Adult Rat Cardiomyocytes Research Articles

LQT1 mutations in KCNQ1 C-terminus assembly domain suppress IKs using different mechanisms.

Long QT syndrome 1 (LQT1) mutations in KCNQ1 that decrease cardiac IKs (slowly activating delayed rectifier K(+) current) underlie ventricular arrhythmias and sudden death. LQT1 mutations may suppress IKs by preventing KCNQ1 assembly, disrupting surface trafficking, or inhibiting gating. We investigated mechanisms underlying how three LQT1 mutations in KCNQ1 C-terminus assembly domain (R555H/G589D/L619M) decrease IKs in heterologous cells and cardiomyocytes. In Chinese hamster ovary (CHO) cells, mutant KCNQ1 + KCNE1 channels either produced no currents (G589D/L619M) or displayed markedly reduced IKs with a right-shifted voltage-dependence of activation (R555H). When co-expressed with wild-type (wt) KCNQ1, the mutant KCNQ1s displayed varying intrinsic dominant-negative capacities that were affected by auxiliary KCNE1. All three mutant KCNQ1s assembled with wt KCNQ1 as determined by fluorescence resonance energy transfer (FRET). We developed an optical quantum dot labelling assay to measure channel surface density. G589D/R555H displayed substantial reductions in surface density, which were either partially (G589D) or fully (R555H) rescued by wt KCNQ1. Unexpectedly, L619M showed no trafficking defect. In adult rat cardiomyocytes, adenovirus-expressed homotetrameric G589D/L619M + KCNE1 channels yielded no currents, whereas R555H + KCNE1 produced diminished IKs with a right-shifted voltage-dependence of activation, mimicking observations in CHO cells. In contrast to heterologous cells, homotetrameric R555H channels showed no trafficking defect in cardiomyocytes. Distinct LQT1 mutations in KCNQ1 assembly domain decrease IKs using unique combinations of biophysical and trafficking mechanisms. Functional deficits in IKs observed in heterologous cells are mostly, but not completely, recapitulated in adult rat cardiomyocytes. A 'methodological chain' combining approaches in heterologous cells and cardiomyocytes provides mechanistic insights that may help advance personalized therapy for LQT1 mutations.

Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation.

Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide's role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field-stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca(2+)]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2-dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC-dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.

A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2 + transients and contractile function in cardiomyocytes

The sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)–phospholamban (PLN) system of sarcoplasmic reticulum plays a pivotal role in regulation of intracellular Ca2+ cycling in ventricular cardiomyocytes. Given that Ca2+ cycling is impaired in heart failure, proteins that contribute to this process are potential targets for the treatment of this condition. We have now isolated PLN-specific aptamers with a phosphorothioate-modified backbone from a library of RNA molecules containing a randomized 40-nucleotide sequence by application of the systematic evolution of ligands by exponential enrichment (SELEX) protocol with a fusion protein containing the cytoplasmic region of human PLN. One of these aptamers was shortened to a 30-nucleotide oligomer (RNA-Apt30) without loss of function. RNA-Apt30 showed a high affinity for the cytoplasmic region of PLN (Kd=11nM), but it did not bind to the phosphorylated form of PLN or to a phosphomimetic mutant. It also increased SERCA2a activity in isolated cardiac SR vesicles with an EC50 of 18nM by relieving PLN-mediated inhibition. Conjugation of RNA-Apt30 to a cell-penetrating peptide allowed its delivery into adult rat cardiomyocytes, in which it enhanced both Ca2+ transients and contractile function. These effects of the aptamer were also apparent in the presence of the β-adrenergic receptor antagonist propranolol. This cell-penetrating PLN aptamer may thus provide a basis for the development of new therapeutic agents for heart failure without the need for gene transfer or a change in endogenous protein expression.

AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-β2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.

Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes.

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10μM for SFLLRN or SLIGRL, 0.1U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.

