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A missense mutation in a highly conserved region of CASQ2 is associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia in Bedouin families from Israel.

Catecholamine-induced polymorphic ventricular tachycardia (PVT) is characterized by episodes of syncope, seizures, or sudden death, in response to physical activity or emotional stress. Two modes of inheritance have been described: autosomal dominant and autosomal recessive. Mutations in the ryanodine receptor 2 gene (RYR2), which encodes a cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel, were recently shown to cause the autosomal dominant form of the disease. In the present report, we describe a missense mutation in a highly conserved region of the calsequestrin 2 gene (CASQ2) as the potential cause of the autosomal recessive form. The CASQ2 protein serves as the major Ca(2+) reservoir within the SR of cardiac myocytes and is part of a protein complex that contains the ryanodine receptor. The mutation, which is in full segregation in seven Bedouin families affected by the disorder, converts a negatively charged aspartic acid into a positively charged histidine, in a highly negatively charged domain, and is likely to exert its deleterious effect by disrupting Ca(2+) binding.

Age of Onset↗

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.

Aequorin↗

Changes in gene expression following short coronary occlusions studied in porcine hearts with run-on assays.

OBJECTIVE: Brief coronary occlusions cause upregulation of expression in a wide variety of genes. These changes in tissue mRNA concentration could have been produced by transcriptional or post-transcriptional events. The aim of this study was to discriminate between increased transcription and changes in mRNA stability using run-on assays with isolated myocyte nuclei. METHODS: Myocyte nuclei isolated from ischaemic/reperfused and normal myocardium were incubated with labelled ribonucleotides. The radioactive RNA was then hybridised with specific cDNA probes and slot blots were autoradiographed. RESULTS: There was increased transcriptional activity for the proto-oncogenes c-myc, c-jun, jun-B, and jun-D. There were marked increases in transcriptional activity for sarcoplasmic Ca(2+)-ATPase, calmodulin, phospholamban, and calsequestrin. Strong transcriptional activity was found for the ubiquitin and heat shock protein (hsp27, hsp70) genes, and for PAI-1 and GAPDH. The transcription for the beta myosin heavy chain gene was not altered. CONCLUSIONS: Changes in the tissue concentration of mRNA species following brief coronary occlusion and reperfusion are most often the result of altered transcriptional activity. Increased c-fos mRNA concentrations observed in earlier studies cannot be explained by transcriptional activity of myocytes during reperfusion. Calmodulin is strongly transcribed but tissue concentration stays constant. The overall pattern of gene expression is indicative of damage at the molecular level, and calcium binding proteins (among perhaps many others) are in need of repair.

Adenosine Triphosphate↗

Multiple quantitative trait loci modify the heart failure phenotype in murine cardiomyopathy.

The variability in outcome of heart failure patients depends on a number of factors including differences in their genetic background. To identify novel genes that modify the human heart failure phenotype, we used a strategy of quantitative trait locus (QTL) mapping in an experimental mouse model of dilated cardiomyopathy induced by cardiac-specific overexpression of calsequestrin and characterized by a strong strain-specific variability in the phenotype. We identified two novel QTLs, Hrtfm3 (heart failure modifier 3) on chromosome (Chr) 4 and Hrtfm4 on Chr 18, significantly linked to survival with likelihood ratio statistics (LRS) of 19.9 and 23.6 respectively (corresponding to LOD scores of 4.3 and 5.1). Two other QTLs, Hrtfm5 on Chr 2 and Hrtfm6 on Chr 13, were significantly linked to cardiac function as measured by echocardiographic fractional shortening (LRS 22.1 and 15.2 respectively, LOD score 4.8 and 3.3) and left ventricular end-diastolic diameter (LRS 23.5 and 18.8, LOD score 5.1 and 4.1). Importantly, Hrtfm5 was not significantly linked to survival. A significant interaction was found between Hrtfm4 and two other QTLs (Hrtfm6 and a QTL near to the marker D19Mit88) for fractional shortening with a LRS of 34.6 and 26.5 respectively (LOD score 7.5 and 5.8). These data show that the effect of genetic background on murine heart failure is complex and result from the action of several loci that differentially modify the cardiac phenotype. The identification of these novel modifier genes will serve as strong candidates for the discovery of modifiers in human heart failure.

