Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) sequesters Ca²⁺ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca²⁺ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca²⁺] but binds DWORF better when [Ca²⁺] is high. In the present study, we demonstrated this opposing Ca²⁺ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca²⁺ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca²⁺]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca²⁺ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca²⁺ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.     

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein,and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Sarco(endo)plasmic reticulum Ca²⁺ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547–554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575–1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the V_max of SERCA-mediated Ca²⁺ uptake but not the pump affinity for Ca²⁺; 2) SLN can bind to SERCA in the presence of high Ca²⁺, but PLB can only interact to the ATP-bound Ca²⁺-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca²⁺ causes uncoupling of the SERCA pump and increased heat production.     

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

SERCA2a in Heart Failure: Role and Therapeutic Prospects

Ca²⁺ is a key molecule controlling several cellular processes, from fertilization to cell death, in all cell types. In excitable and contracting cells, such as cardiac myocytes, Ca²⁺ controls muscle contractility. The spatial and temporal segregation of Ca²⁺ concentrations are central to maintain its concentration gradients across the cells and the cellular compartments for proper function. SERCA2a is a cornerstone molecule for maintaining a balanced concentration of Ca²⁺ during the cardiac cycle, since it controls the transport of Ca²⁺ to the sarcoplasmic reticulum (SR) during relaxation. Alterations of the activity of this pump have been widely investigated, emphasizing its central role in the control of Ca²⁺ homeostasis and consequently in the pathogenesis of the contractile defect seen with heart failure. This review focuses on the molecular characteristics of the pump, its role during the cardiac cycle and the prospects derived from the manipulation of SERCA2a for heart failure treatment.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Characterization of junctate-SERCA2a interaction in murine cardiomyocyte

Junctate is a newly identified sarcoplasmic reticulum (SR) Ca²⁺ binding protein, but its function in cardiac muscle has remained unclear. Our previous study showed that chronic over-expression of junctate in transgenic mice led to altered SR functions and development of severe hypertrophy. In this study, we identified the interaction of junctate with SERCA2a by co-immunoprecipitation and GST-pull-down assay. This interaction was inhibited by higher Ca²⁺ concentration. Immunolocalization assays also showed that junctate and SERCA2a were co-localized in the SR of cardiomyocytes. Direct binding of the C-terminal region of junctate (amino acids 79-270) and luminal domain of SERCA2a (amino acids 70-89) was observed by deletion mutation experiments. Adenovirus-mediated transient over-expression of junctate in cardiomyocytes showed a reduced decay time of Ca²⁺ transients and increased oxalate-supported SERCA2 Ca²⁺ uptake, suggesting an increased activity of SERCA2a. Taken together, according to our data, junctate may play an important role in the regulation of SR Ca²⁺ cycling through the interaction with SERCA2a in the murine heart.     

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca²⁺-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca²⁺-cycling proteins. But it enhanced Ca²⁺ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca²⁺ uptake showed increased Ca²⁺ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca²⁺, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca²⁺-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca²⁺-cycling of cardiomyocytes.     

Genetic modulation of the SERCA activity does not affect the Ca leak from the cardiac sarcoplasmic reticulum

The Ca²⁺ content in the sarcoplasmic reticulum (SR) determines the amount of Ca²⁺ released, thereby regulating the magnitude of Ca²⁺ transient and contraction in cardiac muscle. The Ca²⁺ content in the SR is known to be regulated by two factors: the activity of the Ca²⁺ pump (SERCA) and Ca²⁺ leak through the ryanodine receptor (RyR). However, the direct relationship between the SERCA activity and Ca²⁺ leak has not been fully investigated in the heart. In the present study, we evaluated the role of the SERCA activity in Ca²⁺ leak from the SR using a novel saponin-skinned method combined with transgenic mouse models in which the SERCA activity was genetically modulated. In the SERCA overexpression mice, the Ca²⁺ uptake in the SR was significantly increased and the Ca²⁺ transient was markedly increased. However, Ca²⁺ leak from the SR did not change significantly. In mice with overexpression of a negative regulator of SERCA, sarcolipin, the Ca²⁺ uptake by the SR was significantly decreased and the Ca²⁺ transient was markedly decreased. Again, Ca²⁺ leak from the SR did not change significantly. In conclusion, the selective modulation of the SERCA activity modulates Ca²⁺ uptake, although it does not change Ca²⁺ leak from the SR.     

