Transients In Cardiomyocytes Research Articles

Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

Multiscale computational modeling of the effects of 2'-deoxy-ATP on cardiac muscle calcium handling.

2'-Deoxy-ATP (dATP), a naturally occurring near analog of ATP, is a well-documented myosin activator that has been shown to increase contractile force, improve pump function, and enhance lusitropy in the heart. Calcium transients in cardiomyocytes with elevated levels of dATP show faster calcium decay compared with cardiomyocytes with basal levels of dATP, but the mechanisms behind this are unknown. Here, we design and utilize a multiscale computational modeling framework to test the hypothesis that dATP acts on the sarcoendoplasmic reticulum calcium-ATPase (SERCA) pump to accelerate calcium re-uptake into the sarcoplasmic reticulum during cardiac relaxation. Gaussian accelerated molecular dynamics simulations of human cardiac SERCA2A in the E1 apo, ATP-bound and dATP-bound states showed that dATP forms more stable contacts in the nucleotide binding pocket of SERCA and leads to increased closure of cytosolic domains. These structural changes ultimately lead to changes in calcium binding, which we assessed using Brownian dynamics simulations. We found that dATP increases calcium association rate constants to SERCA and that dATP binds to apo SERCA more rapidly than ATP. Using a compartmental ordinary differential equation model of human cardiomyocyte excitation-contraction coupling, we found that these increased association rate constants contributed to the accelerated rates of calcium transient decay observed experimentally. This study provides clear mechanistic evidence of enhancements in cardiac SERCA2A pump function due to interactions with dATP.

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Light Emitting Diodes Photobiomodulation Improves Cardiac Function by Promoting ATP Synthesis in Mice With Heart Failure.

Heart failure (HF) is the common consequences of various cardiovascular diseases, often leading to severe cardiac output deficits with a high morbidity and mortality. In recent years, light emitting diodes-based therapy (LEDT) has been widely used in multiple cardiac diseases, while its modulatory effects on cardiac function with HF still remain unclear. Therefore, the objective of this study was to investigate the effects of LED-Red irradiation on cardiac function in mice with HF and to reveal its mechanisms. In this study, we constructed a mouse model of HF. We found that LED-Red (630 nm) was an effective wavelength for the treatment of HF. Meanwhile, the application of LED-Red therapy to treat HF mice improved cardiac function, ameliorate heart morphology, reduced pulmonary edema, as well as inhibited collagen deposition. Moreover, LED-Red therapy attenuated the extent of perivascular fibrosis. Besides, LED-Red irradiation promoted calcium transients in cardiomyocytes as well as upregulated ATP synthesis, which may have positive implications for contractile function in mice with HF. Collectively, we identified that LED-Red exerts beneficial effects on cardiac function in HF mice possibly by promoting the synthesis of ATP.

On computational classification of genetic cardiac diseases applying iPSC cardiomyocytes

BackgroundCardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) can be used to study genetic cardiac diseases. In patients these diseases are manifested e.g. with impaired contractility and fatal cardiac arrhythmias, and both of these can be due to abnormal calcium transients in cardiomyocytes. Here we classify different genetic cardiac diseases using Ca2+ transient data and different machine learning algorithms. MethodsBy studying calcium cycling of disease-specific iPSC-CMs and by using calcium transients measured from these cells it is possible to classify diseases from each other and also from healthy controls by applying machine learning computation on the basis of peak attributes detected from calcium transient signals. ResultsIn the current research we extend our previous study having Ca-transient data from four different genetic diseases by adding data from two additional diseases (dilated cardiomyopathy and long QT Syndrome 2). We also study, in the light of the current data, possible differences and relations when machine learning modelling and classification accuracies were computed by using either leave-one-out test or 10-fold cross-validation. ConclusionsDespite more complex classification tasks compared to our earlier research and having more different genetic cardiac diseases in the analysis, it is still possible to attain good disease classification results. As excepted, leave-one-out test and 10-fold cross-validation achieved virtually equal results.

