Mouse Myocytes Research Articles

Abstract 15550: Impact of PET Imaging to Visualize Activated Macrophages in Immune Rejection of Allogenic iPS Derived Cardiac Myocytes in Mice

Introduction: Transplantation of allogeneic cells inevitably induce activated macrophage-related host immune response, which would limit therapeutic effects. Importantly, monitoring magnitude of the immune response is poorly established in the allogeneic cell transplantation therapy for the heart. Hypothesis: In this study, we hypothesized that [18F]-DPA-714-PET imaging may visualize activated macrophages accumulated to the host heart after transplantation of allogeneic induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM). Methods: iPSC-CM cell-sheets of C57BL/6 mice origin were generated by the thermoresponsive dish and transplanted over the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (Group sTx), or Balb/c mice as an allogeneic one (Group aTx). The mice were examined by [18F]-DPA-714-PET to assess maximum standardized uptake value (SUVmax) ratio (A/S ratio) calculated by SUVmax in the anterior and septal wall of the LV. In addition, expression of factors associated with immune rejection was assessed by RT-PCR and the significance of immune rejection was evaluated by Immunochemical staining of CD3 or CD68 positive cell. Results: A/S ratio was comparable in all groups at 1 day (aTx: 1.19±0.05, sTx: 1.16±0.03, sham: 1.20±0.03, p=0.34), though higher in the aTx group than the other groups at 7 day (aTx: 1.38±0.03, p<0.01, sTx: 1.18±0.04, sham: 1.25±0.09) after the cell transplantation. In addition, the expression of IL-2 and MCP-1 of the heart tissue were higher in the aTx group than the other groups at 7 day (aTx: 31.4±3.8, p<0.01, sTx: 1.5±1.2, sham: 2.3±0.9 and aTx: 30.8±12.7, p<0.01, sTx: 4.0±3.4, sham: 5.5±2.0). Furthermore, in the immunochemical histology, the percentages of CD3 and CD68 positive cells were also higher in the aTx group than the other groups at 7day. Conclusions: Accumulation of activated macrophages were identified in the LV surface more intensely after the allogeneic iPSC-CM transplantation than the syngeneic or sham one by the [18F]-DPA-714-PET imaging, warranting that PET imaging may be applicable in clinical setting of the allogeneic cell transplantation therapy to monitor magnitude of host immune response.

Inhibitory effects of SKF96365 on the activities of K(+) channels in mouse small intestinal smooth muscle cells.

In order to investigate the effects of SKF96365 (SKF), which is a non-selective cationic channel blocker, on K+ channel currents, we recorded currents through ATP sensitive K+ (IKATP), voltage-gated K+ (IKv) and Ca2+ activated K+ channels (IBK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished IKATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K+ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC50) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC50 value in IKATP suppression) reversibly inhibited the IKv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient IBK and caffeine-induced IBK. These results indicated that SKF inhibited ATP sensitive K+ channels and voltage-gated K+ channels, with the ATP sensitive K+ channels being more sensitive than the voltage-gated K+ channels. These inhibitory effects on K+ channels should be considered when SKF is used as a cationic channel blocker.

G.P.414 - Contribution of IL-6-dependent signalling mechanism to upregulation of MyHC IIb mRNA but not of MyHC IIa mRNA in mouse myocytes

G.P.414 - Contribution of IL-6-dependent signalling mechanism to upregulation of MyHC IIb mRNA but not of MyHC IIa mRNA in mouse myocytes

Coxsackievirus B3 Induces Autophagic Response in Cardiac Myocytes in vivo.

Viral myocarditis is a common disease that contributes to dilated cardiomyopathy or heart failure. Coxsackievirus B (CVB) is one of the major causative pathogens of viral myocarditis. Previous studies have shown that autophagy is exploited to promote CVB replication in cell lines. To study whether cardiac myocytes respond to CVB infection in a similar way, viral myocarditis was established by the inoculation of 3-week-old BALB/c mice with CVB3. Electron microscopic observation showed that autophagosome-like vesicles were induced in the cardiac myocytes of mice infected by CVB3 at 3, 5, and 7 days after viral infection. The lipidated microtubule-associated protein 1 light chain 3 (LC3), LC3-II, was also significantly increased in both myocardium and the cardiac myocytes extracted from the ventricles of mice infected with CVB3. The increased LC3-II coincided with high level of viral RNA and proteins in both myocardium and isolated cardiac myocytes. Moreover, viral protein synthesis was significantly decreased in primary cardiac myocytes by the treatment with 3-methyladenine, an inhibitor of autophagy. The expression and the phosphorylation of extracellular signal regulated kinase (ERK) were also increased in both myocardium and in the isolated cardiac myocytes of the virus-infected mice, while the interplay of ERK with autophagic response remains to be studied. This study demonstrated that cardiac myocytes respond to CVB3 infection by increased formation of autophagosomes in vivo, which might be exploited for viral replication.

