Anuradha Kalyanasundaram

SERCA pump isoforms: Their role in calcium transport and disease

The sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA) is a pump that transports calcium ions from the cytoplasm into the SR. It is present in both animal and plant cells, although knowledge of SERCA in the latter is scant. The pump shares the catalytic properties of ion‐motive ATPases of the P‐type family, but has distinctive regulation properties. The SERCA pump is encoded by a family of three genes, SERCA1, 2, and 3, that are highly conserved but localized on different chromosomes. The SERCA isoform diversity is dramatically enhanced by alternative splicing of the transcripts, occurring mainly at the COOH‐terminal. At present, more than 10 different SERCA isoforms have been detected at the protein level. These isoforms exhibit both tissue and developmental specificity, suggesting that they contribute to unique physiological properties of the tissue in which they are expressed. The function of the SERCA pump is modulated by the endogenous molecules phospholamban (PLB) and sarcolipin (SLN), expressed in cardiac and skeletal muscles. The mechanism of action of PLB on SERCA is well characterized, whereas that of SLN is only beginning to be understood. Because the SERCA pump plays a major role in muscle contraction, a number of investigations have focused on understanding its role in cardiac and skeletal muscle disease. These studies document that SERCA pump expression and activity are decreased in aging and in a variety of pathophysiological conditions including heart failure. Recently, SERCA pump gene transfer was shown to be effective in restoring contractile function in failing heart muscle, thus emphasizing its importance in muscle physiology and its potential use as a therapeutic agent. Muscle Nerve, 2007

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex

AIMS Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Intra-sarcoplasmic reticulum Ca2+ oscillations are driven by dynamic regulation of ryanodine receptor function by luminal Ca2+ in cardiomyocytes.

During the cardiac cycle, the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR2) channel complex is controlled by the levels of cytosolic and luminal Ca2+ and alterations in these regulatory processes have been implicated in cardiac disease including arrhythmia. To better understand the mechanisms of regulation of SR Ca2+ release by Ca2+ on both sides of the SR membrane, we investigated SR Ca2+ release in a wide range of cytosolic Ca2+ concentrations ([Ca2+]cyt; 1–100 μm) in permeabilized canine ventricular myocytes by monitoring [Ca2+] inside the SR ([Ca2+]SR). Exposing myocytes to activating [Ca2+]cyt resulted in spontaneous oscillations of [Ca2+]SR due to periodic opening and closing of the RyR2s. Elevating [Ca2+]cyt (up to 10 μm) increased the frequency of [Ca2+]SR oscillations; however at higher [Ca2+]cyt (>50 μm) the oscillations diminished due to RyR2s staying perpetually open, resulting in depleted SR. Ablation of cardiac calsequestrin (CASQ2) altered the [Ca2+]cyt dependence of Ca2+ release oscillations such that oscillations were highly frequent at low [Ca2+]cyt (100 nm) but became diminished at moderate [Ca2+]cyt (10 μm), as determined in myocytes from calsequestrin‐null versus wild‐type mice. Our results suggest that under conditions of continuous activation by cytosolic Ca2+, RyR2s can periodically cycle between open and deactivated states due to effects of luminal Ca2+. Deactivation at reduced [Ca2+]SR appears to involve reduction of sensitivity to cytosolic Ca2+ and might be mediated by CASQ2. Inactivation by cytosolic Ca2+ plays no detectable role in controlling SR Ca2+ release.

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.

Upregulation of Adenosine A1 Receptors Facilitates Sinoatrial Node Dysfunction in Chronic Canine Heart Failure by Exacerbating Nodal Conduction Abnormalities Revealed by Novel Dual-Sided Intramural Optical Mapping

