Adult Rabbit Ventricular Myocytes Research Articles

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII—but not PKA, PKC or PKD—raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Location and function of transient receptor potential canonical channel 1 in ventricular myocytes

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.

Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling

Background: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about Mechano-Chemo-Transduction (MCT) mechanisms that underlie heart disease development. We developed a novel Cell-in-Gel system to control mechanical loading on cardiomyocytes during excitation-contraction coupling. Here we investigate mechanical load effects on modulating Ca2+ signaling and contraction dynamics to test the hypothesis NO signaling mediates MCT.Methods: Freshly isolated adult rabbit ventricular myocytes were embedded in polyvinyl alcohol (10%) and crosslinked with tetravalent boronate-PEG (7.5%) to form a 3D viscoelastic hydrogel. The Cell-in-Gel system was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Cardiomyocyte contraction and Ca2+ transients were measured in-gel and compared with load-free cells in solution.Results: Cardiomyocytes contracting in-gel under mechanical load showed augmented systolic Ca2+ transients (Fura-2 fluorescence ratio 1.47 ± 0.07 in-gel vs 0.85 ± 0.03 load-free), revealing MCT transduces mechanical stress to increase intracellular Ca2+ release. NOS inhibition using 1 mM L-NAME reduced the in-gel Ca2+ transient by 33% (n=15), suggesting NO signaling mediates MCT. Cells in-gel showed 8% decrease in contraction (fractional shortening 13.5 ± 0.4% in-gel vs 14.7 ± 0.3% load-free). Cells in-gel treated with L-NAME demonstrated a further reduction in contraction (11.6 ± 0.8%) as a result of decreased MCT by NOS inhibition. Importantly, 12% of cells in-gel showed mechanical load-induced alternans, which was abolished with L-NAME treatment, suggesting NO signaling contributes to load-induced Ca2+ dysregulation.Conclusion: Our findings demonstrate MCT increases the systolic Ca2+ transient to enhance contractility in response to increased afterload, serving a compensatory mechanism to autoregulate contractility. MCT is attenuated by inhibiting NOS. These results suggest NOS mediates increased Ca2+ signaling in cardiomyocytes under mechanical load, providing a mechanistic basis for the Anrep effect.

A Simple Regulation of Cardiomyocyte Excitability

Introduction: Recently, genome wide association studies (GWAS) identified LITAF (lipopolysaccharide-induced TNF factor) as one of the novel loci associated with prolongation of QT interval. Our goal is to investigate the role of LITAF in regulating cardiomyocyte excitability and action potential duration (APD). Methods: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients and transmembrane (TM) potentials. Propagation velocities of depolarizing waves were determined. Action potential (AP) was digitally imaged in adenoviral infected neonatal rabbit cardiomyocytes (NRbCM). Live cell confocal calcium imaging was performed on adult rabbit ventricular myocytes (ARbCM) to determine intracellular Ca2+ handling. A current-voltage (I-V) relationship for the L-type calcium current (ICa,L ) was determined using whole cell patch clamping. To study the effect of LITAF on Cav1.2 channel expression, surface biotinylation and western blot assays were performed using tSA201 cells. Results: Our studies demonstrate that loss-of-function of LITAF in vivo (zebrafish) increases the Ca2+ transient amplitude (which primarily depends on calcium influx), slows atrial-ventricular conduction, and prolongs the APD. In contrast, in vitro gain-of-function experiments in rabbit CM established that overexpression of LITAF downregulates the total and surface pools of Cav1.2 channel polypeptides, decreases Ca2+ transients and L-type Ca2+ conductance, and shorten the APD. Studies in heterologous expression systems revealed that one of the auxiliary subunits of Cav1.2 channels, Cavβ2b, is likely involved in LITAF-mediated inhibition of recombinant Cav1.2 channels. Conclusion: LITAF regulates the expression and function of Cav1.2 channels, thereby determining APD and cardiac cell excitation.