Animals↗

Defective mitochondrial oxidative phosphorylation in myopathies with tubular aggregates originating from sarcoplasmic reticulum.

Abnormalities of the sarcotubular system presenting as tubular aggregates (TAs) have been described in a variety of neuromuscular disorders. Here, we report on immunohistochemical and biochemical findings in 7 patients (2 familial and 5 sporadic cases) suffering from myopathies with TAs. In muscle biopsy specimens from 5 of the 7 patients, TAs were immunopositive for the ryanodine receptor (RYR 1) of the sarcoplasmic reticulum (SR), the SR Ca2+ pump (SERCA2-ATPase), and the intraluminal SR Ca2+ binding protein calsequestrin, indicating an SR origin of these aggregates. Furthermore, these 5 cases showed decreased respiratory chain enzyme activities (NADH:CoQ oxidoreductase. complex I and cytochrome c oxidase [COX], complex IV), while the remaining 2 patients exhibited normal values. Our findings indicate a functional link between mitochondrial dysfunction and the presence of TAs originating from the sarcoplasmic reticulum.

Adult↗

Modulation of cardiac sarcoplasmic reticulum gene expression by lack of oxygen and glucose.

Although ischemia reperfusion has been shown to depress gene expression of the sarcoplasmic reticulum (SR) proteins, such as the ryanodine receptor, Ca2+-pump ATPase, phospholamban, and calsequestrin in the heart, the mechanisms of these changes are not understood. Given the occurrence of hypoxia and the lack of glucose during the ischemic phase, we investigated the effects of these factors on the cardiac SR gene expression. Isolated rat hearts perfused in the absence of oxygen and/or glucose for 30 min showed an increase in the expression of SR genes. However, perfusion of hearts for 60 min with normal oxygenated medium after 30 min of lack of both oxygen and glucose depressed the transcript levels for the SR proteins; these changes did not occur when hearts were deprived of either oxygen or glucose. The effect of intracellular Ca2+-overload, which occurs during reperfusion, was studied by using hearts perfused for 5 min with Ca2+-free medium and then reperfused for 30 min. Ca2+-depletion/repletion induced a dramatic decrease in the transcript levels of the SR genes. These results suggest that the lack of both oxygen and glucose during ischemia are necessary for reperfusion-induced depression in SR gene expression, possibly due to the occurrence of intracellular Ca2+-overload.

Animals↗

Abnormal distribution of calcium-handling proteins: a novel distinctive marker in core myopathies.

Central core disease (CCD) and multi-minicore disease (MmD) are muscle disorders characterized by foci of mitochondria depletion and sarcomere disorganization ("cores") in muscle fibers. Although core myopathies are the most frequent congenital myopathies, their pathogenesis remains elusive and specific diagnostic markers are lacking. Core myopathies are mostly caused by mutations in 2 sarcoplasmic reticulum proteins: the massive Ca-release channel RyR1 or the selenoprotein N (SelN) of unknown function. To search for distinctive markers and to obtain further pathophysiological insight, we identified the molecular defects in 12 core myopathy patients and analyzed the immunolocalization of 6 proteins of the Ca-release complex in their muscle biopsies. In 7 cases with RYR1 mutations (6 CCD, one MmD), RyR1 was depleted from the cores; in contrast, the other proteins of the sarcoplasmic reticulum (calsequestrin, SERCA1/2, and triadin) and the T-tubule (dihydropyridine receptor-alpha1subunit) accumulated within or around the lesions, suggesting an original modification of the Ca-release complex protein arrangement. Conversely, all Ca-related proteins were distributed normally in 5 MmD cases with SelN mutations. Our results provide an appropriate tool to orientate the differential and molecular diagnosis of core myopathies and suggest that different pathophysiological mechanisms lead to core formation in SelN- and in RyR1-related core myopathies.

Adolescent↗

Excitation-contraction coupling from the 1950s into the new millennium.

1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or 'calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a 'voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the 'calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.

Action Potentials↗

Na+-Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+-Ca2+ exchanger.

