-hydroxylase, junctin and junctate, all sharing the same transmembrane domain and a short highly charged acidic luminal region, but carrying distinct intraluminal tails. Interstingly, junctin and junctate are both expressed in skeletal muscle, but only junctin was shown to interact and co-localize with the Ca2+ release complex in the junctional SR, whereas junctate displays a typical longitudinal SR distribution. These two proteins may thus represent an ideal molecular model to investigate the presence of specific regions responsible for protein targeting to the junctional SR.To this aim, junctin deletion mutants were tagged with a green fluorescent protein (GFP) and expressed in primary myoblasts. The results obtained showed that even deletion of small regions within intraluminal domain of junctin resulted in protein mislocalization, thus indicating that the entire luminal sequence is required for protein targeting to the junctional SR. Interestingly, the same region, if inserted downstream the cytoplasmic and trasmembrane domains of sarcolipin, a resident protein of the longitudinal SR, is able to completely re-localize the protein to the junctional SR, further confirming that the intraluminal domain of junctin may actually contain minimal junctional SR targeting sequences.

Skeletal muscle calcium channel ryanodine and the development of pale, soft, and exudative meat in poultry

The development of pale, soft, and exudative (PSE) breast fillet meat has become an economic burden for the poultry industry worldwide. PSE meat results in 1.0-1.5% loss in moisture and carcass weight, and a 2010 estimate of the Brazilian annual production put the economic loss due to PSE at over US$30 million. In the USA, PSE has caused an annual loss of up to US$200 million to the poultry industries. The underlying causes of the color abnormality in PSE meat are not fully understood. However, the likely physiological origin of PSE broiler meat is an excessive release of Ca(2+) promoted by a genetic mutation of the ryanodine receptor (RYR), a Ca(2+)-channel protein in the skeletal muscle sarcoplasmic reticulum. In pigs, the genetic cause of PSE meat has been identified as a point mutation in the RYR1 gene at nucleotide 1843, which causes an amino acid substitution (Arg615 to Cys615) in the RYR. This mutation leads to an alteration in Ca(2+) homeostasis, hypermetabolism, intense muscle contraction, and malignant hyperthermia in pigs susceptible to porcine stress syndrome. An understanding of this process represents the basis for breeding strategies aimed at eliminating the RYR1 mutation from global pig populations, a strategy that the poultry industry intends to emulate. The aim of this study was to review the subject, with an emphasis on the most recent developments in the field.

Exercise training prevents the development of cardiac dysfunction in the low-dose streptozotocin diabetic rats fed a high-fat diet

This study tested the hypothesis that exercise training would prevent the development of diabetes-induced cardiac dysfunction and altered expression of sarcoplasmic reticulum Ca(2 +)-transport proteins in the low-dose streptozotocin-induced diabetic rats fed a high-fat diet (HFD+STZ). Male Sprague-Dawley rats (4 weeks old; 125-150 g) were made diabetic using a high-fat diet (40% fat, w/w) and a low-dose of streptozotocin (35 mg·(kg body mass)(-1)) by intravenous injection. Diabetic animals were divided among a sedentary group (Sed+HFD+STZ) or an exercise-trained group (Ex+HFD+STZ) that accumulated 3554 ± 338 m·day(-1) of voluntary wheel running (mean ± SE). Sedentary animals fed a low-fat diet served as the control (Sed+LFD). Oral glucose tolerance was impaired in the sedentary diabetic group (1179 ± 29; area under the curve (a.u.c.)) compared with that in the sedentary control animals (1447 ± 42 a.u.c.). Although left ventricular systolic function was unchanged by diabetes, impaired E/A ratios (i.e., diastolic function) and rates of pressure decay (-dP/dt) indicated the presence of diastolic dysfunction. Diabetes also reduced SERCA2a protein content and maximal SERCA2a activity (V(max)) by 21% and 32%, respectively. In contrast, the change in each parameter was attenuated by exercise training. Based on these data, it appears that exercise training prevented the development of diabetic cardiomyopathy and the dysregulation of sarcoplasmic reticulum protein content in an inducible animal model of type 2 diabetes.

Age-Associated Changes in Ca2+-ATPase and Oxidative Damage in Sarcoplasmic Reticulum of Rat Heart

Altered Ca(2+) handling may be responsible for the development of cardiac contractile dysfunctions with advanced age. In the present study, we investigated the roles of oxidative damage to sarcoplasmic reticulum (SR) and expression of Ca(2+)-ATPase (SERCA 2a) and phospholamban in age-associated dysfunction of cardiac SR. SR vesicles were prepared from hearts of 2-, 6-, 15-, and 26-month-old Wistar rats. Although activity of Ca(2+)-ATPase decreased with advancing age, no differences in relative amounts of SERCA 2a and phospholamban protein were observed. On the other hand, significant accumulation of protein oxidative damage occurred with aging. The results of this study suggest that age-related alteration in Ca(2+)-ATPase activity in the rat heart is not a consequence of decreased protein levels of SERCA 2a and phospholamban, but could arise from oxidative modifications of SR proteins. Cellular oxidative damage caused by reactive oxygen species could contribute to age-related alternations in myocardial relaxation.

