Thermal modulation of epicardial Ca2+ dynamics uncovers molecular mechanisms of Ca2+ alternans

Ca²⁺ alternans (Ca-Alts) are alternating beat-to-beat changes in the amplitude of Ca²⁺ transients that frequently occur during tachycardia, ischemia, or hypothermia that can lead to sudden cardiac death. Ca-Alts appear to result from a variation in the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variable Ca²⁺ release has been attributed to the alternation of the action potential duration, delay in the recovery from inactivation of RYR Ca²⁺ release channel (RYR2), or an incomplete Ca²⁺ refilling of the SR. In all three cases, the RYR2 mobilizes less Ca²⁺ from the SR in an alternating manner, thereby generating an alternating profile of the Ca²⁺ transients. We used a new experimental approach, fluorescence local field optical mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. In conjunction with a local cold finger, a series of images were recorded within an area where the local cooling induced a temperature gradient. Ca-Alts were larger in colder regions and occurred without changes in action potential duration. Analysis of the change in the enthalpy and Q₁₀ of several kinetic processes defining intracellular Ca²⁺ dynamics indicated that the effects of temperature change on the relaxation of intracellular Ca²⁺ transients involved both passive and active mechanisms. The steep temperature dependency of Ca-Alts during tachycardia suggests Ca-Alts are generated by insufficient SERCA-mediated Ca²⁺ uptake into the SR. We found that Ca-Alts are heavily dependent on intra-SR Ca²⁺ and can be promoted through partial pharmacologic inhibition of SERCA2a. Finally, the FLOM experimental approach has the potential to help us understand how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.     

Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

Regional differences in arrhythmogenesis

JGP study shows that the subendocardium is more susceptible to spontaneous Ca²⁺ release events that can initiate arrhythmias, and this may be reduced by local CaMKII inhibition.

Calcium release and uptake must be carefully controlled in cardiomyocytes to ensure that the heart maintains a regular beat, and spontaneous Ca²⁺ release (SCR) from the sarcoplasmic reticulum—due to leaky ryanodine receptors, for example—can trigger lethal ventricular arrhythmias. In this issue of JGP, Dries et al. demonstrate that the subendocardial layer of the ventricular wall is particularly susceptible to arrhythmogenic SCR, and that this could potentially be treated by local inhibition of calcium/calmodulin-dependent kinase II (CaMKII; 1).Using living myocardial slices, Eef Dries (left), Cesare Terracciano (center), and colleagues show that, following injury, the subendocardial layer of the rat ventricular wall is more susceptible than the subepicardial layer to arrhythmogenic SCR events. High-resolution Ca²⁺ imaging of the subendocardium shows the increased number of SCRs (green dots) in the region bordering the injured tissue. The frequency of SCRs and ectopic contractions can be reduced by CaMKII inhibition.SCRs have been extensively studied in isolated cardiomyocytes, but arrhythmias are multicellular events (2) in which the behavior of individual cells is influenced by their interactions with neighboring cells and the extracellular matrix. “In addition, myocardial electrophysiology changes at different depths of the ventricular wall, and the vast majority of studies do not account for this transmurality,” explains Cesare Terracciano, a professor at the National Heart and Lung Institute, Imperial College London.Terracciano’s group has pioneered the use of living myocardial slices prepared from different layers of the ventricular wall to study regional differences in the electrical and mechanical properties of healthy hearts (3,4). However, it is unclear how these differences are impacted by injury or disease and whether this leaves some layers of the heart wall more susceptible to SCRs and arrhythmogenesis.Terracciano and colleagues, including first author Eef Dries, therefore prepared myocardial slices from different layers of the rat ventricular wall and subjected them to cryoinjury (1). Structural remodeling—in the form of reduced T-tubule density—was similar in both subendocardial and subepicardial slices after injury, but only subendocardial slices showed an increase in spontaneous, arrhythmic contractions.Dries et al. used a fluorescent Ca²⁺ indicator and high-resolution imaging to examine Ca²⁺ signaling in the “border zone” surrounding the cryoinjury, as this region has been implicated in triggering arrhythmias following myocardial infarction. “Intriguingly, and only in subendocardial slices after injury, we observed a reduction in the amplitude of calcium transients that also became slower to decline, changes that are hallmarks of heart failure,” Terracciano says. “SCR events were more frequent and more closely distributed when we cryoinjured the slices but, again, only in the subendocardium.”The clustering of multiple SCRs in both space and time makes them more likely to trigger an ectopic contraction. One possibility is that the open probability of ryanodine receptors is increased in subendocardial slices. This could be caused by enhanced CaMKII-mediated phosphorylation of ryanodine receptors and, indeed, Dries et al. found that, after cryoinjury, receptor phosphorylation is increased in subendocardial, but not subepicardial, slices (1).Accordingly, Terracciano and colleagues found that the CaMKII inhibitor AIP reduced the frequency of SCRs and spontaneous contractions in cryoinjured subendocardial slices. In contrast, AIP had no effect on injured subepicardial slices or on normal, healthy cardiac tissue. CaMKII inhibitors have been proposed as potential therapies for cardiac arrhythmias, but their use has so far been limited by off-target effects. Dries et al.’s results suggest that targeting CaMKII inhibitors to specific regions of the ventricular wall (using localized gene therapy, for example) could greatly improve their efficacy.“A picture is emerging that subendocardial slices are more susceptible to arrhythmogenic stimuli, and this can be important for understanding and treating arrhythmias,” Terracciano says. He now plans to study injured myocardial slices over longer time periods and investigate the molecular changes underlying the enhanced arrhythmogenic susceptibility of the subendocardium, as well as testing localized gene therapy approaches in animal models of disease.     