Effects of Reactive Oxygen Species on NFAT Activation and Translocation in Adult Rabbit Ventricular Myocytes

Nuclear factor of activated T cells (NFAT) transcription factors play a key role during cellular remodeling associated with cardiac hypertrophy and heart failure (HF). Evidence suggests that reactive oxygen species (ROS) are integral to the progression of cardiac hypertrophy and HF. Therefore, we aimed to determine the role of ROS in the activation and translocation of NFAT.Adult rabbit ventricular myocytes were infected with recombinant adenoviruses encoding for NFAT-GFP fusion proteins (isoforms NFATc1 and NFATc3). The subcellular distribution of NFAT was quantified as the ratio of NFATnuc to NFATcyt fluorescence (RNFAT) and nuclear-cytosolic NFAT translocation was expressed as changes of RNFAT. Under basal unstimulated conditions, NFATc3 was predominantly localized in the cytoplasm, whereas NFATc1 displayed a nuclear localization.Acute exposure to the inhibitors of oxidative phosphorylation Rotenone, Antimycin A, Oligomycin and FCCP resulted in the activation and translocation of NFATc1 (60 - 145 % increase of RNFAT) into the nucleus. These inhibitors did not induce translocation of NFATc3; however, exposure to Hydrogen Peroxide (H2O2) or Ruthenium Red (inhibitor for the mitochondrial Ca uniporter), resulted in the activation and translocation of NFATc3 (but not NFATc1). The H2O2-induced NFATc3 translocation was attenuated in the presence of the antioxidant N-acetylcysteine.These data identify a ROS-induced activation and translocation of NFAT in adult ventricular myocytes.

Functional Modulation of Cardiac ATP-Sensitive Potassium Channels by Nitric Oxide via Intracellular Signaling

ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. Nitric oxide (NO) has been shown to stimulate cardiac KATP channels; however, the mechanistic details remain poorly understood. Here we sought to delineate the intracellular mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in combination with pharmacological, genetic and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the cardiac-type KATP) channels in transfected HEK293 cells, which was abolished by selective suppression of cGMP-dependent protein kinase (PKG), extracellular signal-regulated protein kinase (ERK)1/2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and reactive oxygen species (ROS) (hydrogen peroxide H2O2 in particular), respectively. Importantly, NO donors potentiated function of sarcKATP channels preactivated by the channel opener pinacidil in adult rabbit ventricular myocytes, through destabilizing the longest closed state and facilitating opening transitions, and the potentiation was nullified when PKG, calmodulin, CaMKII or ERK1/2 was inhibited. Exogenous H2O2 also stimulated ventricular sarcKATP channels in intact cells in an ERK1/2- and CaMKII-dependent manner. Genetic ablation of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished PKG stimulation of mouse ventricular sarcKATP channels (compared with wild-type controls). Kinase activity and Western blot assays further supported that NO-PKG activation augmented CaMKII activity in ventricular myocytes, which was mediated by ERK1/2. Collectively, we demonstrate that NO stimulates ventricular sarcKATP channels via a cGMP/PKG/ROS(H2O2)/ERK/calmodulin/CaMKII signaling cascade that alters channel gating. This novel signaling pathway may control cardiac excitability and mediate, in part, cytoprotection against ischemia-reperfusion injury, by opening myocardial KATP channels.

The Role of Heart Failure-Causing Phospholamban R9C Mutation in SERCA Dysfunction and Cardiac Ca2+ Mishandling