1. The contribution of the sarcoplasmic reticulum (SR) and Na+-Ca2+ exchanger to intracellular Ca2+ regulation in mouse cardiac myocytes was investigated by measuring contraction after variable rest intervals, rapid cooling contractures (RCCs) and fast application of caffeine. The results obtained showed differences from other species in the roles played by the SR and the Na+-Ca2+ exchanger. They suggest that in mouse ventricular myocytes there is significant Ca2+ entry via the exchanger during rest and during the latter part of the Ca2+ transient. 2. In cardiac myocytes isolated from transgenic mice overexpressing the cardiac Na+-Ca2+ exchanger the time to peak and relaxation of twitches and RCCs were faster than in control littermates. The decline of Ca2+, assessed by indo-1 fluorescence, was faster in transgenic myocytes even in the absence of Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is faster in these myocytes. However, no difference in the expression of SERCA2a, phospholamban or calsequestrin measured with Western blotting could be found in the two groups. 3. We measured SR Ca2+ content by integrating the caffeine-induced transient inward current. The amount of Ca2+ stored in the SR of transgenic mouse myocytes was 69 % greater than in non-transgenic littermates. The increased SR Ca2+ content may be responsible for the faster rate of SR Ca2+ release and uptake in cells from transgenic mice. 4. We performed experiments to assess whether the reversal potential of the Na+-Ca2+ exchanger (ENa-Ca) was different in transgenic cardiac cells. We measured a Ni2+-sensitive current elicited by voltage ramps in non-dialysed myocytes. The current-voltage relationship showed no difference in the reversal potential of the Na+-Ca2+ exchanger in transgenic and control myocytes. This suggests that the effects on the SR Ca2+ content in transgenic cardiac myocytes can be ascribed to the overexpression of the exchanger and are not secondary to changes in intracellular diastolic Ca2+ and Na+.

Animals↗

Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes.

1. Sarco-endoplasmic reticulum Ca2+-ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c-myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. 2. Expression of the two isoforms was similar (approximately 3-fold higher than endogenous SERCA). However, SERCA1 activity was 2-fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. 3. Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30-40 % and half-widths by 10-15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. 4. It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number.

Animals↗

Sarcoplasmic reticulum of human skeletal muscle: age-related changes and effect of training.

The effect of ageing on human skeletal muscle was investigated using needle biopsies from young and aged subjects and from aged subjects trained with different activity patterns. Histochemical staining for myofibrillar ATPase of ageing m. vastus lateralis demonstrated an unchanged fibre type distribution but a selective atrophy of type IIa and type IIb fibres. Analysis of myosin heavy chain (MHC) composition showed that type I MHC increased with ageing (P less than 0.05). The relative content of the MHC isoforms correlated with the relative area of the respective fibre types. Sarcoplasmic reticulum (SR) proteins were investigated in muscle extracts by electrophoretic and immunoblotting techniques. When compared to a young control group (28 +/- 0.1 years old, n = 7) blots of post-myofibrillar supernatant proteins probed with polyclonal antibodies to the rabbit fast SR Ca-ATPase, a marker of extrajunctional SR, showed that the content of Ca-ATPase was significantly lower (P less than 0.05) in the old control group (68 +/- 0.5 years old, n = 8). On the other hand the content of calsequestrin (CS), the major intraluminal protein of SR terminal cisternae (TC), and of the 350-kDa ryanodine-binding protein, which is localized in the junctional regions of TC, did not show a concomitant decrease. These results suggest that ageing differentially affects extrajunctional and junctional SR of human skeletal muscle. These age-related changes were not observed within a group of old strength-trained subjects.

Aged↗

Phospholamban ablation and compensatory responses in the mammalian heart.

Phospholamban is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum. The regulatory role of phospholamban in vivo has recently been elucidated by targeting the gene of this protein in embryonic stem cells and generating phospholamban-deficient mice. The phospholamban knockout hearts exhibited significantly enhanced contractile parameters and attenuated responses to beta-agonists. The hyperdynamic cardiac function of the phospholamban knockout mice was not accompanied by any cytoarchitectural abnormalities or alterations in the expression levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, Na(+)-Ca2+ exchanger, or the contractile proteins. Furthermore, the attenuation of the cardiac responses to beta-agonists was not due to alterations in the phosphorylation levels of the other key cardiac phosphoproteins in the phospholamban knockout hearts. However, ablation of phospholamban was associated with down-regulation of the ryanodine receptor, which suggests that a cross-talk between cardiac sarcoplasmic reticulum Ca2+ uptake and Ca2+ release occurred in an attempt to maintain Ca2+ homeostasis in these hyperdynamic phospholamban knockout hearts.