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na⁺-selective four-domain voltage-gated channels (4D-Na_vs) in animals allowed rapid Na⁺-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na⁺-selective voltage-gated channels (BacNa_v), they typically exhibit much slower kinetics than 4D-Na_vs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na⁺-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na⁺-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Na_v blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na⁺-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Na_vs. The EukCatB channels therefore represent an independent evolution of fast Na⁺-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Electrical signals trigger rapid physiological events that underpin an array of fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt, 2015). In excitable animal cells, Ca²⁺- or Na⁺-selective members of the four-domain voltage-gated cation channel family (4D-Ca_v/Na_v) underpin well-characterized signaling processes (Catterall et al., 2017). The 4D-Ca_v/Na_v family is broadly distributed across eukaryotes, contributing to signaling processes associated with motility in some unicellular protist and microalgal species (Fujiu et al., 2009; Lodh et al., 2016), although these channels are absent from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Ca_v/Na_v channel was Ca²⁺-permeable, with Na⁺-selective channels arising later within the animal lineage (Moran et al., 2015). This shift in ion selectivity represented an important innovation in animals, allowing rapid voltage-driven electrical excitability to be decoupled from intracellular Ca²⁺ signaling processes (Moran et al., 2015).Na⁺-selective voltage-gated channels have not been described in other eukaryotes, although a large family of Na⁺-selective channels (BacNa_v) is present in prokaryotes (Ren et al., 2001; Koishi et al., 2004). BacNa_v are single-domain channels that form homotetramers, resembling the four-domain architecture of 4D-Ca_v/Na_v. Studies of BacNa_v channels have provided considerable insight into the mechanisms of gating and selectivity in voltage-dependent ion channels (Payandeh et al., 2012; Zhang et al., 2012). The range of activation and inactivation kinetics of native BacNa_v are generally slower than observed for 4D-Na_v, suggesting that the concatenation and subsequent differentiation of individual pore-forming subunits may have enabled 4D-Na_v to develop specific properties such as fast inactivation, which is mediated by the conserved intracellular Ile–Phe–Met linker between domains III and IV (Fig. 1A; Irie et al., 2010; Catterall et al., 2017).Open in a separate windowFigure 1.EukCatBs represent a novel class of single-domain channels. A, Schematic diagram of a single-domain EukCatB channel. The voltage-sensing module (S1–S4, blue), including conserved positively charged (++) residues of segment (S4) that responds to changes in membrane potential, is shown. The pore module (S5–S6, red) is also indicated, including the SF motif (Ren et al., 2001). The structure of a 4D-Na_v (showing the SF of rat 4D-Na_v1.4 with canonical “DEKA” locus of Na⁺-selective 4D-Na_v1s) is also displayed (right). The Ile–Phe–Met motif of the fast inactivation gate is indicated (West et al., 1992) B, Maximum likelihood phylogenetic tree of single-domain, voltage-gated channels including BacNa_v and the three distinct classes of EukCat channels (EukCatA–C). Representatives of the specialized family of single-domain Ca²⁺ channels identified in mammalian sperm (CatSpers) are also included. SF for each sequence is shown (right). “Position 0” of the high-field–strength site that is known to be important in determining Na⁺ selectivity (Payandeh et al., 2011), is colored red. Channel sequences selected for functional characterization in this study are shown in bold. EukCatA sequences previously characterized (Helliwell et al., 2019) are also indicated, as is NaChBac channel from B. halodurans (Ren et al., 2001). Maximum likelihood bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are above and below nodes, respectively. Scanning electron micrographs of coccolithophores E. huxleyi (scale bar = 2 μm) and S. apsteinii, (scale bar = 10 μm) are shown.We recently identified several classes of ion channel (EukCats) bearing similarity to BacNa_v in the genomes of eukaryotic phytoplankton. Characterization of EukCatAs found in marine diatoms demonstrated that these voltage-gated channels are nonselective (exhibiting permeability to both Na⁺ and Ca²⁺) and play a role in depolarization-activated Ca²⁺ signaling (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and EukCatCs) were also identified that remain uncharacterized. These channels are present in haptophytes, pelagophytes, and cryptophytes (EukCatBs), as well as dinoflagellates (EukCatCs; Helliwell et al., 2019). Although there is a degree of sequence similarity between the distinct EukCat clades, the relationships between clades are not well resolved, and there is not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may therefore exhibit significant functional differences. Characterization of these different classes of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel structure, function, and evolution, and to our gaining insight into eukaryote membrane physiology more broadly.Notably, EukCatB channels were found in ecologically important coccolithophores, a group of unicellular haptophyte algae that represent major primary producers in marine ecosystems. Coccolithophores are characterized by their ability to produce a cell covering of ornate calcium carbonate platelets (coccoliths; Fig. 1B; Taylor et al., 2017). The calcification process plays an important role in global carbon cycling, with the sinking of coccoliths representing a major flux of carbon to the deep ocean. Patch-clamp studies of coccolithophores indicate several unusual aspects of membrane physiology, such as an inwardly rectifying Cl⁻ conductance and a large outward H⁺ conductance at positive membrane potentials, which may relate to the increased requirement for pH homeostasis associated with intracellular calcification. Here we report that EukCatB channels from two coccolithophore species (Emiliania huxleyi and Scyphosphaera apsteinii) act as very fast Na⁺-selective voltage-gated channels that exhibit many similarities to the 4D-Na_vs, which underpin neuronal signaling in animals. Thus, our findings demonstrate that the capacity for rapid Na⁺-based signaling has evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Dissecting neurotransmission with artificial synapses