Abstract 276: The Regulatory Effects Of Peroxiredoxin II On Phospholamban Phosphorylation And Cardiac Contractile Function In Vivo

Peroxiredoxin II (prxII), a cytosolic form of the anti-oxidant peroxiredoxin family, was recently found to be decreased in failing human hearts. Interestingly, in hyperdynamic hearts of two genetically modified mouse models with: a) phospholamban ablation; and b) overexpression of the active inhibitor-1 of protein phosphatase 1, the levels of this cellular peroxidase (prxII) were markedly increased. Acute overexpression of prxII by adenoviral-delivery in adult rat cardiomyocytes (Ad-prxII) was associated with decreases in the basal rates of contraction and relaxation, as well as calcium kinetics. Accordingly, Ad-prxII-AS infected cardiomyocytes exhibited enhanced contractile parameters and Ca-kinetics. The depressed or increased contractility by Ad-prxII or Ad-prxII-AS was associated with parallel decreases or increases in phosphorylation of phospholamban (Ser16 and Thr17). To determine the in vivo effects of prxII on cardiac contractility, three transgenic lines (TG) with 2-3 fold cardiac-specific overexpression of prxII were generated and their cardiac morphologic and functional phenotypes were characterized. The TG mice exhibited no alterations in cardiac pathology or morphology up to 4 months of age. However, langendorf perfusions revealed that cardiac contractility, including the rates of contraction and relaxation (±dp/dtmax) as well as the left ventricular end systolic pressure (LVESP), were significantly depressed in TG mice (to 75, 76 and 63%, respectively), compared to WTs (100%). The depressed function was not associated with any alterations in the expression levels of key SR calcium handling proteins: SERCA2, total phospholamban, calsequestrin and ryanodine receptor. However, the levels of the phosphorylated PLN at Ser16 were found to be reduced to 50% in the TG mice, compared to WTs. These findings indicate that prxII, an anti-oxidant protein, may regulate basal cardiac contractile performance in vivo through phospholamban phosphorylation.

Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation

Ethnopharmacological relevanceTotal saponins of Aralia elata (Miq) Seem (AS) from the Chinese traditional herb Longya Aralia chinensis L. can improve cardiac function, although the active mechanism remains poorly understood. The present study aimed to determine the direct effect of AS on cardiac function in dogs and the effects on Ca2+ transient and contractions in isolated rat cardiomyocytes. Material and MethodsIn anesthetized dogs, hemodynamic indexes and myocardial oxygen consumption were determined before and after AS was administered. In isolated adult rat cardiomyocytes, contractile and intracellular Ca2+ properties were determined simultaneously in real time by using an IonOptix MyoCam system. ResultsOur results showed that AS directly induced a positive inotropic effect and improved coronary blood flow and energy metabolism, indicating that AS induced a beneficial effect to treat myocardial ischemia/reperfusion injury. Moreover, AS increased sarcomere shortening, maximal velocity of shortening/relengthening (±dL/dt), amplitude of [Ca2+]i transients and SERCA activity in a concentration-dependent manner. PKCε was also activated after the cells were treated with AS. ConclusionThese findings revealed the positive inotropic effect of AS on canine myocardium and isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca2+]i transient and PKCε-dependent signaling pathway.

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

RationaleDihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca2+ channels and their renowned antioxidant properties. MethodsWe synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca2+ channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca2+ channel-blocking activity and antioxidant properties. The Ca2+ channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. ResultsOur docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca2+ channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca2+ channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. ConclusionsOnly substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.

Induction of microRNA-21 with exogenous hydrogen sulfide attenuates myocardial ischemic and inflammatory injury in mice.

