Calcium In Cardiac Myocytes Research Articles

Gene transfer of human cardiomyopathy β-MyHC mutant R403Q directly alters intact cardiac myocyte calcium homeostasis and causes hyper-contractility.

The R403Q mutation of human cardiac β-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human β-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human β-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at ∼40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after β-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

A-Kinase Anchoring Proteins in Cardiac Myocytes and Their Roles in Regulating Calcium Cycling.

The rate of calcium cycling and calcium transient amplitude are critical determinants for the efficient contraction and relaxation of the heart. Calcium-handling proteins in the cardiac myocyte are altered in heart failure, and restoring the proper function of those proteins is an effective potential therapeutic strategy. The calcium-handling proteins or their regulators are phosphorylated by a cAMP-dependent kinase (PKA), and thereby their activity is regulated. A-Kinase Anchoring Proteins (AKAPs) play a seminal role in orchestrating PKA and cAMP regulators in calcium handling and contractile machinery. This cAMP/PKA orchestration is crucial for the increased force and rate of contraction and relaxation of the heart in response to fight-or-flight. Knockout models and the few available preclinical models proved that the efficient targeting of AKAPs offers potential therapies tailor-made for improving defective calcium cycling. In this review, we highlight important studies that identified AKAPs and their regulatory roles in cardiac myocyte calcium cycling in health and disease.

Calcium Buffering in the Heart in Health and Disease.

Changes of intracellular Ca2+ concentration regulate many aspects of cardiac myocyte function. About 99% of the cytoplasmic calcium in cardiac myocytes is bound to buffers, and their properties will therefore have a major influence on Ca2+ signaling. This article considers the fundamental properties and identities of the buffers and how to measure them. It reviews the effects of buffering on the systolic Ca2+ transient and how this may change physiologically, and in heart failure and both atrial and ventricular arrhythmias, as well. It is concluded that the consequences of this strong buffering may be more significant than currently appreciated, and a fuller understanding is needed for proper understanding of cardiac calcium cycling and contractility.

In This Issue

In This Issue

Non-RyR Calcium Leak of the Sarcoplasmic Reticulum is Governed by TRPC1 in Cardiomyocytes

Sarcoplasmic calcium leak in cardiomyocytes has been explained by spontaneous sparks from ryanodine receptors (RyRs), non-spark-mediated RyR leak and non-RyR leak. Ion channels underlying non-RyR leak are currently ill defined. Here, we test the hypothesis that transient receptor potential cation (TRPC) 1 channels contribute to non-RyR leak. We investigated neonatal rat ventricular myocytes (NRVMs) infected with adenoviral eGFP, TRPC1-eGFP, shRNA-TRPC1-eGFP and scrambled RNA-eGFP constructs. NRVMs were studied 4-5 days after infection. NRVMs were fixed and labeled for TRPC1 and sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Three-dimensional image stacks of NRVMs were acquired using a confocal microscope (Leica SP8 TCS). Protein colocalization was measured using the Pearson correlation coefficient (Rr). We also investigated SR calcium load in Rhod-3 loaded NRVMs. In short, NRVMs were paced and then superfused with 0 sodium/0 calcium solution for 2 min. Subsequently caffeine (20 mM) was applied to trigger sarcoplasmic calcium release. Fluorescence signals were acquired using a Leica SP8 TCS microscope and analysed after noise reduction and self-ratioing. We found a mid-level degree of colocalization of SERCA2 and TRPC1 for NRVMs infected with with eGFP (Rr=0.46±0.03), TRPC1-eGFP (0.41±0.03) and shRNA-TRPC1-eGFP (0.47±0.03). Colocalization of eGFP with SERCA2 was higher for TRPC1-eGFP (0.41±0.04) than for eGFP (0.26±0.02) and shRNA-TRPC1-eGFP infected NRVMs (0.22±0.04). We measured decreased and increased sarcoplasmic calcium content in NRVMs infected with TRPC1-eGFP (1.64±0.21) and shRNA-TRPC1-eGFP (4.30±0.49), respectively, versus eGFP (3.18±0.23). Differences between calcium content in eGFP and scrambled RNA-eGFP infected NRVMs were not significant. The investigations support our hypothesis that TRPC1 channels contribute to non-RyR sarcoplasmic calcium leak in cardiomyocytes. Structural data indicate that TRPC1 is located in the sarcoplasmic membrane. We suggest that expression and activity of TRPC1 channels modulate the concentration of sarcoplasmic and cytosolic calcium in cardiac myocytes.

Calcium Channel Mutations in Cardiac Arrhythmia Syndromes.

