Adult Rat Cardiomyocytes Research Articles

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.

Analysis of proteome changes in doxorubicin-treated adult rat cardiomyocyte

Doxorubicin-induced cardiomyopathy in cancer patients is well established. The proposed mechanism of cardiac damage includes generation of reactive oxygen species, mitochondrial dysfunction and cardiomyocyte apoptosis. Exposure of adult rat cardiomyocytes to low levels of DOX for 48h induced apoptosis. Analysis of protein expression showed a differential regulation of several key proteins including the voltage dependent anion selective channel protein 2 and methylmalonate semialdehyde dehydrogenase. In comparison, proteomic evaluation of DOX-treated rat heart showed a slightly different set of protein changes that suggests nuclear accumulation of DOX. Using a new solubilization technique, changes in low abundant protein profiles were monitored. Altered protein expression, modification and function related to oxidative stress response may play an important role in DOX cardiotoxicity.

Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca2+ overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca2+ overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca2+]i levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca2+ stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca2+]i in the absence of [Ca2+]o and the subsequent restoration of [Ca2+]o sustained the increased [Ca2+]i for a few minutes, whereas, the persisting elevation of [Ca2+]i was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl3) or spermine also resulted in the same effect and the duration of [Ca2+]i increase was also shortened in the absence of [Ca2+]o. In adult and neonatal rat cardiomyocytes, GdCl3 increased the expression of TRPC3 mRNA and protein, which were reversed by SKF96365 but not by inhibitors of the L-type channels and the Na+/Ca2+ exchangers. However, GdCl3 had no obvious effect on the expression of TRPC1 protein. These results suggested that CaR stimulation induced activation of TRP channels and promoted the expression of TRPC3, but not TRPC1, that sustained the increased [Ca2+]i.

Mitochondria–cytoskeleton interaction: Distribution of β-tubulins in cardiomyocytes and HL-1 cells

Mitochondria–cytoskeleton interactions were analyzed in adult rat cardiomyocytes and in cancerous non-beating HL-1 cells of cardiac phenotype. We show that in adult cardiomyocytes βII-tubulin is associated with mitochondrial outer membrane (MOM). βI-tubulin demonstrates diffused intracellular distribution, βIII-tubulin is colocalized with Z-lines and βIV-tubulin forms microtubular network. HL-1 cells are characterized by the absence of βII-tubulin, by the presence of bundles of filamentous βIV-tubulin and diffusely distributed βI- and βIII-tubulins. Mitochondrial isoform of creatine kinase (MtCK), highly expressed in cardiomyocytes, is absent in HL-1 cells. Our results show that high apparent K m for exogenous ADP in regulation of respiration and high expression of MtCK both correlate with the expression of βII-tubulin. The absence of βII-tubulin isotype in isolated mitochondria and in HL-1 cells results in increased apparent affinity of oxidative phosphorylation for exogenous ADP. This observation is consistent with the assumption that the binding of βII-tubulin to mitochondria limits ADP/ATP diffusion through voltage-dependent anion channel of MOM and thus shifts energy transfer via the phosphocreatine pathway. On the other hand, absence of both βII-tubulin and MtCK in HL-1 cells can be associated with their more glycolysis-dependent energy metabolism which is typical for cancer cells (Warburg effect).

African Trypanosomes Increase Calcium Wave Frequency in Isolated Adult Rat Cardiomyocytes via Secretion of Cathepsin L

African trypanosomes are blood-borne extracellular parasites which have recently been linked to cardiac dysfunction in ∼70% of sleeping sickness patients. Although this may result from an indirect effect of the parasite (e.g. myocarditis), a direct effect of the parasite on the heart has not been investigated. Adult rat cardiomyocytes were incubated with trypanosome growth media containing Trypanosoma brucei Lister427 (30min). A population assay assessed the percentage of cells demonstrating Ca2+ waves within a 1min period. Incubation with live trypanosomes led to a significant increase in the percentage of cells demonstrating Ca2+ waves (54.8±2.8% vs. 79.2±5.1%; media vs. live trypanosomes, P 0.05), whereas CA074 (specific inhibitor of cathepsin-B) had no effect on Ca2+ wave frequency. These data suggest trypanosomes interact with cardiomyocytes leading to increased Ca2+ wave production via cathepsin-L. This may contribute to the cardiac abnormalities observed in patients with trypanosomiasis.