The naturally occurring, heterozygous Arg9-to-Cys (R9C) missense mutation of phospholamban (PLB) triggers dilated cardiomyopathy, heart failure and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of PLBR9C in regulation of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and cardiomyocyte Ca2+ handling is not clear. The dynamic equilibrium between PLB monomer and pentamer plays a critical role in SERCA regulation. We compared the effect of R9C and pentamer-destabilizing transmembrane triple cysteine mutation (SSS) on PLB pentameric assembly. We co-expressed Cer- or YFP-tagged PLBWT, PLBR9C, PLBSSS, or PLBR9C+SSS in AAV-293 cells and measured intrapentameric Fluorescence Resonance Energy Transfer (FRET). PLBR9C and PLBR9C+SSS exhibited a significant increase in oligomerization as compared to PLBWT and PLBSSS, which was further enhanced by application of 100μM H2O2. In addition, we investigated the stability of PLB pentamer in WT, R9C, SSS, or R9C+SSS background by molecular dynamic simulation studies. Importantly, co-expression of CFP-PLBR9C and YFP-PLBR9C in adult rabbit ventricular myocytes exhibited a large and rapid increase in oligomerization over time after exposure to 100μM H2O2 as compared to PLBWT. To investigate the effect of PLBR9C on SR Ca2+ kinetics, we recorded Ca2+ transients in electrically paced adult cardiomyocytes overexpressing CFP-PLBR9C or CFP-PLBWT. PLBR9C overexpressing cardiomyocytes exhibited faster SR Ca2+ uptake as compared to PLBWT, and a blunted Ca2+ uptake/pacing frequency response. We propose that R9C mutation in PLB increases the stability of the PLB pentameric assembly by crosslinking of adjacent cysteine thiols via disulfide bonds, and these effects are enhanced during oxidative stress. The resultant decrease in availability of PLB monomeric species leads to decrease in inhibition of SERCA, inability to respond to stress, and eventual heart failure.

Abstract 190: Protein Phosphatase 2Ce Is a Novel Phospholamban Phosphatase that Exacerbates Cell Death and Suppresses Cardiac Contractility

Regulation of sarcoplasmic reticulum (SR) calcium-ATPase (SERCA) activity is critical for calcium homeostasis in cardiomyocytes, and has a major impact on contractility and cellular viability of cardiomyocytes. The key regulators for SERCA activity include protein kinases, cAMP dependent protein kinase A and calcium/calmodulin dependent protein kinase II, and protein phosphatase 1. In this report, we have discovered that protein phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted to SR membrane in cardiomyocytes. PP2Ce was detected to interact with phosphlamban in heart. Recombinant PP2Ce protein showed a potent and specific activity towards the calcium/calmodulin dependent protein kinase II dependent phospholamban phosphorylation at threonin 17 site with no significant activity to cAMP dependent protein kinase A dependent phospholamban phosphorylation at serine 16 site. Expression of PP2Ce blunted β-adrenergic stimulated increase of phospholamban phosphorylation without affecting phosphorylation of ryanodine recepter or troponin I. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rabbit ventricular myocytes, and promoted hydrogen peroxide induced cell death in cultured neonatal rat ventricular myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant basal phenotype. However, in isolated perfusion heart preparation, β-adrenergic stimulated contractility was significant reduced in PP2Ce transgenic hearts comparing to wild type controls. Furthermore, we observed significantly larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a novel component of SR calcium regulatory network that has a potentially important role in cell death regulation and cardiac contractility.

Changes in Mitochondrial Calcium and ROS during Ischemia-Reperfusion in Polyphosphate-Depleted Cardiomyocytes

Loss of mitochondrial function plays a critical role for cardiac cell death during ischemia-reperfusion (I/R) injury. Mitochondrial dysfunction during I/R is caused by a combination of Ca2+ overload and increased ROS generation, which lead to permeability transition pore (mPTP) opening and disruption of energy metabolism. We have shown previously that depletion of mitochondrial inorganic polyphosphate (polyP) protected cells from mPTP opening during I/R. The aim of this study was to investigate whether the protective effect of polyP depletion was related to changes in mitochondrial Ca2+ and ROS generation. In adult rabbit ventricular myocytes polyP levels were decreased by adenoviral expression of a mitochondrially targeted polyP hydrolyzing enzyme (PPX). I/R was induced by exposing cells to glucose-free Tyrode solution containing 20 mM 2-deoxyglucose and 2 mM NaCN, pH 6.4, followed by superfusion with standard Tyrode solution. In control cells, a significant increase in mitochondrial Ca2+ ([Ca2+]m), ROS generation and mPTP opening was observed during ischemia together with a depolarized mitochondrial membrane potential. The increase in [Ca2+]m was only partially sensitive to Ru360 (Ca2+ uniporter blocker), but significantly affected by cyclosporine A (mPTP de-sensitizer) indicating possible Ca2+ entry through mPTP. In polyP-depleted cells, however, the opening of the mPTP was significantly diminished despite higher levels of ROS during ischemia. Moreover, in polyP-depleted cells the increase in [Ca2+]m during ischemia was decreased by Ru360, but was not sensitive to CsA. In both control and polyP-depleted cells ROS generation was attenuated by Ru360. These data indicate that mitochondrial Ca2+ uptake during ischemia triggers mPTP opening and ROS generation. The fact that decreased levels of polyP provide protection against mPTP opening despite increased levels of [Ca2+]m and ROS, support the hypothesis that mitochondrial polyP likely acts as a Ca2+ sensor of the mPTP.