Adrenergic beta-Agonists↗

Sarcoplasmic reticulum proteins in heart failure.

Altered calcium homeostasis may play a key role in the pathophysiology of human heart failure. Levels of sarcoplasmic reticulum (SR) proteins and sarcolemmal Na(+)-Ca2+ exchanger were analyzed by Western blot in failing and nonfailing human myocardium and related to myocardial function. Levels of the SR calcium release channel and of calcium storage proteins (calsequestrin and calreticulin) were not different in nonfailing and failing hearts. However, proteins involved in calcium removal were significantly altered in the failing human heart: (1) SR-Ca(2+)-ATPase levels and the ratio of SR-Ca(2+)-ATPase to its inhibitory protein phospholamban were significantly decreased, and (2) Na(+)-Ca2+ exchanger levels and the ratio of Na(+)-Ca2+ exchanger to SR-Ca(2+)-ATPase were significantly increased. SR-Ca(2+)-ATPase levels were closely correlated to systolic function as evaluated by frequency potentiation of contractile force. The frequency-dependent rise of diastolic force was inversely correlated with protein levels of Na(+)-Ca2+ exchanger. These findings indicate that altered expression of SR-Ca(2+)-ATPase and Na(+)-Ca2+ exchanger is relevant for altered systolic and diastolic function in human heart failure.

Animals↗

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction.

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.

Angiotensin-Converting Enzyme Inhibitors↗

Intracellular Ca2+ stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies.

The molecular composition of intracellular Ca2+ stores in developing chicken cerebellum Purkinje neurons from embryonic day 11 (E11) to posthatching day 2 (P2) was studied by immunocytochemistry using specific antibodies for three molecular constituents, the receptor (R) and/or channel sensitive to inositol 1,4,5-trisphosphate (IP3), Ca(2+)-adenosinetriphosphatase (ATPase), and calsequestrin (CS). CS, IP3R, and Ca(2+)-ATPase were first detected by light-microscopic immunofluorescence in migrating Purkinje cells at E11-E12 and throughout late phases of embryonic development. Ontogenesis of CS, IP3R, and Ca(2+)-ATPase accompanied well-defined stages of cerebellum histogenesis and cytogenesis and was accomplished before hatching. High-resolution immunogold electronmicroscopy revealed that, at E18-P1, CS was still largely distributed to the endoplasmic reticulum (ER) lumen and began to be segregated to ER subcompartments (calciosomes) only by P2, whereas the IP3R was concentrated into ER cisternal stacks as early as E18. Both ionotropic and metabotropic plasma membrane receptors were present in dissociated single chicken Purkinje cells from E16 onward, as indicated by measurements of membrane currents (whole cell recording mode) and of cytoplasmic Ca2+ transients monitored with the cell-trappable fluorescent indicator fura 2-acetoxymethyl ester, respectively. Cytoplasmic Ca2+ transients were detected after either activation of glutamate metabotropic receptors, i.e., evidence of IP3-sensitive Ca2+ channels, or application of caffeine, i.e., evidence of ryanodine-sensitive Ca2+ channels. Intracellular Ca2+ stores appear to be functional during embryonic development.

Aging↗

Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat.