SERCA2a ameliorates cardiomyocyte T-tubule remodeling via the calpain/JPH2 pathway to improve cardiac function in myocardial ischemia/reperfusion mice

Transverse-tubules (T-tubules) play pivotal roles in Ca2+-induced, Ca2+ release and excitation–contraction coupling in cardiomyocytes. The purpose of this study was to uncover mechanisms where sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) improved cardiac function through T-tubule regulation during myocardial ischemia/reperfusion (I/R). SERCA2a protein expression, cytoplasmic [Ca2+]i, calpain activity, junctophilin-2 (JPH2) protein expression and intracellular localization, cardiomyocyte T-tubules, contractility and calcium transients in single cardiomyocytes and in vivo cardiac functions were all examined after SERCA2a knockout and overexpression, and Calpain inhibitor PD150606 (PD) pretreatment, following myocardial I/R. This comprehensive approach was adopted to clarify SERCA2a mechanisms in improving cardiac function in mice. Calpain was activated during myocardial I/R, and led to the proteolytic cleavage of JPH2. This altered the T-tubule network, the contraction function/calcium transients in cardiomyocytes and in vivo cardiac functions. During myocardial I/R, PD pretreatment upregulated JPH2 expression and restored it to its intracellular location, repaired the T-tubule network, and contraction function/calcium transients of cardiomyocytes and cardiac functions in vivo. SERCA2a suppressed calpain activity via [Ca2+]i, and ameliorated these key indices. Our results suggest that SERCA2a ameliorates cardiomyocyte T-tubule remodeling via the calpain/JPH2 pathway, thereby improving cardiac function in myocardial I/R mice.

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels.

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.

Antagonism of the Thromboxane-Prostanoid Receptor as a Potential Therapy for Cardiomyopathy of Muscular Dystrophy.

BackgroundMuscular dystrophy (MD) causes a progressive cardiomyopathy characterized by diffuse fibrosis, arrhythmia, heart failure, and early death. Activation of the thromboxane‐prostanoid receptor (TPr) increases calcium transients in cardiomyocytes and is proarrhythmic and profibrotic. We hypothesized that TPr activation contributes to the cardiac phenotype of MD, and that TPr antagonism would improve cardiac fibrosis and function in preclinical models of MD.Methods and ResultsThree different mouse models of MD (mdx/utrn double knockout, second generation mdx/mTR double knockout, and delta‐sarcoglycan knockout) were given normal drinking water or water containing 25 mg/kg per day of the TPr antagonist ifetroban, beginning at weaning. After 6 months (10 weeks for mdx/utrn double knockout), mice were evaluated for cardiac and skeletal muscle function before euthanization. There was a 100% survival rate of ifetroban‐treated mice to the predetermined end point, compared with 60%, 43%, and 90% for mdx/utrn double knockout, mdx/mTR double knockout, and delta‐sarcoglycan knockout mice, respectively. TPr antagonism improved cardiac output in mdx/utrn double knockout and mdx/mTR mice, and normalized fractional shortening, ejection fraction, and other parameters in delta‐sarcoglycan knockout mice. Cardiac fibrosis in delta‐sarcoglycan knockout was reduced with TPr antagonism, which also normalized cardiac expression of claudin‐5 and neuronal nitric oxide synthase proteins and multiple signature genes of Duchenne MD.Conclusions TPr antagonism reduced cardiomyopathy and spontaneous death in mouse models of Duchenne and limb‐girdle MD. Based on these studies, ifetroban and other TPr antagonists could be novel therapeutics for treatment of the cardiac phenotype in patients with MD.