Effect of Acidosis on Ventricular L-Type Calcium Current during Action Potentials

Changes in extra- and intracellular pH (pHo and pHi) accompany myocardial ischemia and protons have major effects on ventricular ion currents. However, the separate action of low pHo and low pHi on L-type Ca2+ current (ICa,L) during action potentials is unresolved. ICa,L was measured (at 37oC) by applying action potential voltage-clamps (AP-clamp) to adult rabbit ventricular myocytes bathed in CO2/HCO3--free solution, with sarcolemmal sodium-hydrogen exchange blocked (30μM cariporide). pHi and Ca2+i were measured with SNARF-1 and fluo-4, respectively. Starting from control conditions (pHo 7.4; pHi 7.2) each type of acidosis was applied for 2 min: low pHo (pHo 6.5, pHi 7.1), low pHi (pHo 7.4; pHi 6.7, achieved with 80mM acetate superfusion) and combined acidosis (pHo 6.5; pHi 6.7). In current-clamped cells,low pHo shortened AP duration, whereas low pHi or combined acidosis lengthened AP duration and slowed phase 1 repolarization. During AP-clamps, initial peak ICa,L was reduced by each type of acidosis. For low pHo, this effect was mediated largely by direct channel pore block. For intracellular and combined acidosis, it resulted largely from: a) increased steady-state inactivation of ICa,L produced by an H+i-induced rise in diastolic Ca2+i and b) by slowing of phase 1 repolarization. Net Ca2+ influx via ICa,L was reduced by low pHo but markedly increased by low pHi or combined acidosis, the latter two effects mediated by proton-induced slowing of ICa,L inactivation kinetics. Whole-cell modeling successfully predicted the AP changes induced by low pHo and low pHi plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid-base disturbances in the heart.

Arrhythmogenic consequences of myofibroblast–myocyte coupling

Fibrosis is known to promote cardiac arrhythmias by disrupting myocardial structure. Given recent evidence that myofibroblasts form gap junctions with myocytes at least in co-cultures, we investigated whether myofibroblast-myocyte coupling can promote arrhythmia triggers, such as early afterdepolarizations (EADs), by directly influencing myocyte electrophysiology. Using the dynamic voltage clamp technique, patch-clamped adult rabbit ventricular myocytes were electrotonically coupled to one or multiple virtual fibroblasts or myofibroblasts programmed with eight combinations of capacitance, membrane resistance, resting membrane potential, and gap junction coupling resistance, spanning physiologically realistic ranges. Myocytes were exposed to oxidative (1 mmol/L H(2)O(2)) or ionic (2.7 mmol/L hypokalaemia) stress to induce bradycardia-dependent EADs. In the absence of myofibroblast-myocyte coupling, EADs developed during slow pacing (6 s), but were completely suppressed by faster pacing (1 s). However, in the presence of myofibroblast-myocyte coupling, EADs could no longer be suppressed by rapid pacing, especially when myofibroblast resting membrane potential was depolarized (-25 mV). Analysis of the myofibroblast-myocyte virtual gap junction currents revealed two components: an early transient-outward I(to)-like current and a late sustained current. Selective elimination of the I(to)-like component prevented EADs, whereas selective elimination of the late component did not. Coupling of myocytes to myofibroblasts promotes EAD formation as a result of a mismatch in early vs. late repolarization reserve caused by the I(to)-like component of the gap junction current. These cellular and ionic mechanisms may contribute to the pro-arrhythmic risk in fibrotic hearts.

