Overexpression Of Calsequestrin Research Articles

Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.

Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model.

Sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and its inhibitory protein called phospholamban (PLN) are pivotal for Ca2+ handling in cardiomyocyte and are known that their expression level and activity were changed in the heart failure patients. To examine whether PLN inhibition can improve survival rate as well as cardiac function in heart failure, we performed PLN ablation in calsequestrin overexpressing (CSQ-Tg) mice, a severe heart failure model, using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system. According this method, generation rate of PLN wild type mice (PLN copy >0.95) and PLN homozygous knockout (KO) mice (PLN copy <0.05) were 39.1% and 10.5%, respectively. While CSQ overexpression causes severe heart failure symptoms and premature death, a significant ameliorating effect on survival rate was observed in PLN homozygous KO/CSQ-Tg mice compared to PLN wild type/CSQ-Tg mice (median survival days are 55 and 50 days, respectively). Measurement of cardiac function with cardiac catheterization at the age of 5 weeks revealed that PLN ablation improved cardiac function in CSQ-Tg mice without affecting heart rate and blood pressure. Furthermore, increases in atrial and lung weight, an index of congestion, were significantly inhibited by PLN ablation. These results suggest that PLN deletion would be a promising approach to improve both mortality and cardiac function in the heart failure.

Modulation of SRCa2+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.

Severe Cardiac Hypertrophy and Depressed Contractility in Transgenic Mice with Co-Overexpression of Calsequestrin and SERCA2a

Cardiac-specific overexpression of calsequestrin (CSQ) is associated with hypertrophy, depressed contractility, impaired SR Ca2+ release and signs of heart failure. The Ca2+ATPase (SERCA2a) represents the major determinant of myocardial contractility. Its stable or transient expression results in increased contractile parameters and higher Ca2+ transients. Hence, we tested whether the additional expression of SERCA2a in CSQ-overexpressing hearts results in beneficial functional effects. For this purpose, we generated double transgenic mice overexpressing both CSQ and SERCA2a (TGCxS) by cross-breeding of single transgenic mouse lines (TGCSQ and TGSER). SERCA2a protein expression was increased by 1.4-fold in both TGSER and TGCxS compared to wild-type (WT) hearts. Consistently, at submaximal free Ca2+ levels (30 nmoL/L), 45Ca2+ uptake reached 12%, 25%, 14% and 30% of maximal Ca2+ levels in WT, TGSER, TGCSQ and TGCxS, respectively (n=5, P<0.05). CSQ was overexpressed by 10-fold in both TGCSQ and TGCxS compared to WT. The expression of triadin was decreased by 53% in both TGCSQ and TGCxS compared to WT (P<0.05). The ryanodine receptor expression was reduced by 44%, 33% and 86% in TGCSQ, TGSER and TGCxS, respectively, compared to WT (P<0.05). In addition, co-overexpression of CSQ and SERCA2a was associated with severe cardiac hypertrophy. We observed a heart weight to body weight ratio of 4.4±0.1, 4.9±0.1, 8.9±0.3 and 12.5±1.0 mg/g in WT, TGSER, TGCSQ and TGCxS, respectively (n=7, P<0.05). Stained tissue sections revealed an enhanced interstitial fibrosis in ventricles of both TGCSQ and TGCxS. Functional analysis was performed on isolated working heart preparations. Contractility (+dP/dt) was depressed in TGCxS (1378±144 mmHg/s) compared to WT, TGSER and TGCSQ (2461±119, 3644±161 and 1879±105 mmHg/s, respectively, n=6, P<0.05). We conclude that the additional overexpression of SERCA2a failed to rescue the impaired cardiac phenotype of CSQ transgenic mice.

Sarcoplasmic reticulum Ca 2+ release in neonatal rat cardiac myocytes

In the neonatal mammalian heart, the role of ryanodine receptor (= Ca 2+ release channel)-mediated sarcoplasmic reticulum (SR) Ca 2+ release for excitation–contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca 2+ transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca 2+ content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca 2+ storage but unchanged Ca 2+ signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation–contraction coupling in the developing mammalian heart.