Abstract 388: High-throughput Single Cell Tracking of Mitochondrial Function in Cardiomyocytes

Mitochondria play a significant role in the regulation of multiple functions in the heart, ranging from metabolism to cell death. Most mitochondrial assays require either isolating the organelle, thus disrupting the intracellular signaling or using intact cells with average measurements for the entire population neglecting the ability to distinguish cell heterogeneity. Here we describe a novel method for tracking single cell mitochondrial function in cell populations over time. Adult mouse myocytes were exposed to the mitochondrial uncoupler FCCP and hydrogen peroxide to induce changes in membrane potential and oxidative stress, respectively. TMRM and mitosox fluorescence were used to quantify mitochondrial membrane potential and ROS production, Fluo-4 fluorescence was used to assess intracellular calcium. Tracking of single cells was performed using Matlab and Imaris software. After FCCP exposure TMRM signal intensity decreased before there was a significant change in myocyte length that led to hypercontracture and cell death within 10 minutes. To better understand the dynamics of mitochondrial function and its relationship with cell death a dose response curve was established for hydrogen peroxide at 100, 500 and 1000 μM. The higher doses of hydrogen peroxide induced hypercontracture faster than lower doses. For further studies 100 μM was used to assess how homogenous the response to hydrogen peroxide was in cell populations. We found 3 distinct populations of cells responding at different times, a population of cells hypercontracted between 7-10 min, another between 10-15 min and a third population only after 15 min. Changes in membrane potential, oxidative stress and intracellular calcium were simultaneously assessed for every single cell during these time points. In addition, given the organized structure of the mitochondria in the myocyte we have adapted our technique to track individual mitochondria to study heterogeneous responses at the single mitochondrion level. This method provides a unique tool to simultaneously assess multiple parameters of mitochondrial function in single cells over time. In doing so, we have unmasked a complex heterogeneity of single cell behavior that is lost in methods than only average cell populations.

Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes.

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.

Effect of novel dietary supplement on metabolism in vitro and in vivo

Obesity is an increasingly prevalent and preventable morbidity with multiple behavioral, surgical and pharmacological interventions currently available. Commercial dietary supplements are often advertised to stimulate metabolism and cause rapid weight and/or fat loss, although few well-controlled studies have demonstrated such effects. We describe a commercially available dietary supplement (purportedly containing caffeine, catechins, and other metabolic stimulators) on resting metabolic rate in humans, and on metabolism, mitochondrial content, and related gene expression in vitro. Human males ingested either a placebo or commercially available supplement (RF) in a randomized double-blind placebo-controlled cross-over fashion. Metabolic rate, respiratory exchange ratio, and blood pressure were measured hourly for 3 h post-ingestion. To investigate molecular effects, human rhabdomyosarcoma cells (RD) and mouse myocytes (C2C12) were treated with various doses of RF for various durations. RF enhanced energy expenditure and systolic blood pressure in human males without altering substrate utilization. In myocytes, RF enhanced metabolism, metabolic gene expression, and mitochondrial content suggesting RF may target common energetic pathways which control mitochondrial biogenesis. RF appears to increase metabolism immediately following ingestion, although it is unclear if RF provides benefits beyond those provided by caffeine alone. Additional research is needed to examine safety and efficacy for human weight loss.

Distinct Etiological Roles for Myocytes and Motor Neurons in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy.