Voltage gated calcium channels are essential for cardiac physiology by serving as sarcolemma- restricted gatekeepers for calcium in cardiac myocytes. Activation of the L-type voltagegated calcium channel provides the calcium entry required for excitation-contraction coupling and contributes to the plateau phase of the cardiac action potential. Given these critical physiological roles, subtle disturbances in L-type channel function can lead to fatal cardiac arrhythmias. Indeed, numerous human arrhythmia syndromes have been linked to mutations in the L-type channel leading to gain-of-function or loss-of-function mutations. In this review, we discuss the current state of knowledge regarding these mutations present in Timothy Syndrome, Long and Short QT Syndromes, Brugada Syndrome and Early Repolarization Syndrome. We discuss the pathological consequences of the mutations, the biophysical effects of the mutations on the channel as well as possible therapeutic considerations and challenges for future studies.

Abstract 402: Selective Protein Secretion Upon Endoplasmic Reticulum Stress Protects Cardiac Myocytes

Protein secretion is important for proper cardiac myocyte function. Many secreted proteins are synthesized and folded in the sarco- endo-plasmic reticulum (SR/ER). A number of diseases, including heart disease, alter the ER in ways that impair ER protein folding, causing ER stress, which can result in cardiac myocyte dysfunction and decreased viability. In studies aimed at assessing the effects of ER stress on cardiac myocyte viability, heart disease-related ER stress was mimicked by treating neonatal rat ventricular myocytes (NRVM) with either tunicamycin (TM) or thapsigargin (TG), which inhibit SR/ER protein glycosylation or decrease SR/ER calcium, respectively. When treated in high culture media volumes, both TM and TG caused cardiac myocyte death; however, in low culture media volumes, while TM still caused death, remarkably, TG was protective, suggesting that potentially protective factors were secreted in response to TG but not TM. To characterize these factors, the identities of proteins in control-, TM-, and TG-conditioned medium from NRVM were determined by proteomic approaches using high performance liquid chromatography and mass spectrometry. Twenty-four different proteins, known to be synthesized in the ER, were identified in control-conditioned medium. The levels of eighteen of these proteins, including extracellular matrix proteins, hormones, and growth factors were decreased in TM- and TG-conditioned medium. However, the levels of three SR/ER-resident, calcium-binding chaperones, glucose regulated protein 78 (GRP78), glucose regulated protein 94 (GRP94), and calreticulin were increased in TG-conditioned medium but not in TM-conditioned medium. Furthermore, we found that ischemia/reperfusion, which decreases SR/ER calcium, upregulated secretion of the proteins selectively secreted in response to TG. Thus, while ER stress mediated by TM or TG decreases the movement of most proteins through the secretory pathway, TG, which mimics the effects of heart disease on SR/ER calcium in cardiac myocytes, selectively enhances the secretion of a subset of proteins, which confer protection. Therefore, proteins once thought to be permanent residents of the SR/ER may have novel, extracellular, protective roles in the diseased heart.

Nuclear calcium in cardiac myocytes.

Calcium (Ca) is a universal second messenger involved in the regulation of various cellular processes, including electrical signaling, contraction, secretion, memory, gene transcription, and cell death. In heart, Ca governs cardiomyocyte contraction, is central in electrophysiological properties, and controls major signaling pathway implicated in gene transcription. How cardiomyocytes decode Ca signal to regulate gene expression without interfering with, or being controlled by, "contractile" Ca that floods the entire cytosol during each heartbeat is still elusive. In this review, we summarize recent findings on nuclear Ca regulation and its downstream signaling in cardiomyocytes. We will address difficulties in reliable quantification of nuclear Ca fluxes and discuss its role in the development and progression of cardiac hypertrophy and heart failure. We also point out key open questions to stimulate future work.

Calcium Concentration Fluctuations and Subspace Volume Influence Calcium-Regulated Calcium Channel Gating and Subspace Dynamics

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough that local signaling may involve 1-100 calcium ions. Therefore, the question arises: is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of subspace calcium concentration and channel open probability (a result that accounts for the small subspace volume and concentration fluctuations) with the corresponding deterministic model (an approximation that assumes large system size and ignores concentration fluctuations). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium-binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast, demonstrating that the calcium concentration fluctuations and subspace volume influence channel gating and subspace dynamics. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers [Tadross, Dick, Yue. Cell 133: 1228-40, 2008]. Additional work will consider the influence of slow and rapid buffers present in the subspace and whether and under what conditions these buffers mitigate the effects of concentration fluctuations on channel gating and subspace dynamics.

Discrete-State Stochastic Models of Calcium-Regulated Calcium Influx and Subspace Dynamics Are Not Well-Approximated by ODEs That Neglect Concentration Fluctuations

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough (e.g., 10−17 liters) that local signaling may involve 1–100 calcium ions. Is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of fluctuating subspace calcium concentration (a result that accounts for the small subspace volume) with the corresponding deterministic model (an approximation that assumes large system size). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers.

