Adult Rat Cardiomyocytes Research Articles

Fission Promotes Respiration and ROS Production in Individual Mitochondria

The balance between fission and fusion essentially participates in mitochondrial biogenesis, transportation and maturation. Whether endogenous fission and fusion processes regulate mitochondrial respiration and ROS production in the heart is not well studied. Cardiac myocytes are excitable cells that featured by dynamic intracellular calcium regulations. Increased intracellular calcium is known to activate dynamin-like protein 1 (DLP1), a key regulator of mitochondrial fission. In this study, we used adenovirus mediated gene transfer of a mitochondrial targeted superoxide indicator and confocal microscopy to investigate the role of fission in mitochondrial respiration and ROS production regulation. We monitored the mitochondrial superoxide flashes (SOFs), which are quantal events of superoxide generation coupled with single mitochondrial respiration, in cultured cardiomyocytes. Acute application of Mdivi-1 (50 µM), a DLP inhibitor suppressed resting SOF activity in rat adult cardiomyocytes and H9C2 cardiac myoblasts. Furthermore, metabolic substrates (20 mM Pyruvate or 20 mM Glucose)-induced SOF events were blocked by Mdivi-1, indicating an essential role of fission in maintaining normal mitochondrial function. Acute stimulation of fission by KCl (50 mM), which induced calcium transients, dramatically stimulated the SOF (SOF frequency from 0.97±0.36 to 2.46±0.34 per 1000 µm2 per 100 s) in H9C2 cells. The effect of KCl is blocked by Mdivi-1. Interestingly, long-term incubation of high glucose (35 mM for 48 hr), which has been shown to induce mitochondrial fragmentation, failed to stimulate SOF activity in H9C2 cells. These results reveal an essential role of mitochondrial fission in regulating physiological functions of individual mitochondria and also highlight that chronically disturbing fission/fusion dynamics may have detrimental effects.

Mitochondrial Dynamics in Neonatal and Adult Cardiomyocytes

Mitochondrial function is central to heart physiology and pathology. The function of mitochondria is dynamically regulated by their fusion and fission in many cell types. However, the study of mitochondrial dynamics in cardiomyocytes (CM) has been difficult. Using photoactivatable (PA) fluorescent proteins we characterized the mitochondrial dynamics in normal neonatal and adult rat CM. CM were transduced by mitochondrial matrix targeted DsRed and PA-GFP encoding adenoviruses and evaluated by confocal miscroscopy 36-48 h post infection. With this approach, we studied an early developmental stage (neonatal) and fully differentiated (adult) CM. We also studied the same cells upon prolonged exposure to ethanol that is known to cause mitochondrial dysfunction and cardiomyopathy.We show that mitochondria form a highly connected network in neonatal CM, and mostly discrete structures with some connectivity mostly in longitudinal orientation in adult CM. Neonatal CM displayed considerable fusion activity (0.25 events/25 square micrometers/min). Eighty two % of the events resulted in complete merge of the organelles, whereas 18% appeared as fusion followed by separation. By contrast, the fusion events were scarce in the adult CM. Mitochondrial movements were also more frequent and elapsed longer distances in neonatal than in adult CM.Neonatal CM exposed to 50 mM ethanol for 48h showed 40% decrease in the network continuity and 75% decrease in the fusion event rate. In adult CM isolated from ethanol-fed (6 months) rats, mitochondrial continuity decreased to 50% of the control.Thus, mitochondria are highly dynamic in neonatal CM. Some stable intermitochondrial connections allow content exchange in the adult CM but less fusion activity is present, probably due to the spatial restriction presented by the myofilaments. Chronic ethanol exposure suppresses mitochondrial dynamics in CM, which effect might provide a possible mechanism for the impaired contractility.