Paradoxical Loss of Sarcoplasmic Reticulum Calcium Release Refractoriness Caused by Overexpressing Calsequestrin in Cardiac Muscle

L-type Ca current (ICa) triggers Carelease from the sarcoplasmic reticulum (SR) by activating RyR2 Ca release channels. Here we probe the role of the SR protein calsequestrin in the regulation of SR Ca2+ release. Voltage-clamped cardiomyocytes loaded with Fluo-4 were stimulated with 1 Hz train (S1 stimuli). Ca2+ release was activated with ICa tail currents to maintain constant trigger. Application of premature extra stimuli (S2) at successively shorter S1-S2 coupling interval resulted in significant depression of Ca2+ release during S2 stimuli in wild-type myocytes. In calsequestrin knockout myocytes (Casq2 KO), the amplitudes of S2 and S1 were the same even at shortest S1-S2 intervals (Figure). This result suggests that calsequestrin is the primary regulator of release refractoriness. To test this hypothesis further, we next used myocytes from calsequestrin overexpressing mice (Casq2 OverX). Casq2 OverX cardiomyocytes exhibit significantly depressed Ca release during S1 transients despite increased SR Ca2+ content. Surprisingly, Ca release amplitudes during the premature S2 stimulus exceeded S1 at short S1-S2 intervals (Figure). Thus, either too little or too much calsequestrin causes loss of SR Ca release refractoriness. Supported by NIH-RO1-HL88635.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Losartan reduces mortality in a genetic model of heart failure

Altered Ca(2+) homoeostasis accompanies heart failure. As a model of heart failure, transgenic mice (TG) with selective overexpression of calsequestrin (CSQ) in the heart were used. CSQ is the main Ca(2+) binding protein in the lumen of the junctional sarcoplasmic reticulum. Overexpression of CSQ leads to hypertrophy, fibrosis, heart failure, cardiac arrhythmias, and ultimately premature death compared to littermate controls (WT). In the present study, cardiac hypertrophy was noted at 2 months of age (relative heart weight 6.4 +/- 0.2 mg/g in WT and 11.2 +/- 0.3 mg/g in TG, n = 7, p < 0.05) which progressed at 5 months of age (relative heart weight 15.5 +/- 1.1 mg/g in TG, n = 11). Furthermore, an increased degree of fibrosis (from 0.29 +/- 0.04 in WT to 0.77 +/- 0.06 in TG, n = 8, p < 0.05) was quantified by sirius red staining. Cardiac function was greatly impaired in TG as exemplified by reduced pressure development and cardiac arrhythmias. It is hypothesized that losartan, an inhibitor of angiotensin II receptors, might be able to attenuate these detrimental effects. Hence, TG and WT were treated for 1 or 4 months perorally with losartan (5 mg/kg/day) or solvent alone (control conditions) starting at 4 weeks of age. Under control conditions, none of the WT died within the observation period whereas all TG died within 9 months. Losartan treatment reduced the mortality of TG: Mean life span was raised from 116 to 193 days (n = 18 end, p < 0.05). Likewise, losartan reduced relative heart weight and the degree of fibrosis. In addition, losartan improved hemodynamic parameters, like left ventricular pressure and its first derivative. However, losartan treatment did not modify overexpression of CSQ in the heart of TG. These results imply that the angiotensin II receptor (type 1) contributes to heart failure due to CSQ overexpression, as its blockade improved survival.

Losartan reduces mortality in a model of heart failure

Calsequestrin (CSQ) is the main Ca2+ store in the cardiac sarcoplasmic reticulum (SR). Overexpression of CSQ selectively in the heart of transgenic mice (TG) under the control of the alpha myosin heavy chain promoter leads to hypertrophy, fibrosis, heart failure, cardiac arrhythmias and ultimately premature death compared to littermate controls (WT). We hypothesized that losartan, an inhibitor of angiotensin II receptors, may be able to abrogate these detrimental effects. Hence, TG and WT were treated for about 2 or 5 months perorally with losartan (5 mg/Kg body weight) or solvent alone. Under control conditions (solvent alone), none of the WT died within the observation period whereas all TG died within 9 months. In the treatment group the mortality of TG was significantly reduced: mean life span was raised from 116 d to 192.5 d (n=18). Likewise, the degree of fibrosis was reduced. Hemodynamic parameters (pressure; dp/dt) were improved. However, the relative heart weight remained unchanged. Likewise, the expression of CSQ in the heart was unchanged by losartan treatment. In conclusion, the renin angiotensin system is apparently involved in heart failure due to CSQ overexpression, because its blockade improves survival (supported by the DFG).

