Sarcoplasmic Reticulum Calcium Uptake Research Articles

Use of Inotropic Agents in Patients with Advanced Heart Failure

Abnormalities of cardiac function, with high intraventricular filling pressure and low cardiac output, play a central role in patients with heart failure. Agents with inotropic properties are potentially useful to correct these abnormalities. However, with the exception of digoxin, no inotropic agent has been associated with favourable effects on outcomes. This is likely related to the mechanism of action of current agents, which is based on an increase in intracellular cyclic adenosine monophosphate and calcium concentrations. Novel agents acting through different mechanisms, such as sarcoplasmic reticulum calcium uptake, cardiac myosin and myocardial metabolism, have the potential to improve myocardial efficiency and lower myocardial oxygen consumption. These characteristics might allow a haemodynamic improvement in the absence of untoward effects on the clinical course and prognosis of the patients.

Gene transfer of the sarcoplasmic reticulum pump in the treatment of heart failure

This editorial refers to ‘Augmentation of left ventricularmechanics by recirculation-mediated AAV2/1-SERCA2agene delivery in experimental heart failure’ by JustinMariani et al. published in this issue on page 247–253.Therapy of congestive heart failure was revolutionized more than20 years ago by the introduction of angiotensin-convertingenzyme inhibitors and b-adrenoceptor blocking agents. Bothhave resulted in the improvement of symptoms and prognosis ofpatients with chronic heart failure. In the meantime, device-basedtherapies have been added to the therapeutic armamentarium.However, all attempts to directly address molecular alterations rel-evant to heart failure pathophysiology as treatment strategies havefailed. Because morbidity and mortality of heart failure patients arestill unacceptably high, there is an urgent need for new treatmentstrategies. Alterations of calcium cycling have been identified as asignificant contributor to heart failure pathophysiology, and there-fore, therapeutic strategies addressing calcium homeostasis seemlogical.

Life of mice - development of cardiac energetics

Life of mice - development of cardiac energetics

Caspase-dependent protein phosphatase 2A activation contributes to endotoxin-induced cardiomyocyte contractile dysfunction*

Several studies report calcium mishandling, sarcomere disarray, and caspase activation during heart failure. Although active caspases have been shown to cleave myofibrillar proteins, little is known regarding their effects on calcium handling proteins. Therefore, we aimed to explore how endotoxin-induced caspase activation disrupts intracellular calcium regulation. Randomized controlled trial. Small animal research laboratory. Adult male Sprague-Dawley rats. Sepsis was induced by injection of endotoxin (10 mg/kg, intravenously). Caspase inhibition was achieved by coinjection with zVAD.fmk (3 mg/kg, intravenously). We first isolated adult rat ventricular myocytes from control, endotoxin, and (endotoxin + zVAD)-treated rats to characterize contractile parameters and cellular calcium homeostasis. Underlying molecular mechanisms responsible for calcium mishandling were explored on sarcoplasmic reticulum vesicles and mitochondria prepared from treated animals. All experiments were performed 4 hrs postendotoxin treatment. zVAD normalized reductions in fractional cell shortening and relaxation rate triggered by endotoxin treatment. Both sarco-/endoplasmic reticulum Ca-ATPase and mitochondria-dependent calcium uptakes were impaired after endotoxin treatment and prevented when myocytes were isolated from zVAD-treated endotoxinic rat hearts. zVAD blocked endotoxin-induced phospholamban dephosphorylation, protein phosphatase 2A activation, and mitochondrial calcium retention capacity reduction. To strengthen these results, control sarcoplasmic reticulum vesicles and mitochondria were incubated with active recombinant caspase-3. Although no effects were observed on mitochondria, caspase-3 directly exerts detrimental effects on sarcoplasmic reticulum calcium uptake capacity by activating protein phosphatase 2A, leading to phospholamban dephosphorylation. Caspase inhibition protects from endotoxin-induced sarcoplasmic reticulum calcium uptake capacity reduction and mitochondrial dysfunction.

