Neonatal Mouse Cardiac Myocytes Research Articles

P16ink4a overexpression ameliorates cardiac remodeling of mouse following myocardial infarction via CDK4/pRb pathway

BackgroundP16ink4a can accumulate in senescent cells and can be induced by different oncogenic stimulations. These functions make p16ink4a a biomarker of senescence and cancer. However, the exact role of p16ink4a remains unclear in cardiovascular disease. This study was aimed to investigate the role of p16ink4a in cardiac remodeling after myocardial infarction (MI). MethodsIn vivo, gain and loss of function experiments using p16ink4a overexpression and knockdown adenovirus were induced to determine the effect of p16ink4a on cardiac structure and function after MI. The in vitro effects of p16ink4a were evaluated by overexpression and knockdown adenovirus of p16ink4a on isolated neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). ResultsExpression level of p16ink4a was increased after MI and enriched in the infarction area. In vivo, overexpression of p16ink4a protected, while knockdown of p16ink4a worsened cardiac function. In vitro, p16ink4a did not influence the hypertrophy of NMCMs. Overexpression of p16ink4a inhibited the proliferation and migration of NMCFs and reduced the level of collagen I and α-SMA. Consistently, knockdown of p16ink4a in vitro displayed the opposite effects. Further mechanism studies revealed that p16ink4a affected the expression level of cyclin-dependent kinase 4 (CDK4) and phosphorylation of retinoblastoma (pRb), which could be a potential pathway in regulating cardiac remodeling after MI. ConclusionOverexpression of 16ink4a in cardiac fibroblasts can ameliorate cardiac dysfunction and attenuate pathological cardiac remodeling in mice after MI by regulating the p16ink4a/CDK4/pRb pathway.

Sulphenylation of CypD at Cysteine 104: A Novel Mechanism by Which SO2 Inhibits Cardiomyocyte Apoptosis.

Objectives: The study was designed to explore the role of endogenous gaseous signaling molecule sulfur dioxide (SO2) in the control of cardiomyocyte apoptosis and its molecular mechanisms. Methods: Neonatal mouse cardiac myocytes (NMCMs) and H9c2 cells were used in the cell experiments. The endogenous SO2 pathway including SO2 level and the expression of SO2-generating enzyme aspartate aminotransferase 1/2 (AAT1/2) were detected in NMCMs. The apoptosis of cardiomyocytes was examined by a TUNEL assay. The cleavage and the activity of apoptotic proteins caspase9 and caspase3 were measured. The content of ATP, the opening of mitochondrial permeability transition pore (mPTP), and the cytochrome c (cytc) leakage were detected by immunofluorescence. The sulphenylation of cyclophilin-D (CypD) was detected by biotin switch analysis. The four CypD mutant plasmids in which cysteine sites were mutated to serine were constructed to identify the SO2-affected site in vitro. Results: ISO down-regulated the endogenous SO2/AAT pathway of cardiomyocytes in association with a significant increase in cardiomyocyte apoptosis, demonstrated by the increases in apoptosis, cleaved-caspase3/caspase3 ratio, and caspase3 activity. Furthermore, ISO significantly reduced ATP production in H9c2 cells, but the supplement of SO2 significantly restored the content of ATP. ISO stimulated mPTP opening, resulting in an increase in the release of cytc, which further increased the ratio of cleaved caspase9/caspase9 and enhanced the protein activity of caspase9. While, the supplementation of SO2 reversed the above effects. Mechanistically, SO2 did not affect CypD protein expression, but sulphenylated CypD and inhibited mPTP opening, resulting in an inhibition of cardiomyocyte apoptosis. The C104S mutation in CypD abolished SO2-induced sulphenylation of CypD, and thereby blocked the inhibitory effect of SO2 on the mPTP opening and cardiomyocyte apoptosis. Conclusion: Endogenous SO2 sulphenylated CypD at Cys104 to inhibit mPTP opening, and thus protected against cardiomyocyte apoptosis.