Abstract 8142: By Virtue of Its Interaction with the β 2 -adrenergic Receptor, CXCR4 Plays a Role in the Regulation of β 2 -adrenergic Receptor Signaling Pathway Affecting Calcium and Contractility in Cardiac Myocytes

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α (SDF-1α) and its G-protein coupled receptor, CXCR4. SDF-1 α /CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. We hypothesis that β 2 adrenergic receptor in the heart is negatively modulated by interactions with CXCR4 chemokine receptor. For this we have used biophysical techniques such as bioluminescence resonance energy transfer assay and biochemical techniques such as immunoprecipitation and signaling assays to identify the CXCR4-β2AR interaction and determine the physiological significance of this interaction. Our studies demonstrate activation of CXCR4 by SDF-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. In conclusion, we demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal switching from Gαs to Gαi-mediated pathway and suggests the interaction is a novel mechanism for regulating cardiac myocyte calcium handling and subsequent contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4- β2AR complex could represent a hitherto unknown target for therapeutic intervention.

Hypoxia and HIF-1 suppress SERCA2a expression in embryonic cardiac myocytes through two interdependent hypoxia response elements

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is an essential component of cardiomyocyte excitation–contraction (EC)-coupling. Suppression of SERCA2a expression induces contractile dysfunction and has been reported in various forms of ischemic cardiac disease as well as in hypobaric hypoxia. The present study investigated whether SERCA2a expression is regulated by hypoxia in embryonic mouse cardiomyocytes and explored the underlying mechanism. We show that in cultured embryonic cardiomyocytes hypoxia (1% O2) induce time-dependent downregulation of SERCA2a expression. This mechanism manifested as specific changes in cardiac myocyte calcium signals induced by reduced expression and activity of SERCA2a. Chemical activation of hypoxia-inducible factor-1 (HIF-1) by DFO or overexpression of normoxia-stabile HIF-1α (HIF-1α/VP16) suppressed endogenous SERCA2a expression as well as the activity of the SERCA2a-promoter-luciferase reporter. Analysis of the SERCA2a promoter found two putative HIF-1 binding HRE-sites. Site-specific promoter mutagenesis revealed that co-operative HIF-1 binding to both of these hypoxia response elements on the SERCA2a promoter is required for expressional suppression. This mechanism establishes a link between oxygen supply and calcium activity in embryonic cardiac myocytes that is exploited in cardiac development, and further may offer a possible explanation for the functional depression of SERCA2a seen in ischemic and hypoxic myocardium.

Location of Transmembrane Segments of Na+/ca2+ Exchanger NCX1 Investigated with Chemical Crosslinkers

The sodium-calcium exchanger (NCX1) is a plasma membrane protein important in regulating calcium in cardiac myocytes. The topological model is comprised of nine transmembrane segments (TMs) and a large intracellular loop, which has two Ca2+ binding domains (CBD1 and CBD2), between TMs five and six. CBD1 and CBD2 have been crystallized recently and are important in regulating the function of NCX1. On the other hand, the three dimensional structure of the full length NCX1 is unknown. To gain insights into that 3-D structure, we performed cysteine crosslinking experiments. Pairs of amino acids in different TMs were mutated to cysteine on the backbone of cysteineless NCX1. The mutated NCXs were expressed in an insect cell line and treated with cysteine-specific chemical crosslinkers followed by SDS-PAGE to determine the proximity of the introduced cysteines. The results allow us to place TMS I, IV and IX into the context of the other TMS. By combining our new results with our previous work ( J Biol Chem. 2006, 281: 22808-14; J Biol Chem. 2001, 276:194-9.) , we propose that TMs II and VII, which contain a number of hydrophilic residues, are surrounded by the remaining TMs.

Glucocorticoids Improve Calcium Cycling in Cardiac Myocytes After Cardiopulmonary Bypass

