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PubMed2002

Altered function in atrium of transgenic mice overexpressing triadin 1.

Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, proteins that are important for Ca(2+) release. To better understand the role of triadin 1 in SR-Ca(2+) release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac triadin 1 under control of the alpha-myosin heavy chain (MHC) promoter. Three-week-old transgenic atria exhibited mild hypertrophy. Finally, atrial weight was increased by 110% in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR Ca(2+)-ATPase isoform. Force of contraction was already decreased in 3-wk-old transgenic atria. The application of caffeine led to a positive inotropic effect in transgenic atria of 3-wk-old mice. Rest pauses resulted in an increased potentiation of force of contraction after restimulation in 3- and 6-wk-old mice and a reduced potentiation of force of contraction in 18-wk-old transgenic mice. Hence, triadin 1 overexpression triggered time-dependent alterations in SR protein expression, Ca(2+) homeostasis, and contractility, indicating for the first time an inhibitory function of triadin 1 on SR-Ca(2+) release in vivo.

Age Factors↗
PubMed2003

Effects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes.

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca(2+)](i) transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger (NCX1), Na(+)-K(+)-ATPase, and calsequestrin levels. SR Ca(2+) uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca(2+)](o), rASPLM myocytes had significantly (P < 0.003) lower contraction and [Ca(2+)](i) transient amplitudes than control GFP myocytes. At 5 mM [Ca(2+)](o), both contraction and [Ca(2+)](i) transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca(2+)](i) transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca(2+)](i) transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

Adenoviridae↗
PubMed1997

Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart.

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

Animals↗
PubMed1998

Effects of aging on sarcoplasmic reticulum Ca2+-cycling proteins and their phosphorylation in rat myocardium.

Diminished Ca2+-sequestering activity of the sarcoplasmic reticulum (SR) is implicated in the age-associated slowing of cardiac muscle relaxation. In attempting to further define the underlying mechanisms, the present study investigated the impact of aging on the contents of major SR Ca2+-cycling proteins and SR protein phosphorylation by endogenous Ca2+/calmodulin-dependent protein kinase (CaM kinase). The studies were performed using homogenates and SR vesicles derived from the ventricular myocardium of adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. Western immunoblotting analysis showed no significant age-related difference in the relative amounts of ryanodine receptor-Ca2+-release channel (RyR-CRC), the Ca2+-storage protein calsequestrin, Ca2+-pumping ATPase (Ca2+-ATPase), and Ca2+-ATPase-regulatory protein phospholamban (PLB) in SR or homogenate. On the other hand, the relative amount of immunoreactive CaM kinase II (delta-isoform) was approximately 50% lower in the aged heart. CaM kinase-mediated phosphorylation of RyR-CRC, Ca2+-ATPase, and PLB was reduced significantly ( approximately 25-40%) in the aged compared with adult rat. ATP-dependent Ca2+-uptake activity of SR and the stimulatory effect of calmodulin on Ca2+ uptake were also reduced significantly with aging. Treatment of SR vesicles with anti-PLB antibody (PLBab) invoked relatively less stimulation of Ca2+ uptake in the aged (</=26%) compared with the adult (</=65%) rat. Ca2+-ATPase but not PLB underwent phosphorylation by CaM kinase in PLBab-treated SR with resultant stimulation of Ca2+ uptake. The rates of Ca2+ uptake by PLBab-treated SR were significantly lower (45-55%) in the aged compared with adult rat in the absence and presence of calmodulin. These findings imply that changes in the intrinsic functional properties of SR Ca2+-cycling proteins and/or their phosphorylation-dependent regulation contribute to impaired SR function in the aging heart.

Aging↗
PubMed2000

Biochemical mechanism(s) of stunning in conscious dogs.

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% (P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.