JGP study demonstrates how recordings from neuron–HEK cell cocultures provide a clearer picture of the factors involved in synaptic transmission.

Resolving the rapid series of steps involved in synaptic transmission and assessing the contributions of different molecules to each of them is an enormous challenge. In this issue of JGP, Chiang et al. show that the process can be studied with greater resolution at the artificial synapses formed between neurons and cocultured human embryonic kidney (HEK) cells (1).Chung-Wei Chiang (left), Meyer Jackson (right), and colleagues studied synaptic transmission between hippocampal neurons and cocultured HEK cells. Mutations in the SNARE protein synaptobrevin 2 alter the shape of mEPSCs generated in HEK cells, an effect made clearer by the absence of dendritic filtering in this artificial system.Meyer Jackson and colleagues at the University of Wisconsin School of Medicine and Public Health are particularly interested in exocytosis. Though this process can be measured directly in endocrine cells, its role in controlling the dynamics of synaptic transmission can be difficult to separate from all the downstream steps required to elicit a response in the postsynaptic neuron. “We wanted to study a surrogate synapse with a simplified response to neurotransmitter that would allow us to focus on vesicle release with greater resolution,” Jackson explains.For years, researchers have studied synaptogenesis by transfecting HEK cells with a handful of postsynaptic factors that enable them to assemble functional synapses when cocultured with neurons (2, 3). Jackson realized that these artificial synapses lack two key sources of variability that can obscure the contribution of vesicle release to synaptic transmission. First, the postsynaptic apparatus of neuron–HEK synapses is consistent and can be precisely controlled (in contrast to regular synapses, where the molecular composition may vary from synapse to synapse). Second, the compact shape of HEK cells greatly reduces the influence of dendritic filtering, the phenomenon by which synaptic inputs take varying lengths of time to reach the cell body, depending on the location of the synapse within the dendritic arbor.Jackson and colleagues, including first author Chung-Wei Chiang, transfected HEK cells with four postsynaptic proteins—the AMPA receptor subunit GluA2, the adhesion molecule neuroligin 1, the scaffold protein PSD-95, and the accessory factor stargazin—and cocultured them with hippocampal neurons (1). The researchers then measured the miniature excitatory postsynaptic currents (mEPSCs) evoked in the HEK cells by the spontaneous release of single synaptic vesicles from neighboring neurons.The mEPSCs generated at these artificial synapses were larger and faster than mEPSCs produced by regular, neuron–neuron synapses (though they involved comparable amounts of charge, suggesting that the vesicle populations are similar at both types of synapse).Notably, the rise rate of mEPSCs in HEK cells was faster and less variable, in keeping with the absence of dendritic filtering and the consistent, shorter distance between the artificial synapses and the HEK cell body. This allowed Chiang et al. (1) to resolve the contribution of vesicle release to mEPSC dynamics using mutant versions of the SNARE protein synaptobrevin 2 that impede the flux of neurotransmitters through synaptic fusion pores (4). These mutant proteins decreased the rise rate and decay rate of mEPSCs at artificial synapses. “However, the effect was much clearer in HEK cells than we’d previously seen in neurons,” Jackson says.Chiang et al. (1) were also able to resolve the contribution of postsynaptic receptors to mEPSC shape, but Jackson is most interested in using the neuron–HEK coculture system to investigate synaptic vesicle release in more detail. “It opens up a new approach that will allow us to study synaptic exocytosis more precisely and look for much subtler effects,” Jackson says. For example, Jackson hopes to explain why mutations in some exocytotic proteins have major effects on endocrine cells but little to no effect at synapses.     

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

Background

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias.

Methods

Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca²⁺ was measured using Indo-1AM and Ca²⁺ alternans (Ca-ALT) was induced with a standard ramp pacing protocol.

Results

As predicted, SR Ca²⁺ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca²⁺ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20).

Conclusions

Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.     

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. (Mol Cell Biochem 261: 245–249, 2004)     

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.     

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.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

Altered Ca2+ Kinetics Associated with α-Actinin-3 Deficiency May Explain Positive Selection for ACTN3 Null Allele in Human Evolution

Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more “energy efficient” in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca²⁺ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca²⁺ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca²⁺-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the twitch transient; (ii) a fourfold increase in the rate of SR Ca²⁺ leak; (iii) a threefold increase in the rate of SR Ca²⁺ pumping; and (iv) enhanced maintenance of tetanic Ca²⁺ during fatigue. The SR Ca²⁺ pump, SERCA1, and the Ca²⁺-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca²⁺ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent evolution.     

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Ca²⁺ (sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA)) and Cu⁺ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca²⁺, demonstrated by the addition of ATP and Ca²⁺ to SERCA deprived of Ca²⁺ (E2) as compared with ATP to Ca²⁺-activated enzyme (E1·2Ca²⁺). Activation by Ca²⁺ is slower at low pH (2H⁺·E2 to E1·2Ca²⁺) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca²⁺ translocation. A “H⁺-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca²⁺ release by H⁺/Ca²⁺ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu⁺/H⁺ exchange. As opposed to SERCA after Ca²⁺ chelation, ATP7A/B does not undergo reverse phosphorylation with P_i after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

Comparison of Insulins Glargine and Degludec in Diabetic Rhesus Macaques (Macaca mulatta) with CGM Devices