Polyglutamine (polyQ) expansion of the androgen receptor (AR) causes Kennedy's disease/spinobulbar muscular atrophy (KD/SBMA) through poorly defined cellular mechanisms. Although KD/SBMA has been thought of as a motor neuron disease, recent evidence indicates a key role for skeletal muscle. To resolve which early aspects of the disease can be caused by neurogenic or myogenic mechanisms, we made use of the tet-On and Cre-loxP genetic systems to selectively and acutely express polyQ AR in either motor neurons (NeuroAR) or myocytes (MyoAR) of transgenic mice. After 4 weeks of transgene induction in adulthood, deficits in gross motor function were seen in NeuroAR mice, but not MyoAR mice. Conversely, reduced size of fast glycolytic fibers and alterations in expression of candidate genes were observed only in MyoAR mice. Both NeuroAR and MyoAR mice exhibited reduced oxidative capacity in skeletal muscles, as well as a shift in fast fibers from oxidative to glycolytic. Markers of oxidative stress were increased in the muscle of NeuroAR mice and were reduced in motor neurons of both NeuroAR and MyoAR mice. Despite secondary pathology in skeletal muscle and behavioral deficits, no pathological signs were observed in motor neurons of NeuroAR mice, possibly due to relatively low levels of polyQ AR expression. These results indicate that polyQ AR in motor neurons can produce secondary pathology in muscle. Results also support both neurogenic and myogenic contributions of polyQ AR to several acute aspects of pathology and provide further evidence for disordered cellular respiration in KD/SBMA skeletal muscle.

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes.

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.

Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2.

In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca(2+) homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 (ADA/ADA) ) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca(2+) sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344-1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 (ADA/ADA) hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 (ADA/ADA) mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.

Effect of α-Allocryptopine on Delayed Afterdepolarizations and Triggered Activities in Mice Cardiomyocytes Treated with Isoproterenol.

Objective. To investigate the effect of α-allocryptopine (ALL) on delayed afterdepolarization (DAD) incidence and triggered activity (TA) in mice administered isoproterenol (ISO). Methods. Mouse ventricular myocytes were isolated. And the cellular electrophysiological properties of ventricular myocytes were investigated. Results. We found that the incidences of DADs and TA in mouse myocytes were increased by ISO treatment. In sharp contrast, triggered arrhythmia events were rarely observed in myocytes with 10 μM ALL treatment. Transient inward current (I ti) was reduced significantly with ALL treatment, which contributed to DAD-related triggered arrhythmia. Compared to Iso-treated group, the L-type calcium current (I Ca,L) densities were decreased after exposure to ALL, along with slower activation, quicker inactivation, and longer time constant of recovery from inactivation kinetics. Conclusion. There is less triggered arrhythmia events in ventricular myocytes treated with ALL. This effect may be associated with the inhibition of I ti and I Ca,L.

Mitochondrial Abnormalities in a Mouse CPVT Model with RyR2 Loss-Of-Function Mutation

Mitochondria exchange matrix content in a unique and dynamic way by one of two mechanisms: a short-range direct interaction via closely spaced kissing junctions or long-range propagation of signal by the extrusion of extended nanotubular extensions (nanotunneling) (Huang et al 2013. Proc Natl Acad Sci U S A, 110(8): 2846-51). Both are normal, but infrequent features of cardiac myocytes. It is interesting to know whether this behavior may be affected by changes in Ca2+ homeostasis. We evaluated the cardiac ultrastructure in 7-month old heterozygous mice with a cardiac ryanodine receptor (RyR2) mutation (RyR2-A4860G) that depresses channel function and leads to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) without hypertrophy (Zhao et al., submitted). The mutation is embryonic lethal in mice homozygous for the mutation. In ventricular myocytes of heterozygotes mice (RyR2-A4860G+/−) it has a dual effect on SR Ca2+ release: an initial reduction due to lower RyR2 activity, linked to random bursts due to SR Ca2+ overload. The most striking and only structural alteration in cardiac myocytes in the mutant left ventricle is a clear increase in the frequency of long thin nano-tunneling mitochondrial extensions. The frequency of nanotunneling was ∼3 fold higher in mutant than in WT mice. Numerous small mitochondrial profiles are more frequent in mutant mice resulting in an average surface area of 0.17± 0.15 µm2 in mutant versus 0.28 ± 0.22 µm2 in WT (p<0.00001). This decrease is mostly due to sections through the thin tunneling extensions. The increased nanotunneling frequency would indicate an enhanced rate of long range intermitochondrial signal transfer. Interestingly, no activation of nanotunneling has been reported by EM in other CPVT models and/or in other alterations affecting proteins of CRU and Ca2+ homeostasis, making this a unique mitochondrial response.

Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice

BackgroundMetabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions.Methods and ResultsMice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C.ConclusionsThese results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.

Maximal acceleration of Ca2+ release refractoriness by β-adrenergic stimulation requires dual activation of kinases PKA and CaMKII in mouse ventricular myocytes.

Refractoriness of calcium release in heart cells is altered in several disease states, but the physiological mechanisms that regulate this process are incompletely understood. We examined refractoriness of calcium release in mouse ventricular myocytes and investigated how activation of different intracellular signalling pathways influenced this process. We found that refractoriness of calcium release is abbreviated by stimulation of the 'fight-or-flight' response, and that simultaneous activation of multiple intracellular signalling pathways contributes to this response. Data obtained under several conditions at the subcellular, microscopic level were consistent with results obtained at the cellular level. The results provide insight into regulation of cardiac calcium release and how alterations to this process may increase arrhythmia risk under different conditions. Time-dependent refractoriness of calcium (Ca(2+)) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca(2+) spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca(2+) spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca(2+) sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol (isoprenaline) accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark-to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca(2+) release refractoriness in mouse myocytes.

Cardiac P2X purinergic receptors as a new pathway for increasing Na⁺ entry in cardiac myocytes.

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes.

Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo.

Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined. Define the in vivo mechanisms underlying Nav1.5 membrane regulation. Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G(-/-) mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin-dependent kinase II to the intercalated disc. Mechanistically, βIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ; however, βIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation. Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

Enhanced late INa induces proarrhythmogenic SR Ca leak in a CaMKII-dependent manner

ObjectiveEnhanced late Na current (late INa) induces Na-dependent Ca overload as well as proarrhythmogenic events on the cellular level that include spatio-temporally uncoordinated diastolic Ca release from the sarcoplasmic reticulum (SR) and delayed afterdepolarizations (DADs). The Ca/calmodulin-dependent protein kinase II (CaMKII) gets activated upon increases in [Ca]i and mediates diastolic SR Ca leak as well as DADs. RationaleWe hypothesized that increased late INa (in disease-comparable ranges) exerts proarrhythmogenic events in isolated ventricular mouse myocytes in a manner depending on CaMKII-dependent SR Ca leak. We further tested whether inhibition of disease-related late INa may reduce proarrhythmogenic SR Ca leak in myocytes from failing human hearts. MethodsVentricular myocytes were isolated from healthy wildtype (WT), failing CaMKIIδC transgenic (TG) mouse, and failing human hearts. ATX-II (0.25–10nmol/L) was used to enhance late INa. Spontaneous Ca loss from the SR during diastole (Ca sparks), DADs, non-triggered diastolic Ca transients in myocytes and premature beats of isometrically twitching papillary muscles were used as readouts for proarrhythmogenic events. CaMKII autophosphorylation was assessed by immunoblots. Late INa was inhibited using ranolazine (Ran, 10μmol/L) or TTX (2μmol/L), and CaMKII by KN-93 (1μmol/L) or AIP (1μmol/L). ResultsIn WT myocytes, sub-nanomolar ATX-II exposure (0.5nmol/L) enhanced late INa by ~60%, which resulted in increased diastolic SR Ca loss despite unaltered SR Ca content. In parallel, DADs and non-triggered diastolic Ca transients arose. Inhibition of enhanced late INa by RAN or TTX significantly attenuated diastolic SR Ca loss and suppressed DADs as well as mechanical alternans in mouse and diastolic SR Ca loss in failing human myocytes. ATX-II caused Ca-dependent CaMKII-activation without changes in protein expression, which was reversible by Ran or AIP. Conversely, CaMKII-inhibition decreased diastolic SR Ca loss, DADs and non-triggered diastolic Ca transients despite ATX-II-exposure. Finally, failing mouse myocytes with increased CaMKII activity (TG CaMKIIδC) showed an even aggravated diastolic SR Ca loss that was associated with an increased frequency of non-triggered diastolic Ca transients upon enhanced late INa. ConclusionsIncreased late INa (in disease-comparable ranges) induces proarrhythmogenic events during diastole in healthy and failing mouse myocytes, which are mediated via CaMKII-dependent SR Ca loss. Inhibition of late INa not only attenuated these cellular arrhythmias in mouse myocytes but also in failing human myocytes indicating some antiarrhythmic potential for an inhibition of the elevated late INa/CaMKII signaling pathway in this setting.