Abstract 14114: Role of Toxic Protein Sequestration by Aggregates in Cardiac Myocytes in Ischemic Cardiomyopathy

Introduction: Proteotoxic aggregation causes cardiomyopathy (CMP) associated with rare genetic mutations. The role of protein aggregates in ischemic cardiomyopathy is unclear. Hypothesis: Cardiac myocyte protein aggregation is deleterious in cardiomyopathy. Methods: Human (ischemic) and murine (ischemic and proteotoxic) CMP models were profiled by immunohistochemistry (IHC) and proteomic characterization of the detergent-insoluble fraction. Aggregates were evaluated by immunocytochemistry in p62 deficient neonatal mouse cardiac myocytes (NMCMs) with adenoviral transduction of mutant alpha-B crystallin (CRYABR120G). Mice with cardiac myocyte selective p62 deficiency (p62ckO) were generated and characterized (echocardiography, myocardial structure, autophagic flux, mitophagy, and biochemical analyses). p62cKO and floxed controls were subjected to closed-chest ischemia (90 minutes) or sham followed by reperfusion (IR) and evaluated 4 weeks later. Cardiac selective (AAV9-cTnT) reconstitution of p62, its aggregation-deficient K7R mutant or GFP, was performed. Wild-type mice transduced with a fungal disaggregase (AAV9-Hsp104 or control) were modeled for IR as above. Results: Protein aggregation increases in ischemic CMP, but not controls, with increased insoluble CRYAB with phosphorylation at serine 59 (pS59CRYAB). p62-deficient NMCMs are unable to form aggregates following CRYABR120G transduction, indicating the necessity of p62 in protein aggregation. p62cKO mice show preserved baseline cardiac structure/function, without significantly decreased mitophagy or autophagic flux. Compared to controls, p62cKO mice (post-IR) show infarct expansion and impaired LV remodeling at 4 weeks, associated with decreased sequestration of pS59CRYAB in insoluble fraction. Reconstitution with AAV9 p62, but not aggregation-deficient K7R, shows partial functional rescue after IR. Finally, disruption of protein aggregation by AAV9 Hsp104 (but not controls) results in decreased sequestration of pS59CRYAB, and decreased function post-IR. Conclusions: Contrary to the hypothesis, preventing or disrupting protein aggregation, associated with decreased pS59CRYAB sequestration, is maladaptive in ischemic cardiomyopathy.

Long Noncoding RNA FTX Reduces Hypertrophy of Neonatal Mouse Cardiac Myocytes and Regulates the PTEN/PI3K/Akt Signaling Pathway by Sponging MicroRNA-22.

BackgroundCardiac myocyte hypertrophy results from clinical conditions that include hypertension and valvular heart disease, and can result in heart failure. This study aimed to investigate the expression and role of the long noncoding RNA FTX (lnc-FTX), an X-inactive-specific transcript (XIST) regulator transcribed from the X chromosome, in hypertrophy of neonatal mouse cardiac myocytes induced by angiotensin II (Ang II) in vitro.Material/MethodCardiac myocytes were isolated from neonatal mice and cultured with and without Ang II. Immunofluorescence, with localization of an antibody to alpha-smooth muscle actin (α-SMA), was used to identify the neonatal mouse cardiac myocytes. Quantitative real-time polymerase chain reaction (qRT-PCR) measured gene expression levels. The cell counting kit-8 (CCK-8) assay was used to determine cell viability, and Western blot measured protein expression. StarBase v2.0 bioinformatics software was used for target gene prediction and was confirmed with the luciferase reporter assay.ResultsThe expression of lnc-FTX was reduced in mouse cardiac myocytes treated with Ang II. Overexpression of lnc-FTX reduced cell apoptosis, cardiomyocyte contractility, and the expression of c-Jun, A-type natriuretic peptide (ANP), and B-type natriuretic peptide (BNP) induced by Ang II. The target of lnc-FTX was micro-RNA 22 (miRNA-22). The mechanism of action of lnc-FTX in neonatal mouse cardiac myocytes was through suppression of the PI3K/Akt signaling pathway by promoting the release of PTEN by sponging miRNA-22.ConclusionsThe expression of lnc-FTX was associated with reduced hypertrophy of neonatal mouse cardiac myocytes and regulated the PTEN/PI3K/Akt signaling pathway by sponging miRNA-22.