Abstract 397: Preventing Cardiomyopathy of Muscular Dystrophy Through Antagonism of the Thromboxane/Prostanoid Receptor

Muscular dystrophy causes mechanical damage and increased membrane permeability of cardiomyocytes, leading to progressive cardiomyopathy and diffuse myocardial fibrosis that typically begins in the free wall of the LV. Cardiac dysfunction is a primary cause of death in Duchenne (DMD) and other muscular dystrophy (MD) patients. Activation of the thromboxane/prostanoid (TP) receptor increases calcium transients in cardiomyocytes, causes arrhythmia, and is pro-fibrotic. We thus hypothesized that TP receptor activation contributes to the cardiac phenotype of MD, and that blockade of the TP receptor would improve cardiac fibrosis and function in mouse MD models. We gave 3 different mouse models of MD either normal drinking water or water containing 25 mg/kg/day of the TP receptor antagonist ifetroban, from weaning to the predetermined endpoint. TP receptor antagonism improved 10-week survival from 60% to 100% in utrophin/dystrophin double knockout mice, a model of severe DMD, and increased cardiac output compared with surviving vehicle-treated mice. In the mdx/mTR mouse model of DMD, treatment with ifetroban likewise improved 6-month survival from 43% to 100% and increased cardiac output. Finally, we examined delta-sarcoglycan knockout (dSG KO) male mice, a model of limb-girdle muscular dystrophy (LGMD) that replicates the DMD cardiac phenotype with improved survival. TP receptor antagonism normalized fractional shortening, ejection fraction, and LVSP in dSG KO mice, and decreased plasma ANP. However, it had no effect on the contraction deficits of isolated cardiomyocytes other than to normalize the slowed relaxation of dSG KO. Ifetroban-treated mice had improved myocardial, but not skeletal muscle fibrosis. This was most noticeable in the LV free wall and occurred in conjunction with decreased TGF-beta activity and normalized plasma WBC. Typical of DMD cardiomyopathy, dSG KO hearts had reduced expression of neuronal nitric oxide synthase (nNOS) and claudin-5, which was improved with TPr antagonism. The results of our studies indicate that TP receptor activation may contribute to MD cardiomyopathy, and oral antagonism of the TP receptor may be a novel therapeutic for the cardiac phenotype in DMD and LGMD patients.

Increased Cardiomyocyte Alignment and Intracellular Calcium Transients Using Micropatterned and Drug-Releasing Poly(Glycerol Sebacate) Elastomers.

Myocardial tissue engineering is a promising therapy for myocardial infarction recovery. The success of myocardial tissue engineering is likely to rely on the combination of cardiomyocytes, prosurvival regulatory signals, and a flexible biomaterial structure that can deliver them. In this study, poly(glycerol sebacate) (PGS), which exhibits stable elasticity under repeated tensile loading, was engineered to provide physical features that aligned cardiomyocytes in a similar manner to that seen in native cardiac tissue. In addition, a small molecule mimetic of brain derived neurotrophic factor (BDNF) was polymerized into the PGS to achieve a continuous and steady release. Micropatterning of PGS elastomers increased cell alignment, calcium transient homogeneity, and cell connectivity. The intensity of the calcium transients in cardiomyocytes was enhanced when cultured on PGS which released a small molecule BDNF mimetic. This study demonstrates that robust micropatterned elastomer films are a potential candidate for the delivery of functional cardiomyocytes and factors to the injured or dysfunctional myocardium, as well as providing novel in vitro platforms to study cardiomyocyte physiology.

Physical Exercise and Regulation of Intracellular Calcium in Cardiomyocytes of Hypertensive Rats.