Treating and monitoring type 2 diabetes mellitus (T2DM) in NHP can be challenging. Multiple insulin and hypoglycemic therapies and management tools exist, but few studies demonstrate their benefits in a NHP clinical setting. The insulins glargine and degludec are long-acting insulins; their duration of action in humans exceeds 24 and 42 h, respectively. In the first of this study''s 2 components, we evaluated whether insulin degludec could be dosed daily at equivalent units to glargine to achieve comparable blood glucose (BG) reduction in diabetic rhesus macaques (Macaca mulatta) with continuous glucose monitoring (CGM) devices. The second component assessed the accuracy of CGM devices in rhesus macaques by comparing time-stamped CGM interstitial glucose values, glucometer BG readings, and BG levels measured by using an automated clinical chemistry analyzer from samples that were collected at the beginning and end of each CGM device placement. The CGM devices collected a total of 21,637 glucose data points from 6 diabetic rhesus macaques that received glargine followed by degludec every 24 h for 1 wk each. Ultimately, glucose values averaged 29 mg/dL higher with degludec than with glargine. Glucose values were comparable between the CGM device, glucometer, and chemistry analyzer, thus validating that CGM devices as reliable for measuring BG levels in rhesus macaques. Although glargine was superior to degludec when given at the same dose (units/day), both are safe and effective treatment options. Glucose values from CGM, glucometers, and chemistry analyzers provided results that were analogous to BG values in rhesus macaques. Our report further highlights critical clinical aspects of using glargine as compared with degludec in NHP and the benefits of using CGM devices in macaques.