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.

All Preconditioning-Related G Protein-Coupled Receptors Can Be Demonstrated in the Rabbit Cardiomyocyte

G protein-coupled receptors for adenosine (A(1), A(3), A(2A), and A(2B)), bradykinin (B(1)) and opioids (δ) are all involved in the mechanism of ischemic preconditioning. Although the heart is comprised of many tissue types, it has been assumed that preconditioning's protective signaling occurs in the cardiomyocyte. We critically tested that hypothesis by testing for the presence of each of these receptors in isolated adult rabbit ventricular myocytes that had been transfected with cyclic nucleotide-gated (CNG) ion channels. Because subsarcolemmal cyclic adenosine monophosphate (cAMP) opens the CNG channels, we could monitor cAMP levels within a single cardiomyocyte by measuring channel current with a patch pipette. The presence of a receptor would be confirmed if we could alter cAMP in the cell with a selective agonist to the receptor being studied. Superfusion with the β-adrenergic G(s)-coupled receptor agonist isoproterenol (50 nmol/L) transiently increased cAMP levels and, therefore, channel current. Pretreatment with selective agonists to A(1) or A(3) adenosine receptors (ARs) that are G(i)-coupled markedly attenuated the response to isoproterenol, indicating inhibition of adenylyl cyclase by increased G(i) activity. Agonists to bradykinin or δ-opioid receptors also attenuated isoproterenol's response. A(2A)AR and A(2B)AR are G(s)-coupled. The A(2A)AR-selective agonist CGS21680 increased current through CNG channels but only in the presence of phosphodiesterase (PDE) inhibitors, indicating low surface receptor activity and high intracellular PDE activity. As we previously reported, BAY 60-6583, an A(2B)AR-selective agonist which mimics preconditioning's protection in rabbit heart, neither increased nor decreased membrane current in transfected cardiomyocytes, suggesting the absence or a markedly limited number of A(2B)AR in the sarcolemma. However, reverse transcription polymerase chain reaction (RT-PCR) of purified cardiomyocytes yielded an A(2B)AR band, implying that rabbit cardiomyocytes do indeed express A(2B)AR. These data reveal that all receptors reported to be involved in ischemic preconditioning do exist on or within the cardiomyocyte.

Beta-Adrenergic Activation Enhances Histone Deacetylase 4 nuclear Localization via Pka and Counters Camkii-Dependent Effects in Adult Rabbit Cardiac Myocytes

Nuclear localization of histone deacetylase 4 (HDAC4), that represses transcription and possibly heart failure by suppressing the transcription factor MEF2, is dependent upon its phosphorylation status. Phosphorylation at three key serines can trigger the nuclear export of HDAC4, while dephosphorylation results in nuclear accumulation. HDAC4 is known to bind CaMKII and phosphorylation by this kinase facilitates nuclear export. A probable PKA phosphorylation site has been identified, suggesting the hypothesis that HDAC4 localization is modulated by both CaMKII and PKA. We tested whether B-adrenergic-induced PKA activity alters HDAC4 localization in adult rabbit ventricular myocytes using confocal imaging and GFP-tagged HDAC4. At baseline, the nuclear to cytosolic ratio of HDAC4 (Nuc/Cyto) was 2.19±.04 (n=638). Myocytes treated with 1µM isoproterenol (Iso) had a 58±8% increase in HDAC4 Nuc/Cyto compared to baseline (n=123). Activation of adenylyl cyclase via forskolin (10µM) produced a similar 50±5% increase in Nuc/Cyto ratio (n=107). CaMKII inhibition at rest with 1µM KN-93 also raised the Nuc/Cyto by 40±6% (n=99), whereas PKA inhibition (2 µM H-89) did not alter baseline HDAC4 localization (n=91). Conversely, phosphatase inhibition (1 µM okadaic acid) decreased basal Nuc/Cyto by 57±5% (n=97). Thus, baseline CaMKII activity limits the degree of nuclear HDAC4 localization, and preventing HDAC4 dephosphorylation exacerbates this. Furthermore, B-adrenergic activation causes HDAC4 nuclear import via PKA (vs other cAMP or B-adrenergic signaling). In conclusion, while CaMKII activates HDAC4 nuclear export, B-adrenergic signaling via PKA favors nuclear retention/import and transcriptional repression in cardiac hypertrophic signaling.

