Adult Mouse Cardiac Myocytes Research Articles

Role of Ca2+ in the Control of H2O2-Modulated Phosphorylation Pathways Leading to eNOS Activation in Cardiac Myocytes

Nitric oxide (NO) and hydrogen peroxide (H2O2) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H2O2–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H2O2 treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca2+ ([Ca2+]i), and document that activity of the L-type Ca2+ channel is necessary for the H2O2-promoted increase in sarcomere shortening and of [Ca2+]i. Using the chemical NO sensor Cu2(FL2E), we discovered that the H2O2-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca2+ channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H2O2-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca2+]i as well as the activation of protein kinase C (PKC). We also found that H2O2-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca2+ channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H2O2-promoted eNOS phosphorylation. Here we present evidence documenting that H2O2-promoted Akt phosphorylation is dependent on activation of the L-type Ca2+ channel, but is independent of PKC. These studies establish key roles for Ca2+- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H2O2.

Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli’s salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.

The A-kinase Anchoring Protein Yotiao Facilitates Complex Formation between Adenylyl Cyclase Type 9 and the IKs Potassium Channel in Heart

The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (I(Ks)) and recruits both PKA and PP1 to regulate I(Ks) phosphorylation and gating. Human mutations that disrupt I(Ks)-Yotiao interaction result in reduced PKA-dependent phosphorylation of the I(Ks) subunit KCNQ1 and inhibition of sympathetic stimulation of I(Ks), which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the I(Ks)-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous I(Ks)-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the I(Ks) channel to achieve localized temporal regulation of β-adrenergic stimulation.

H11 Kinase/Hsp22‐mediated activation of the valosin‐containing protein promotes the expression of the inducible isoform of nitric oxide synthase in the heart

Expression of H11 kinase/Hsp22 (Hsp22) in the heart stimulates cell growth and survival, and activates the expression of the inducible nitric oxide synthase (iNOS) mediating ischemic preconditioning, the most powerful prophylaxis against cardiac cell death, through activation of the Akt pathway. Our goal was to elucidate the downstream effector by which Hsp22 and Akt increase iNOS expression. We hypothesized that such effector is the valosin‐containing protein (VCP), an Akt substrate activating the transcription factor NF‐kB by promoting the poly‐ubiquitination of the NF‐kB inhibitor, IkBa. Using two‐dimensional gel electrophoresis and mass spectrometry, and immunoprecipitation, we found that Hsp22 and Akt co‐localize and interact together with the valosin‐containing protein (VCP). This interaction predominated in the nuclear fraction of adult mouse cardiac myocytes, and it was markedly increased in a transgenic mouse with cardiac‐specific over‐expression of Hsp22. Adeno‐mediated over‐expression of VCP in isolated cardiomyocytes activated NF‐kB and dose‐dependently increased the expression of iNOS, which was abolished by the NF‐kB inhibitor SN50. Over‐expression of a dominant‐negative mutant of VCP lacking the domain of poly‐ubiquitination did not increase iNOS expression. Therefore, VCP mediates the expression of iNOS downstream Hsp22 and Akt through an NF‐kB‐dependent mechanism.

Phosphatidylinositol 3,5 bisphosphate increases contractility and intracellular calcium in cardiac muscle by directly activating the cardiac ryanodine receptor.

We have previously reported that treatment of left ventricular cardiac muscle strips with phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) during field stimulation enhanced the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. In addition, PI(3,5)P2 caused an increase in intracellular calcium (Ca) in cardiac myocytes. These data suggested that PI(3,5)P2 increased contractile force by promoting extracellular Ca entry or by facilitating intracellular Ca release. To elucidate the mechanism responsible for the effects of PI(3,5) on intracellular Ca, we have performed ratiometric Ca imaging in isolated adult mouse cardiac myocytes. We have found that PI(3,5)P2 induced a dramatic increase in intracellular Ca levels, which was inhibited by preincubation with ryanodine. To confirm that PI(3,5)P2 was acting on RyR2 we performed electrophysiological and biochemical studies. PI(3,5)P2 increased [3H]ryanodine binding and this effect was prevented by preincubation with an antibody against PI(3,5)P2. Furthermore, single RyR2 channels reconstituted in lipid bilayers were activated by PI(3,5)P2 in a dose‐dependent manner, indicating that PI(3,5)P2 binds directly to RyR2. We provide inaugural evidence that PI(3,5)P2 regulates cardiac contractility via modulation of intracellular Ca by direct binding and activation of the RyR2 channel.