Prominent Heart Organ-Level Performance Deficits in a Genetic Model of Targeted Severe and Progressive SERCA2 Deficiency

The cardiac SERCA2 Ca²⁺ pump is critical for maintaining normal Ca²⁺ handling in the heart. Reduced SERCA2a content and blunted Ca²⁺ reuptake are frequently observed in failing hearts and evidence implicates poor cardiac Ca²⁺ handling in the progression of heart failure. To gain insight into mechanism we investigated a novel genetic mouse model of inducible severe and progressive SERCA2 deficiency (inducible Serca2 knockout, SERCA2 KO). These mice eventually die from overt heart failure 7-10 weeks after knockout but as yet there have been no reports on intrinsic mechanical performance at the isolated whole heart organ level. Thus we studied whole-organ ex vivo function of hearts isolated from SERCA2 KO mice at one and four weeks post-knockout in adult animals. We found that isolated KO heart function was only modestly impaired one week post-knockout, when SERCA2a protein was 32% of normal. At four weeks post-knockout, function was severely impaired with near non-detectable levels of SERCA2. During perfusion with 10 mM caffeine, LV developed pressures were similar between 4-week KO and control hearts, and end-diastolic pressures were lower in KO. When hearts were subjected to ischemia-reperfusion injury, recovery was not different between control and KO hearts at either one or four weeks post-knockout. Our findings indicate that ex vivo function of isolated SERCA2 KO hearts is severely impaired long before symptoms appear in vivo, suggesting that physiologically relevant heart function in vivo can be sustained for weeks in the absence of robust SR Ca²⁺ flux.     

Calmodulin can induce and control damped oscillations in plasma membrane Ca<Superscript>2+</Superscript>-ATPase activity: A kinetic model

Plasma membrane Ca²⁺-ATPase is the pump that extrudes calcium ions from cells using ATP hydrolysis to maintain low Ca²⁺ concentrations in the cell. Calmodulin stimulates Ca²⁺-ATPase by binding to the autoinhibitory enzyme domain, which allows the access of cytoplasmic ATP and Ca²⁺ to the catalytic and transport sites. Our kinetic model predicts damped oscillations of the enzyme activity and interprets the known nonmonotonic kinetic behavior of the enzyme in the presence of calmodulin. For parameters close to experimental data, the kinetic model explains the dependence of the frequency and damping factor of the oscillatory enzyme activity on the calmodulin concentration. The calculated pre-steady-state curves fit well to known experimental data. Kinetic analysis allows us to assign Ca²⁺-ATPase to hysteretic enzymes exhibiting activity oscillations in open systems.     

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca²⁺ affinity of SERCA2a by competing for Ca²⁺ binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca²⁺-ATPase activity and E1∼P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca²⁺ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca²⁺ for binding to SERCA2a. This was confirmed with the Ca²⁺ pump mutant, D351A, which is catalytically inactive but retains strong Ca²⁺ binding. Increasingly higher Ca²⁺ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca²⁺ binding. Finally, the specific conformation of E2 (Ca²⁺-free state of SERCA2a) that binds PLB was investigated using the Ca²⁺-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca²⁺ showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca²⁺ binding to SERCA2a by stabilizing a unique E2·ATP state that is unable to bind thapsigargin or vanadate.     

A Mathematical Study of the Differential Effects of Two SERCA Isoforms on Ca2+ Oscillations in Pancreatic Islets

Cytosolic Ca²⁺ dynamics are important in the regulation of insulin secretion from the pancreatic β-cells within islets of Langerhans. These dynamics are sculpted by the endoplasmic reticulum (ER), which takes up Ca²⁺ when cytosolic levels are high and releases it when cytosolic levels are low. Calcium uptake into the ER is through sarcoendoplasmic reticulum Ca²⁺-ATPases, or SERCA pumps. Two SERCA isoforms are expressed in the β-cell: the high Ca²⁺ affinity SERCA2b pump and the low affinity SERCA3 pump. Recent experiments with islets from SERCA3 knockout mice have shown that the cytosolic Ca²⁺ oscillations from the knockout islets are characteristically different from those of wild type islets. While the wild type islets often exhibit compound Ca²⁺ oscillations, composed of fast oscillations superimposed on much slower oscillations, the knockout islets rarely exhibit compound oscillations, but produce slow (single component) oscillations instead. Using mathematical modeling, we provide an explanation for this difference. We also investigate the effect that SERCA2b inhibition has on the model β-cell. Unlike SERCA3 inhibition, we demonstrate that SERCA2b inhibition has no long-term effect on cytosolic Ca²⁺ oscillations unless a store-operated current is activated.     

