Ivar Sjaastad

T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction

In cardiac myocytes, initiation of excitation–contraction coupling is highly localized near the T‐tubule network. Myocytes with a dense T‐tubule network exhibit rapid and homogeneous sarcoplasmic reticulum (SR) Ca2+ release throughout the cell. We examined whether progressive changes in T‐tubule organization and Ca2+ release synchrony occur in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by ligation of the left coronary artery, and CHF was diagnosed by echocardiography (left atrial diameter >2.0 mm). CHF mice were killed at 1 or 3 weeks following MI (1‐week CHF, 3‐week CHF) and cardiomyocytes were isolated from viable regions of the septum, excluding the MI border zone. Septal myocytes from SHAM‐operated mice served as controls. T‐tubules were visualized by confocal microscopy in cells stained with di‐8‐ANEPPS. SHAM cells exhibited a regular striated T‐tubule pattern. However, 1‐week CHF cells showed slightly disorganized T‐tubule structure, and more profound disorganization occurred in 3‐week CHF with irregular gaps between adjacent T‐tubules. Line‐scan images of Ca2+ transients (fluo‐4 AM, 1 Hz) showed that regions of delayed Ca2+ release occurred at these gaps. Three‐week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca2+ release. A common pattern of Ca2+ release in 3‐week CHF was maintained between consecutive transients, and was not altered by forskolin application. Thus, progressive T‐tubule disorganization during CHF promotes dyssynchrony of SR Ca2+ release which may contribute to the slowing of SR Ca2+ release in this condition.

Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity

OBJECTIVE Heart failure is associated with alterations in contractile parameters and accompanied by abnormalities in intracellular calcium homeostasis. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and phospholamban (PLB) are important in intracellular calcium cycling. The aim of the present study was to examine mechanisms causing reductions in SERCA2 activity in the failing heart. METHODS Myocardial infarction (MI) was induced in male Wistar rats, and animals with congestive heart failure were examined 6 weeks after the primary operation. RESULTS Serine(16) monomeric and pentameric phosphorylated PLB were significantly downregulated (50 and 55%, respectively), whereas threonine(17) phosphorylated PLB was unchanged in failing compared to sham hearts. Protein phosphatases 1 and 2A were significantly upregulated (26 and 42%, respectively) and phosphatase 2C significantly downregulated (29%), whereas the level of protein kinase A regulatory subunit II remained unchanged during heart failure. Increasing PLB phosphorylation by forskolin in isolated cardiomyocytes after inhibition of the Na(+)-Ca(2+) exchanger activity had significantly greater effect on SERCA2 activity in failing than in sham cells (49 and 20% faster transient decline, respectively). Decreasing PLB phosphorylation by the protein kinase A inhibitor H89 had significantly less effect on SERCA2 activity in failing compared to sham cardiomyocytes (20 and 75% slower transient decline, respectively). CONCLUSION The observed changes in SERCA2 activity after increasing and decreasing serine(16) PLB phosphorylation in cardiomyocytes from sham and failing hearts, suggest that the observed reduction in serine(16) PLB phosphorylation is one major factor determining the reduced SERCA2 activity in heart failure after MI.

Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca(2+) from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca(2+) necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca(2+) content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca(2+) transients were predominantly generated by enhanced Ca(2+) flux through L-type Ca(2+) channels and the Na(+)-Ca(2+) exchanger. Moreover, equivalent increases in cytosolic [Ca(2+)] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca(2+) transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure.

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase α2-isoform in heart failure