The impact of aging on the Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) was investigated using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle (SM) and the relatively fast-twitch gastrocnemius muscle (GM) isolated from adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. In addition, isometric twitch characteristics of SM and GM were determined in situ in adult and aged rats under anesthesia. The rates of ATP-supported Ca2+ uptake by SM SR was markedly lower ( approximately 50%) in the aged compared with adult at varying Ca2+ (0.11-8.24 microM) concentrations. Kinetic analysis of the data revealed age-associated decrease in maximum activity reached (Vmax) and increase in the concentration of Ca2+ giving half of Vmax. In contrast, no significant age-related difference was observed in ATP-supported Ca2+ uptake activity of GM SR. The Ca(2+)-stimulated adenosinetriphosphatase (ATPase) activities and the amount of Ca(2+)-ATPase protein did not vary significantly with aging in SM or GM SR. Also, no significant age-related difference was observed in the content of the ryanodine receptor (Ca(2+)-release channel) or the Ca2+ binding protein, calsequestrin in SM and GM SR. In isometrically contracting SM, the time to peak force, half-relaxation time, and contraction duration were significantly prolonged in the aged compared with adult, whereas there was no age-related difference in maximum developed force. None of these isometric twitch parameters differed significantly with age in the GM. These results demonstrate that the effects of aging on skeletal muscle contractile properties and SR function are muscle specific. Furthermore, the data strongly suggest that impairment in SR Ca2+ pump function, apparently due to uncoupling of ATP hydrolysis from Ca2+ transport, contributes to the age-associated slowing of relaxation in the soleus muscle.

Aging↗

Expression of endoplasmic reticulum stress proteins during skeletal muscle disuse atrophy.

Disuse atrophy of skeletal muscle leads to an upregulation of genes encoding sarcoplasmic reticulum (SR) calcium-handling proteins. Because many of the proteins that are induced with endoplasmic reticulum (ER) stress are ER calcium-handling proteins, we sought to determine whether soleus muscle atrophy was associated with a prototypical ER stress response. Seven days of rat hindlimb unloading did not alter expression of ubiquitous ER stress proteins such as Grp78, calreticulin, and CHOP/GADD-153, nor other proteins that have been shown to be activated by ER stressors such as vinculin, the type I D-myo-inositol 1,4,5-trisphosphate receptor, or protein kinase R, a eukaryotic initiation factor 2 alpha kinase. On the other hand, expression of heme oxygenase-1 (HO-1), an antioxidant ER stress protein, was significantly increased 2.2-fold. In addition, unloading led to an increase in calsequestrin, the muscle-specific SR calcium-binding protein, at both the mRNA (68%) and protein (24%) levels. Although disuse atrophy is associated with a significant remodeling of muscle-specific proteins controlling SR calcium flux, it is not characterized by a prototypical ER stress response. However, the upregulation of HO-1 may indicate ER adaptation to oxidative stress during muscle unloading.

Animals↗

Chronic cardiotrophin-1 stimulation impairs contractile function in reconstituted heart tissue.

Cardiotrophin-1 (CT-1) is known to promote survival but also to induce an elongated morphology of isolated cardiac myocytes, leading to the hypothesis that CT-1, which is chronically augmented in human heart failure, might induce eccentric cardiac hypertrophy and contractile failure. To address this, we used heart tissues reconstituted from neonatal rat cardiac myocytes (engineered heart tissue, EHT) as multicellular in vitro test systems. CT-1 dose-dependently affected contractile function in EHTs. After treatment with 0.1 nM CT-1 (corresponds to plasma levels in humans) for 10 days, twitch tension significantly decreased to 0.30 +/- 0.04 mN (n = 15) vs. 0.45 +/- 0.04 mN (n = 16) in controls. Furthermore, positive inotropic effects of cumulative concentrations of Ca2+ and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension amounted to 0.27 +/- 0.04 mN (n = 15) vs. 0.47 +/- 0.06 mN (n = 16) in controls (P < 0.001). When EHTs were treated for only 5 days, qualitatively similar results were obtained but changes were less pronounced. Immunostaining of whole mount EHT preparations revealed that after CT-1 treatment, the number of nonmyocytes significantly increased by 98% (1 nM, 10 days), and myocytes did not form compact, longitudinally oriented muscle bundles. Interestingly, expression of the Ca2+-handling protein calsequestrin was markedly reduced (69 +/- 7% of control) by treatment with CT-1 (0.1 nM, 10 days). In summary, long-term exposure to CT-1 induces contractile dysfunction in EHTs. Structural changes due to impaired differentiation and/or remodeling of heart tissue may play an important role.

Adrenergic beta-Agonists↗