Ca V 1.2 β-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations

Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing disordered cell membrane excitability and myocardial dysfunction remain uncertain. Failing human cardiomyocytes exhibit increased CaMKII and voltage-gated Ca(2+) channel (Ca(V)1.2) activity, and enhanced expression of a specific Ca(V)1.2 beta-subunit protein isoform (beta(2a)). We recently identified Ca(V)1.2 beta(2a) residues critical for CaMKII phosphorylation (Thr 498) and binding (Leu 493), suggesting the hypothesis that these amino acids are crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT beta(2a) expression causes cellular Ca(2+) overload, arrhythmia-triggering cell membrane potential oscillations called early afterdepolarizations (EADs), and premature death in paced adult rabbit ventricular myocytes. Prevention of intracellular Ca(2+) release by ryanodine or global cellular CaMKII inhibition reduced EADs and improved cell survival to control levels in WT beta(2a)-expressing ventricular myocytes. In contrast, expression of beta(2a) T498A or L493A mutants mimicked the protective effects of ryanodine or global cellular CaMKII inhibition by reducing Ca(2+) entry through Ca(V)1.2 and inhibiting EADs. Furthermore, Ca(V)1.2 currents recorded from cells overexpressing CaMKII phosphorylation- or binding-incompetent beta(2a) subunits were incapable of entering a CaMKII-dependent high-activity gating mode (mode 2), indicating that beta(2a) Thr 498 and Leu 493 are required for Ca(V)1.2 activation by CaMKII in native cells. These data show that CaMKII binding and phosphorylation sites on beta(2a) are concise but pivotal components of a molecular and biophysical and mechanism for EADs and impaired survival in adult cardiomyocytes.

Endothelin and Phenylephrine Both Trigger Nuclear IP3 Elevation, but Differ in Ability to Activate Nuclear HDAC5 Export

We previously found that both ET-1 and phenylephrine (PE) induced similar HDAC5 phosphorylation and nuclear export in adult cardiac myocytes (which contributes to hypertrophic signaling). However, ET-1 requires IP3 receptor (IP3R) activation (at the nucleus) and CaM-CaMKII activation to mediate this full effect, while PE does not. That is, IP3R inhibition or CaMKII block do not prevent PE-induced HDAC5 nuclear export, despite the fact that both agonists can activate IP3 production. Here we test whether the apparent IP3-independence of PE signaling is due to a failure of IP3 elevation in the nucleus (i.e. IP3 produced at the plasma membrane may be degraded before reaching the nucleus). Using a nuclear targeted FRET-based IP3 sensor (Fire-1-Nuc) we assessed changes in nuclear [IP3] upon ET-1 and PE application in adult rabbit ventricular myocytes. Both ET-1 and PE induce rapid and robust elevation of nuclear [IP3] reaching an early peak in <1 min. While the ET-1-induced a slightly larger peak nuclear [IP3], the PE-induced rise is more sustained (lasting more than 10 min). These results demonstrate that a PE induces a strong rise in nuclear [IP3], and does not support the hypothesis that PE fails to induce a nuclear IP3 signal (compared to ET-1). We cannot rule out the possibility that the kinetic differences in nuclear [IP3] between these agonists contribute to different downstream signaling. Another explanation is that PE-induced nuclear IP3 is less effective than ET-1-induced IP3 in driving activation of nuclear CaM and CaMKII to phosphorylate HDAC5. That is, an IP3-independent effect of one of these agonists could prevent or promote the ability of IP3 to signal in the nucleus.