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Rate-dependent action potential alternans in human heart failure implicates abnormal intracellular calcium handling

Alternans in action potential voltage (APV-ALT) at heart rates <110 bpm is a novel index to predict ventricular arrhythmias. However, the rate dependency of APV-ALT and its mechanisms in failing versus nonfailing human myocardium are poorly understood. It is hypothesized that APV-ALT in human heart failure (HF) reflects abnormal calcium handling. Using a modeling and clinical approach, our objectives were to (1) determine how APV-ALT varies with pacing rate and (2) ascertain whether abnormalities in calcium handling explain the rate dependence of APV-ALT in HF. APV-ALT was analyzed at several cycle lengths (CLs) using a dynamic pacing protocol applied to a human left ventricle wedge model with various alterations in calcium handling. Modeled APV-ALT was used to predict APV-ALT in left ventricle monophasic action potentials recorded from HF (n = 3) and control (n = 2) patients with the same pacing protocol. Reducing the sarcoplasmic reticulum calcium uptake current < or =25%, the release current < or =11%, or the sarcolemmal L-type calcium channel current < or =43% of control predicted APV-ALT to arise at CL > or =600 ms and then increase in magnitude by >400% for CL <400 ms. In HF patients, APV-ALT arose at CL = 600 ms and then increased in magnitude by >500% at CL <350 ms. For all other model alterations and for control patients, APV-ALT occurred only at CL <500 ms. APV-ALT shows differing rate dependence in HF versus control patients, arising at slower rates in HF and predicted by models with abnormal calcium handling. Future studies should investigate whether APV-ALT at slow rates identifies patients with deranged calcium handing, including HF patients before decompensation or at risk for arrhythmias.

Adenosine A3 receptor-mediated cardioprotection against doxorubicin-induced mitochondrial damage

Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective agonist of A3 adenosine receptor (A3R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca2+ accumulation induced by DOX.Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5mg/kg i.p. DOX, (3) 33μg/kg i.v. Cl-IB-MECA, (4) DOX+Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca2+ content was determined by exposing cultured rat cardiomyocytes to caffeine.Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35±5% vs. 54±5%, p<0.01) in DOX-treated animals, compared to the control group. DOX increased Ca2+ levels in the cytosol and in mitochondria by diminishing the SR Ca2+ uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca2+ overload.We conclude that the adenosine A3 receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca2+ homeostasis and prevention of mitochondrial damage that occurs as a result of Ca2+ overload.

The Effect of 2- and 4-Week Ovariectomy on Female Rabbit Urinary Bladder Function

To evaluate the effect ovariectomy (OVX) after 2 and 4 weeks on bladder function and biochemistry of the adult female rabbit urinary bladder. Twelve mature female rabbits were divided into 3 groups: control, 2-week ovariectomized, and 4-week ovariectomized. At the end of the experimental period, the following studies were performed: contractile studies on isolated strips; examinations of the activity of citrate synthase (a marker for mitochondrial function) and thapsigargin-sensitive calcium ATPase (a marker for sarcoplasmic reticular calcium uptake function); and quantification of Rho-kinase (ROK) alpha and beta and myosin light chain kinase by Western blot analyses. By 28 days after OVX, there were significant decreases in bladder weight, contractile responses, and citrate synthase and sarcoplasmic reticular calcium uptake activity. In addition, by 28 days following OVX the relative concentration of ROK alpha was significantly increased, whereas ROK beta concentration was significantly decreased. Myosin light chain kinase was significantly reduced. Our study demonstrated that OVX contributed significantly to chronically decreased contractile function in the detrusor muscle of the female rabbit bladder, and this decrease, in turn, was mediated by decreased mitochondrial and sarcoplasmic reticulum function. These specific bladder dysfunctions could be related to the demonstrated decreased blood flow to the bladder muscle and mucosa and the increased generation of free radicals. Changes in smooth muscle regulatory proteins, especially myosin light chain kinase, may also play a role in contractile dysfunctions.