8 Expression and Activity of Braf in Cardiomyocytes

<h3>Rationale</h3> The Raf family (Raf1, ARaf, BRaf) are upstream kinases for the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade which is involved in cell proliferation and survival. Dysregulation of BRaf is associated with cancer, and BRaf inhibitors are in clinical use. In the heart, ERK1/2 signalling is required for cardioprotection and is associated with hypertrophic growth. Although Raf1 and ARaf have been shown to be activated by hypertrophic stimuli in cardiomyocytes, BRaf was not previously detected in cardiac tissue. <h3>Methodology</h3> Expression of BRaf protein in adult rat heart and neonatal cardiomyocytes was studied by immunoblotting with N- or C-terminal antibodies. Following immunoprecipitation, activities of Raf1 and BRaf were compared using GST-MKK1 as substrate. Co-immunoprecipitation experiments were performed. <h3>Results</h3> BRaf protein was detected in extracts from adult rat hearts or cardiomyocytes as a doublet of ~90 kDa (predicted 89 kDa) using antibodies to either the N- or C-terminus. Both bands were immunoprecipitated with any of the antibodies. Activity assays confirmed that Raf1 exhibits low basal activity that is substantially increased by epidermal growth factor (EGF). However, BRaf had substantially greater basal activity that was further increased in response to EGF. As in other cells, BRaf associated with Raf1/ARaf in cardiomyocytes as demonstrated by co-immunoprecipitation. <h3>Conclusions</h3> BRaf is expressed in cardiomyocytes and adult hearts at significant levels, it exhibits high basal activity, its activity can be increased by EGF, and it forms complexes with ARaf and Raf1. Thus, the BRaf inhibitors in use for cancer therapy have the potential to affect cardiac function.

Cardiac Selective Modulator of Human Myosin for the Treatment of Genetic Hypertrophic Cardiomyopathy

Genetic hypertrophic cardiomyopathy (HCM) results from mutations in the cardiac sarcomere, including β-cardiac myosin, with HCM afflicting about 1 out of every 500 people in the United States. HCM is characterized by hyper-contractility and myocyte hypertrophy. Current agents used to treat HCM include β-blockers and Ca2+ channel blockers to decrease the hyper-contractility and improve cardiac relaxation. A novel and more direct approach to decreasing hyper-contractility and improving diastolic relaxation in HCM patients is by modulating β-cardiac myosin to produce less force. We believe modulation at the sarcomere level provides a focused strategy while lowering the potential for adverse drug events. In this study we examined the effects of MY461, a cardiac myosin selective inhibitor, on adult rat cardiomyocytes to fully understand the mechanism of action on excitation-contraction (E-C) coupling. Cellular contractility was assessed using edge detection and the calcium transient was measured using fura-2 loaded myocytes. MY461 decreased contractility in a dose dependent manner with an IC50 of 250nM without altering the calcium transient. This inhibitory effect can be reversed by stimulation of the β-adrenergic pathway with the known agonist isoproterenol. We hypothesize that modulation of mutant β-cardiac myosin activity with a small molecule agent such as MY461 could potentially treat disorders resulting from hyper-contractility such as HCM.

A New Ca2+ Probe, Calstabi-Cam, Targeted to Ryanodine Receptors of Cardiomyocytes

The contractile force of cardiomyocytes is controlled by Ca2+ cross-signaling between L-type Ca2+ channels and ryanodine receptors (RyR2) across the narrow dyadic cleft. To detect the junctional Ca2+ signal, we designed a peptide probe (Calstabi-Cam) with calmodulin as its Ca2+ sensor, yellow fluorescent protein (EYFP) as reporting fluorophor, and FKBP12.6 (calstabin2) as subunit of RyR2. Effective adenoviral expression in cultured adult rat cardiomyocytes was verified after 48 hours when Calstabi-Cam co-localized with fluorescent RyR antibodies in a sarcomeric z-lines pattern. Dissociation constant (kd) of Calstabi-Cam for Ca2+ measured in permeablized myocytes was 80 nM. The kinetic of Ca2+ signals was measured in voltage-clamped cells with a Leica TIRF microscope which allowed comparison of rapidly interlaced images of cytosolic Ca2+ probes (fura-2 or fluo-4) and Calstabi-Cam. Fluo-4 Ca2+ sparks were detected superimposed on the sarcomeric fluorescence patterns of Calstabi-Cam. On activation of Ca2+ release by caffeine or membrane depolarization, Calstabi-Cam fluorescence signals had slower rise times compared to fura-2, but had much slower decay kinetics. Scans of focal Calstabi-Cam signals at different sites, occurring spontaneously or at the onset of evoked Ca2+ releases, appeared to have a significant distribution of magnitudes and latencies. We conclude that Ca2+-sensing biological peptides may be targeted to the cleft-space occupied by DHPR/RyRs complex as to make it possible to record the variance of Ca2+ signals at different dyadic junctions.

4 Characterisation of Mitochondrial Morphology in the Adult Rodent Heart

RationaleIn adult cardiomyocytes there are known to be 3 different sub-populations of mitochondria: Subsarcolemmal (SS), Interfibrillar (IF), and Perinuclear (PN), which appear to have distinctive properties and shapes. Here, we...