In Situ Calibration of Cytoplasmic and Nucleoplasmic Calcium Concentration in Adult Rat and Mouse Cardiac Myocytes

Quantifying subcellularly resolved Ca2+ signals in cardiac myocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. Translation of changes in Ca2+-dependent fluorescence into changes in [Ca2+] relies on the indicator's behavior in situ, but properties of fluorescent indicators in different intracellular compartments may differ. Thus, we determined the in situ calibration of a frequently used Ca2+ indicator, Fluo-4, and evaluated its use in reporting cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiac myocytes.Calibration solutions were made by mixing known quantities of EGTA and CaEGTA solutions and the free [Ca2+] was confirmed with a Ca2+-sensitive electrode. Solutions contained metabolic inhibitors and cyclopiazonic-acid (5μM) to block active Ca2+ transport and the Ca2+ ionophore A-23187 (10μM) to allow equilibration of [Ca2+] between bath solution and cell interior. Ventricular rat and mouse myocytes were loaded with Fluo-4/AM (8μM, 20min). Fluo-4 fluorescence (excitation/emission: 488/>505nm) was recorded using a Nipkow dual disc-based confocal microscope.Concentration-response curves were obtained and a significant difference in the apparent Ca2+ binding affinities (Kd) of Fluo-4 between cytoplasmic (993±56nM; 1026±65nM) and nucleoplasmic (1211±73nM; 1251±71nM) compartments was observed for both mouse and rat cells, respectively (both n=15, P<0.01). The established curves were used to transform raw Fluo-4 fluorescence signals during electrically stimulated [Ca2+] transients (1Hz, room temperature) into nucleoplasmic and cytoplasmic [Ca2+]. There was a significant difference in diastolic (121±24nM vs 149±35nM; 99±17nM vs 121±26nM) and systolic (420±148nM vs 364±102nM; 787±172nM vs 491±157nM) [Ca2+] between cytoplasmic and nucleoplasmic compartments in mouse and rat cells, respectively (both n=15; P<0.01).The results reveal that, in cardiac myocytes, the Ca2+-dependent fluorescent properties of Fluo-4 differ between cytoplasm and nucleoplasm and that significant differences between cytoplasmic and nucleoplasmic [Ca2+] exist during diastole as well as systole.

Abstract 2375: Regulation of Cardiac Myocyte Contractility by Phospholemman

Phospholemman (PLM) regulates cardiac contractility by modulating Na + /Ca 2+ exchanger (NCX1) and/or Na + -K + -ATPase activities. PLM, when phosphorylated at serine68, disinhibits Na + -K + -ATPase but inhibits NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 hours had normal surface membrane area and t-tubule appearance, and exhibited near normal contractility. Contractile differences between wild-type (WT) and PLM knockout (KO) myocytes were preserved after 48h of culture. Infection with adenovirus overexpressing GFP did not affect contractility at 48h. When WT PLM was overexpressed in PLM-KO myocytes, part of PLM was phosphorylated, both Na + -K + -ATPase current (I pump ) and Na + /Ca 2+ exchange current (I NaCa ) were depressed, and contractility and [Ca 2+ ] i transients reverted back to those observed in cultured WT myocytes. Overexpressing PLMS68E mutant (phosphomimetic) in PLM-KO myocytes resulted in suppression of I NaCa but no effect on I pump . Contractility and [Ca 2+ ] i transient amplitudes in PLM-KO myocytes overexpressing PLMS68E mutant were depressed when compared to PLM-KO myocytes overexpressing GFP. Overexpressing PLMS68A mutant (mimicking unphosphorylated PLM) in PLM-KO myocytes had no effects on I NaCa but decreased I pump . Contractility and [Ca 2+ ] i transient amplitudes in PLM-KO myocytes overexpressing S68A mutant were similar to PLM-KO myocytes overexpressing GFP. Neither WT PLM nor its mutants had any effect on SR Ca 2+ uptake in KO myocytes. We conclude that at the single myocyte level, PLM affects cardiac contractility and [Ca 2+ ] i homeostasis primarily by its direct inhibitory effects on Na + /Ca 2+ exchange.