Glucocorticoids can reduce myocardial dysfunction associated with ischemia and reperfusion injury following cardiopulmonary bypass (CPB) and circulatory arrest. The hypothesis was that maintenance of cardiac function after CPB with methylprednisolone therapy results, in part, from preservation of myocyte calcium cycling. Piglets (5-7 kg) underwent CPB and 120 min of hypothermic circulatory arrest with (CPB-GC) or without (CPB) methylprednisolone (30 mgkg(-1)) administered 6h before and at CPB. Controls (No-CPB) did not undergo CPB or receive glucocorticoids (n=6 per treatment). Myocardial function was monitored in vivo for 120 min after CPB. Calcium cycling was analyzed using rapid line-scan confocal microscopy in isolated, fluo-3-AM-loaded cardiac myocytes. Phospholamban phosphorylation and sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a) protein levels were determined by immunoblotting of myocardium collected 120 min after CPB. Calpain activation in myocardium was measured by fluorometric assay. Preload recruitable stroke work in vivo 120 min after reperfusion decreased from baseline in CPB (47.4±12 versus 26.4±8.3 slope of the regression line, P<0.05), but was not different in CPB-GC (41±8.1 versus 37.6±2.2, P=0.7). In myocytes isolated from piglets, total calcium transient time remained unaltered in CPB-GC (368±52.5 ms) compared with controls (434.5±35.3 ms; P=0.07), but was prolonged in CPB myocytes (632±83.4 ms; P<0.01). Calcium transient amplitude was blunted in myocytes from CPB (757±168 nM) compared with controls (1127±126 nM, P<0.05) but was maintained in CPB-GC (1021±155 nM, P>0.05). Activation of calpain after CPB was reduced with glucocorticoids. Phospholamban phosphorylation and SERCA2a protein levels in myocardium were decreased in CPB compared with No-CPB and CPB-GC (P<0.05). The glucocorticoid-mediated improvement in myocardial function after CPB might be due, in part, to prevention of calpain activation and maintenance of cardiac myocyte calcium cycling.

A role for NAADP in mouse cardiac myocyte calcium signaling?

A role for NAADP in mouse cardiac myocyte calcium signaling?

A role for NAADP in mouse cardiac myocyte calcium signalling?

A role for NAADP in mouse cardiac myocyte calcium signalling?

Transcriptional activation of the SERCA2 gene by calcium in cardiac myocytes

Transcriptional activation of the SERCA2 gene by calcium in cardiac myocytes

Effects of uremic serum on isolated cardiac myocyte calcium cycling and contractile function.

Diastolic dysfunction occurs in patients with chronic renal failure. Moreover, serum from uremic patients contains one or more inhibitors of the plasmalemmal Na,K-ATPase (sodium pump). We hypothesized that a circulating substance present in uremic sera contributes to both sodium pump inhibition and diastolic dysfunction. Serum samples were obtained from six patients with chronic renal failure and diastolic dysfunction. Their serum samples caused marked inhibition of Na,K-ATPase purified from dog kidney at all concentrations studied (all P < 0.01) and also impaired ouabain-sensitive rubidium uptake by myocytes isolated from Sprague-Dawley rats (P < 0.01). These cardiac myocytes were studied for their contractile function with video-edge detection and calcium metabolism with indo-1 fluorescence spectroscopy after exposure to these uremic sera. These uremic sera caused increases in myocyte fractional shortening (P < 0.01) as well as an increase in the time constant of relengthening (P < 0.01). Examining the calcium transient, the time constant for calcium recovery was also increased (P < 0.01). Exposure of these cells to sera from age- and sex-matched healthy subjects did not result in significant changes in contraction or calcium cycling. Extracts of uremic serum samples inhibited isolated Na,K-ATPase whereas extracts of normal serum samples did not. The effect of uremic serum extracts on contractile function and calcium cycling were quite similar to that of intact serum or the addition of ouabain. Co-incubation of uremic serum extract with an antibody fragment directed against digoxin markedly attenuated the inhibition of Na,K-ATPase activity and completely prevented any effects on calcium cycling or contractile function. These data show that one or more substances are present in uremic sera that acutely cause increased force of contraction and impaired recovery of cardiac myocyte calcium concentration as well as impaired relaxation. As these effects are similar to that seen with ouabain and can be prevented by co-incubation with an antibody fragment to digitalis, which also attenuates the sodium pump inhibitory effect, we suggest that this (these) substance(s) circulating in uremic sera and inhibiting the sodium pump also causes the acute diastolic dysfunction seen in our system.

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of intracellular free calcium ([Ca(2+)](i)) was investigated in indo-1 loaded ventricular myocytes from the rat, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-1 at different temperatures was calibrated both at pH-stat and at alpha-stat, and the result demonstrated that the alpha-stat calibration should be preferred. Analysis of the fluorescent image showed a striking increase of [Ca(2+)](i) as well as spontaneous calcium waves in rat cells, indicating an overloaded calcium. In contrast, cardiac myocytes of the ground squirrel were found to keep a constant [Ca(2+)](i) without calcium overload regardless of temperature variation. It is believed that understanding of the mechanisms underlying the intercellular calcium homeostasis of hibemators may lead to solutions of some medical questions.

Cytokines increase neonatal cardiac myocyte calcium concentrations: the involvement of nitric oxide and cyclic nucleotides.

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.