Animals↗
PubMed2000

Sprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca(2+)](i) decline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca(2+) regulation, the present study examined whether 6-8 wk of high-intensity sprint training (HIST) would restore [Ca(2+)](i) dynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant (P </= 0.05) decreases in whole cell capacitances [Sham-Sed 179 +/-12 (n = 20); MI-Sed 226 +/- 7 (n = 20); MI-HIST 183 +/- 11 pF (n = 19)]. HIST significantly (P < 0.0001) restored both systolic [Ca(2+)](i) [Sham-Sed 421 +/- 9 (n = 79); MI-Sed 350 +/- 6 (n = 70); MI-HIST 399 +/- 9 nM (n = 70)] and half-time of [Ca(2+)](i) decline (Sham-Sed 0. 197 +/- 0.005; MI-Sed 0.247 +/- 0.006; MI-HIST 0.195 +/- 0.006 s) toward normal. Compared with Sham-Sed myocytes, SR Ca(2+)-ATPase expression significantly (P < 0.001) decreased by 44% in MI-Sed myocytes. Surprisingly, expression of SR Ca(2+)-ATPase was further reduced in MI-HIST myocytes to 26% of that measured in Sham-Sed myocytes. There were no differences in calsequestrin expression among the three groups. Expression of phospholamban was not different between Sham-Sed and MI-Sed myocytes but was significantly (P < 0.01) reduced in MI-HIST myocytes by 25%. Our results indicate that HIST instituted shortly after MI improves [Ca(2+)](i) dynamics in surviving myocytes. Improvement in SR function by HIST is mediated not by increased SR Ca(2+)-ATPase expression, but by modulating phospholamban regulation of SR Ca(2+)-ATPase activity.

Animals↗
PubMed2004

Sprint training improves contractility in postinfarction rat myocytes: role of Na+/Ca2+ exchange.

Previous studies in adult myocytes isolated from rat hearts 3-9 wk after myocardial infarction (MI) demonstrated abnormal contractility and decreased Na(+)/Ca(2+) exchanger (NCX1) activity. In addition, a program of high-intensity sprint training (HIST) instituted shortly after MI restored both contractility and NCX1 activity toward normal. The present study examined the hypotheses that reduced NCX1 activity caused abnormal contractility in myocytes isolated from sedentary (Sed) rat hearts 9-11 wk after coronary artery ligation and that HIST ameliorated contractile dysfunction in post-MI myocytes by increasing NCX1 activity. The approach was to upregulate NCX1 in MI-sedentary (MISed) myocytes and downregulate NCX1 in MI-exercised (MIHIST) myocytes by adenovirus-mediated gene transfer. Overexpression of NCX1 in MISed myocytes did not affect sarco(endo)plasmic reticulum Ca(2+)-ATPase and calsequestrin levels but rescued contractile abnormalities observed in MISed myocytes. That is, at 5 mM extracellular Ca(2+) concentration, the subnormal contraction amplitude in MISed myocytes (compared with Sham myocytes) was increased toward normal by NCX1 overexpression, whereas at 0.6 mM extracellular Ca(2+) concentration the supernormal contraction amplitude in MISed myocytes was lowered. Conversely, NCX1 downregulation by antisense in MIHIST myocytes abolished the beneficial effects of HIST on contraction amplitudes in MI myocytes. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in post-MI myocytes and that HIST ameliorated contractile dysfunction in post-MI myocytes partly by enhancing NCX1 activity.

Adenoviridae↗
PubMed2002

Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.

The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes.

Animals↗
PubMed2001

Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

Animals↗
PubMed2002

Genetic modifier loci affecting survival and cardiac function in murine dilated cardiomyopathy.

BACKGROUND: Understanding the role for genetic factors in human heart failure is difficult because environmental factors cannot be standardized and genetic variation is great. One approach to identify genes that modify disease outcome is to use mouse models that show strong genetic variation of the disease phenotype. METHODS AND RESULTS: In this study, we used transgenic mice that develop severe dilated cardiomyopathy due to the cardiac-specific overexpression of calsequestrin. Transgenic mice showed marked strain-specific variation of cardiac function and survival, independent of transgene expression. A reciprocal backcross strategy was employed using two inbred strains showing distinct differences in survival and cardiac function. To map the genes that modified the heart failure phenotype, progeny from the 2 reciprocal backcrosses were used in a genome-wide scan for linkage. We identified two loci significantly linked to survival with a maximum likelihood ratio statistic of 36.2 (LOD score approximately 7.8) on chromosome 2 and of 26.5 (LOD score approximately 5.7) on chromosome 3. The chromosome 3 locus was also significantly linked to cardiac function with a maximum likelihood ratio statistic of 42.9 (LOD score approximately 9.3). Because only a single strong modifier locus was found in each backcross, we applied a haplotype analysis to map crossovers and successfully narrowed the critical intervals for each locus. CONCLUSION: Using a sensitized mouse model, we identified major modifier loci that affect the genetically complex disease of heart failure. This approach should allow the rapid identification of candidate genes involved in disease susceptibility in human populations and new insights into the pathogenesis of heart failure.