Interferon-γ Induces Chronic Active Myocarditis and Cardiomyopathy in Transgenic Mice

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-alpha is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine interferon (IFN)-gamma remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-gamma transgenic mice, which constitutively express IFN-gamma in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-gamma mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4(+) and CD8(+) T cells but also Mac2(+) (galectin 3(+)) macrophages and CD11c(+) dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-gamma overexpression. IFN-gamma-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-alpha. Myotoxic IFN-gamma effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-gamma effect. The precise mechanism of IFN-gamma cardiotoxicity remains to be elucidated.

Oxidative Phenotype Protects Myofibers from Pathological Insults Induced by Chronic Heart Failure in Mice

The fiber specificity of skeletal muscle abnormalities in chronic heart failure (CHF) has not been defined. We show here that transgenic mice (8 weeks old) with cardiac-specific overexpression of calsequestrin developed CHF (50.9% decrease in fractional shortening and 56.4% increase in lung weight, P<0.001), cachexia (37.8% decrease in body weight, P<0.001), and exercise intolerance (69.3% decrease in running distance to exhaustion, P<0.001) without a significant change in muscle fiber-type composition. Slow oxidative soleus muscle maintained muscle mass, whereas fast glycolytic tibialis anterior and plantaris muscles underwent atrophy (11.6 and 13.3%, respectively; P<0.05). In plantaris muscle, glycolytic type IId/x and IIb, but not oxidative type I and IIa, fibers displayed significant decreases in cross-sectional area (20.3%, P<0.05). Fast glycolytic white vastus lateralis muscle showed sarcomere degeneration and decreased cytochrome c oxidase IV (39.5%, P<0.01) and peroxisome proliferator-activated receptor gamma co-activator 1alpha protein expression (30.3%, P<0.01) along with a dramatic induction of the MAFbx/Atrogin-1 mRNA. These findings suggest that exercise intolerance can occur in CHF without fiber type switching in skeletal muscle and that oxidative phenotype renders myofibers resistant to pathological insults induced by CHF.

Cardiac Overexpression of the Human P2X4 Receptor Exhibits an Anti‐hypertrophic Phenotype

Cardiac-specific overexpression of the human P2X4 receptor (hP2X4R) rescues the calsequestrin (CSQ) overexpression model of heart failure (Am. J. Physiol. 287: H1096-H1103, 2004). The mechanism by which P2X4R overexpression rescues heart failure is unknown. Our objective was to test the hypothesis that reversal or attenuation of hypertrophy is a mechanism that underlies the beneficial effect of the P2X4R overexpression. In age-matched animals (~ 12 weeks), the CSQ mice showed significantly enlarged hearts with elevated heart/body weight ratio (15.1±0.9 mg/g, n=6, ± SD) as compared to the binary (9.1 ± 1.4), non-transgenic (NTG) (4.8±0.1, n=8) or P2X4R (4.7±0.3, n=8) animals (P<0.01). Cell length and cross-sectional area of cardiac myocytes were quantified as indices of myocyte hypertrophy. Cell length of binary mice (150.6 ± 1.6 μm, n=54) was significantly less than that of the CSQ mice (157.7 ± 2.1 μm, n=60 cells, ± SE, P<0.05). Similarly, myocyte cross section area was significantly reduced in binary (229.6 ± 4.1) vs. CSQ (349.6 ± 5.9) mice (P<0.01). Conclusion: Reversal of hypertrophy, arising from decreases in both myocyte size and extracellular collagen content, is a mechanism by which the heart failure is rescued in this model of severe cardiomyopathy.