Maintaining physiological levels of hydrogen sulfide during ischemia is necessary to limit injury to the heart. Because of the anti-inflammatory effects of hydrogen sulfide, we proposed that the hydrogen sulfide donor, sodium sulfide (Na2S), would attenuate myocardial injury through upregulation of protective microRNA-21 (miR-21) and suppression of the inflammasome, a macromolecular structure that amplifies inflammation and mediates further injury. Na2S-induced miR-21 expression was measured by quantitative polymerase chain reaction in adult primary rat cardiomyocytes and in the mouse heart. We measured inflammasome formation and activity in cardiomyocytes challenged with lipopolysaccharide and ATP or simulated ischemia/reoxygenation and in the heart after regional myocardial ischemia/reperfusion, in the presence or absence of Na2S. To assess the direct anti-inflammatory effects of hydrogen sulfide in vivo, we used a peritonitis model by way of intraperitoneal injection of zymosan A. Na2S attenuated inflammasome formation and activity, measured by counting cytoplasmic aggregates of the scaffold protein apoptosis speck-like protein containing a caspase-recruitment domain (-57%) and caspase-1 activity (-50%) in isolated cardiomyocytes and in the mouse heart (all P<0.05). Na2S also inhibited apoptosis (-38%) and necrosis (-43%) in cardiomyocytes in vitro and reduced myocardial infarct size (-63%) after ischemia/reperfusion injury in vivo (all P<0.05). These protective effects were absent in cells treated with the miR-21 eraser, antagomiR-21, and in miR-21 knockout mice. Na2S also limited the severity of inflammasome-dependent inflammation in the model of peritonitis (P<0.05) in wild-type but not in miR-21 knockout mice. Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes.

0341: AMPK exerts an insulin-sensitizing effect on cardiac glucose uptake by multiple molecular mechanisms including cytoskeleton reorganization

Insulin-resistant cardiomyocytes are characterized by a decreased ability of insulin to stimulate glucose uptake. We have previously shown that the activation of AMPK by metformin or phenformin restores insulin-sensitivity in insulin-resistant cardiomyocytes. The aim of our present work is to understand by which molecular mechanisms AMPK exerts its insulin sensitizing effect. In this study we focused on the mTOR/p70S6K pathway and on cytoskeleton reorganization. mTOR/p70S6K, which is known to be inhibited by AMPK, is able to reduce insulin signaling via a negative feedback loop involving serine phosphorylation of IRS-1. On the other hand, cytoskeleton reorganization, which is a known target of AMPK, is responsible for the translocation of the glucose transporter GLUT4 to the plasma membrane. Adult rat cardiomyocytes were primary cultured and treated with different agents including insulin, AMPK activator (phenformin), mTOR inhibitor rapamycin and/or actin cytoskeleton inhibitor latrunculin B. Glucose uptake was assessed by detritiation of 2- 3 H-glucose. First, we tested if rapamycin was able to mimic AMPK activators. Similarly to phenformin, rapamycin increased the insulin-dependent phosphorylation of Akt involved in the regulation of glucose uptake. Despite the ability of rapamycin to induce this Akt over-phosphorylation, rapamycin was not able to restore the insulin-dependent stimulation of glucose uptake like phenformin did. On the other hand, latrunculin B abolished the insulin-sensitizing action of phenformin on glucose uptake, in insulin-sensitive as well as in insulinresistant cells. actin cytoskeleton reorganization but not mTOR/p70S6K, is involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake. The role played by Small G proteins, known to be involved in the regulation of actin cytoskeleton is under investigation.