Mitochondrial Interactosome in Energy Metabolism in Healthy and Cancer Cells

In this study we investigated intracellular distribution of β-tubulin isotypes, expression of mitochondrial creatine kinase (MtCK) and their roles in Mitochondrial Interactosome (supercomplex formed by ATP synthasom, ANT, MtCK, VDAC-tubulin and other VDAC-linker proteins) in adult rat cardiomyocytes, in cancerous HL-1 cells of cardiac phenotype and several other cancer cells lines. Immunofluorescent and western blot analysis of adult cardiac muscle cells revealed the presence of all β-tubulin isotypes with the very regular localization of tubulin II(β2) corresponding to mitochondrial “crystal like” pattern. Antibodies against tubulin IV(β4) revealed characteristic staining of microtubular network. Polymerized transversal lines of tubulin III(β3) matched with Z-lines (alpha-actinin) fixing, probably, microtubular network to sarcomer structure. Tubulin I(β5) distribution was diffusely spotted and fragmentary polymerized. In cancer cardiac HL-1 and other cancer cell types MI remodelling is characterized by the absence of II(β2). Additionally, these cells do not contain tubulin IV(β4) isotype creating micro-tubular network in adult cardiomyocytes and have diffusely distributed tubulin I(β5) and III(β3) isotypes. Other characteristic of cancer cells was the absence of both sarcomeric and ubiquitous mitochondrial isoform of creatine kinase (MtCK). In cardiac cells respiration is controlled by creatine via activation of MtCK within MI. Knowing that the extremely low apparent Km for free ADP in cancer cells as well as in isolated mitochondria is associated with the lack of tubulin II(β2) isotype, we can assume that tubulin II(β2) can specifically control the voltage dependent anion channel (VDAC) selective permeability resulting in adenine nucleotides micro-compartmentation within the MI. The absence of II(β2) tubulin isotype and MtCK isoforms induce significant alterations of phenotype and regulatory mechanisms of cancer cells energy metabolism. By these mechanisms, all mitochondrial ATP can be captured to maintain activated glycolysis and lactate production, known as Warburg effect.

Mutations that Alter cTnC Ca 2+ Binding Affect Interactions with cTnI and Cardiomyocyte Contraction

We are using a series of cardiac TnC (cTnC) variants with increased (L48Q) or decreased (L57Q or I61Q) Ca2+ binding affinity (in solution) that alter Ca2+ regulation of force development in skinned myocardium to study 1) molecular mechanisms of the thin filament Ca2+ signaling pathway, and 2) their influence on intracellular Ca2+ metabolism. Solution binding studies indicated increased affinity of the cTnC variants for cTnI in the order L48Q > wild type (WT) > L57Q > I61Q. We previously reported for stopped flow experiments that L48Q cTnC-Tn slows calcium dissociation (by about 4 fold compared to WT), and L57Q and I61Q increases the rate of release. Ca2+ dissociation from reconstituted thin filaments containing cTnC variants was increased in all cases, with L48Q again slowing release and I61Q increasing calcium release compared to WT. Previously, our molecular dynamics (MD) simulations suggested increased Ca2+ and cTnI affinity of L48Q vs. WT cTnC resulting from increased mobility of the B-helix, greater exposure of the hydrophobic patch and stronger interactions of Ca2+ with some of the coordinating residues at Ca2+ binding site II. We now report ‘lifting’ of B helix of L48Q cTnC(1-89) in apo states simulations. There were no similar movements with L57Q and I61Q simulations. Adenoviral mediated expression of cTnC variants in quiescent cultured adult rat cardiomyocytes resulted in shortening rate and magnitude in the order I61Q > L57Q > WT > L48Q. Ca2+ transients (Fura-2) were not affected by L48Q, but reduced with L57Q or I61Q cTnC. Together these data suggest that cTnC variants with altered Ca2+ binding affinity can influence interactions with cTnI and alter myocardial Ca2+ metabolism. HL091368, HL65497 (MR), AHA-09PRE2090056 (DW).