Background— Although sinoatrial node (SAN) dysfunction is a hallmark of human heart failure (HF), the underlying mechanisms remain poorly understood. We aimed to examine the role of adenosine in SAN dysfunction and tachy-brady arrhythmias in chronic HF. Methods and Results— We applied multiple approaches to characterize SAN structure, SAN function, and adenosine A1 receptor expression in control (n=17) and 4-month tachypacing-induced chronic HF (n=18) dogs. Novel intramural optical mapping of coronary-perfused right atrial preparations revealed that adenosine (10 &mgr;mol/L) markedly prolonged postpacing SAN conduction time in HF by 206±99 milliseconds (versus 66±21 milliseconds in controls; P=0.02). Adenosine induced SAN intranodal conduction block or microreentry in 6 of 8 dogs with HF versus 0 of 7 controls (P=0.007). Adenosine-induced SAN conduction abnormalities and automaticity depression caused postpacing atrial pauses in HF versus control dogs (17.1±28.9 versus 1.5±1.3 seconds; P<0.001). Furthermore, 10 &mgr;mol/L adenosine shortened atrial repolarization and led to pacing-induced atrial fibrillation in 6 of 7 HF versus 0 of 7 control dogs (P=0.002). Adenosine-induced SAN dysfunction and atrial fibrillation were abolished or prevented by adenosine A1 receptor antagonists (50 &mgr;mol/L theophylline/1 &mgr;mol/L 8-cyclopentyl-1,3-dipropylxanthine). Adenosine A1 receptor protein expression was significantly upregulated during HF in the SAN (by 47±19%) and surrounding atrial myocardium (by 90±40%). Interstitial fibrosis was significantly increased within the SAN in HF versus control dogs (38±4% versus 23±4%; P<0.001). Conclusions— In chronic HF, adenosine A1 receptor upregulation in SAN pacemaker and atrial cardiomyocytes may increase cardiac sensitivity to adenosine. This effect may exacerbate conduction abnormalities in the structurally impaired SAN, leading to SAN dysfunction, and potentiate atrial repolarization shortening, thereby facilitating atrial fibrillation. Atrial fibrillation may further depress SAN function and lead to tachy-brady arrhythmias in HF.

Fibrosis: a structural modulator of sinoatrial node physiology and dysfunction

Heart rhythm is initialized and controlled by the Sinoatrial Node (SAN), the primary pacemaker of the heart. The SAN is a heterogeneous multi-compartment structure characterized by clusters of specialized cardiomyocytes enmeshed within strands of connective tissue or fibrosis. Intranodal fibrosis is emerging as an important modulator of structural and functional integrity of the SAN pacemaker complex. In adult human hearts, fatty tissue and fibrosis insulate the SAN from the hyperpolarizing effect of the surrounding atria while electrical communication between the SAN and right atrium is restricted to discrete SAN conduction pathways. The amount of fibrosis within the SAN is inversely correlated with heart rate, while age and heart size are positively correlated with fibrosis. Pathological upregulation of fibrosis within the SAN may lead to tachycardia-bradycardia arrhythmias and cardiac arrest, possibly due to SAN reentry and exit block, and is associated with atrial fibrillation, ventricular arrhythmias, heart failure and myocardial infarction. In this review, we will discuss current literature on the role of fibrosis in normal SAN structure and function, as well as the causes and consequences of SAN fibrosis upregulation in disease conditions.

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart.

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca2+ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca2+. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca2+ activation.

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

Background: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). Methods: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Results: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10–100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. Conclusions: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine.Background: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels ( I K,Ado). Methods: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Results: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P <0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P <0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10–100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P <0.01) and GIRK4 (1.7±0.8-fold; P <0.05) protein expression than lateral/posterior LA. Conclusions: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. # Clinical Perspective {#article-title-40}

Detrimental effects of thyroid hormone analog DITPA in the mouse heart: increased mortality with in vivo acute myocardial ischemia-reperfusion.

There is emerging evidence that treatment with thyroid hormone (TH) can improve postischemic cardiac function. 3,5-Diiodothyropropionic acid (DITPA), a TH analog, has been proposed to be a safer therapeutic agent than TH because of its negligible effects on cardiac metabolism and heart rate. However, conflicting results have been reported for the cardiac effects of DITPA. Importantly, recent clinical trials demonstrated no symptomatic benefit in patients with DITPA despite some improved hemodynamic and metabolic parameters. To address these issues, dose-dependent effects of DITPA were investigated in mice for baseline cardiovascular effects and postischemic myocardial function and/or salvage. Mice were treated with subcutaneous DITPA at 0.937, 1.875, 3.75, or 7.5 mg·kg(-1)·day(-1) for 7 days, and the results were compared with untreated mice for ex vivo and/or in vivo myocardial ischemia-reperfusion (I/R). DITPA had no effects on baseline body temperature, body weight, or heart rate; however, it mildly increased blood pressure. In isolated hearts, baseline contractile function was significantly impaired in DITPA-pretreated mice; however, postischemic recovery was comparable between untreated and DITPA-treated groups. In vivo baseline cardiac parameters were significantly affected by DITPA, with increased ventricular dimensions and decreased contractile function. Importantly, DITPA-treated mice demonstrated high prevalence of fatal cardiac rhythm abnormalities during in vivo ischemia and/or reperfusion. There were no improvements in myocardial infarction and postischemic fractional shortening with DITPA. Myocardial sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and heat shock protein (HSP) levels remained unchanged with DITPA treatment. Thus DITPA administration impairs baseline cardiac parameters in mice and can be fatal during in vivo acute myocardial I/R.