Animals↗
PubMed1999

Subunit expression of the cardiac L-type calcium channel is differentially regulated in diastolic heart failure of the cardiac allograft.

BACKGROUND: Left ventricular diastolic dysfunction is a major cause of cardiac allograft failure. Multimeric L-type calcium channels (alpha1-, alpha2/delta-, and beta-subunits) are essential for excitation/contraction coupling in the heart. Their gene expression was studied in allografts that developed diastolic heart failure. METHODS AND RESULTS: mRNA levels of calcium channel subunits were measured by competitive reverse transcriptase-polymerase chain reaction in microbiopsy samples from the interventricular septum. Size and tissue variabilities between biopsy samples were assessed by determination of cardiac calsequestrin mRNA levels. In the cardiac allografts studied, mRNA levels in microbiopsy samples were considered to represent left ventricular gene expression, because septal and left ventricular gene expression in Northern blots was equivalent, and left ventricles contracted homogeneously. Biopsy samples (n=72) were taken from allografts with normal left ventricular end-diastolic pressure (LVEDP; 8 to 13 mm Hg; n=30), moderately elevated LVEDP (14 to 18 mm Hg; n=26), and elevated LVEDP (19 to 28 mm Hg; n=16). Increased LVEDP was related to slowed diastolic relaxation determined by the time constant tau (r2=0.86), whereas systolic performance (dP/dt; ejection fraction) was preserved. With increasing LVEDP, mRNA levels of the pore-forming alpha1c-subunit (n=15) and of the regulatory alpha2/delta-subunit (n=17) remained unchanged but decreased exponentially (r2=-0.83) for the regulatory beta-subunit (n=40). Compared with cardiac allografts with normal LVEDP (n=15), beta-subunit mRNA level was reduced by 75% at elevated LVEDP (n=9; P=0.012). In an explanted, diastolically failing cardiac allograft, beta-subunit expression was reduced correspondingly by 72% and 76% on the mRNA level in septal and left ventricular myocardium and by 80% on the protein level. CONCLUSIONS: The downregulated expression of the calcium channel beta-subunit might contribute to altered calcium handling in diastolically failing cardiac allografts.

Adult↗
PubMed1999

Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na+,K+-ATPase activity but unchanged Na+-Ca2+ exchanger protein levels in human heart failure.

BACKGROUND: Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+, K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. METHODS AND RESULTS: Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic alpha1-, alpha2-, alpha3-, and glycoprotein beta1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain-binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase alpha1- (0.62+/-0.06 of control), alpha3- (0.70+/-0.09), and beta1- (0.61+/-0.04) but not alpha2-isoforms were significantly reduced (P<0.01), whereas levels of NCE (0.92+/-0.13 of control) and calsequestrin (0.98+/-0.06) remained unchanged. Both Na+,K+-ATPase activity (NF: 1.9+/-0.29; NYHA class IV: 1.1+/-0.17 micromol ATP/min per milligram of protein) and the 3H-ouabain binding sites (Bmax NF: 15.9+/-1.9 pmol/mg protein; NYHA class IV: 9.7+/-1.5) were reduced in NYHA class IV and correlated significantly to each other (r2=0. 73; P<0.0001), as did beta1-subunit expression. In left ventricular papillary muscle strips from NYHA class IV compared with nonfailing tissue the Na+-channel modulator BDF 9198 exerted an increase in force of contraction with unchanged effectiveness but enhanced potency. CONCLUSIONS: The enhanced sensitivity of failing human myocardium toward cardiac glycosides may be, at least in part, attributed to a reduced protein expression and activity of the sarcolemmal Na+,K+-ATPase without a change in Na+-Ca2+ exchanger protein expression.