Inhibition of MMP-2 expression with siRNA increases baseline cardiomyocyte contractility and protects against simulated ischemic reperfusion injury.

Matrix metalloproteinases (MMPs) significantly contribute to ischemia reperfusion (I/R) injury, namely, by the degradation of contractile proteins. However, due to the experimental models adopted and lack of isoform specificity of MMP inhibitors, the cellular source and identity of the MMP(s) involved in I/R injury remain to be elucidated. Using isolated adult rat cardiomyocytes, subjected to chemically induced I/R-like injury, we show that specific inhibition of MMP-2 expression and activity using MMP-2 siRNA significantly protected cardiomyocyte contractility from I/R-like injury. This was also associated with increased expression of myosin light chains 1 and 2 (MLC1/2) in comparison to scramble siRNA transfection. Moreover, the positive effect of MMP-2 siRNA transfection on cardiomyocyte contractility and MLC1/2 expression levels was also observed under control conditions, suggesting an important additional role for MMP-2 in physiological sarcomeric protein turnover. This study clearly demonstrates that intracellular expression of MMP-2 plays a significant role in sarcomeric protein turnover, such as MLC1 and MLC2, under aerobic (physiological) conditions. In addition, this study identifies intracellular/autocrine, cardiomyocyte-produced MMP-2, rather than paracrine/extracellular, as responsible for the degradation of MLC1/2 and consequent contractile dysfunction in cardiomyocytes subjected to I/R injury.

Recommendations for Short Term Culturing of Viable Rod Shaped Rat Cardiomyocytes

In vitro primary cultures of isolated adult rat cardiomyocytes are becoming an increasingly popular model to study heart muscle stressors. This model can easily be manipulated in a controlled environment and results obtained can provide valuable insights into the pathophysiology of heart disease. Over the past years several improved methodologies have been described in the development of a robust technique to help maintain cardiomyocytes in culture. However, despite these advances, culturing of primary cardiomyocytes remains a challenge. In this study, we present a simple yet reproducible method for isolation and culture of viable rod shaped cardiomyocytes. Cardiomyocytes were maintained in supplemented Media 199 with or without foetal bovine serum. Their viability was assessed using trypan blue while the metabolic activity was measured using an Adenosine 5’-triphosphate assay. Results obtained in this study were used to provide general guidelines to evade pitfalls related to low cardiomyocyte yields and subsequent poor culturability of cardiomyocytes. Isolated cardiomyocytes cultured in the presence of foetal bovine serum maintained their in vitro striated rod shaped morphology for 72 hours in culture, after which, they flattened and spread out. Whereas, cardiomyocytes cultured in the absence of foetal bovine serum remained rod shaped for up to 120 hours.

Abstract 340: Marked Attenuation of Cell Injury by Substrate Availability During Ischemia

Background: Ischemic injury in cardiomyocytes results in rigor contracture (reduction of length by approximately one third) and reperfusion injury results in hypercontracture (change of morphology from rod-shaped to round forms). Rapid reoxygenation during reperfusion at high oxygen concentrations during limited substrate availability could augment cell injury. We therefore hypothesized that substrate availability during ischemia delays rigor and delayed and gradual reoxygenation reduces hypercontracture. Then, we compared the effects of normocarbic immediate (NIR) and hypercarbic delayed reoxygenation (HDR) following aglycemic anoxia (AA) and normoglycemic anoxia (NA) in isolated adult rat cardiomyocytes. Methods: The set-up for creating anoxic environment incorporated (i) a gas blender, (ii) equilibration of ischemic buffer with anoxic gas mixture and perfusing the chamber, and (iii) an acrylic hood, constantly flushed with anoxic gas mixture, to cover the chamber on the stage of the microscope. This set-up resulted in PO2 of 0 mmHg constantly over a period of time. The cells were subjected to 15 min of baseline, 45 min of simulated ischemia and 120 min of simulated reoxygenation (AA-NIR, NA-NIR, AA-HDR, and NA-HDR) with or without glucose as a substrate. Results: Substrate availability significantly reduced the occurrence of rigor and hypercontracture (AA vs NA followed by any reoxygenation protocol: p&lt;0.0001, Gehan Breslow survival analysis). Survival from rigor and hypercontracture were not different in AA-NIR and AA-HDR groups while they were significantly higher in NA-NIR compared to NA-HDR group (p&lt;0.0001, Gehan Breslow survival analysis). There was no difference in viability among the groups. Conclusions: Substrate availability during ischemia ameliorates ischemia and reperfusion injury while hypercarbic delayed reoxygenation does not appear to be beneficial.

