Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin

Calsequestrin (CSQ) is the major Ca2+ binding protein of the cardiac sarcoplasmic reticulum (SR). Transgenic mice overexpressing CSQ at the age of 7 weeks exhibit concentric cardiac hypertrophy, and by 13 weeks the condition progresses to dilated cardiomyopathy. The present study used a differential display analysis to identify genes whose expressions are modulated in the CSQ-overexpressing mouse hearts to provide information on the mechanism of transition from concentric cardiac hypertrophy to failure. Cardiac ankyrin repeat protein (CARP), glutathione peroxidase (Gpx1), and genes which participate in the formation of extracellular matrix including decorin, TSC-36, Magp2, Osf2, and SPARC are upregulated in CSQ mouse hearts at 7 and 13 weeks of age compared to those of non-transgenic littermates. In addition, two novel genes without sequence similarities to any known genes are upregulated in CSQ-overexpressing mouse hearts. Several genes are downregulated at 13 weeks, including SR Ca2+-ATPase (SERCA2) and adenine nucleotide translocase 1 (Ant1) genes. Further, a functionally yet unknown gene (NM_026586) previously identified in the mouse wolffian duct is dramatically downregulated in CSQ mice with dilated hearts. Thus, CARP, Gpx1, and genes encoding extracellular matrix proteins may participate in the development of cardiac hypertrophy and fibrosis, and changes in SERCA2, Ant1, and NM_026586 mRNA expression may be involved in transition from concentric to dilated cardiac hypertrophy.     

Knocking down type 2 but not type 1 calsequestrin reduces calcium sequestration and release in C2C12 skeletal muscle myotubes

We examined the roles of type 1 and type 2 calsequestrins (CSQ1 and CSQ2) in stored Ca2+ release of C2C12 skeletal muscle myotubes. Transduction of C2C12 myoblasts with CSQ1 or CSQ2 small interfering RNAs effectively reduced the expression of targeted CSQ protein to near undetectable levels. As compared with control infected or CSQ1 knockdown myotubes, CSQ2 and CSQ1/CSQ2 knockdown myotubes had significantly reduced stored Ca2+ release evoked by activators of intracellular Ca2+ release channel/ryanodine receptor (10 mM caffeine, 200 microM 4-chloro-m-cresol, or 10 mM KCl). Thus, CSQ1 is not essential for effective stored Ca2+ release in C2C12 myotubes despite our in vitro studies suggesting that CSQ1 may enhance ryanodine receptor channel activity. To determine the basis of the reduced stored Ca2+ release in CSQ2 knockdown myotubes, we performed immunoblot analyses and found a significant reduction in both sarco/endoplasmic reticulum Ca2+-ATPase and skeletal muscle ryanodine receptor proteins in CSQ2 and CSQ1/CSQ2 knockdown myotubes. Moreover, these knockdown myotubes exhibited reduced Ca2+ uptake and reduced stored Ca2+ release by UTP (400 microM) that activates a different family of intracellular Ca2+ release channels (inositol 1,4,5-trisphosphate receptors). Taken together, our data suggest that knocking down CSQ2, but not CSQ1, leads to reduced Ca2+ storage and release in C2C12 myotubes.     

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes

Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.     

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.     

Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated.

Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.     

Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment

This study was designed to test the hypothesis that blockade of the renin-angiotensin system improves cardiac function in congestive heart failure by preventing changes in gene expression of sarcoplasmic reticulum (SR) proteins. We employed rats with myocardial infarction (MI) to examine effects of an angiotensin-converting enzyme inhibitor, imidapril, on SR Ca(2+) transport, protein content, and gene expression. Imidapril (1 mg.kg(-1).day(-1)) was given for 4 wk starting 3 wk after coronary artery occlusion. Infarcted rats exhibited a fourfold increase in left ventricular end-diastolic pressure, whereas rates of pressure development and decay were decreased by 60 and 55%, respectively. SR Ca(2+) uptake and Ca(2+) pump ATPase, as well as Ca(2+) release and ryanodine receptor binding activities, were depressed in the failing hearts; protein content and mRNA levels for Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also decreased by approximately 55-65%. Imidapril treatment of infarcted animals improved cardiac performance and attenuated alterations in SR Ca(2+) pump and Ca(2+) release activities. Changes in protein content and mRNA levels for SR Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also prevented by imidapril treatment. Beneficial effects of imidapril on cardiac function and SR Ca(2+) transport were not only seen at different intervals of MI but were also simulated by another angiotensin-converting enzyme inhibitor, enalapril, and an ANG II receptor antagonist, losartan. These results suggest that blockade of the renin-angiotensin system may increase the abundance of mRNA for SR proteins and, thus, may prevent the depression in SR Ca(2+) transport and improve cardiac function in congestive heart failure due to MI.     

Voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter.

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.     

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar heart-to-body weight ratios but express lower amounts of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice. alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF, beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1, GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+ mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump removal caused the agonist-treated hearts and gene expression to regress to control or near-control levels. Thus Egr-1 deficiency caused a blunted catecholamine-induced hypertrophy response and increased sensitivity to stress.     

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.     

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.     

Differential localization and functional role of calsequestrin in growing and differentiated myoblasts

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin- monensin, appear to be incompetent for Ca2+ accumulation.     

A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.