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.

Vincristine attenuates doxorubicin cardiotoxicity

Our aim was to test the hypothesis that the vinca alkaloid vincristine could prevent doxorubicin-induced cardiomyocyte death and to identify the mechanisms involved. Adult mouse cardiac myocytes were incubated for 24h with doxorubicin, with and without concurrent vincristine. Trypan blue exclusion showed that 50–60% of myocytes treated with doxorubicin alone survived. Concurrent vincristine treatment increased survival to ⩾85%. Treatment with doxorubicin+vincristine activated the prosurvival signal Akt and diminished cytochrome C release. The PI3K/Akt inhibitor LY294002 and the MEK/ERK inhibitor PD98059 augmented doxorubicin cardiotoxicity and attenuated salvage during concurrent vincristine treatment, indicating that the mechanism of vincristine cardioprotection involves activation of specific survival signals. Vincristine retarded the onset of apoptosis in association with a delay in poly(ADP) ribose polymerase activation. Vincristine also exhibited greater protection than the antioxidant MPG. These novel findings may have clinical implications for the prevention of doxorubicin cardiomyopathy.

GATA4 is a survival factor in adult cardiac myocytes but is not required for α1A-adrenergic receptor survival signaling

Recently, we defined an alpha1A-adrenergic receptor-ERK (alpha1A-AR-ERK) survival signaling pathway in adult cardiac myocytes. Previous studies in neonatal cardiac myocytes indicated that the cardiac-specific transcription factor GATA4 is a downstream mediator of alpha1-ERK signaling and that phosphorylation of GATA4 by ERK increases DNA binding and transcriptional activity. Therefore, we examined GATA4 as a potential downstream effector of alpha1A-ERK survival signaling in adult cardiac myocytes. We measured norepinephrine (NE)-induced cell death in cultured cardiac myocytes lacking alpha1-ARs (cultured from alpha1A/B-AR double-knockout mice, alpha1ABKO mice) that are susceptible to cell death induced by several proapoptotic stimuli, including NE. Our results show that overexpression of GATA4 is sufficient to protect alpha1ABKO cardiac myocytes from NE-induced cell death. However, we found that the alpha1A-subtype did not induce phosphorylation or increase the activity of GATA4 in adult mouse cardiac myocytes in culture or in vivo. Furthermore, we examined the effect of siRNA-mediated knockdown of GATA4 on alpha1A-survival signaling. In alpha1B-knockout cardiac myocytes, which express only the alpha1A-subtype and are protected from NE-induced cell death, GATA4 knockdown did not reverse alpha1A-survival signaling in response to NE. In summary, we found that GATA4 acted as a survival factor by preventing cell death in alpha1ABKO cardiac myocytes, but GATA4 was not activated by alpha1-AR stimulation and was not required for alpha1A-survival signaling in adult cardiac myocytes. This also identifies an important mechanistic difference in alpha1-signaling between adult and neonatal cardiac myocytes.

Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.