Abstract 186: NHE-1 Inhibition Reduces Rigor and Hypercontracture in Isolated Cardiomyocytes During Ischemia and Reperfusion

Background: During ischemia, anaerobic metabolism prompts intracellular acidosis with activation of the sodium hydrogen exchanger isoform-1 (NHE-1), resulting in intracellular Na+ overload consequent to reductions in Na+-K- ATPase activity. The Na+ excess prompts Ca2+ entry through the sodium-calcium exchanger, leading to Ca+ overload. Ca+ overload, predisposes cells to hypercontracture upon reoxygenation. NHE-1 inhibition reduces Na+ overload and the subsequent Ca+ overload. We developed a programmable system to model ischemia and reperfusion by dynamically blending O2, CO2, and N2 and used it to model changes in gas tension at the tissue levels during ischemia and reperfusion. We validated the model by comparing the effects of reoxygenation with and without the NHE-1 inhibitor zoniporide in isolated adult rat cardiomyocytes. Methods: The set-up included: (i) a programmable dynamic gas blender developed by us, (ii) equilibration of ischemic buffer with anoxic gas mixture at 3-5 LPM and perfusion within an imaging chamber at 2.5 ml/min, (iii) a glass reservoir and stainless steel tubing to avoid gas diffusion, (iv) an acrylic hood, constantly flushed with anoxic gas mixture, covering the chamber on the stage of the microscope, and (v) an O2 microelectrode to continuously monitor PO2 in the chamber. The set-up resulted in PO2 of 0 mmHg during ischemia. The cells were subjected to 15 min of baseline stabilization, 45 min of ischemia, and 120 min of reperfusion. Zoniporide (at a final concentration of 3 μM) was administered starting at 10 min of ischemia and was maintained throughout reperfusion. Results: Zoniporide significantly attenuated development of rigor and subsequent hypercontracture (P=0.001, Gehan Breslow survival analysis). Conclusion: The newly develop system reproduced at the cell level effects of ischemia and reperfusion. These effects were attenuated by NHE-1 inhibition as it has been reported in isolated organs and in the in situ heart.

Abstract 163: Inhibition of p38a Regulates SERCA2a Function

Rationale: p38 MAPK is a negative regulator of cardiac contractile function, but the underlying molecular mechanism is not well understood. Objective: To determine the molecular mechanism mediating the positive inotropic effect of p38 inhibition. Methods and Results: The effect of p38 inhibition on calcium regulatory proteins and calcium cycling was investigated in isolated neonatal and adult rodent cardiomyocytes using chemical inhibitors, recombinant adenoviruses and RNAi. Chemical inhibition of p38 enhanced phospholamban (PLB) phosphorylation at serine 16 residue. This was recapitulated with dominant negative p38α adenovirus and by p38α RNAi, but not with dominant negative p38β adenovirus. Chemical inhibition of p38 and dominant negative p38α resulted in significant decrease in Ca 2+ -transient decay time in adult rat cardiomyocytes. Inhibition of p38α, but not p38β, induced an increase in phosphorylation of protein phosphatase inhibitor-1 at the protein kinase A site threonine 35. Analysis of underlying mechanisms revealed that p38 inhibition increased phosphorylation of G protein-coupled receptor kinase 2 (GRK2), but not GRK3 or GRK5. p38 inhibition- induced PLB phosphorylation was abolished in cardiomyocytes overexpressing wild type GRK2. In contrast, increased PLB phosphorylation upon p38 inhibition was not reduced in cardiomyocytes overexpressing GRK2 harboring a non-phosphorylatable mutation in serine 670 residue. Finally, p38 inhibition-induced shortening of Ca 2+ -transient decay time was absent in cardiomyocytes overexpressing wild type GRK2. Conclusions: Inhibition of p38α regulates protein phosphatase inhibitor-1 resulting in increased PLB phosphorylation and enhanced SERCA2a function.

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4–GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types.Using an adenoviral system we successfully expressed PMCA4–GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4–GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4mutGCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4–GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4mutGCaMP2. In addition, PMCA4–GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4–GCaMP2 and PMCA4mutGCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively.In summary, the PMCA4–GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation–contraction coupling in the heart.