Diabetes mellitus attenuates myocardial preconditioning of desflurane in ischemia-reperfused rat heart

Desflurane has been commonly used in anesthesia that possesses a cardioprotective effect against ischemia-reperfusion. We evaluated whether diabetes mellitus affected desflurane preconditioning induced cardioprotection in ischemia-reperfusion rat hearts in view of pro- and anti-apoptotic signaling pathways and Ca2+ homeostasis. Streptozotocin-induced, diabetic rats and age-matched wild-type Sprague-Dawley rats were subjected to a left anterior descending coronary artery occlusion for 30 min followed by 1 hr of reperfusion. Myocardial infarct size, activities of Akt, ERK1/2, Bcl-2, Bax and cytochrome c, and gene expression influencing Ca2+ homeostasis were examined. Desflurane preconditioning significantly reduced myocardial infarct size compared to ischemia-reperfusion in wild-type rats but not in diabetic rats. In addition, decrease in Akt, ERK1/2, and Bcl-2, and the increase in Bax and cytochrome c by ischemia-reperfusion was reduced by desflurane preconditioning. In diabetic rat hearts, however, desflurane preconditioning failed to recover the phosphorylation state of Akt and ERK1/2 and to repress apoptotic signaling in association with impoverished modulation of abnormal changes in sarcoplasmic reticulum proteins in ischemia-reperfusion rat hearts. These results suggest that diabetes mellitus attenuated desflurane preconditioning against ischemia-reperfusion, which might be associated with reduced recovery of the activities of proteins involved in anti-apoptotic signaling pathways and sarcoplasmic reticulum.

Hypertensive heart disease

Hypertensive heart disease

Abstract 281: Long-Term Therapy with a Partial Adenosine A1-Receptor Agonist Upregulates mRNA and Protein Expression of Glucose Transporters in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: Recruitment of glucose transport proteins GLUT-1 and -4 is a mechanism by which the heart increases glucose transport for metabolism in response to increased energy demands. In the failing LV, mRNA and protein levels of GLUT-1 and -4 are down-regulated and the response to increased demands is met by increased dependence on fatty acid oxidation. We previously showed that treatment with the partial adenosine A1-receptor agonist capadenoson (CAP) improves LV systolic function in dogs with advanced heart failure (HF). This study examined the effects of CAP on the regulation of GLUT-1 and -4 in LV myocardium of dogs with chronic HF. Methods: Studies were performed in LV tissue from 12 HF dogs randomized to 3 months oral therapy with the CAP (7.5 mg twice daily, n=6) or to no therapy at all (Control, n=6). LV tissue from 6 normal (NL) dogs was used for comparison. mRNA expression of GLUT-1 and -4 was measured in LV tissue homogenate using real time polymerase chain reaction (RT-PCR). Protein levels of GLUT-1 and -4 and calsequestrin (CSQ), a sarcoplasmic reticulum protein that does not change in HF (internal control), were measured in SDS-extract using Western blotting coupled with chemiluminescent method. Band intensity was quantified in densitometric units (du). Results: Compared to NL dogs, Control HF dogs showed a 3.6 fold decrease in mRNA expression of GLUT-1 and a 2.7 fold decrease in the expression of GLUT-4. CAP-treatment in HF dogs improved mRNA expression of GLUT-1 (1.38 fold decrease) and GLUT-4 (0.8 fold decrease). Similarly, compared to NL dogs, Control HF dogs showed a significant decrease of protein levels of GLUT-1 (0.64 ± 0.04 vs. 0.26 ± 0.04 du, p<0.05) and GLUT-4 (1.42 ± 0.06 vs. 0.40 ± 0.05 du, p<0.05). CAP treatment significantly increased protein levels of both GLUT-1 (0.49 ± 0.03 du) and GLUT-4 (0.82 ± 0.03) compared to Control HF dogs (p<0.05). CSQ levels were similar in all study groups. Conclusions: In dogs with chronic HF, long-term therapy with CAP improves mRNA and protein expression of GLUT-1 and -4. This improvement in expression of glucose transport proteins is likely to restore partial dependency of the failing heart to more efficient glucose metabolism, and therefore, lead to improved myocardial energetics and pump function.

Accelerated Junctional Rhythm and Nonalternans Repolarization Lability Precede Ventricular Tachycardia in Casq2−/− Mice

Calsequestrin-2 (CASQ2) is a Ca(2+) buffering protein of myocardial sarcoplasmic reticulum. CASQ2 mutations underlie a form of catecholaminergic polymorphic ventricular tachycardia (CPVT). The CPVT phenotype is recapitulated in Casq2 -/- mice. Repolarization lability (RL)-beat-to-beat variability in the T wave morphology-has been reported in long-QT syndrome, but has not been evaluated in CPVT. ECG from Casq2 -/- mice was evaluated with respect to heart rate (HR) and RL changes prior to onset of ventricular tachycardia (VT) to gain insight into arrhythmogenesis in CPVT. Telemetry from unrestrained mice (3-month-old males, 5 animals of each genotype) and ECG before and after isoproterenol administration in anesthetized mice was analyzed. Average HR in sinus rhythm (SR), occurrence of nonsinus rhythm and RL were quantified. HR was slower in Casq2 -/- animals. Accelerated junctional rhythm (JR) occurred more frequently in Casq2 -/- mice and often preceded VT. In Casq2 -/- mice, HR increased prior to VT onset, prior to onset of JR and on transition from JR to VT. RL increased during progression from SR to VT and after isoproterenol administration in Casq2 -/-, but not in Casq2+/+ animals. Isoproterenol did not increase repolarization alternans in either genotype. Accelerated JR, likely caused by triggered activity in His/Purkinje system, occurs frequently in Casq2 -/- mice. The absence of CASQ2 results in increased RL. The increase in HR and in RL precede onset of arrhythmias in this CPVT model. Nonalternans RL precedes ventricular arrhythmia in wider range of conditions than previously appreciated.

Lethal, Hereditary Mutants of Phospholamban Elude Phosphorylation by Protein Kinase A

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.