Abstract 537: Sildenafil Suppresses Cardiac Hypertrophy through Inhibition of Canonical Transient Receptor Potential Cation Channel 6 Expression in Cardiac Myocytes

Background: We have shown that a phosphodiesterase 5A (PDE5A) inhibitor, sildenafil blocks cardiac pathological hypertrophy through inhibition of Gαq mediated signaling and calcineurin (Cn) signaling. However, the precise molecular mechanism is still unknown. Recently canonical transient receptor potential cation channel 6 (TRPC6) and its up-regulation have been shown to play a critical role for Gαq-mediated Cn activation in pathogenesis of cardiac hypertrophy. Therefore, we hypothesized that PDE5A inhibition blocks TRPC6 gene induction in cardiac myocytes and thus prevents cardiac hypertrophy. Methods and Results: We examined TRPC6 expression levels using real-time RT-PCR in hypertrophied mouse heart created by transverse aortic constriction (TAC). TRPC6 mRNA expression was significantly increased after TAC. Sildenafil effectively attenuated TAC-induced hypertrophy, Cn protein level and upregulation of TRPC6 mRNA. To investigate role of TRPC6 in sildenafil’s anti-hypertrophic effects, we studied cultured adult mouse cardiac myocytes (AMCM) and neonatal rat cardiac myocytes (NRCM). Stimulation with Endothelin 1 (ET1) or angiotensin II (AngII) increased cell surface area, leucine uptake, Cn protein expression and TRPC6 gene expression. Sildenafil dose-dependently blocked these increases. 8-bromo cGMP also blocked TRPC6 induction by ET1 and AngII. In addition, 8-bromo cGMP stimulation following adenovirus-mediated overexpression of cGMP-dependent protein kinase I-α (PKG) showed a marked decrease in TRPC6 expression. These results suggest that PKG activation regulates TRPC6 gene in cardiac myocytes. Using adenovirus-based expression of artificial PDE5A-gene silencing miRNA, PDE5 protein was effectively knocked down both in NRCM and AMCM. Importantly, PDE5-miRNA completely blocked ET1-mediated increase of Cn and TRPC6 same as sildenafil. Conclusion: Sildenafil effectively blocks TRPC6 induction both in vivo and in vitro . TRPC6 gene regulation is PKG and PDE5A dependent in cardiac myocytes. Given that TRPC6 induction triggers Gαq-mediated maladaptive signaling, especially Cn, these data show that the inhibition of TRPC6 gene expression contributes to the Gαq-coupled anti-hypertrophic mechanism of sildenafil.

Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to G(i) protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P(1) receptors were observed to transduce signals from exogenous S1P, an S1P(1) receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P(1) receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P(1) receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P(1) receptor function was closely associated with activation of Akt, inactivation of GSK-3beta, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P(1) receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

Acute vincristine pretreatment protects adult mouse cardiac myocytes from oxidative stress

Vincristine is a chemotherapeutic agent that disrupts microtubules. We noted that paclitaxel (Taxol), which stabilizes microtubules, protected cultured adult mouse cardiac myocytes from oxidative stress induced by H 2O 2. We hypothesized that vincristine, which disrupts microtubules, should have the opposite effect. To our surprise, we found that pretreatment with concentrations of vincristine ranging from 30 to 120 μmol/L for 60 min preserved myocyte viability and morphology after incubation with 30 μmol/L of H 2O 2 for 35 min as measured by trypan blue exclusion. The cardioprotective effects of vincristine were also observed during prolonged hypoxia. With continuous exposure to vincristine, survival lasted for as long as 24 h, but longer periods of exposure up to 42 h resulted in extensive cell death. Despite microtubule disruption evidenced on deconvolution microscopy, vincristine activated a prosurvival pathway resulting in increased phosphorylation of Akt, ERK and GSK-3β and in reduced cytochrome C release into the cytosol. Pharmacological inhibitors of Akt and Erk attenuated the cardioprotective effect of vincristine. We conclude that short-term pretreatment with vincristine exerts dramatic protective effects in cultured adult mouse myocytes subjected to acute oxidative stress. Despite causing microtubule disruption, vincristine initiates a prosurvival signaling pathway. As vincristine and doxorubicin are often used in conjunction to treat patients, it is possible that vincristine could be used to modify the cardiotoxicity of doxorubicin.