Activin A impairs insulin action in cardiomyocytes via up-regulation of miR-143

Enhanced activin A release from epicardial adipose tissue (EAT) has been linked to the development of cardiac dysfunction in type 2 diabetes (T2D). This study examined whether the inhibition of insulin action induced by epicardial adipokines in cardiomyocytes can be ascribed to alterations in miRNA expression. Expression levels of miRNAs were assessed by real-time PCR in primary adult rat cardiomyocytes (ARC) exposed to conditioned media generated from EAT biopsies (CM-EAT) from patients with and without T2D. CM-EAT-T2D altered the expression of eight miRNAs in ARC vs. CM-EAT from patients without T2D. Of these, only expression of the miR-143/145 cluster was affected by activin A in the same direction as CM-EAT-T2D. Accordingly, activin A neutralizing antibodies prevented the induction of the miR-143/145 cluster by CM-EAT-T2D. Subsequently, the impact of the miR-143/145 cluster on insulin action was investigated. Transfection of HL-1 cells with precursor-miR-143 (pre-miR-143), but not pre-miR-145, blunted the insulin-mediated phosphorylation of Akt and its substrate proline-rich Akt substrate of 40 kDa (PRAS40), and reduced insulin-stimulated glucose uptake. Also lentivirus-mediated expression of pre-miR-143 in ARC reduced insulin-induced Akt phosphorylation. These effects were ascribed to down-regulation of the miR-143 target and regulator of insulin action, the oxysterol-binding protein-related protein 8 (ORP8) in both ARC and HL-1 cells. Finally, LNA-anti-miR-143 protected against the detrimental effects of CM-EAT-T2D on insulin action in ARC. Activin A released from EAT-T2D inhibits insulin action via the induction of miR-143 in cardiomyocytes. This miRNA inhibits the Akt pathway through down-regulation of the novel regulator of insulin action, ORP8.

TWEAK/Fn14 axis is a positive regulator of cardiac hypertrophy

Cardiac pressure overload-induced hypertrophy and pathological remodelling frequently leads to right ventricular dysfunction, which is the most frequent cause of death in patients with pulmonary arterial hypertension. Nowadays, accumulating reports support the concept that proinflammatory cytokines and growth factors play crucial roles in the failing heart. We recently identified Fn14 as an endogenous key regulator in cardiac fibrosis in the PAB (Pulmonary Artery Banding) pressure-overload model. Right ventricular overload after PAB is also characterized by hypertrophy. The aim of this study was to determine whether right ventricular (RV) cardiac hypertrophy induced by PAB is mediated by the TWEAK/Fn14 axis. After baseline MRI, Fn14−/− mice and wild-type (WT) littermates were randomly assigned to two groups: (1) SHAM-operated (n⩾4, per genotype) and (2) PAB (n⩾11, per genotype). The results of MRI and histological analysis demonstrated that Fn14−/− mice exhibit less PAB-induced cardiac hypertrophy compared to WT littermates. Moreover, Fn14 overexpression in cultured adult rat cardiomyocytes enhanced cardiomyocyte size. Collectively, our studies demonstrate that Fn14 ablation attenuates RV hypertrophy after PAB and that activation of TWEAK/Fn14 signaling promotes cardiomyocyte growth in vitro. These results nominate Fn14 as a potential novel target for the treatment of heart hypertrophy.

221 REDUCING SARCOLEMMAL SODIUM CURRENT DECREASES SPONTANEOUS SR CA2+ RELEASE

Aim Ca2+ waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There is some evidence that INa blocking agents can reduce spontaneous sarcoplasmic reticulum (SR) Ca2+ release via effects on the RyR. We tested the hypothesis that direct modulation of INa may also be important in altering SR Ca2+ release. Methods and Results Imaging of spontaneous SR Ca2+ release events in healthy adult rat cardiomyocytes was performed. Variation in frequency of stimulation was used to produce Ca2+ sparks or waves. When SR Ca2+ content was held constant, spark frequency, wave frequency and wave velocity were reduced by a variety of INa blockers (including flecainide, lidocaine, propafenone and TTX). To assess the contribution of INa to spark and wave production voltage clamping was used to activate contraction from holding potentials of -80mV or -40mV. This confirmed that reducing Na+ influx during myocyte stimulation is sufficient to reduce waves and that such agents only cause Ca2+ wave reduction when INa is active. It was found that Na+/Ca2+-exchanger (NCX)-mediated Ca2+ efflux was significantly enhanced by INa blockade and that the effects of INa blockade on wave frequency could be reversed by reducing [Na+]o, suggesting an important downstream role for NCX function in the changes in SR Ca2+ release. Veratridine, an INa activator, increased wave frequency and the effects were abrogated by increasing [Na]o during the wave-detection period. Conclusions Our results show, for the first time, that reducing Na+ influx reduces spontaneous SR Ca2+ release. We have also shown that alterations in [Na+]i modulate wave frequency through alterations in NCX function. A reduction in INa, for example, can increase the [Na+]o:[Na+]i gradient, hence causing more effective Ca2+ efflux at resting membrane potentials. This not only suggests a clear mechanism for the reduction in waves seen with INa blockers, but also emphasises the detrimental effects of high [Na]i seen in disease states such as heart failure. It also allows us to understand an alternative way in which INa blockers exert their antiarrhythmic actions.