Enzymatic and non-enzymatic post-translational modifications linking diabetes and heart disease.

Enzymatic and non-enzymatic post-translational modifications linking diabetes and heart disease. Diabetes-related morbidity and mortality is evident in the increased prevalence of cardiovascular diseases and the high-risk of heart failure. Diabetic cardiomyopathy is a well-known complication of diabetes, and is defined as a ventricular dysfunction independent of coronary heart diseases and hypertension. The Framingham study firmly established an epidemiological link between diabetes and heart failure1. Diabetes causes metabolic disturbances, cardiac fibrosis, endothelial and vascular smooth muscle cell dysfunctions, altered contractile performance, and arrhythmia. The underlying mechanisms of the adverse effects of diabetes on the heart remain poorly understood. However, it is certain that diabetic heart disease is a consequence of multiple factors including uncontrolled hyperglycemia, insulin resistance and dyslipidemia. Chronic hyperglycemia causes glucose toxicity, which mediates the detrimental effects of diabetes through the activation of a number of pathways, such as the polyol pathway, oxidative stress, mitochondrial dysfunction, activation of protein kinases, hexosamine biosynthesis pathway and non-enzymatic glycation reaction. The hexosamine biosynthesis pathway generates uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is an activated precursor for both N-linked and O-linked glycosylation of proteins, and the substrate for β-O-linked GlcNAc (O-GlcNAc) modification of proteins (Figure 1). Recently, O-GlcNAc covalent modification of cardiac proteins has received considerable attention as a key factor in diabetic cardiac pathology2, 3. In contrast to non-enzymatic glycation reactions, O-GlcNAc glycosylation is a reversible and labile post-translational modification. After entering a cell, glucose is phosphorylated to glucose-6-phosphate (Figure 1). It is further metabolized during glycolysis to fructose-6-phosphate, which leads to accessory pathways of glucose metabolism; one such pathway is the hexosamine biosynthesis pathway. Under normal physiological conditions, approximately 5% of intracellular glucose enters the hexosamine biosynthesis pathway; however, its flux is upregulated in diabetic cardiomyocytes4. The rate-limiting enzyme glutamine, fructose-6-phosphate aminotransferase (GFAT), converts fructose-6-phosphate to glucosamine-6-phosphate, which is then converted into UDP-GlcNAc and transferred onto target proteins by O-GlcNAc transferase (OGT). The enzyme, O-GlcNAcase, catalyzes the removal of the O-GlcNAc adduct from O-GlcNAcylated proteins (Figure 1)2. O-GlcNAcylation is reported to be involved in a number of cellular processes including signal transduction, protein–protein interactions, translation, protein degradation and gene expression alteration4. Recently, O-GlcNAcylation has been shown in the regulation of cardiomyocyte excitation-contraction coupling by influencing intracellular Ca2+ homeostasis5. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is a serine-threonine kinase that is activated by an increase in cellular Ca2+. The disruption of normal intracellular Ca2+ homeostasis is the event mainly responsible for initiating and perpetuating myocardial dysfunction, electrical instability, and arrhythmia. The CaMKII regulates Ca2+ homeostasis proteins in the myocardium. Recently, it has been reported that O-GlcNAc modification of CaMKII leads to cardiac arrhythmias associated with diabetes, which are linked to heart failure and sudden cardiac death2. CaMKII fusion with O-GlcNAc leads to chronic overactivation of CaMKII, triggering arrhythmias, and inhibition of O-GlcNAcylation of CaMKII prevented the occurrence of arrhythmias2. In addition to direct O-GlcNAc modification of proteins involved in excitation-contraction coupling, nuclear O-GlcNAcylation of transcription factors, histones and ribonucleic acid (RNA) polymerase II has been known to alter gene transcription, resulting in cardiac deterioration in patients with diabetes3. Compared with the dynamic and enzymatic regulation of O-GlcNAcylation, non-enzymatic glycation-modification is generally irreversible and obstructive. Both extracellular and intracellular formation of advanced glycation end-products (AGE) occurs in proteins such as collagen and other matrix components, and in cardiomyocyte intracellular proteins. The involvement of AGE-modification in excitation-contraction coupling has been described in type 1 diabetic rat myocardium, including the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase; SERCA2) and ryanodine receptor 2 (RYR2), a Ca2+ release channel6. Remodeling and degradation of the extracellular matrix is also affected in diabetes as a result of reduced expression of matrix metalloproteinases and consequent lower collagen turnover7. Binding of AGE to the receptor for AGE (RAGE) on fibroblasts stimulates collagen production, further compromising ventricular compliance8. We also reported that AGE and RAGE impaired Ca2+ homeostasis in diabetic mouse myocytes; thus, accounting for the abnormal contractile and relaxation functions9. Diabetic cardiomyopathy involves various complex functional and structural abnormalities. Recent findings highlight the dynamic O-GlcNAcylation and irreversible glycation of fundamental cardiomyocyte proteins in the context of diabetes. This can potentially be used to study enzymatic and non-enzymatic post-translational modifications associated with glucose metabolism for diabetic cardiomyopathy interventions.