Adult cardiac fibroblasts null for sphingosine kinase-1 exhibit growth dysregulation and an enhanced proinflammatory response

Cardiac fibroblasts are critical for the maintenance of extracellular matrix deposition and turnover in the normal heart and are key mediators of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. Sphingosine kinase (SphK) activation is a well-recognized determinant of cell fate in cardiac myocytes and other cells, but SphK responses have not previously been studied in cardiac fibroblasts. Initially we found that total SphK activity is over 10-fold higher in cardiac fibroblasts than in adult mouse cardiac myocytes. SphK is composed of two major isoforms, SphK-1 and SphK-2. In fibroblasts isolated from SphK-1 knockout mice, SphK activity was greatly reduced indicating that SphK-1 is the major isoform expressed in these cells. To determine whether SphK regulates cell proliferation and the proinflammatory protein inducible nitric oxide synthase (iNOS), we exposed cultured cardiac fibroblasts to the cytokine interleukin-1β (IL-1β) and/or hypoxia. Both hypoxia and IL-1β alone and in combination enhanced fibroblast SphK activity. In wild-type fibroblasts, hypoxia induced proliferation, but in SphK-1 null fibroblasts this response was blunted even in the presence of serum. In contrast, we found that iNOS expression and NO production were enhanced in SphK-1 null fibroblasts during hypoxia. In wild-type fibroblasts, IL-1β was only a weak inducer of iNOS and of NO accumulation and hypoxia alone had no significant effect on iNOS activation. However, IL-1β in combination with hypoxia extensively stimulated iNOS and NO production, and this stimulation was enhanced in SphK-1 null fibroblasts. We conclude that activation of endogenous SphK-1 serves a dual regulatory function: it is required for optimal cardiac fibroblast proliferation but is a negative modulator of proinflammatory responses during hypoxia.

Deletion of the Sphingosine Kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes

Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C epsilon, is ineffective in SphK1-null myocytes; 3] S1P generated by SphK activation requires cellular export to be cardioprotective. We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3-6) and measured cell viability by trypan blue exclusion. In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress+glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S1P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 microM of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P(1) receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 h before exposure to hypoxia+glucose deprivation. As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S1P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor.

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled‐coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.

The yin and yang of increased β-adrenergic signaling: β1-adrenergic genetic polymorphism and protection against acute myocardial ischemic injury

significant improvements in the treatment and prevention of cardiac diseases have led to declining morbidity and mortality over the past three decades ([2][1]), and drugs targeting adrenergic signaling have played a key role in the beneficial effects recently afforded to these patients. However,

Heterodimerization of β 1 - and β 2 -Adrenergic Receptor Subtypes Optimizes β-Adrenergic Modulation of Cardiac Contractility

Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant beta-adrenergic receptor (betaAR) subtypes expressed in the heart, beta1AR, and beta2AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native beta1AR and beta2AR, coexpression of both betaAR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to betaAR agonist stimulation with isoproterenol (ISO) was shifted leftward by approximately 1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of betaAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of beta1AR greatly suppressed ligand-independent spontaneous activity of coexisting beta2ARs. Thus, heterodimerization of beta1AR and beta2AR in intact cardiac myocytes creates a novel population of betaARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing beta-adrenergic modulation of cardiac contractility.

The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice

We tested the hypothesis that chronic changes in intracellular Ca(2+) (Ca(2+)(i)) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca(2+) cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K(+) current (I(to,f)), a slowly inactivating K(+) current (I(K,slow)) and the steady state current (I(SS)) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca(2+)(i), were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca(2+)(i) on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca(2+) handling proteins without cardiac hypertrophy or failure may produce profound changes in K(+) channel expression and activity as well as cardiac AP.