Low-dose nicotine promotes autophagy of cardiomyocytes by upregulating HO-1 expression

Nicotine as a major component of addiction in cigarettes has been reported to play protective roles in some pathological processes. It is reported that activation of the nicotinic acetylcholine receptor also has a cardioprotective effect. Thus, in our study, we investigated the effect and mechanism of nicotine on the autophagy of cardiomyocytes, and whether nicotine protects cardiomyocytes against palmitic acid (PA) injury. The results indicated that low-dose nicotine promoted neonatal mouse cardiac myocytes (NMCMs) autophagy and accelerated autophagic flux while inhibiting NMCMs apoptosis, but high-dose nicotine inhibited autophagy and promoted apoptosis. Moreover, low-dose nicotine upregulated heme oxygenase-1 (HO-1) expression and knocking down HO-1 abolished the effects of nicotine on the autophagy and apoptosis of NMCMs. Methyllycaconitine citrate (α7-nAChR blocker, MLA) inhibited HO-1 expression and the effects of nicotine on autophagy and apoptosis of NMCMs. Furthermore, low-dose nicotine improved the inhibited autophagy and increased apoptosis induced by palmitic acid (PA) in NMCMs and these effects were reversed by knocking down HO-1. In conclusion, our data suggested that low-dose nicotine promoted autophagy and inhibited apoptosis of cardiomyocytes by upregulating HO-1.

MiR-207 inhibits autophagy and promotes apoptosis of cardiomyocytes by directly targeting LAMP2 in type 2 diabetic cardiomyopathy

Autophagy dysfunction plays a critical role in diabetic cardiomyopathy (DCM). MiR-207 regulates the expression of lysosomal-associated membrane protein 2 (LAMP2), an autophagy-related protein, following ischemic stroke in neurocytes. However, the roles and mechanisms of miR-207 in DCM remain unknown. Therefore, in this study, we aim to investigate the roles and mechanisms of miR-207 in type 2 DCM. The results showed that autophagic dysfunction with increased LC3II and P62 expression and decreased LAMP2 expression, and increased cell apoptosis with up-regulated cleaved-caspase3 expression were noted in the myocardium of type 2 DCM mice and neonatal mouse cardiac myocytes (NMCMs) stimulated with PA. In addition, miR-207 was significantly upregulated in the myocardium of DCM mice and NMCMs stimulated with PA. In NMCMs, miR-207 inhibited LAMP2 mRNA and protein expression. MiR-207 mimics significantly inhibited autophagy by increasing P62 and LC3II expression and promoted cell apoptosis by increasing cleaved-caspase3 expression, and these effects were reversed by LAMP2 overexpression. In conclusion, miR-207 inhibited autophagy and promoted apoptosis of cardiomyocytes by directly targeting LAMP2, which participated in the development of type 2 diabetic cardiomyopathy.

TNFR2 Stimulation Promotes Mitochondrial Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expression.

Mitochondria are important cellular organelles and play essential roles in maintaining cell structure and function. Emerging evidence indicates that in addition to having proinflammatory and proapoptotic effects, TNFα (tumor necrosis factor α) can, under certain circumstances, promote improvements in mitochondrial integrity and function, phenomena that can be ascribed to the existence of TNFR2 (TNFα receptor 2). The present study aimed to investigate whether and how TNFR2 activation mediates the effects of TNFα on mitochondria. Freshly isolated neonatal mouse cardiac myocytes treated with shRNA targeting TNFR1 were used to study the effects of TNFR2 activation on mitochondrial function. Neonatal mouse cardiac myocytes exhibited increases in mitochondrial fusion, a change that was associated with increases in mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption capacity. Importantly, TNFR2 activation-induced increases in OPA1 (optic atrophy 1) protein expression were responsible for the above enhancements, and these changes could be attenuated using siRNA targeting OPA1. Moreover, both Stat3 and RelA bound to the promoter region of OPA1 and their interactions synergistically upregulated OPA1 expression at the transcriptional level. Stat3 acetylation at lysine 370 or lysine 383 played a key role in the ability of Stat3 to form a supercomplex with RelA. Meanwhile, p300 modulated Stat3 acetylation in HEK293T (human embryonic kidney 293T) cells, and p300-mediated Stat3/RelA interactions played an indispensable role in OPA1 upregulation. Finally, TNFR2 activation exerted beneficial effects on OPA1 expression in an in vivo transverse aortic constriction model, whereby TNFR1-knockout mice exhibited better outcomes than in mice with both TNFR1 and TNFR2 knocked out. TNFR2 activation protects cardiac myocytes against stress by upregulating OPA1 expression. This process was facilitated by p300-mediated Stat3 acetylation and Stat3/RelA interactions, leading to improvements in mitochondrial morphology and function.