AIMS The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout

Alterations in trans‐sarcolemmal and sarcoplasmic reticulum (SR) Ca2+ fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte‐specific excision of the SR Ca2+‐ATPase (SERCA) gene. At 4 weeks following gene deletion (4‐week KO) cardiac function remained near normal values. However, end‐stage heart failure developed by 7 weeks (7‐week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4‐ and 7‐week KO, and relaxation was slowed. Ca2+ transients were similarly altered. Reduction in Ca2+ transient magnitude resulted from complete loss of SR Ca2+ release between 4‐ and 7‐week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca2+ release was partly offset by increased L‐type Ca2+ current, which was facilitated by AP prolongation in 7‐week KO. Ca2+ entry via reverse‐mode Na+–Ca2+ exchange (NCX) was also enhanced. Up‐regulation of NCX and plasma membrane Ca2+‐ATPase increased Ca2+ extrusion rates in 4‐week KO. Diastolic dysfunction in 7‐week KO resulted from further SERCA2 loss, but also impaired NCX‐mediated Ca2+ extrusion following Na+ accumulation. Reduced Na+‐K+‐ATPase activity contributed to the Na+ gain. Normalizing [Na+] by dialysis increased the Ca2+ decline rate in 7‐week KO beyond 4‐week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na+ accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na+ gain is prevented, up‐regulated Ca2+ extrusion mechanisms can maintain near‐normal diastolic function in the absence of SERCA2.

Cumulative organ damage and prognostic factors in juvenile dermatomyositis: a cross-sectional study median 16.8 years after symptom onset

OBJECTIVE To describe cumulative organ damage in juvenile dermatomyositis (JDM) patients and to identify patient characteristics and early disease variables that predict organ damage. METHODS An inception cohort of 60 patients diagnosed with JDM from 1970 to 2006 was examined, median 16.8 (2.0-38.1) years after disease onset. Disease activity was measured by the disease activity score (DAS), organ damage by the myositis damage index (MDI) and physical function by the childhood or adult HAQ (CHAQ/HAQ). Medical records were reviewed for early disease variables at diagnosis, and 6 and 12 months post-diagnosis. RESULTS Fifty-four (90%) patients had a cumulative MDI total score >or=1 at follow-up (mean 4.2 +/- 3.1). Damage occurred most frequently in cutaneous, muscular and skeletal domains (77, 65 and 57%, respectively). Early predictors of damage were DAS and MDI 6 months post-diagnosis (beta = 0.334; P = 0.002 and 0.382, P < 0.001, respectively). Follow-up time also correlated with MDI (P = 0.010). Calcinosis, seen in 47% of the patients, was predicted by male gender [odds ratio (OR) 3.8; 95% CI 1.2, 12.1], and DAS 6 months post-diagnosis (OR 1.2; 95% CI 1.1, 1.4). The MDI score correlated with CHAQ/HAQ and DAS at follow-up (r(s) = 0.355; P = 0.005 and 0.446, P < 0.001, respectively). The DAS decreased during the first-year post-diagnosis, whereas the MDI increased over time. CONCLUSIONS The majority of JDM patients had cumulative organ damage at follow-up, which was predicted by high disease activity and organ damage 6 months post-diagnosis.

Mutual antagonism between IP3RII and miRNA-133a regulates calcium signals and cardiac hypertrophy

IP3RII-induced calcium release decreases miR-133a expression, which further increases IP3RII levels and calcium release and thereby promotes hypertrophic heart remodeling.

Heart failure – a challenge to our current concepts of excitation–contraction coupling

Development of novel therapeutic strategies for congestive heart failure (CHF) seems to be hampered by insufficient knowledge of the molecular machinery of excitation‐contraction (EC) coupling in both normal and failing hearts. Cardiac hypertrophy and failure represent a multitude of cardiac phenotypes, and available invasive and non‐invasive techniques, briefly reviewed here, allow proper quantification of myocardial function in experimental models even in rats and mice. Both reduced fractional shortening and reduced velocity of contraction characterize myocardial failure. Only when myocardial function is depressed in vivo can meaningful studies be done in vitro of contractility and EC coupling. Also, we point out potential limitations with the whole cell patch clamp technique. Two main factors stand out as explanations for myocardial failure. First, a basic feature of CHF seems to be a reduced Ca2+ load of the sarcoplasmic reticulum (SR) mainly due to a low phosphorylation level of phospholamban. Second, there seems to be a defect of the trigger mechanism of Ca2+ release from the SR. We argue that this defect only becomes manifest in the presence of reduced Ca2+ reuptake capacity of the SR and that it may not be solely attributable to reduced gain of the Ca2+‐induced Ca2+ release (CICR). We list several possible explanations for this defect that represent important avenues for future research.

Dual Serotonergic Regulation of Ventricular Contractile Force Through 5-HT2A and 5-HT4 Receptors Induced in the Acute Failing Heart

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.