Cardiac force-frequency relationship and frequency-dependent acceleration of relaxation are impaired in LPS-treated rats

IntroductionFrequency-dependent acceleration of relaxation (FDAR) ensures appropriate ventricular filling at high heart rates and results from accelerated sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity independent of calcium removal from the cell. Because lipopolysaccharide (LPS) challenge may induce aberrations in calcium trafficking and protein phosphorylation, we tested whether LPS would abolish FDAR in rats.MethodsFollowing LPS injection, changes in force-frequency relationship and FDAR were studied in cardiomyocytes, isolated hearts and in vivo by echocardiography. Calcium uptake and phosphatase activities were studied in sarcoplasmic reticulum (SR) vesicle preparations. Western blots of phospholamban and calcium/calmodulin-dependent protein kinase II, and serine/threonine phosphatase activity were studied in heart preparations.ResultsIn cardiomyocytes and isolated heart preparations, reductions in time constant of relaxation (τ) and time to 50% relaxation at increasing rate of pacing were blunted in LPS-treated rats compared with controls. Early diastolic velocity of the mitral annulus (Ea), a relaxation parameter which correlates in vivo with τ, was reduced in LPS rats compared with control rats. LPS impaired SR calcium uptake, reduced phospholamban phosphorylation and increased serine/threonine protein phosphatase activity. In vivo inhibition of phosphatase activity partially restored FDAR, reduced phosphatase activity and prevented phospholamban dephosphorylation in LPS rat hearts.ConclusionsLPS impaired phospholamban phosphorylation, cardiac force-frequency relationship and FDAR. Disruption of frequency-dependent acceleration of LV relaxation, which normally participates in optimal heart cavity filling, may be detrimental in sepsis, which is typically associated with elevated heart rates and preload dependency.

Action Potential Dynamics Explain Arrhythmic Vulnerability in Human Heart Failure: A Clinical and Modeling Study Implicating Abnormal Calcium Handling

The purpose of this study was to determine whether abnormalities of calcium cycling explain ventricular action potential (AP) oscillations and cause electrocardiogram T-wave alternans (TWA). Mechanisms explaining why heart failure patients are at risk for malignant ventricular arrhythmias (ventricular tachycardia [VT]/ventricular fibrillation [VF]) are unclear. We studied whether oscillations in human ventricular AP explain TWA and predict VT/VF, and used computer modeling to suggest potential cellular mechanisms. We studied 53 patients with left ventricular ejection fraction 28 +/- 8% and 18 control subjects. Monophasic APs were recorded in the right ventricle (n = 62) and/or left ventricle (n = 9) at 109 beats/min. Alternans of AP amplitude, computed spectrally, had higher magnitude in study patients than in controls (p = 0.03), particularly in AP phase II (p = 0.02) rather than phase III (p = 0.10). The AP duration and activation restitution (n = 11 patients) were flat at 109 beats/min and did not explain TWA. In computer simulations, only reduced sarcoplasmic reticulum calcium uptake explained our results, causing calcium oscillations, AP amplitude alternans, and TWA that were all abolished by calcium clamping. On prospective follow-up for 949 +/- 553 days, 17 patients had VT/VF. The AP amplitude alternans predicted VT/VF (p = 0.04), and was 78% concordant with simultaneous TWA (p = 0.003). Patients with systolic dysfunction show ventricular AP amplitude alternans that prospectively predicted VT/VF. Alternans in AP amplitude, but not variations in AP duration or conduction, explained TWA at < or =109 beats/min. In computer models, these findings were best explained by reduced sarcoplasmic reticulum calcium uptake. Thus, in heart failure patients, in vivo AP alternans may reflect cellular calcium abnormalities and provide a mechanistic link with VT/VF.

Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin‐free skeletal muscle

Previous work suggested that altered Ca(2+) homeostasis might contribute to dysfunction of nebulin-free muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-inhibitor sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as expression of other Ca(2+)-handling proteins. Ca(2+) uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca(2+) transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca(2+), as well as in electrically stimulated intact muscle. We found profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of other Ca(2+)-handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca(2+) uptake was >3-fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact myofibers. Ca(2+)-activated stress in skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent compared with wild type. Half-relaxation time was significantly longer in nebulin-free compared with wild-type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca(2+) handling of the SR-myofibrillar system.

Reply: Does the adenosine A2A receptor stimulate the ryanodine receptor?

In our paper on the expression and function of the adenosine A2A receptor (A2AR) in human atrial myocytes [1], we show that agonist stimulation of this receptor induces a protein kinase A (PKA)-dependent increase in the frequency of calcium sparks and spontaneous calcium waves. We therefore investigated whether A2AR stimulation affected the L-type calcium current, sarcoplasmic reticulum (SR) calcium uptake, or SR calcium release, as these mechanisms are known to be modulated by PKA-dependent phosphorylation [2–4]. Since only the fast (SR calcium release-dependent) time constant for I Ca inactivation was affected by A2AR stimulation, we proposed that a PKA-mediated increase in ryanodine receptor (RyR) opening may account for our results. However, according to the letter …

Alterations in cardiac contractile performance and sarcoplasmic reticulum function in sucrose-fed rats is associated with insulin resistance

Diabetes mellitus (DM) causes the development of a specific cardiomyopathy that results from the metabolic derangements present in DM and manifests as cardiac contractile dysfunction. Although myocardial dysfunction in Type 1 DM has been associated with defects in the function and regulation of the sarcoplasmic reticulum (SR), very little is known about SR function in Type 2 DM. Accordingly, this study examined whether abnormalities in cardiac contractile performance and SR function occur in the prestage of Type 2 DM (i.e., during insulin resistance). Sucrose feeding was used to induce whole body insulin resistance, whereas cardiac contractile performance was assessed by echocardiography and SR function was measured by SR calcium (Ca(2+)) uptake. Sucrose-fed rats exhibited hyperinsulinemia, hyperglycemia, and hyperlipidemia relative to control rats. Serial echocardiographic assessments in the sucrose-fed rats revealed early abnormalities in diastolic function followed by late systolic dysfunction and concurrent alterations in myocardial structure. The hearts of the 10-wk sucrose-fed rats showed depressed SR function demonstrated by a significant reduction in SR Ca(2+) uptake. The decline in SR Ca(2+) uptake was associated with a significant decrease in the cAMP-dependent protein kinase and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of phospholamban. The results show that abnormalities in cardiac contractile performance and SR function occur at an insulin-resistant stage before the manifestation of overt Type 2 DM.

Altered energy transfer from mitochondria to sarcoplasmic reticulum after cytoarchitectural perturbations in mice hearts