SUMOylation determines the voltage required to activate cardiac IKs channels

IKs channels open in response to depolarization of the membrane voltage during the cardiac action potential, passing potassium ions outward to repolarize ventricular myocytes and end each beat. Here, we show that the voltage required to activate IKs channels depends on their covalent modification by small ubiquitin-like modifier (SUMO) proteins. IKs channels are comprised of four KCNQ1 pore-forming subunits, two KCNE1 accessory subunits, and up to four SUMOs, one on Lys424 of each KCNQ1 subunit. Each SUMO shifts the half-maximal activation voltage (V1/2) of IKs ∼ +8 mV, producing a maximal +34-mV shift in neonatal mouse cardiac myocytes or Chinese hamster ovary (CHO) cells expressing the mouse or human subunits. Unexpectedly, channels formed without KCNE1 carry at most two SUMOs despite having four available KCNQ1-Lys424 sites. SUMOylation of KCNQ1 is KCNE1 dependent and determines the native attributes of cardiac IKs in vivo.

Lipophilic And Hydrophilic Statins Differentially Modulate Endoplasmic Reticulum Stress Response In Cardiac Myocytes

Statins inhibit HMG‐CoA reductase, target mevalonic acid synthesis, and limit cholesterol biosynthesis. HMG‐CoA reductase is expressed in the membrane of the endoplasmic reticulum (ER). Statins are prescribed to prevent cardiovascular events.In cultured neonatal mouse cardiac myocytes the lipophilic statin atorvastatin and the hydrophilic statin pravastatin both up‐regulated PDI, indicating unfolded protein response (UPR) meant to relieve ER stress. Only atorvastatin increased ER stress, growth arrest, and induced apoptosis via induction of CHOP, Puma, active Caspase‐3 and PARP. Dose‐dependent release of LDH was only observed in atorvastatin treated cells (1–10 μM). Hearts of mice treated with atorvastatin (5mg/kg/day for 7 months) showed protein aggresomes and autophagosomes when compared to vehicle treated controls. While atorvastatin changed mitochondrial ultrastructure, no differences in cardiac function, exercise ability or creatine kinase levels were found.We show differential activation of ER stress by atorvastatin and pravastatin in cardiac myocytes. Our results provide a novel mechanism through which specific statins therapeutically modulate the balance of UPR/ER stress responses thereby possibly influencing cardiac remodeling.

Imperatorin prevents cardiac hypertrophy and the transition to heart failure via NO-dependent mechanisms in mice

Augmented endothelial nitric oxide (NO) synthase (eNOS) signaling has been reported to be associated with improvements in cardiac remodeling, and NO levels have been shown to be related to cardiac hypertrophy and heart failure. Imperatorin, a dietary furanocoumarin, has been shown to prevent cardiac hypertrophy in the spontaneous hypertension rats (SHR). Thus, we aimed to clarify whether imperatorin attenuates both cardiac hypertrophy and heart failure via the NO-signaling pathway. In neonatal mouse cardiac myocytes, imperatorin inhibited protein synthesis stimulated by either isoproterenol or phenylephrine, which was unchanged by NG-nitro-L-arginine methyl ester (L-NAME). Four weeks after transverse aortic constriction (TAC) on Kunming (KM) male mice, the ratio of heart weight to body weight was lower after imperatorin treatment than in controls (6.60 ± 0.35 mg/g in TAC, 4.54 ± 0.29 mg/g with imperatorin 15 mg kg −1 d −1, ig, P < 0.01); similar changes in the ratio of lung weight to body weight (7.30 ± 0.85 mg/g in TAC, 5.42 ± 0.51 mg/g with imperatorin 15 mg kg − 1 d − 1 , ig) and the myocardial fibrosis. All of these improvements were blunted by L-NAME. Imperatorin treatment significantly activated phosphorylation of eNOS. Myocardial mRNA levels of natriuretic peptide precursor type B and protein inhibitor of NO synthase, which were increased in the TAC mice, were decreased in the imperatorin-treated ones. Imperatorin can attenuate cardiac hypertrophy both in vivo and in vitro, and halt the process leading from hypertrophy to heart failure by a NO-mediated pathway.

Specific PKG inhibitors: do they really exist?