BackgroundRegulation of intracellular calcium (Ca2+) in cardiomyocytes is altered by hypertension; and aerobic exercise brings benefits to hypertensive individuals.Objective To verify the effects of aerobic exercise training on contractility and intracellular calcium (Ca2+) transients of cardiomyocytes and on the expression of microRNA 214 (miR-214) in the left ventricle of spontaneously hypertensive rats (SHR).MethodsSHR and normotensive Wistar rats of 16 weeks were divided into 4 groups -sedentary hypertensive (SH); trained hypertensive (TH); sedentary normotensive (SN); and trained normotensive (TN). Animals of the TH and TN groups were subjected to treadmill running program, 5 days/week, 1 hour/day at 60-70% of maximum running velocity for 8 weeks. We adopted a p ≤ 0.05 as significance level for all comparisons.ResultsExercise training reduced systolic arterial pressure in hypertensive rats. In normotensive rats, exercise training reduced the time to 50% cell relaxation and the time to peak contraction and increased the time to 50% decay of the intracellular Ca2+ transients. In SHR, exercise increased the amplitude and reduced the time to 50% decay of Ca2+ transients. Exercise training increased the expression of miR-214 in hypertensive rats only.ConclusionThe aerobic training applied in this study increased the availability of intracellular Ca2+ and accelerated the sequestration of these ions in left ventricular myocytes of hypertensive rats, despite increased expression of miR-214 and maintenance of cell contractility.

Protective Effects of Dinitrosyl Iron Complexes under Oxidative Stress in the Heart.

Background. Nitric oxide can successfully compete with oxygen for sites of electron-transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia-reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca2+ transients in cardiomyocytes during hypoxia-reoxygenation. Results. The isolated rat hearts were subjected to 30 min hypoxia followed by 30 min reoxygenation. The presence of 30 nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia-induced elevation of diastolic Ca2+ level and eliminated Ca2+ transport alterations manifested by slow Ca2+ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia-reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca2+ transport. Oxacom demonstrates potential for cardioprotection during hypoxia-reoxygenation.

Left ventricular deformation associated with cardiomyocyte Ca(2+) transients delay in early stage of low-dose of STZ and high-fat diet induced type 2 diabetic rats.

BackgroundIn the early stage of diabetes, the cardiac ejection fraction is preserved, despite the existence of the subclinical cardiac dysfunction to some extent. However, the detailed phenotype of this dysfunction and the underlying mechanism remain unclear. To improve our understanding of this issue, we used low-dose STZ and high-fat diet to induce type 2 diabetic models in rats. The effects and the mechanism associated with the early stages of the disease were analyzed.MethodsThe type 2 diabetic mellitus (T2DM) in SD rats were induced through 30 mg/kg STZ and high-fat diet. Two-dimensional spackle-tracking echocardiography (STE) and the dobutamine test were performed to examine the cardiac function. Calcium transients of left ventricular myocytes were detected and the related intracellular signalling factors were analyzed by western blotting.ResultsAfter 6-weeks, T2DM rats in left ventricular (LV) diastole showed decreased global and segment strain(S) levels (P < 0.05), both in the radial and circumferential directions. Strain rate (Sr) abatement occurred in three segments in the radial and circumferential directions (P < 0.05), and the radial global Sr also decreased (P < 0.05). In the systolic LV, radial Sr was reduced, except the segment of the anterior septum, and the Sr of the lateral wall and post septum decreased in the circumferential direction (P < 0.05). Conventional M-mode echocardiography failed to detect significant alterations of cardiac performance between the two groups even after 12 weeks, and the decreased ejection fraction (EF%), fractional shortening (FS%) and end-systolic diameters (ESD) could be detected only under stress conditions induced by dobutamine (P < 0.05). In terms of calcium transients in cardiac myocytes, the Tpeak in model rats at 6 weeks was not affected, while the Tdecay1/2 was higher than that of the controls (P < 0.05), and both showed a dose-dependent delay after isoproterenol treatment (P < 0.05). Western blot analysis showed that in 6-week T2DM rats, myocardial p-PLB expression was elevated, whereas p-CaMKII, p-AMPK and Sirt1 were significantly down-regulated (P < 0.05).ConclusionA rat model of T2DM was established by low dose STZ and a high-fat diet. LV deformation was observed in the early stages of T2DM in association with the delay of Ca2+ transients in cardiomyocytes due to the decreased phosphorylation of CaMKII. Myocardial metabolism remodeling might contribute to the early LV function and calcium transportation abnormalities.