Sarcoplasmic reticulum (SR) calcium pump function requires a high local ATP/ADP ratio, which can be maintained by direct nucleotide channelling from mitochondria, and by SR-bound creatine kinase (CK)-catalysed phosphate-transfer from phosphocreatine. We hypothesized that SR calcium uptake supported by mitochondrial direct nucleotide channelling, but not bound CK, depends on the juxtaposition of these organelles. To test this, we studied a well-described model of cytoarchitectural disorganization, the muscle LIM protein (MLP)-null mouse heart. Subcellular organization was characterized using electron microscopy, and mitochondrial, SR and myofibrillar function were assessed in saponin-permeabilized fibres by measuring respiration rates and caffeine-induced tension transients. MLP-null hearts had fewer, less-tightly packed intermyofibrillar mitochondria, and more subsarcolemmal mitochondria. The apparent mitochondrial Km for ADP was significantly lower in the MLP-null heart than in control (175 +/- 15 and 270 +/- 33 microM, respectively), indicating greater ADP accessibility, although maximal respiration rate, mitochondrial content and total CK activity were unaltered. Active tension in the myofibres of MLP-null mice was 54% lower than in controls (39 +/- 3 and 18 +/- 1 mN mm(-2), respectively), consistent with cytoarchitectural disorganization. SR calcium loading in the myofibres of MLP-null mice was similar to that in control myofibres when energy support was provided via Bound CK, but approximately 36% lower than controls when energy support was provided by mitochondrial (P < 0.05). Mitochondrial support for SR calcium uptake was also specifically decreased in the desmin-null heart, which is another model of cytoarchitectural perturbation. Thus, despite normal oxidative capacity, direct nucleotide channelling to the SR was impaired in MLP deficiency, concomitant with looser mitochondrial packing and increased nucleotide accessibility to this organelle. Changes in cytoarchitecture may therefore impair subcellular energy transfer and contribute to energetic and contractile dysfunction.

“Ryanogate”

See related article, pages 235–244 The sarcoplasmic reticulum (SR) is the major intracellular calcium storage depot in cardiac muscle. Cycling of calcium between the lumen of the SR and the myoplasmic space occurs repetitively during each heart beat. Excitation–contraction coupling in the heart begins when calcium entry through voltage-gated L-type calcium channels in the sarcolemma induces the opening of calcium release channels (also known as ryanodine receptors, or RyR) in the adjacent SR. The majority of the calcium that enters the cytosol during the early portion of each cycle is then resequestered into the SR lumen via the actions of the calcium uptake protein, (sarco)endoplasmic reticulum calcium adenosine triphosphatase (SERCA). Tight regulation of the timing and quantity of these intracellular calcium fluxes is critical to achieve graded contractility and relaxation of the heart muscle. Such control allows optimal matching of cardiac function to heart rate and metabolic needs of the body. The mechanisms of excitation–contraction coupling have been reviewed more thoroughly elsewhere.1 There has been a great deal of interest in the possibility that altered calcium homeostasis in the cardiac myocyte is the fundamental abnormality in the failing heart.2 The most widely accepted hypothesis to explain myocyte dysfunction is that reduced abundance or activity of SERCA protein directly results in a lower rate of calcium uptake by the SR. In this scenario, reduced SR uptake results in lower SR calcium content, which then produces lower SR calcium release and reduced contractility. However, the “SERCA-centric” view of heart failure is at best only a partial explanation of the cellular abnormalities that occur in failing hearts.3–5 We now recognize that many other highly complex mechanisms in addition to SERCA expression regulate SR calcium uptake and release. In particular, posttranslational modifications of SR proteins are known to be extremely …

Adenylyl cyclase type V deletion increases basal left ventricular function and reduces left ventricular contractile responsiveness to β–adrenergic stimulation

We tested the hypothesis that deletion of adenylyl cyclase type V (AC(V)) would be associated with decreased left ventricular (LV) contractile function and responsiveness to beta-adrenergic receptor (betaAR) stimulation. Absence of cardiac AC(V) expression was confirmed by RT-PCR and immunoblotting in AC(V)-deleted mice (AC(V) (-/-)). Compared to sibling mice with normal amounts of AC(V) (CON), basal and water-soluble forskolin derivative NKH477-stimulated cAMP production was reduced in both LV homogenates and in isolated cardiac myocytes. Basal LV +dP/dt (isolated perfused hearts) was increased (CON: 3,649 +/- 247 mmHg/s; AC(V) (-/-): 4,625 +/- 350 mmHg/s; p = 0.035, n = 10), but the potency of dobutamine on LV +dP/dt was decreased by AC(V) deletion (log EC(50): CON: -6.83 +/- 0.14 M; AC(V) (-/-): -5.99 +/- 0.15 M; p = 0.0007, n = 10). The initial rates of ATP-dependent sarcoplasmic reticulum calcium uptake, assessed in LV homogenates, showed that AC(V) deletion increased SERCA2a affinity for Ca(2+) (log EC(50): CON: -5.94 +/- 0.03 M; AC(V) (-/-): -6.09 +/- 0.02 M; p = 0.001, n = 8). AC(V) deletion is also associated with increased phospholamban phosphorylation, decreased type 1 protein phosphatase catalytic subunit content and activity, and reduced cardiac Galphas protein content. In conclusion, AC(V) deletion has a favorable effect on basal LV function despite reduced cAMP levels. Increased SERCA2a affinity for Ca(2+) and increased phospholamban phosphorylation are contributing factors. However, AC(V) deletion is associated with reduced LV contractile responsiveness to betaAR stimulation, an effect that is associated with reduced Galphas protein content and reduced cAMP generating capacity in cardiac myocytes.