Background Cellular cGMP effects can be mediated by a number of effectors including cGMP-dependent protein kinases (PKGs), cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMPgated channels. Pharmacological inhibitors of PKG are often used to discriminate between these diverse cGMP effects. Currently used PKG inhibitors can be divided into three classes: cyclic nucleotide binding site inhibitors like Rp-phosphorothioate analogs, ATP binding site inhibitors like KT5823, and substrate binding site inhibitors like the recently described DT-oligopeptides. However, several studies have observed no PKG inhibition by KT5823 in intact cells [1] or by Rp-cGMPS analogs in smooth muscle cells [2], as well as unspecific (PKG-independent) effects of Rp-cGMPS analogs in platelets [3]. Results We tested the inhibitory effects of (D)-DT2 and DT3 on PKG and its effects on intact cells. Our data show that (D)-DT2 selectively inhibited 2 nM purified PKG Ia and Ib with an IC50 of 8n M, and that up to 1µ M (D)-DT2 did not inhibit PKG II or PKA. In broken platelet cell experiments, PKG activity was inhibited by (D)-DT2 starting at 5 µM, with complete inhibition at 20 µM, but we also observed inhibition of PKA activity at these concentrations. However, concentrations of up to 200 µM of compounds failed to inhibit PKG activity (assessed by phosphorylation of the established PKG substrates VASP, PDE5 and GRP2) in intact human platelets, rat mesangial cells and neonatal mouse cardiac myocytes. It should be noted that the measured PKG concentration is about 7 µM in platelets and 0.1 – 0.5 µM in all other tested cells. (D)-DT2 effects on platelet function did not correlate with PKG activity. Preincubation of platelets with 10 nM (D)-DT2 strongly inhibited thrombin-induced platelet aggregation and calcium mobilization, whereas it potentiated these effects in collagen-stimulated platelets. Conclusion Interpretations of results based on PKG inhibitors require caution. None of the commercially available PKG inhibitors should be used without control experiments in intact cells since they may have unpredictable functional effects not mediated by PKG activity.

Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels

L-type Ca(2+) channels (LTCCs) play a critical role in Ca(2+)-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca(2+) signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Ca(v)1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba(2+) current (I(Ba,L)) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak I(Ba,L) by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact I(Ba,L). Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Ca(v)1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Ca(v)1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak I(Ba,L) by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Ca(v)1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K(+) channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.

Transcription Factor CHF1/Hey2 Regulates Specific Pathways in Serum Stimulated Primary Cardiac Myocytes: Implications for Cardiac Hypertrophy

We have previously found that overexpression of CHF1/Hey2 in the myocardium prevents the development of phenylephrine-induced hypertrophy. To identify transcriptional pathways regulated by CHF1/Hey2, we cultured primary neonatal mouse cardiac myocytes from wild type and transgenic mice overexpressing CHF1/Hey2 and treated them with serum, a potent hypertrophic stimulus. We verified that overexpression of CHF1/Hey2 suppressed cardiac myocyte hypertrophy induced by serum and then determined transcriptional profiles by microarray hybridization. We identified and verified important downstream target genes by single gene analysis and qRT-PCR and then identified important biological processes by Gene Set Analysis using Biological Process Gene Sets from the Gene Ontology Consortium. We found that CHF1/Hey2 suppresses pathways involved in water transport, adenylate cyclase activity, embryonic eye morphogenesis, gut development and fluid transport after serum stimulation. Genes involved in protein dephosphorylation, demonstrate increased expression in myocytes overexpressing CHF1/Hey2, independent of serum treatment. Genes overexpressed prior to serum treatment are involved in regulation of transcription factor activity, nuclear protein export and steroid hormone receptor signaling. Genes overexpressed after serum treatment are involved in autophagy, apoptosis and mitochondrial biogenesis.