Cardioprotective benefits of adenosine triphosphate-sensitive potassium channel opener diazoxide are lost with administration after the onset of stress in mouse and human myocytes.

Adenosine triphosphate-sensitive (KATP) potassium channel opener diazoxide (DZX) maintains myocyte volume and contractility during stress via an unknown mechanism when administered at the onset of stress. This study was performed to investigate the cardioprotective potential of DZX when added after the onset of the stresses of hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress. Isolated mouse ventricular and human atrial myocytes were exposed to control Tyrode's solution (TYR) for 10 to 20 minutes, test solution for 30 minutes (hypothermic hyperkalemic cardioplegia [CPG], CPG + 100uM diazoxide [CPG+DZX], metabolic inhibition [MI], MI+DZX, mild hypo-osmotic stress [0.9T], or 0.9T + DZX), with DZX added after 10 or 20 minutes of stress, followed by 20 minutes of re-exposure to TYR (±DZX). Myocyte volume (human + mouse) and contractility (mouse) were compared. Mouse and human myocytes demonstrated significant swelling during exposure to CPG, MI, and hypo-osmotic stress that was not prevented by DZX when administered either at 10 or 20 minutes after the onset of stress. Contractility after the stress of CPG in mouse myocytes significantly declined when DZX was administered 20 minutes after the onset of stress (p < 0.05 vs TYR). Contractility after hypo-osmotic stress in mouse myocytes was not altered by the addition of DZX. To maintain myocyte volume homeostasis and contractility during stress (hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress), KATP channel opener diazoxide requires administration at the onset of stress in this isolated myocyte model. These data have potential implications for any future clinical application of diazoxide.

Tubulin polymerization disrupts cardiac β-adrenergic regulation of late INa

The anticancer drug paclitaxel (TXL) that polymerizes microtubules is associated with arrhythmias and sinus node dysfunction. TXL can alter membrane expression of Na channels (NaV1.5) and Na current (INa), but the mechanisms are unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can be activated by β-adrenergic stimulation and regulates INa gating. We tested whether TXL interferes with isoproterenol (ISO)-induced activation of CaMKII and consequent INa regulation. In wild-type mouse myocytes, the addition of ISO (1 µmol/L) resulted in increased CaMKII auto-phosphorylation (western blotting). This increase was completely abolished after pre-treatment with TXL (100 µmol/L, 1.5 h). The mechanism was further investigated in human embryonic kidney cells. TXL inhibited the ISO-induced β-arrestin translocation. Interestingly, both knockdown of β-arrestin2 expression using small interfering RNA and inhibition of exchange protein directly activated by cAMP (Epac) blocked the ISO-induced CaMKII auto-phosphorylation similar to TXL. The generation of cAMP, however, was unaltered (Epac1-camps). CaMKII-dependent Na channel function was measured using patch-clamp technique in isolated cardiomyoctes. ISO stimulation failed to induce CaMKII-dependent enhancement of late INa and Na channel inactivation (negative voltage shift in steady-state activation and enhanced intermediate inactivation) after pre-incubation with TXL. Consistent with this, TXL also inhibited ISO-induced CaMKII-specific Na channel phosphorylation (at serine 571 of NaV1.5). Pre-incubation with TXL disrupts the ISO-dependent CaMKII activation and consequent Na channel regulation. This may be important for patients receiving TXL treatments, but also relevant for conditions of increased CaMKII expression and enhanced β-adrenergic stimulation like in heart failure.