The TRPV4 Ion Channel Alters Intracellular Calcium Transients in Cardiomyocytes of Aged Mice

Cardiac excitation-contraction coupling (ECC) occurs via calcium-induced calcium release and is therefore critically dependent on precise regulation of intracellular calcium homeostasis. Cardiomyocytes of the aged heart exhibit alterations in calcium homeostasis which predisposes the heart to calcium overload, contractile dysfunction, and arrhythmia. In this investigation we tested the hypothesis that the transient receptor potential vanilloid member 4 (TRPV4) osmotically-sensitive cation channel is expressed in cardiomyocytes of the aged mouse heart and alters intracellular calcium transients. Western blot analysis of whole heart homogenates of Young (3-6 month) and Aged (24-26 month) C57BL/6 male mice revealed increased expression of TRPV4 protein in hearts of Aged. Enzymatically isolated left-ventricular cardiomyocytes were loaded with the calcium indicator dye fluo-4/AM and electrically stimulated at 0.5 Hz to induce intracellular calcium transients. Pharmacological activation of TRPV4 with the agonist RN1747 (100 nM) produced an increase in calcium transient amplitude in cardiomyocytes of Aged (F/F0:3.3±0.4 RN1747 versus 2.6±0.4 control, P<0.05), but not Young (F/F0:2.8±0.3 RN1747 versus 2.9±0.2 control) mice. Following hypo-osmotic stress (250 mOsm, 40 minutes), cardiomyocytes of Aged exhibited enhanced responsiveness to RN1747 (F/F0:4.3±0.6 RN1747 versus 3.5±0.4 control, P<0.05) which associated with altered transient decay kinetics and cellular calcium overload (7/8 cells); these effects were prevented by the TRPV4 antagonist HC067047 (1 uM; F/F0:2.9±0.3 RN1747+HC versus 3.0±0.2 HC, 0/5 cells). In conclusion, the TRPV4 ion channel is expressed in cardiomyocytes of the aged heart and alters calcium transients during ECC.

Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)β binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)βs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation.

Monitoring of intracellular free Zn2+ and Ca2+ changes in cardiomyocytes from metabolic syndrome rats

Metabolic syndrome (MetS) is the name for a group of risk factors that raises important risk for heart disease and other health problems. The main features of metabolic syndrome include insülin resistance and hypertension. Although evidence is emerging that changes in cardiac energy metabolism might contribute to the development of contractile dysfunction, a depressed sarcoplasmic reticulum (SR) function seems to be a good candidate to underlie MetS‐induced cardiac dysfunction. Therefore, the present study was designed to determine whether the properties of Zn2+ (with FluoZin‐3 loading) and Ca2+ (with Fluo‐3 loading) sparks might provide insight into the role of SR in the altered Zn2+ and Ca2+ signaling in MetS cardiomyocytes when they were analyzed together with Zn2+ and Ca2+ transients. MetS was induced with high‐sucrose drinking water during 16‐week period in male rats. Basal Zn2+ and Ca2+ levels as well as frequencies of Zn2+ and Ca2+ sparks in cardiomyoctes significantly increased compared to aged‐matched controls. The Ca2+ transients exhibited significantly reduced amplitude and prolonged time course while no change in amplitude and prolongation in time course of Zn2+ transients in cardiomyocytes from MetS rats. Spatio‐temporal properties of both Zn2+ and Ca2+ sparks were also significantly altered to being almost parallel to the changes of Zn2+ and Ca2+ transients. In addition, RyR2 from MetS group was hyperphosphorylated while its protein level was depressed, similar to the hyperphosphorylation of RyR2 with Zn2+ in a dose‐dependent manner. These data show that cardiomyocytes from MetS rats exhibit altered local Zn2+ and Ca2+ signaling with increased basal Zn2+ and Ca2+ levels.

Life after the birth of the mitochondrial Na+/Ca2+ exchanger, NCLX.