Voltage-gated sodium channel modulation by σ-receptors in cardiac myocytes and heterologous systems

The sigma-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated K(+) and Ca(2+) channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated Na(+) channel (Na(v)1.5) by sigma-receptors. The sigma(1)-receptor ligands [SKF-10047 and (+)-pentazocine] and sigma(1)/sigma(2)-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited Na(v)1.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the sigma(1)-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited Na(+) current in neonatal mouse cardiac myocytes. In sigma(1)-receptor knockout myocytes, the sigma(1)-receptor-specific ligands were far less effective in modulating Na(+) current, but the sigma(1)/sigma(2)-receptor ligands modulated Na(+) channels as well as in wild type. Photolabeling with the sigma(1)-receptor photoprobe [(125)I]-iodoazidococaine demonstrated that sigma(1)-receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of sigma(1)-receptor-specific ligands in HEK-293 cells than in COS-7 cells. sigma-Receptors modulated Na(+) channels despite the omission of GTP and ATP from the patch pipette solution. sigma-Receptor-mediated inhibition of Na(+) current had little if any voltage dependence and produced no change in channel kinetics. Na(+) channels represent a new addition to the large number of voltage-gated ion channels modulated by sigma-receptors. The modulation of Na(v)1.5 channels by sigma-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity.

Loss of O‐GlcNAc transferase activity sensitizes cardiac myocytes to post‐hypoxic death

We recently reported that diabetic hearts have a paradoxical reduction in β‐O‐linked‐N‐acetylglucosamine (O‐GlcNAc) signaling. Because enhancement of O‐GlcNAc signaling is cardioprotective, we hypothesized that loss of OGT activity is sufficient to sensitize nondiabetic cardiac myocytes to post‐hypoxic injury. Here, loxP‐ flanked OGT neonatal mouse cardiac myocytes (n=3/group) were infected with an adenovirus carrying cre recombinase gene (AdCre, 72hours; 50 moi). Expression of cre recombinase reduced OGT expression (44+/−16%, p&lt;0.05) and O‐GlcNAc levels (28+/−5%, p&lt; 0.05) according to western blotting. Furthermore, such transcriptional silencing of OGT significantly (122+/−7%, p&lt; 0.05) exacerbated post‐hypoxic injury according to LDH release. To further confirm these findings, neonatal rat cardiac myocytes were transfected with 30nM OGT siRNA, to knockdown OGT. OGT siRNA‐treatment significantly reduced OGT (49+/−8%, p&lt;0.05) and O‐GlcNAc levels (75+/−13%, p&lt;0.05) compared with control siRNA. Such decrement in O‐GlcNAc signaling corresponded with exacerbated post‐hypoxic LDH release (120+/−2%, p&lt;0.05). We conclude that reduced OGT activity diminishes O‐GlcNAc levels and sensitizes cardiac myocytes to lethal cell injury. Such insights support the hypothesis that defects in O‐GlcNAc signaling are at least partially responsible for the sensitivity of the diabetic myocardium to ischemia‐reperfusion injury. Moreover, basal OGT expression is elemental for the ability of cardiac myocytes to withstand hypoxic injury. NIH (HL0833220), AHA (0535270N) and AHA (0715493B).

Distinct PDE4D splice variants regulate beta‐adrenergic signaling in neonatal mouse cardiac myocytes

We have shown that inhibition of the cAMP-specific type 4 cyclic nucleotide phosphodiesterases (PDE4s) modifies beta-adrenergic signaling in mouse neonatal cardiac myocytes. In the present study, we further elucidated the role of individual PDE4 variants in the control of b1- and b2-adrenergic receptor (b1-AR and b2-AR) signaling. Stimulation of b2-AR resulted in a selective PKA-mediated activation of the PDE4D splice variant PDE4D5. Conversely, the variant PDE4D8 was predominantly activated by PKA upon b1-AR stimulation. Cyclic AMP-hydrolyzing (PDE) activity was co-immunoprecipiated with bARs from extracts of myocytes overexpressing b1-AR or b2-AR. This activity is due to PDE4D, because it was sensitive to PDE4-selective inhibitors and PDE activity was still recovered when cells deficient in PDE4A or PDE4B but not PDE4D were used. Moreover, the physical association of PDE4D variants and b-ARs was reproduced by co-IPs of recombinant PDE4D splice variants with b1- and b2-AR. Importantly, treatment with b-adrenergic agonists such as Isoproterenol diminished the PDE4D/b-AR co-IP indicating that this complex is dynamically regulated by b-AR signaling. Taken together, our results indicate that distinct PDE4D splice variants form macromolecular signaling complexes with and control the cAMP signals from b1- and b2-ARs. This research was supported by NIH grants HD20788 to M.C. and HL71078-01 to B.K.