0347: Leucine, a potent inhibitor of cardiac glucose uptake

It was demonstrated that branched-chain amino acids like leucine induce insulin resistance in muscle and adipose tissues. The mechanism proposed to explain leucine action involves mTOR/p70S6K signaling. This pathway can be activated by leucine and is implicated in the stimulation of an insulin negative feedback loop. Knowing that insulin-resistance participates in diabetic cardiomyopathy, we were interested in studying leucine effect in cardiomyocytes. Primary cultured adult rat cardiomyocytes were pretreated with different concentrations of leucine (from 1 to 10 mM) during different periods of time (up to 20h) before being exposed to insulin (3x10 −9 M, 30 min). In absence of leucine, insulin induced a 6-fold increase in glucose uptake (0.31+/−0.04 vs. 0.05+/−0.01 μmoles/mg.h). This correlated with the increase in phosphorylation state of PKB and AS160, both known to regulate glucose transport downstream of insulin. Pre-incubation with leucine for 1 h stimulated mTOR/p70S6K pathway resulting in the inhibiting phosphorylation of IRS-1 located in the proximal insulin signaling pathway. This is accompanied by a significant decrease in PKB and AS160 phosphorylation but, surprisingly, insulin-stimulated glucose uptake was preserved (0.31+/–0.05 μmoles/mg.h). On the other hand, a longer incubation (14h) with leucine induced a drastic decrease in glucose transport (0.056+/–0.01 μmoles/mg.h). The mTOR/p70S6K inhibitor rapamycin did not prevent this inhibition. Moreover, the non-metabolized leucine analog BCH was able to stimulate mTOR/p70S6K pathway but had no effect on the insulin-mediated stimulation of glucose uptake. By contrast, intermediates of leucine catabolism, alpha-ketoisocaproate, acetoacetate and betahydroxybutyrate, inhibited glucose uptake similarly to leucine. Leucine catabolism reduces insulin-dependent glucose transport independently of insulin signaling.

0338: Glucagon-like peptide 1 or GLP-1 counteracts NADPH oxidase activation during hyperglycemia through an AMPK-dependent pathway in adult cardiomyocytes

Exposure to high glucose (HG) stimulates NADPH oxidase (NOX2) dependent-ROS production in cardiomyocytes. NOX2 activation is not triggered by an increased glucose metabolism but results from a glucose transport through a sodium-glucose co-transporter (SGLT). The aim of this work is to identify potential therapeutic approaches to counteract glucotoxicity. Primary cultures of adult rat cardiomyocytes were exposed to high glucose concentration (HG, 21mM). AMP-activated protein kinase (AMPK) activation by A769662 or Phenformin nearly suppressed ROS production under hyperglycemia. GLP-1, a new anti-diabetic drug, exerted a similar effect, blocking hyperglycemia-mediated ROS production. Interestingly, GLP-1 treatment induced a AMPK activation, the maximal activation being observed at 100 nM. α2AMPK, the major isoform expressed in cardiomyocytes (but not α1AMPK) was activated in response to GLP-1. Anti-ROS properties of AMPK were not related to change in glucose uptake or glycolysis. Using in situ proximity ligation assay technology, we demonstrated that AMPK activation prevented p47phox translocation to the caveolar structure after high glucose concentration exposure, whatever the AMPK activators used. NOX2 activation by α-methyl-D-glucopyranoside, a glucose analog exclusively transported through SGLT, or by Angiotensin 2 was also counteracted by GLP-1. The crucial role of AMPK in limiting glucotoxicity was demonstrated by overexpressing a constitutively active form of AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG. Finally, in mice cardiomyocytes, GLP-1 did not exert protective action in the absence of α2AMPK. GLP-1 induces α2AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, limiting glucotoxicity.

Influence of araloside C on cardiac contractile function (850.7)

The present study aimed to determine the direct effect of araloside C on Ca2+ transient and contractions in isolated rat cardiomyocytes. In isolated adult rat cardiomyocytes, contractile and intracellular Ca2+ properties were determined simultaneously in real time by using an IonOptix MyoCam system. Western blot analysis was performed to evaluate the expressions of Ca2+ cycling proteins, including sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), phospholamban (PLB), and Na+‐Ca2+ exchanger (NCX). Our results showed that araloside C directly increased sarcomere shortening, maximal velocity of shortening/relengthening (±dL/dt) and amplitude of [Ca2+]i transients in a concentration‐dependent manner. SERCA activity was also increased by araloside C. The expression of SERCA2a was not significantly upregulated, whereas PLB and NCX expressions were downregulated. These findings revealed the positive inotropic effect of araloside C on isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca2+]i transient.Grant Funding Source: This study was supported by the National Natural Sciences Foundation of China (Grant No. 81173589).

Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure

Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment of heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and RT-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.