Powered by the mitochondrial membrane potential, Ca(2+) permeates the mitochondria via a Ca(2+) channel termed Ca(2+) uniporter and is pumped out by a Na(+)/Ca(2+) exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca(2+) transients are critical for metabolic activity and regulating global Ca(2+) responses. On the other hand, failure to control mitochondrial Ca(2+) is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca(2+) stores required for antigen-dependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca(2+) influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca(2+) stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca(2+) transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na(+) and Ca(2+) signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca(2+) efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.

Adiponectin regulates SR Ca2 + cycling following ischemia/reperfusion via sphingosine 1-phosphate-CaMKII signaling in mice

The adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent studies have demonstrated that APN induces a marked Ca2+ influx in skeletal muscle. However, whether APN modulates [Ca2+]i activity, especially [Ca2+]i transients in cardiomyocytes, is still unknown. This study was designed to determine whether APN modulates [Ca2+]i transients in cardiomyocytes. Adult male wild-type (WT) and APN knockout (APN KO) mice were subjected to myocardial ischemia/reperfusion (I/R, 30min/30min) injury. CaMKII-PLB phosphorylation and SR Ca2+-ATPase (SERCA2) activity were downregulated in I/R hearts of WT mice and further decreased in those of APN KO mice. Both the globular domain of APN and full-length APN significantly reversed the decrease in CaMKII-PLB phosphorylation and SERCA2 activity in WT and APN KO mice. Interestingly, compared with WT littermates, single myocytes isolated from APN KO mice had remarkably decreased [Ca2+]i transients, cell shortening, and a prolonged Ca2+ decay rate. Further examination revealed that APN enhances SERCA2 activity via CaMKII-PLB signaling. In in vivo and in vitro experiments, both APN receptor 1/2 and S1P were necessary for the APN-stimulated CaMKII-PLB-SERCA2 activation. In addition, S1P activated CaMKII-PLB signaling in neonatal cardiomyocytes in a dose dependent manner and improved [Ca2+]i transients in APN KO myocytes via the S1P receptor (S1PR1/3). Further in vivo experiments revealed that pharmacological inhibition of S1PR1/3 and SERCA2 siRNA suppressed APN-mediated cardioprotection during I/R. These data demonstrate that S1P is a novel regulator of SERCA2 that activates CaMKII-PLB signaling and mediates APN-induced cardioprotection.

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca2+]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na+/Ca2+ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca2+]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.

A modified local control model for Ca2 + transients in cardiomyocytes: Junctional flux is accompanied by release from adjacent non-junctional RyRs

Excitation–contraction coupling in cardiomyocytes requires Ca2+ influx through dihydropyridine receptors in the sarcolemma, which gates Ca2+ release through sarcoplasmic ryanodine receptors (RyRs). Ca2+ influx, release and diffusion produce a cytosolic Ca2+ transient. Here, we investigated the relationship between Ca2+ transients and the spatial arrangement of the sarcolemma including the transverse tubular system (t-system). To accomplish this, we studied isolated ventricular myocytes of rabbit, which exhibit a heterogeneously distributed t-system. We developed protocols for fluorescent labeling and triggered two-dimensional confocal microscopic imaging with high spatiotemporal resolution. From sequences of microscopic images, we measured maximal upstroke velocities and onset times of local Ca2+ transients together with their distance from the sarcolemma. Analyses indicate that not only sarcolemmal release sites, but also those that are within 1μm of the sarcolemma actively release Ca2+. Our data also suggest that release does not occur at sites further than 2.5μm from the sarcolemma. The experimental data are in agreement with results from a mathematical model of Ca2+ release and diffusion. Our findings can be explained by a modified local control model, which constrains the region of regenerative activation of non-junctional RyR clusters. We believe that this model will be useful for describing excitation–contraction coupling in cardiac myocytes with a sparse t-system, which includes those from diseased heart tissue as well as atrial myocytes of some species.