Blockade of HERG channels by HIV protease inhibitors

The HIV protease inhibitor class of antiretroviral drug causes unpredicted adverse effects by changing elements of normal cellular metabolism. A case of QT prolongation in a patient receiving protease inhibitors made us question whether these drugs might be responsible. We identified 24 patients with QT prolongation or torsade de pointes, or both, associated with protease inhibitors, using the Food and Drug Administration's voluntary adverse event reporting system. Attending physicians thought that protease inhibitors were the most probable cause of these symptoms in 14 of the patients. Drug-induced QT prolongation is usually caused by block of human ether-a-go-go-related gene (HERG) potassium channels, and we showed that lopinavir, nelfinavir, ritonavir, and saquinavir caused dose-dependent block of HERG channels heterologously expressed in HEK293 cells in vitro. We also recorded block by lopinavir of repolarising potassium current (I(Kr)) channels in neonatal mouse cardiac myocytes. Our data show that four protease inhibitors block HERG channels, suggesting that protease inhibitors could predispose individuals to QT prolongation and torsade de pointes.

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.

Natriuretic Peptide Gene Expression after β-Adrenergic Stimulation in Adult Mouse Cardiac Myocytes

The role of A- and B-type natriuretic peptides (ANP and BNP) in cardiac pathophysiology are of increasing interest. Isolated neonatal mouse cardiac myocytes express increased levels of ANP mRNA in the absence of growth factors in culture. Expression of ANP and BNP mRNA has not been studied in isolated adult mouse cardiac myocytes (AMCM). We examined expression of ANP and BNP mRNA in isolated AMCM with and without stimulation with β-adrenergic receptor agonists and antagonists. AMCM were isolated and maintained in culture for 24–48 h with and without stimulation with the β-adrenergic receptor agonist isoproterenol (Iso), the β1-antagonist CGP20712A (CGP), or the β2-antagonist ICI-118,551 (ICI). Northern blot analysis was performed using probes for mouse ANP and BNP mRNA. TUNEL assay was performed after β-adrenergic receptor stimulation of AMCM. BNP mRNA expression was increased fivefold (P < 0.001) after 48 h in culture without adrenergic stimulation. BNP mRNA expression was reduced (P < 0.0001) after stimulation with Iso while ANP expression remained similar to unstimulated cells. CGP prevented the Iso reduction in BNP mRNA. Iso stimulation at doses that reduced BNP mRNA expression increased TUNEL positive nuclei, an effect blocked by the β1-antagonist CGP. In conclusion, we have demonstrated differential gene expression of ANP and BNP in AMCM in culture. Expression of BNP mRNA increases in AMCM in culture and β1-adrenergic receptor stimulation attenuates increased BNP gene expression and results in apoptosis.

Natriuretic peptide gene expression after beta-adrenergic stimulation in adult mouse cardiac myocytes.

The role of A- and B-type natriuretic peptides (ANP and BNP) in cardiac pathophysiology are of increasing interest. Isolated neonatal mouse cardiac myocytes express increased levels of ANP mRNA in the absence of growth factors in culture. Expression of ANP and BNP mRNA has not been studied in isolated adult mouse cardiac myocytes (AMCM). We examined expression of ANP and BNP mRNA in isolated AMCM with and without stimulation with beta-adrenergic receptor agonists and antagonists. AMCM were isolated and maintained in culture for 24-48 h with and without stimulation with the beta-adrenergic receptor agonist isoproterenol (Iso), the beta1-antagonist CGP20712A (CGP), or the beta2-antagonist ICI-118,551 (ICI). Northern blot analysis was performed using probes for mouse ANP and BNP mRNA. TUNEL assay was performed after beta-adrenergic receptor stimulation of AMCM. BNP mRNA expression was increased fivefold (P < 0.001) after 48 h in culture without adrenergic stimulation. BNP mRNA expression was reduced (P < 0.0001) after stimulation with Iso while ANP expression remained similar to unstimulated cells. CGP prevented the Iso reduction in BNP mRNA. Iso stimulation at doses that reduced BNP mRNA expression increased TUNEL positive nuclei, an effect blocked by the beta1-antagonist CGP. In conclusion, we have demonstrated differential gene expression of ANP and BNP in AMCM in culture. Expression of BNP mRNA increases in AMCM in culture and beta1-adrenergic receptor stimulation attenuates increased BNP gene expression and results in apoptosis.