Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.     

On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca(2+) release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca(2+) sensitivity of the Ca(2+) release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca(2+) transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca(2+) homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca(2+) transients showed a decreased maximum Ca(2+) amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca(2+) homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.     

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling

In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).     

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.     

Regulation of myocardial function by histidine-rich, calcium-binding protein

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.     

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.     

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca(2+) transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val(49) --> Gly mutant (2-fold), which is a superinhibitor of SR Ca(2+)-ATPase affinity for Ca(2+), were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC(50) level of SR Ca(2+) uptake for Ca(2+) (0.67 +/- 0.09 microm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca(2+) signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired beta-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca(2+) cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.     

Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin

Junctin is a transmembrane protein of the cardiac junctional sarcoplasmic reticulum (SR) that binds to the ryanodine receptor, calsequestrin, and triadin 1. This quaternary protein complex is thought to facilitate SR Ca2+ release. To improve our understanding of the contribution of junctin to the regulation of SR function, we examined the age-dependent effects of junctin overexpression in the atrium of 3-, 6-, and 18-wk-old transgenic mice. The ratio of atrial weight and body weight was unchanged between junctin-overexpressing (JCN) and wild-type (WT) mice at all ages investigated (n=6-8). The protein expression of triadin 1 was decreased starting in 3-wk-old JCN atria (by 69%), whereas the expression of the ryanodine receptor was diminished in 6- (by 48%) and 18-wk-old (by 57%) JCN atria compared with age-matched WT atria. Force of contraction was decreased by 35% in 18-wk-old JCN compared with age-matched WT left atrial muscle strips, which was accompanied by a prolonged time of relaxation (48.1 +/- 0.9 vs. 44.2 +/- 0.8 ms, respectively, n=6-8, P <0.05). The spontaneous beating rate of isolated right atria was higher in 18-wk-old JCN mice compared with age-matched WT mice (389 +/- 10 vs. 357 +/- 6 beats/min, respectively, n=6-8, P <0.05). Heart rate was lower by 9% in telemetric ECG recordings in 18-wk-old JCN mice during stress tests. Three-week-old JCN atria exhibited a higher potentiation of force of contraction at rest pauses of 30 s (by 13%) and of 300 s (by 35%), suggesting increased SR Ca2+ content. This was consistent with the higher force of contraction in 3-wk-old JCN atria (by 29%) compared with age-matched WT atria (by 10%) under the administration of caffeine. We conclude that in 3-wk-old atria, junctin overexpression was associated with a reduced expression of triadin 1 resulting in a higher SR Ca2+ load without changes in contractility or heart rate. In 6-wk-old JCN atria, the compensatory downregulation of the ryanodine receptor may offset the effects of junctin overexpression. Finally, the progressive decrease in ryanodine receptor density may contribute to the decreased atrial contractility and lower heart rate during stress in 18-wk-old JCN mice.     

Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction

ANG II type 2 receptor (AT(2)) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca(2+) concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT(2) was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT(2) in the ventricular myocytes and partial colocalization of AT(2) with AT(1). Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca(2+) concentration, 25 degrees C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca(2+)-dependent contractile dysfunction in myocytes from AT(2) transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT(2) expression. Second, activity of the Na(+)/H(+) exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to beta-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to beta-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca(2+) in wild-type myocytes but caused a negative inotropic effect in myocytes from AT(2) transgenic mice. In contrast with beta-adrenergic response, the depressed response to ANG II was related to level of AT(2) overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT(2) has the potential to cause Ca(2+)- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.     

The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.     

Changes in ionic currents and beta-adrenergic receptor signaling in hypertrophied myocytes overexpressing G alpha(q)

Transgenic overexpression of G alpha(q) causes cardiac hypertrophy and depressed contractile responses to beta-adrenergic receptor agonists. The electrophysiological basis of the altered myocardial function was examined in left ventricular myocytes isolated from transgenic (G alpha(q)) mice. Action potential duration was significantly prolonged in G alpha(q) compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K(+) currents, transient outward K(+) currents (I(to)), and Na(+)/Ca(2+) exchange currents were reduced in G alpha(q) myocytes. Consistent with functional measurements, Na(+)/Ca(2+) exchanger gene expression was reduced in G alpha(q) hearts. Kinetics or sensitivity of I(to) to 4-aminopyridine was unchanged, but 4-aminopyridine prolonged the action potential more in G alpha(q) myocytes. Isoproterenol increased L-type Ca(2+) currents (I(Ca)) in both groups, with a similar EC(50), but the maximal response in G alpha(q) myocytes was approximately 24% of that in NTG myocytes. In NTG myocytes, the maximal increase of I(Ca) with isoproterenol or forskolin was similar. In G alpha(q) myocytes, forskolin was more effective and enhanced I(Ca) up to approximately 55% of that in NTG myocytes. These results indicate that the changes in ionic currents and multiple defects in the beta-adrenergic receptor/Ca(2+) channel signaling pathway contribute to altered ventricular function in this model of cardiac hypertrophy.     

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.     

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.     

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.     

The effects of overexpression of the Na+/Ca2+ exchanger on calcium regulation in hypertrophied mouse cardiac myocytes

In cardiac hypertrophy and failure it has been shown that the amount of Na/Ca exchanger protein can increase. Several studies have investigated this modification in overt heart failure. However, the role of Na/Ca exchanger overexpression during the development of hypertrophy is unknown. To address this question we investigated Ca2+ regulation in an early stage of cardiac hypertrophy before signs of heart failure occurred and evaluated the role of Na/Ca exchanger overexpression. Cardiac hypertrophy was induced by a constant infusion of angiotensin II (Ang, 1 microg/min/kg) via an osmotic pump for 14 days. Thereafter, ventricular myocytes from either wild type (NON) or transgenic mice overexpressing the Na/Ca exchanger (TR) were isolated. Myocytes were loaded with indo-1 AM or fluo-4 AM to monitor cytoplasmic [Ca2+] with all experiments performed at 37 degrees C. In myocytes exposed to Ang there was an increase in cell capacitance of more than 20% indicating cellular hypertrophy. Ca2+ transients were prolonged in hypertrophied NON myocytes but not in TR myocytes. Action potentials had a less negative plateau in TR myocytes. Sarcoplasmic reticulum (SR) Ca2+ content, measured using rapid caffeine application, was greater in TR myocytes but unaffected by hypertrophy. Ca2+ spark frequency was significantly greater in TR. Na/Ca exchanger overexpression prevented the prolongation of the Ca2+ transient observed in hypertrophy and maintained a similar SR Ca2+ leak suggesting a compensatory role in Ca2+ regulation in hypertrophied cardiac myocytes from transgenic mice. We suggest this compensatory effect is mediated by increased SR Ca2+ content and faster Ca2+ removal via the Na/Ca exchanger.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.     

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin

Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.     

Transgenic overexpression of the Ca2+-binding protein S100A1 in the heart leads to increased in vivo myocardial contractile performance

S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with approximately 4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after beta-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in beta-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.