Erk1-Mediated Development of Left Ventricular Cardiac Hypertrophy in a P21 Activated Kinase-1 (Pak1) Knockout Mouse Model

Pak1, a serine/threonine protein kinase, plays a critical role in cardiac excitation-contraction. We hypothesize that Pak1 prevents or attenuates the development of left ventricular (LV) cardiac hypertrophy. Six wild-type (WT) and six Pak1-knockout (KO) mice were randomized into four groups of three mice each to receive subcutaneously 25μg/g/day of isoproterenol (ISO) or saline (CTRL) for seven days. Transthoracic echocardiography showed in the Pak1-KO/ISO group vs. WT/ISO group, respectively: i) increased LV fractional shortening (%)(61.65 ± 2.72 vs. 48.02 ± 4.01; p = 0.048); ii) reduced LV chamber volume (μL) in diastole (36.77 ± 4.19 vs. 73.92 ± 3.95; p = 0.003) and systole (4.14 ±0.54 vs. 16.24 ± 2.19; p = 0.0058); iii) increased isoproterenol-induced LV cardiac hypertrophy in Pak1-KO mice (40% increase in LV mass, p = 0.049) vs. WT mice (15.2 % increase in LV mass, p = 0.07); iv) enhanced early filling deceleration time (mm/s2) (−80460 ± 13620 vs. −21550 ± 3560; p = 0.014). We also generated novel evidence that Erk1 and Pak1 establish protein-protein interaction in whole cardiac tissue, as assessed by co-immunoprecipitation. Western immunoblotting of Erk1 phosphorylation in whole cardiac tissue showed maximal Erk1 activation in Pak1-KO/ISO mice vs. all other groups (phosphoErk1/totalErk1 ratio in Pak1-KO/ISO 0.80 ± 0.005 vs.: i) WT/CTRL 0.042 ± 0.02171, p<0.0001; ii) WT/ISO 0.3086 ± 0.0924, p=0.006; iii) Pak1-KO/CTRL 0.6020 ± 0.046, p=0.013), whereas Erk1 phosphorylation was consistently reduced in lysates obtained from adenovirally-infected adult rat cardiomyocytes that express constitutively active Pak1 vs. dominant negative Pak1 (phosphoErk1/actin ratio: 7.06% vs. 73.73%, respectively). In conclusion, Pak1-null mice develop LV cardiac hypertrophy due to increased Erk1 activation, indicating a role for Pak1 as a natural inhibitor of Erk1 and a novel anti-hypertrophic signaling molecule.

The Cytosolic Protein Kinase CK2 is the Physiological Cardiac Calsequestrin Kinase

The luminal sarcoplasmic reticulum (SR) protein cardiac calsequestrin (CSQ2) contains phosphate on roughly half of its cardiac-specific C-terminal serines. CSQ2 kinase has been assumed to be protein kinase CK2 despite its being only cytoplasmic and nuclear. In CSQ2 from heart tissue, phosphate distribution differs among CSQ2 molecules depending upon how far CSQ2 has trafficked through the cardiac secretory pathway, with the more recently synthesized containing the most phosphate. To establish the identity of CSQ2 kinase as protein kinase CK2, specific inhibitors of CK2 were used along with RNAi. In vitro, tetrabromocinammic acid (TBCA, 100 µM) inhibited CSQ2 kinase and CK2 activities by about 85% at equal concentrations. In intact cultured primary adult rat cardiomyocytes and in COS cells, 24 h of TBCA (100 µM) treatment produced a 75% decrease in phosphorylation of CSQ2. To further confirm the identity of CSQ2 kinase as protein kinase CK2, CK2α and CK2α′ subunits were down-regulated which led to a 90% decrease in their protein levels. CSQ2 kinase activity in the same cells was reduced by 50%. CSQ2 protein expressed in CK2-silenced cells showed reduced phosphate. These findings suggest that cytosolic CK2 phosphorylates luminalCSQ2, a reaction expected to occur only during nascent protein translocation. CSQ2 phosphorylation site mutants analyzed in COS cells, had their characteristic rough ER form CSQ2 glycan (GlcNAc2Man9,8) undergo phosphorylation-site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lesser and greater levels of ER retention, respectively. Our data support a hypothesis whereby CK2 is able to phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, leading to changes in its trafficking through the secretory pathway.

Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity

In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1)and human ventricular (hVLC-1)light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or β-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rβ-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Targeting sigma-1 receptor signaling by endogenous ligands for cardioprotection

Introduction: The sigma receptors, initially described as a subtype of opioid receptors, are now considered to be a unique receptor expressed in neonatal rat cardiomyocytes and in the plasma membrane of adult rat cardiomyocytes. A number of sigma receptor ligands influence cardiovascular function and the heart has binding sites for sigma receptor ligands that alter contractility both in vivo and in vitro. The human sigma-1 receptor gene contains a steroid-binding component and gonadal steroid dehydroepiandrosterone (DHEA) which interacts with the sigma-1 receptor.Areas covered: We recently documented that the pathophysiological role of the sigma-1 receptor in the heart and its modulation using DHEA, was cardioprotective. Moreover, agonist-induced activation of the sigma-1 receptor modulates diverse ion channels and thereby regulates heart function. Novel concepts for understanding the pathophysiological relevance of sigma-1 receptors in the progression of heart failure, and developing clinical therapeutics targeting for the receptor in cardiovascular diseases are discussed.Expert opinion: Future studies should attempt to develop cardiac-specific knockdown of the sigma-1 receptor to observe its downstream signaling. We expect that these observations will lead to a novel therapeutic target for which a new class of antihypertrophic drugs can be designed.

Calibrated micropost arrays for biomechanical characterisation of cardiomyocytes

The study of biomechanics of isolated cardiomyocytes can allow us to understand the cardiac function and disease development in the absence of viscoelastic or contractile properties of the surrounding tissue. However, popular techniques such as micropipettes and carbon fibre-based measurements require serial, single-cell measurements and limit the amount of data passing through a system or process. The authors utilise elastomer micropost arrays microfabricated by a replica molding technique for precise and quantitative force measurements of cardiomyocytes with the potential for high throughput. The authors also present a calibration system using a piezoresistive force sensor and video-analysis technique to improve the resolution and validate the analysis of these low-force measurements. Calibrated microposts arrays are applied to measure the contractile forces of rat neonatal cardiomyocytes. Using image processing, the contractile forces for a sample of cardiomyocytes are extracted. It was found that isolated rat neonatal myocytes generate 39±5 nN average contractile force per post and an integrated axial contractile force of 189±20 nN, which is four times smaller than isolated adult Wistar rat cardiomyocytes and 30 times smaller than isolated adult rat cardiomyocytes. With calibration and quantitative image analysis, this study demonstrates that micropost systems can provide precise high-throughput test beds for myocyte mechanics using pharmacologic, small peptide, gene therapies or heart disease models.

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Background/Aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor RORα have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and RORα and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORα were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erbα expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erbα mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erbα mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes

Cardiomyocyte substrate utilization is important in maintaining optimal cardiac function. Adiponectin has been shown to confer cardioprotective effects in part via regulating glucose and fatty acid uptake and oxidation in cardiomyocytes. Here we investigated mechanisms whereby adiponectin mediates a particular metabolic effect by focusing on lipoprotein lipase (LPL), an enzyme that increases free fatty acid availability to the heart by breakdown of chylomicrons and very-low-density lipoproteins in circulation. We used primary adult rat cardiomyocytes and demonstrate that adiponectin increased LPL translocation to the cell surface where it could be released at least partly in its active form, as evidenced by measuring basal and heparin-releasable LPL activity. Furthermore, these effects of adiponectin were mediated via remodeling of the actin cytoskeleton. We quantitatively assessed the filamentous to globular actin ratio and show that increased stress fiber formation, visualized by rhodamine-phalloidin immunofluorescence, in response to adiponectin, is achieved via stimulating Ras homolog gene family A (RhoA) activity, determined using G-LISA RhoA activation assay kit. We also demonstrate that adiponectin induces phosphorylation and inhibition of cofilin, leading to a reduction in actin treadmilling. Increased cofilin phosphorylation and stress fiber formation in response to adiponectin were prevented by inhibition of either RhoA or its downstream kinase Rho-associated protein kinase. Importantly, inhibition of cytoskeletal remodeling prevented adiponectin-stimulated plasma membrane LPL content detected by immunofluorescence and also subsequent LPL activity. In summary, we show that adiponectin mediates actin cytoskeleton remodeling to translocate LPL and allow subsequent activation.

Redox regulation of the mitochondrial K ATP channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK ATP) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK ATP regulation. mK ATP activity was measured by both osmotic swelling and Tl + flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia–reperfusion (IR) injury was also used to determine the role of mK ATP in cardioprotection by nitroxyl. Key findings were as follows: (i) mK ATP was activated by O 2 − and H 2O 2 but not other peroxides. (ii) mK ATP was inhibited by NADPH. (iii) mK ATP was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK ATP-dependent manner. Overall, these results suggest that the mK ATP channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK ATP may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Inhibition of Rho–ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho–ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho–ROCK–flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.

Interference of hawthorn on serum digoxin measurements by immunoassays and pharmacodynamic interaction with digoxin.

Abstract Context.—Hawthorn is an herb indicated for treating cardiac illness. Because a patient taking digoxin may also take hawthorn, we investigated potential interference of hawthorn in serum digoxin measurements using immunoassays as well as pharmacodynamic interaction between hawthorn and digoxin. Hawthorn contains alkaloids that are structurally similar to digoxin and may interfere with serum digoxin measurement using immunoassays. In addition, hawthorn has cardioactive properties similar to digoxin. Objective.—To study potential pharmacodynamic interaction between hawthorn and digoxin. Design.—The effects of hawthorn extract on serum digoxin measurements using Digoxin III (Abbott Laboratories, Abbott Park, Illinois) and the Tina-Quant digoxin assay (Roche Diagnostics, Indianapolis, Indiana) were investigated using 2 different brands of extract. To study the pharmacodynamic interaction between hawthorn and digoxin, we used an isolated adult rat cardiomyocyte system, measuring calcium transients by r...

Interference of Hawthorn on Serum Digoxin Measurements by Immunoassays and Pharmacodynamic Interaction With Digoxin

Hawthorn is an herb indicated for treating cardiac illness. Because a patient taking digoxin may also take hawthorn, we investigated potential interference of hawthorn in serum digoxin measurements using immunoassays as well as pharmacodynamic interaction between hawthorn and digoxin. Hawthorn contains alkaloids that are structurally similar to digoxin and may interfere with serum digoxin measurement using immunoassays. In addition, hawthorn has cardioactive properties similar to digoxin. To study potential pharmacodynamic interaction between hawthorn and digoxin. The effects of hawthorn extract on serum digoxin measurements using Digoxin III (Abbott Laboratories, Abbott Park, Illinois) and the Tina-Quant digoxin assay (Roche Diagnostics, Indianapolis, Indiana) were investigated using 2 different brands of extract. To study the pharmacodynamic interaction between hawthorn and digoxin, we used an isolated adult rat cardiomyocyte system, measuring calcium transients by real-time fluorescence spectrophotometry. Hawthorn interfered only with the Digoxin III immunoassay but had no effect on the Tina-Quant assay. Both hawthorn extracts increased intracellular calcium levels, but the lack of additive response with digoxin suggests both may bind to the same site of Na, K adenosine triphosphatase. Because of interference of hawthorn with a digoxin immunoassay and pharmacodynamic interaction with digoxin, a patient receiving digoxin should avoid hawthorn.