µ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants.

µ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of µ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to µ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) µ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) µ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after µ-calpain treatment. The ability of µ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.

Extensive autolytic fragmentation of membranous versus cytosolic calpain following myocardial ischemia-reperfusion.

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Distribution of omega-3 fatty acids in tissues of rabbits fed a flaxseed-supplemented diet.

Diets rich in omega-3 polyunsaturated fatty acids are associated with decreased incidences of cardiovascular disease. The extent of incorporation and distribution of these beneficial fats into body tissues is uncertain. Rabbits were fed regular rabbit chow or a diet containing 10% ground flaxseed that is highly enriched with the omega-3 polyunsaturated fatty acid alpha-linolenic acid (ALA). The high-flaxseed diet resulted in an incorporation of ALA in all tissues, but mostly in the heart and liver with little in the brain. Docosahexaenoic and eicosapentaenoic acid levels were also selectively increased in some tissues, and the effects were not as large as ALA. Arachidonic acid and the ratio of omega-6/omega-3 fatty acids were decreased in all tissues obtained from the flax-supplemented group. Consumption of dietary flaxseed appears to be an effective means to increase ALA content in body tissues, but the degree will depend upon the tissues examined.

Dietary flaxseed protects against ventricular fibrillation induced by ischemia-reperfusion in normal and hypercholesterolemic Rabbits.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the (n-3) PUFA found in fish oils, exert antiarrhythmic effects during ischemia. Flaxseed is the richest plant source of another (n-3) PUFA, alpha-linolenic acid (ALA), yet its effects remain largely unknown. Our objective was to determine whether a flaxseed-rich diet is antiarrhythmic in normal and hypercholesterolemic rabbits. Male New Zealand White (NZW) rabbits (n = 14-16) were fed as follows: regular diet (REG group); diet containing 10% flaxseed (FLX group); 0.5% cholesterol (CHL group); or 0.5% cholesterol + 10% flaxseed (CHL/FLX group) for up to 16 wk. Plasma cholesterol was significantly elevated in the CHL and CHL/FLX groups. Plasma triglycerides were unchanged. ALA levels increased significantly in plasma and hearts of the FLX and CHL/FLX groups. After the feeding period, rabbit hearts were isolated and subjected to global ischemia (30 min) and reperfusion (45 min). Ventricular fibrillation (VF) occurred during ischemia in 33% of REG but in none of FLX hearts, and 28% of CHL but only 6% of CHL/FLX hearts. VF incidence during reperfusion was 28% and 26% in REG and FLX hearts, respectively. The incidence significantly increased to 64% in CHL hearts, and was significantly attenuated (18%) in CHL/FLX hearts. CHL markedly prolonged the QT interval, whereas FLX significantly shortened the QT interval and reduced arrhythmias in the FLX and CHL/FLX hearts. In vitro application of (n-3) PUFA shortened the action potential duration, an effect consistent with the QT data. This study demonstrates that dietary flaxseed exerts antiarrhythmic effects during ischemia-reperfusion in rabbit hearts, possibly through shortening of the action potential.

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles.

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.

Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells.

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY FL thapsigargin and BODIPY FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 microM) and activatory concentrations of ryanodine (1 microM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.

Nucleolin is a calcium-binding protein.

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.

Effects of chronic, rapid right atrial pacing on cardiac hemodynamics and myofibrillar ATPase activity in piglets.

OBJECTIVES: To determine whether chronic, rapid right atrial pacing in newborn neonatal piglets has any effects on cardiac hemodynamics, and whether these changes are associated with intrinsic alterations in cardiac contractile potential as shown by cardiac myofibrillar calcium ATPase activity. BACKGROUND: Although many studies have examined aspects of heart function in models of supraventricular tachycardia, far less is known about its effects in neonatal animals. It is thought that rapid pacing induces a dilated cardiomyopathy in immature pigs. ANIMALS AND METHODS: Two-week-old piglets underwent rapid right atrial pacing (250 beats/min) for 10 days, and their cardiac hemodynamic response was monitored. To obtain subcellular mechanistic information regarding systolic dysfunction, cardiac myofibrils were isolated and calcium adenosine triphosphatase activity was measured. RESULTS: Control piglets had a heart rate of 185 beats/min at the end of the experimental period. Pulmonary artery flow, pulmonary artery flow index and left ventricular end-diastolic diameter were unchanged as a function of rapid, chronic right atrial pacing. Aortic pressure decreased in the paced piglets. Left atrial pressure increased approximately threefold in the paced animals. Left ventricular end-systolic diameter was also significantly higher after pacing, but left ventricular end-diastolic diameter was unchanged. Left ventricular shortening fraction was depressed approximately 50%. Myofibrillar calcium adenosine triphosphatase activity was significantly depressed as a function of pacing. CONCLUSIONS: Neonatal piglets undergoing chronic supraventricular tachycardia exhibit systolic dysfunction in the absence of dilation. The depression in contractile protein calcium adenosine triphosphatase activity provides information at a subcellular level regarding the mechanism responsible for this cardiomyopathy.