Sodium Current-Induced Release of Calcium from the Sarcoplasmic Reticulum in Rabbit Ventricular Myocytes

The hypothesis that Na current (INa) can induce release of Ca from the sarcoplasmic reticulum (SR) by activating reverse Na-Ca exchange (NCX) has been debated since 1990. We tested this hypothesis with epi-fluorescence imaging of adult rabbit ventricular myocytes loaded with the Ca indicator fluo-4. Ca release was triggered with an action potential clamp with and without an initial voltage ramp from −80 to −40 mV, for a duration of 1.5s. We confirmed that this protocol selectively blocked INa without altering Ca influx through L-type Ca channels (LCCs) and SR Ca load. With 0 mM Na in the pipette (to reduce intracellular Na), inactivating INa reduced SR Ca release flux by 27%±4% (n=9). With 5 mM Na in the pipette, the Ca release upon inactivation of INa was reduced by 33%±5% (n=4). We suggest that increased activation of reverse NCX by increased intracellular Na concentration mainly produced by INa explains these findings. These conclusions are in agreement with studies on normal and NCX knockout mice, which show that INa affects SR Ca release only in normal, but not in NCX knockout mice. In similar experiments, we applied 100 nM TTX to selectively block brain isoforms of Na channels. In the presence of TTX, the SR Ca release flux was reduced by 35%±3% (n=6). This effect of INa on Ca release can be explained by early reverse NCX, activated by TTX sensitive INa, which could prime the dyadic cleft with Ca. Furthermore, the results can be explained if INa activation of NCX, and subsequent priming of the dyadic cleft with Ca, increases the coupling fidelity between LCCs and ryanodine receptors within a couplon. Thus the presence of INa increases the likelihood that couplons are activated.

Modulation of the Mitochondrial Permeability Transition Pore of Cardiac Myocytes by Inorganic Polyphosphate

Background: Inorganic polyphosphate (polyP) is a long polymer made of up to several hundred orthophosphates linked together by phosphoanhydride bonds.Previously we found that polyP of rat liver mitochondria participates in formation of a channel with properties similar to the mitochondrial permeability transition pore (mPTP) suggesting a possible role in pathophysiology. The aim of this study was to investigate the role of polyP in the regulation of mitochondrial Ca homeostasis and Ca-induced opening of mPTP in cardiac myocytes. Methods: We used primary cultures of adult rabbit ventricular myocytes with enzymatically reduced levels of mitochondrial polyP achieved by adenoviral expression of polyP hydrolyzing enzyme from yeast (scPPX). Cytosolic Ca ([Ca]i), mitochondrial Ca ([Ca]m), mitochondrial membrane potential, and mPTP activity were measured using the fluorescent dyes indo-1, rhod-2, TMRM, or calcein red, respectively. Results: 1) No difference was detected in amplitude, rise and decay time of [Ca]i transients induced by electrical field stimulation (1 Hz) in control and scPPX expressing intact myocytes. 2) In permeabilized cells under conditions of mitochondrial Ca overload, mitochondrial Ca uptake in control cells was followed by fast Ca release which was prevented by the mPTP inhibitor cyclosporine A. The rate of mitochondrial Ca release was significantly slower in scPPX cells. 3) Similar levels of basal mitochondrial membrane potential were observed in both cell types, however Ca-induced mitochondrial membrane depolarization was more pronounced in control cells. 4) Mitochondria of permeabilized myocytes expressing scPPX were less sensitive to Ca-induced mPTP opening as estimated by the kinetics of calcein red release and the degree of Ca-induced mitochondrial membrane depolarization. Conclusion: Our data indicate that reducing of the mitochondrial polyP levels decreases Ca-induced opening of the mPTP in cardiac myocytes.

Coupling of Isolated Adult Rabbit Ventricular Myocytes to Fibroblasts Under Stress Induces Afterdepolarizations

Coupling of Isolated Adult Rabbit Ventricular Myocytes to Fibroblasts Under Stress Induces Afterdepolarizations

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K+ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (Ito) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (IK1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca2+] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected Ito and IK1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in IK1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with IK1.