Effects of calsequestrin over-expression on excitation–contraction coupling in isolated rabbit cardiomyocytes

This study investigated the role of calsequestrin (CSQ) in the control of excitation-contraction (E-C) coupling in the heart. CSQ over-expression was induced in isolated rabbit ventricular cardiomyocytes using an adenovirus coding for rabbit CSQ (Ad-CSQ). After 24 h of culture, CSQ protein expression was increased by 58+/-18% (n=10). An adenovirus coding for beta-galactosidase (Ad-LacZ) was used as a control. In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (I(Ca,L)) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by approximately 78%. Doubling the external Ca2+ concentration in the control group (Ad-LacZ) increased the LTCC amplitude to a similar degree (85+/-6%), but increased the Ca2+ transient amplitude by 149+/-13%. This suggests that SR Ca2+ release may be inhibited upon CSQ over-expression. Alternatively, nifedipine (0.5 microM) was used to reduce I(Ca,L) in Ad-CSQ-transfected cells to values comparable to control (Ad-LacZ). Under these conditions, Ca2+ transient amplitude was not different from Ad-LacZ, but the SR Ca2+ content was approximately 60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (I(NCX)). The cause of the increased I(Ca,L) is unknown. No change in the expression level of the alpha1-subunit of the L-type Ca channel was observed. beta-Escin-permeabilized cardiomyocytes were used to study Ca2+ sparks imaged with Fluo-3 at 145-155 nmol/L [Ca2+]. Spontaneous Ca2+ spark frequency, duration, width, and amplitude were unchanged in the Ad-CSQ group, but SR Ca2+ content was 48% higher than Ad-LacZ. CSQ over-expression increased SR Ca2+ content but reduced the gain of E-C coupling in rabbit cardiomyocytes.

QTL mapping in a mouse model of cardiomyopathy reveals an ancestral modifier allele affecting heart function and survival

The progression from myocardial hypertrophy to heart failure is a complex process, involving genetic and environmental factors. Elucidating the genetic components contributing to heart failure has been difficult, largely because of the heterogeneity of human populations. We have employed a strategy to map genetic loci that modify the heart failure phenotype in a transgenic mouse model of cardiomyopathy caused by cardiac-specific overexpression of calsequestrin. Strain-specific differences in both cardiac function and survival are observed when the transgene is moved into different inbred mouse strains. We have previously reported linkage results from mapping in reciprocal backcrosses between C57/BL6 (BL6) and DBA/2J (DBA) and a backcross between DBA/AKR and AKR. Here we report the results of a genome-wide linkage scan in the reciprocal backcross between DBA/AKR and DBA. We identified one novel locus on Chromosome (Chr) 18 that affects heart function and a second on Chr 3 that shows significant linkage to both survival and heart function. Intriguingly, the Chr 3 allele of AKR shows a susceptibility effect on phenotype, whereas the overall effect of the AKR genetic background is protective. The Chr 3 locus also completely overlaps the Hrtfm2 locus, which was previously mapped in crosses between DBA and BL6. Mapping the same QTL in two different crosses allowed us to use ancestral haplotypes to narrow the candidate gene interval from 9 to 2 Mb. Identification of the genes at these QTLs in the mouse will provide novel candidate genes that can be evaluated for their role in human heart failure.

Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression

Desensitization and down-regulation of beta-adrenergic receptors (betaARs) are prominent features of heart failure largely mediated by increased levels of betaAR kinase-1 (betaARK1). beta-adrenergic receptor kinase 1 interacts with phosphoinositide-3 kinase (PI3K), and upon agonist stimulation, the betaARK1/PI3K complex is recruited to agonist-stimulated betaARs. Here we tested the hypothesis that in vivo selective inhibition of betaARK1-associated PI3K activity would preserve betaAR signaling and, therefore, improve cardiac function and survival in experimental heart failure. We used a murine model of heart failure induced by calsequestrin (CSQ) cardiac-specific overexpression; CSQ mice were crossed with mice overexpressing in the heart a catalytically inactive PI3Kgamma (PI3Kgamma(inact)) to competitively displace endogenous PI3K from betaARK1. Catalytically inactive PI3KgammaPI3K overexpression in CSQ mice inhibited betaARK1-associated PI3K activity, normalized betaAR levels, and preserved betaAR responsiveness to isoproterenol (ISO). Restoration of betaAR signaling via PI3Kgamma(inact) overexpression resulted in marked improvement of cardiac function and a significant prolongation of survival. Importantly, the effects of PI3Kgamma(inact) overexpression were restricted to betaAR signaling, because cellular PI3K signaling was unaltered, as shown by the similar activation of multiple downstream signaling pathways in both CSQ and CSQ/PI3Kgamma(inact) mice. These data in the CSQ model of cardiac dysfunction indicate that membrane-targeted PI3K activity plays a detrimental role in heart failure, and its inhibition represents a novel therapeutic approach to ameliorate cardiac dysfunction and improve survival.