Diabetes is a group of metabolic diseases that cause hyperglycemia secondary to deficient insulin response, secretion, or both.⁴ Diabetes is categorized by the American Diabetes Association into 4 types: 1) type 1 diabetes mellitus, in which the pancreas is unable to produce insulin for glucose absorption; 2) type 2 diabetes mellitus (T2DM), when the body does not use insulin correctly; 3) gestational diabetes, in which the body is insulin-intolerant during pregnancy (or is first discovered then); and 4) other specific forms of diabetes in which the patient is particularly predisposed to becoming diabetic due to various comorbidities or to inadvertent induction caused by some medications.⁴ In 2018, 34.2 million (10.5%) Americans of all ages were diagnosed with diabetes.^22,23,30 Approximately 90% to 95% of Americans with diabetes have T2DM,²⁴ making T2DM the most common form of diabetes diagnosed in humans.T2DM is a multifactorial disease primarily determined by genetics, behavioral and environmental factors (for example, age, diet, sedentary lifestyle, obesity).^4,46,50,74 As a consequence of these factors, the pancreas increases insulin secretion to maintain normal glucose tolerance.⁷⁴ Over time, the high insulin demand causes pancreatic β-cell destruction, resulting in reduced production of insulin.^39,50,74 As β-cell destruction increases, hyperglycemia and T2DM develop. Insulin resistance and hyperglycemia are tolerated for a period of time^19,82,83 before clinical signs associated with T2DM develop (e.g., polydipsia, polyuria, polyphagia with concurrent weight loss).⁴ Once clinical signs develop, T2DM is most commonly diagnosed as a fasting blood glucose level (FBG) of 126 mg/dL or greater,^2,4 2-h plasma glucose value of 200 mg/d or greater during a 75-g oral glucose tolerance test,^2,4 and/or glycosylated hemoglobin (HbA1c) of 6.5% or greater.^2,4 Depending on the FBG, oral glucose tolerance test, and HbA1c results, various treatment options are recommended by the American Diabetes Association. Most importantly, lifestyle changes, including diet and exercise, are recommended as the first line of treatment, along with oral antihyperglycemic drugs such as metformin.^5,25,46 Treatment efficacy is evaluated with self-monitoring blood glucose or continuous glucose monitoring (CGM) devices.³ Human patients using CGM devices have achieved considerable reductions in HbA1c compared with patients not using them.³ As CGM devices have become more readily available, user friendly, and affordable, they have become an essential tool in managing T2DM.Similar to humans, most NHP affected by diabetes are diagnosed with T2DM.^80,83 NHP are predisposed to similar genetic, behavioral and environmental factors (e.g., age, diet, sedentary lifestyle, obesity);^{6,18,19,37,44,52,82,83} have similar pathophysiology;^38,81-83 are diagnosed via FBG,^39,83 HbA1c,^21,31,49,56 fructosamine,^20,83,87 and weight loss;^49,80,83,86 and are treated with exercise and diet modifications as a first line of treatment.^{11,19,39,53,79} Although the human and NHP conditions are similar, the treatment and management of T2DM is somewhat different, especially when NHP have restricted physical activity due to housing constraints.Previous studies indicate that daily dosing with insulin glargine achieves appropriate glycemic control in NHP.⁴⁸ Therefore, we implemented glargine, along with some diet modification, to improve glycemic control in our diabetic colony. Other noninsulin therapies, such as metformin, had been used, but compliance was low (for example, due to large pill size, unpleasant taste, etc.). However, achieving glycemic control using diet modification, insulin glargine treatment, monthly scheduled FBG, quarterly HbA1c, and regular weight monitoring was challenging in a large colony. Monthly FBG and fructosamine testing were performed due to affordability and practicality for NHP in a research setting. Given that fructosamine levels correlate with BG concentrations for the preceding 2 to 3 wk and HbA1c percentages relate to BG concentration over 1.5 to 3 mo,^49,87 HbA1C was selected over fructosamine for T2DM management in our colony. Determining which T2DM treatment and diagnostics are most effective can be difficult in large colonies of NHP. Therefore, improved treatment and management strategies would help to manage T2DM in NHP more efficiently.Insulin glargine is a long-acting insulin, with a half-life of 12 h and duration of action of 12 to 24 h in humans^40,55 and 12 h in dogs.