Adolescent↗
PubMed2006

Clinical phenotype and functional characterization of CASQ2 mutations associated with catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. METHODS AND RESULTS: We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2(G112+5X)) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2(L167H)) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2(G112+5X) did not bind Ca2+, whereas CASQ2(L167H) had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2(G112+5X). CONCLUSIONS: CASQ2(L167H) and CASQ2(G112+5X) alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2(G112+5X) displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Amino Acid Substitution↗
PubMed1996

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

Animals↗
PubMed2002

The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model.

Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy, an inherited human disorder with increased ventricular wall thickness, myocyte hypertrophy, and disarray. To understand the early consequences of mutant sarcomere proteins, we have studied mice (designated alphaMHC(403/+)) bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain. We demonstrate that Ca(2+) is reduced in the sarcoplasmic reticulum of alphaMHC(403/+) mice, and levels of the sarcoplasmic reticulum Ca(2+)-binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca(2+) in these animals is seen in their decreased expression of the ryanodine receptor Ca(2+)-release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca(2+) channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in alphaMHC(403/+) mice. We conclude that disruption of sarcoplasmic reticulum Ca(2+) homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca(2+) channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations.

Animals↗
PubMed2006

Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia.

Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca2+ storage protein essential for SR Ca2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca2+ leak, resulting in premature spontaneous SR Ca2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca2+ storage, lack of Casq2 also causes increased diastolic SR Ca2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

Alleles↗
PubMed2005

The assembly of calcium release units in cardiac muscle.

Calcium release units (CRUs) are constituted of specialized junctional domains of the sarcoplasmic reticulum (jSR) that bear calcium release channels, also called ryanodine receptors (RyRs). In cardiac muscle, CRUs come in three subtypes that differ in geometry, but have common molecular components. Peripheral couplings are formed by a junction of the jSR with the plasmalemma; dyads occur where the jSR is associated with transverse (T)-tubules; corbular SR is a jSR domain that is located within the cells and bears RyRs but does not associate with either plasmalemma or T-tubules. Using transmission electron microscopy, this study followed the formation of CRUs and their accrual of four components: the L-type channel dihydropyridine receptors (DHPRs) of plasmalemma/T-tubules; the RyRs of jSR; triadin (Tr) and junctin (JnC), two homologous components of the jSR membrane; and calsequestrin (CSQ), the internal calcium binding proteins. During differentiation, peripheral couplings are formed first and the others follow. RyRs and DHPRs are targeted to subdomains of the CRUs that face each other and are acquired in a concerted manner. Overexpressions of either junction (JnC or Tr) and of CSQ, singly or in conjunction, shed light on the specific role of JnC in the structural development, organization, and maintenance of jSR cisternae and on the independent synthetic pathways and targeting of JnC and CSQ. In addition, the structural cues provided by the overexpression models allow us to define sequential steps in the synthetic pathway for JnC and CSQ and their targeting to the CRUs of differentiating myocardium.

Animals↗
PubMed2006

The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1.

JP-45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), colocalizes with its Ca2+ -release channel (the ryanodine receptor), and interacts with calsequestrin and the skeletal-muscle dihydropyridine receptor Cav1. We have identified the domains of JP-45 and the Cav1.1 involved in this interaction, and investigated the functional effect of JP-45. The cytoplasmic domain of JP-45, comprising residues 1-80, interacts with Cav1.1. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1, the I-II loop and the C-terminal region. Interaction between JP-45 and the I-II loop occurs through the alpha-interacting domain in the I-II loop. beta1a, a Cav1 subunit, also interacts with the cytosolic domain of JP-45, and its presence drastically reduces the interaction between JP-45 and the I-II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge-movement and shifted VQ1/2 to a more negative potential (-10 mV). JP-45 depletion decreased both the content of Cav1.1 and peak charge-movements. Our data demonstrate that JP-45 is an important protein for functional expression of voltage-dependent Ca2+ channels.

Animals↗