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Caveolin-3 suppresses late sodium current by inhibiting nNOS-dependent S-nitrosylation of SCN5A

Aims: Mutations in CAV3-encoding caveolin-3 (Cav3) have been implicated in type 9 long QT syndrome (LQT9) and sudden infant death syndrome (SIDS). When co-expressed with SCN5A-encoded cardiac sodium channels these mutations increased late sodium current (INa) but the mechanism was unclear. The present study was designed to address the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A. Methods and results: HEK-293 cells expressing SCN5A and LQT9 mutation Cav3-F97C resulted in a 2-fold increase in late INa compared to Cav3-WT. This increase was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. Based on these findings, we hypothesized that an nNOS complex mediated the effect of Cav3 on SCN5A. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin (SNTA1), nNOS, and Cav3. Compared with Cav3-WT, Cav3-F97C produced significantly larger peak INa amplitudes, and showed 3.3-fold increase in the late INa associated with increased S-nitrosylation of SCN5A. L-NMMA reversed both the Cav3-F97C induced increase in late and peak INa and decreased S-nitrosylation of SCN5A. Overexpression of Cav3-F97C in adult rat cardiomyocytes caused a significant increase in late INa compared to Cav3-WT, and prolonged the action potential duration (APD90) in a nNOS-dependent manner. Conclusions: Cav3 is identified as an important negative regulator for cardiac late INa via nNOS dependent direct S-nitrosylation of SCN5A. This provides a molecular mechanism for how Cav3 mutations increase late INa to cause LQT9. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

X-ROS signalling is enhanced and graded by cyclic cardiomyocyte stretch

A sustained, single stretch of a cardiomyocyte activates a transient production of reactive oxygen species by membrane-located NADPH oxidase 2 (Nox2). This NoX2-dependent ROS (X-ROS) tunes cardiac Ca(2+) signalling by reversibly sensitizing sarcoplasmic reticulum Ca(2+) release channels. In contrast to static length changes, working heart cells are cyclically stretched and shortened in the living animal. Additionally, this stretch cycle is constantly varied by changes in the pre-load and heart rate. Thus, the objective of this study was (i) to characterize X-ROS signalling during stretch-shortening cycles and (ii) to evaluate how the amplitude (pre-load) and frequency (heart rate) of cell stretch affects X-ROS and Ca(2+) signalling. Single adult rat cardiomyocytes were attached to MyoTak™-coated micro-rods and stretched, while ROS production and Ca(2+) signals were monitored optically. Although a sustained stretch led to only a transient burst of ROS, cyclic stretch-shortening cycles led to a steady-state elevation of ROS production. Importantly, this new redox state was graded by both the amplitude of stretch (3-15%) and cycle frequency (1-4 Hz). Elevated ROS production enhanced Ca(2+) signalling sensitivity as measured by the Ca(2+) spark rate. The steady-state level of ROS production in a cardiomyocyte is graded by the amplitude and frequency of cell stretch. Thus, mechanical changes that depend on the pre-load and heart rate regulate a dynamic redox balance that tunes cellular Ca(2+) signalling.

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption. A variety of specific antigens have already been identified and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca(2+)) contents. In the last step, the effect of patient IgG on the cell shortening and Ca(2+) transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

Interaction of epoxyeicosatrienoic acids and adipocyte fatty acid-binding protein in the modulation of heart function

Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we verified in previous studies a direct bioactive impact of FABP4 on heart function. We demonstrated that human adipocytes release factors that directly suppress heart contraction in vitro. Subsequently, we identified FABP4 as the main cardiodepressant factor. Since FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergetic way. Therefore, we investigated in the present study a possible involvement of lipid ligands on the negative inotropic effect of adipocyte-factors in interaction with FABP4 in vitro. First, we verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte-conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6-EET represented about 45% of total EET-concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocytes contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner. These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and the interaction of these factors modulates the cardiac function. Therefore, elevated levels of FABP4 and 5,6-EET in obese subjects may partially explain the development of heart dysfunction in those subjects.