Enhancement of Cardiac Function and Suppression of Heart Failure Progression By Inhibition of Protein Phosphatase 1

Abnormal calcium cycling, characteristic of experimental and human heart failure, is associated with impaired sarcoplasmic reticulum calcium uptake activity. This reflects decreases in the cAMP-pathway signaling and increases in type 1 phosphatase activity. The increased protein phosphatase 1 activity is partially due to dephosphorylation and inactivation of its inhibitor-1, promoting dephosphorylation of phospholamban and inhibition of the sarcoplasmic reticulum calcium-pump. Indeed, cardiac-specific expression of a constitutively active inhibitor-1 results in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility at the cellular and intact animal levels. Furthermore, the beta-adrenergic response is enhanced in the transgenic hearts compared with wild types. On aortic constriction, the hypercontractile cardiac function is maintained, hypertrophy is attenuated and there is no decompensation in the transgenics compared with wild-type controls. Notably, acute adenoviral gene delivery of the active inhibitor-1, completely restores function and partially reverses remodeling, including normalization of the hyperactivated p38, in the setting of pre-existing heart failure. Thus, the inhibitor 1 of the type 1 phosphatase may represent an attractive new therapeutic target.

Myotonic Dystrophy Protein Kinase Phosphorylates Phospholamban and Regulates Calcium Uptake in Cardiomyocyte Sarcoplasmic Reticulum

Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology.

Mechanisms of Interleukin-4 Effects on Calcium Signaling in Airway Smooth Muscle Cells

In airway smooth muscle cells, interleukin (IL)-4 inhibited both carbachol- and caffeine-induced calcium mobilization from the sarcoplasmic reticulum (SR). Because of the known signaling pathways for IL-4 and importance of calcium uptake in maintaining SR calcium stores shared by agonists and caffeine, it was hypothesized that this rapid inhibitory effect might depend on phosphatidylinositol 3-kinase (PI3K) and on inhibition of calcium uptake by the SR. Enzyme-dispersed bovine trachealis cells were loaded with Fura-2/acetoxymethyl ester, and changes in cytosolic calcium were imaged in single cells. Cells were pretreated with inhibitors of PI3K, either wortmannin (100 nM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (50 microM), or deguelin (100 nM). Calcium transients in response to carbachol (10 microM) were significantly decreased to 0.34 +/- 0.10 of control after 20-min treatment with IL-4 but were 1.10 +/- 0.26 and 1.08 +/- 0.23 when wortmannin or deguelin, respectively, was added along with IL-4. LY294002 alone had nonspecific effects on transients. In other experiments, cyclopiazonic acid (CPA) (5 microM), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 +/- 0.08 of control (n = 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 +/- 0.05 (n = 6; p < 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 microM) gave similar results. It was concluded that IL-4 did not inhibit transients in the presence of PI3K antagonists but that it did in the presence of CPA. This suggested that IL-4 inhibited calcium transients by mechanisms dependent upon a wortmannin-sensitive PI3K but not by inhibition of calcium uptake into the SR.

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart.

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.