^34,43,60 Once injected subcutaneously, insulin glargine forms a microprecipitate in the neutral pH environment, which delays and prolongs absorption in subcutaneous tissues.¹² Insulin degludec is a newer form of long-acting insulin, with a half-life of 25 h^41,63,62,77 and duration of action that exceeds 42 h in humans.^40,41,68,77 Insulin degludec forms a soluble and stable dihexamer in the pharmaceutical formulation, which includes phenol and zinc.^63,78 The phenol diffuses away, leading to the formation of a soluble depot in the form of long multihexamer chains in which zinc slowly diffuses from the end of the multihexamers, causing a gradual, continuous, and extended-release of monomers from the depot of the injection site.^63,78 Pharmacodynamic studies in humans, demonstrate that the “glucose-lowering effect” of insulin degluc⁴⁰ is evenly distributed over 24 h, allowing a more stable steady-state and improved wellbeing.⁷⁸ This approach could potentially reduce the number of hypoglycemic events and provide a less rigid daily injection schedule,⁵⁸ thus potentially making insulin degludec—compared with insulin glargine—a safer, alternative diabetes therapy.In addition to medical intervention, glycemic control is achieved through regular management and monitoring of BG. Self-monitoring blood glucose checks in humans^3,5 and glucose curves in animals¹⁰ are some of the management tools used to determine or evaluate therapy for T2DM patients. Telemetry systems like CGM devices are used to monitor interstitial glucose and have been used extensively in humans^3,17,33 and animals^{16,27,36,42,47,84,85} to monitor BG in real-time. Using CGM devices 1) reduces or eliminates the number of blood draws needed to collect FBG,⁶¹ 2) accurately assesses insulin therapy via a real-time glucose curve,^72,84,85 3) allows patients and clinicians to titrate treatment^61,73 as indicated, and 4) obtains continuous glucose data with reduced manipulation and subsequent decreased stress.^72,84,85 Therefore, CGM devices can be a safe and informative tool in monitoring spontaneous T2DM in NHP.Between 2015 and 2030, the prevalence of diabetes is predicted to increase by 54% to more than 54 million Americans affected by diabetes (i.e., diabetes mellitus types 1 and 2).⁷⁰ NHP are an essential model for human T2DM because of their similar pathophysiology, diagnostics, treatment, and management. As more people develop diabetes, novel therapies will continue to be developed. Studying new treatments and management tools in NHP can further human and NHP T2DM research to prevent the progression of T2DM and hopefully diminish projections for the number of future diabetes cases. Human medical literature, American Diabetes Association, and drug manufacturers all recommend giving equal doses (i.e., number of units/day) of long-acting insulins when changing from one long-acting insulin to degludec.^26,63,67 Therefore, we hypothesized that insulin degludec would provide effective glycemic control for rhesus macaques with T2DM when dosed at equivalent doses (that is, the same number of units/day) as insulin glargine. In addition, we hypothesized that CGM devices would provide accurate BG readings as compared with chemistry analyzer and glucometer BG readings, making it a more efficient and effective tool for measurement of BG levels in rhesus macaques with T2DM.     

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.     

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.