Increased calcium leak associated with reduced calsequestrin expression in hyperthyroid cardiomyocytes

IntroductionCalcium (Ca²⁺) leak during cardiac diastole is chiefly mediated by intracellular Ca²⁺ channel/Ryanodine Receptors. Increased diastolic Ca²⁺ leak has been proposed as the mechanism underlying the appearance of hereditary arrhythmias. However, little is known about alterations in diastolic Ca²⁺ leak and the specific roles played by key intracellular Ca²⁺-handling proteins in hyperthyroidism, a known arrhythmogenic condition.AimWe sought to determine whether there were modifications in diastolic Ca²⁺ leak, based on the recording of Ca²⁺ sparks and Ca²⁺ waves; we also investigated changes in the expression and activity of key Ca²⁺ handling proteins, including ryanodine receptors, Sarco-Endoplasmic Reticulum Ca²⁺ ATPase pump and calsequestrin in isolated left-ventricular cardiomyocytes isolated from hyperthyroid rats.Materials and methodsElectrocardiography (ECG) recordings were performed in control and hyperthyroid rats. Ca²⁺ sparks, Ca²⁺ waves, and electrically-stimulated Ca²⁺ transients were recorded in Fluo-3-loaded cardiomyocytes from both experimental groups using confocal microscopy. In addition, left-ventricular homogenates and Ryanodine Receptor-enriched membrane fractions were prepared for assessing [³H]-ryanodine binding, hydrolytic ATPase activity of SERCA pump and expression levels of key proteins by Western blot, and cDNA for real-time qPCR.Results and conclusionsExtrasystoles were observed in hearts of hyperthyroid rats by ECG recordings. Arrhythmogenic activity, high incidence of Ca²⁺ waves, and de novo Ca²⁺ wavelets −in the absence of sarcoplasmic reticulum Ca²⁺ overload- were recorded in these cardiomyocytes. The exacerbated diastolic Ca²⁺ leak and arrhythmogenic activities were related to a diminished expression of calsequestrin along with increased SERCA pump activity, which, in effect, promoted a gain-of-function in RyRs without alterations in SR Ca²⁺ load, RyR expression or its Ca²⁺ sensitivity.     

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in the intracellular Ca²⁺ homeostasis of cardiac myocytes, pumping Ca²⁺ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca²⁺ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca²⁺-ATPase and Ca²⁺ transport activities in cardiac SR vesicles, and on Ca²⁺ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca²⁺-ATPase and Ca²⁺ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca²⁺ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca²⁺ load by enhancing SERCA2a-mediated Ca²⁺ transport. Analysis of cytosolic Ca²⁺ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca²⁺ transients and SR Ca²⁺ load, with negligible effects on L-type Ca²⁺ channels and Na⁺/Ca²⁺ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca²⁺ transients and SR Ca²⁺ load, but it decreased the propensity toward Ca²⁺ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [³H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca²⁺ and a small increase in μM Ca²⁺. Accordingly, compound 1 slightly decreased Ca²⁺ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca²⁺ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca²⁺ uptake, as found in failing hearts.     

Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy

Pig coronary artery smooth muscle expresses the Na⁺–Ca²⁺-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca²⁺ pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca²⁺ pool. Caveolae may also direct Ca²⁺ traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca²⁺ for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca²⁺ signaling pathways but not others.     

Three-dimensional spatio-temporal modelling of store operated Ca2+ entry: Insights into ER refilling and the spatial signature of Ca2+ signals

The spatial organisation of Orai channels and SERCA pumps within ER-PM junctions is important for enhancing the versatility and specificity of sub-cellular Ca²⁺ signals generated during store operated Ca²⁺ entry (SOCE). In this paper, we present a novel three dimensional spatio-temporal model describing Ca²⁺ dynamics in the ER-PM junction and sub-PM ER during SOCE. We investigate the role of Orai channel and SERCA pump location to provide insights into how these components shape the Ca²⁺ signals generated and affect ER refilling. We find that the organisation of Orai channels within the ER-PM junction controls the amplitude and shape of the Ca²⁺ profile but does not enhance ER refilling. The model shows that ER refilling is only weakly affected by the location of SERCA2b pumps within the ER-PM junction and that the placement of SERCA2a pumps within the ER-PM junction has much greater control over ER refilling.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.