Role for Myosin-V Motor Proteins in the Selective Delivery of Kv Channel Isoforms to the Membrane Surface of Cardiac Myocytes

Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Novel Insights into the Cardio-Protective Effects of FGF21 in Lean and Obese Rat Hearts

AimsFibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia.Methods and ResultsFGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased.ConclusionIn an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia.

Possible Role for the Phoshorylated Tail in Retaining CSQ2 to Specific Sites Within the Secretory Pathway

Each of the two calsequestrin (CSQ) genes expresses muscle-type specificity; CSQ1 is present only in skeletal muscle, while CSQ2 is found in cardiac tissue and slow-type skeletal muscle. The two forms of the protein share about 60% identity and have similar 3D structures. Yet, differences in distribution of negative surface charges lead to differences in intracellular polymerization and localization. A second difference in structure is the CSQ2-specific C-terminal extension, a tail that contains a cluster of protein kinase CK2-sensitive serines. In this study, we examined the subcellular localization of CSQ1 and CSQ2 following their acute overexpression in adult rat cardiomyocytes, using isoform specific antibodies. The native rat CSQ2 showed the expected junctional SR distribution, co-staining with ryanodine receptor. Canine and human CSQ2 localized within 48 h to the same puncta as the native protein, with subtle differences in distribution. In contrast to all CSQ2 proteins, CSQ1 was not observed in jSR, rather accumulating in punctae near the cell surface. To test whether the C-terminal 23-mer extension on CSQ2 (cT) underlied this difference we constructed a chimeric cDNA encoding CSQ1-cT. This protein accumulated in yet a third pattern, distinct from both the transverse jSR localization (CSQ2 compartment) and the subsarcolemmal accumulation (CSQ1 compartment). CSQ1-cT appeared to assemble in an array of punctae aligned longitudinally across the myoplasm. We conclude that CSQ2, following its perinuclear biosynthesis, encounters junctional SR sites where it can accumulate by polymerization. In contrast, CSQ1 proceeds to cortical sites along microtubules to concentrate near the cell surface. Thus, the cardiac-specific cT may interact with an upstream sorting protein, suggesting a possible mechanism of CSQ retention beyond its polymerization. Studies in cultured non-muscle cells support the hypothesis for cT-dependent CSQ retention.

Letters to the Editor

Letters to the Editor

Microtubule Integrity is Essential to Junctional SR Protein Delivery

Cardiac muscle contraction has special reliance upon ER subcompartments to regulate Ca2+ cycling and homeostasis. A unique ER subcompartment, the junctional sarcoplasmic reticulum (jSR) plays a critical role in this process, through the concentration of a unique group of resident proteins that regulate Ca2+ release. Impaired function of jSR has been shown to lead to cardiac pathologies; however, cellular mechanisms and pathways via which proteins traffic to jSR remain undiscovered. To investigate their delivery mechanisms, canine forms of junctin (JCT) or triadin (TRD) were overexpressed in adult rat cardiomyocytes, and their accumulation was visualized by confocal fluorescence microscopy using species-specific antibodies. Both JCT and TRD exhibited patterns of juxtanuclear buildup after 24h. By 48h, higher-intensity punctae were detected across the width of the cell. With microtubule depolymerization, anterograde movement ceased, and newly synthesized proteins did not migrate away from the nucleus. CSQ2-DsRed forms a polymeric complex that localizes early in biosynthesis to juxtanuclear cisternae. To determine whether all jSR proteins follow a common biosynthetic and transport route to jSR, TRD or JCT were co-overexpressed with CSQ2-DsRed. Both TRD and JCT transport from juxtanuclear sites was prohibited. In contrast, triadin in which CSQ2 binding sites were deleted proceeded to the cell periphery. These data suggest that TRD and JCT are biosynthesized at juxtanuclear sites and can interact in situ with CSQ2 at early stages in biosynthesis, and are delivered to jSR sites along a microtubule dependent pathway that lies close to Z-lines, with little or no accumulation at other sarcomeric locations. We conclude that TRD and JCT share the same direct microtubule dependent route, not via free SR compartments.