A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy.

The P2X4 purinergic receptor (P2X4R) is a ligand-gated ion channel. Its activation by extracellular ATP results in Ca2+ influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 +/- 91 days for the binary CSQ/P2X4R mouse, n = 35) of the CSQ mouse (71.3 +/- 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored beta-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and +/-dP/dt. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure.

Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene.

Several mouse models have already proved valuable for investigating hypertrophic responses to cardiac stress. Here, we characterize one caused by a well defined single copy transgene, RenTgMK, that genetically clamps plasma renin and thence angiotensin II at high levels. All of the transgenic males develop concentric cardiac hypertrophy with fibrosis but without dilatation. Over half die suddenly aged 6-8 months. Telemetry showed disturbances in diurnal rhythms a few days before death and, later, electrocardiographic disturbances comparable to those in humans with congestive heart failure. Expression of seven hypertrophy-related genes in this and two categorically different models (lack of atrial natriuretic peptide receptor A; overexpression of calsequestrin) were compared. Statistical analyses show that ventricular expressions of the genes coding for atrial natriuretic peptide, beta myosin heavy chain, medium chain acyl-CoA dehydrogenase, and adrenomedullin correlate equally well with the degree of hypertrophy, although their ranges of expression are, respectively, 50-, 30-, 10-, and 3-fold.

Manipulating cardiac contractility in heart failure: data from mice and men.

In the year 1990, cardiovascular disease surpassed infectious diseases as the leading cause of death worldwide, and by the year 2020 it is predicted to be the leading cause of all disability.1,2 In the United States, cardiovascular disease has been estimated to account for 44% of the nation’s mortality and is a leading cause of morbidity.3,4 Heart failure afflicts an estimated 5 million Americans, with ≈400 000 new individuals diagnosed each year at an annual cost of more than $20 billion.4–6 The alarming reality behind these statistics is our current lack of an effective therapy to repair or otherwise reverse severe forms of cardiac dysfunction and pathological remodeling associated with heart failure. Typically, heart failure is the final culmination of protracted disease states precipitated by underlying hypertension, ischemic disease and atherosclerosis, valvular insufficiency, viral myocarditis, or mutations in genes encoding sarcomeric proteins.6 Given these diverse etiologies, it is not surprising that the final phenotypic manifestations of heart failure can also vary considerably, although dilated cardiomyopathy is the most common. This syndrome is characterized by a progressive loss in contractility and ejection fraction, ventricular chamber dilatation, ventricular wall thinning, increased peripheral vascular resistance, and dysregulated fluid homeostasis. The predominant therapeutic strategy used over the past two decades for treating such patients has been based in pharmacological manipulation of cardiac contractility.7–9 Initially, positive inotropic agents were used as a means of enhancing cardiac pump function aimed at alleviating congestive symptomology. However, use of positive inotropes is now indicated only as a means of acutely bridging patients in severe heart failure because these agents actually worsen prognosis in individuals with somewhat more stable heart failure.8 More recently, pharmacological blockade of β-adrenergic receptors has emerged as the favored treatment for individuals in heart failure. β-Adrenergic receptor antagonists initially …

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.

Two quantitative trait loci on chromosomes 4 and 18 modify mice survival in a model of heart failure

Survival of heart failure patients depends on several factors including the etiology of the disease and the modality of its treatment. However, a significant component of the variability of heart failure phenotype is due to differences in the genetic background of patients. To identify the genes that modify the outcome of the disease, we have used an experimental model of heart failure induced by cardiac specific overexpression of calsequestrin (CSQ) in mice. CSQ overexpression leads to a dilated cardiomyopathy with a strong strain-specific variability and this model can be used to identify quantitative trait loci (QTL) modifying heart failure phenotype.

Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins

Ca 2+ storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca 2+-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.