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The current therapeutic options for acute decompensated heart failure are limited to afterload reducers and positive inotropes. The latter increases myocardial contractility through changes in myocyte calcium (Ca2+) handling (mostly through stimulation of the β-adrenergic pathways [β-ADR]) and is associated with paradoxical effects of arrhythmias, cell death, and subsequently increased mortality. We have previously demonstrated that probenecid can increase cytosolic Ca2+ levels in the cardiomyocyte resulting in an improved inotropic response in vitro and in vivo without activating the β-ADR system. We hypothesize that, in contrast to other commonly used inotropes, probenecid functions through a system separate from that of β-ADR and hence will increase contractility and improve function without damaging the heart. Furthermore, our goal was to evaluate the effect of probenecid on cell death in vitro and its use in vivo as a positive inotrope in a mouse model of ischemic cardiomyopathy. Herein, we demonstrate that probenecid induced an influx of Ca2+ similar to isoproterenol, but does not induce cell death in vitro. Through a series of in vivo experiments we also demonstrate that probenecid can be used at various time points and with various methods of administration in vivo in mice with myocardial ischemia, resulting in improved contractility and no significant difference in infarct size. In conclusion, we provide novel data that probenecid, through its activity on cellular Ca2+ levels, induces an inotropic effect without causing or exacerbating injury. This discovery may be translatable if this mechanism is preserved in man.

Introduction Probenecid, previously used for the treatment of gout, is a transient receptor potential vanilloid 2 (TRPV2) agonist. We have found TRPV2 in murine cardiomyocytes and when stimulated by probenecid, results in a positive inotropic response through Ca 2+ influx independent of β-adrenergic signaling. Our hypothesis is that probenecid will increase contractility without affecting cell survival. Methods We studied the role of probenecid as a positive inotrope in a mouse model of ischemic heart disease. We administered probenecid via intraperitoneal injections (100mg/kg) before and after ischemia/reperfusion injury (I/R) and orally via treated water (100mg/kg daily for 4 weeks), using saline injections and untreated water as respective controls. Ischemia was induced by a 45 minute ligation of the LAD followed by reperfusion and all mice were followed serially via echocardiography. We also evaluated the effects of probenecid on HL-1 cell apoptosis by conducting cell viability assays. Results Treatment with probenecid before I/R had no effect on subsequent infarct size (51.6±3.71% vs. 53.4±1.54%;P=NS). However, post-I/R probenecid caused an increase in ejection fraction (EF) of 10.9±1.98% in mice with initial EF between 40-50% (n=6; P Conclusions These experiments demonstrate 1) probenecid increases myocardial contractility at baseline and significantly more so after I/R injury; 2) probenecid therapy improves function when used chronically; 3) the increase in contractility is not associated with either increased infarct size in vivo or apoptosis in vitro. Thus, unlike other positive inotropes, such as isoproterenol, probenecid increases contractility without resulting in significant cell death.

TRPV2 is a member of transient receptor potential vanilloid (TRPV) family. As a Ca 2+ channel, it can detect various stimuli such as noxious heat (>52°C), membrane stretching, as well as a number of exogenous chemicals, including probenecid, 2-aminoethoxydiphenyl borate, and lysophospholipids. TRPV2 has been found in many tissue types, including neuron and kidney, but the function of TRPV2 in the heart is poorly understood. Here we show TRPV2 is involved in the Ca 2+ cycling process and then regulates the function of the cardiomyocyte. We identified the mRNA expression of TRPV2 in the cardiac tissues of mice using real-time PCR. By performing echocardiography we found that administration of probenecid, a selective TRPV2 agonist, increased cardiac ejection fraction in mice. This positive inotropic effect of probenecid was also shown in Langendorff perfused mice hearts as increased peak +dP/dt. In isolated ventricular myocytes, we found that probenecid significantly increased myocyte fractional shortening in a dose-dependent manner, which was fully blocked by ruthenium red, a non-selective TRPV2 blocker. We also performed fluorescent studies to examine myocyte Ca 2+ cycling. We found that probenecid significantly increased Ca 2+ transient and resting-state Ca 2+ sparks and this effect was eliminated by ruthenium red. When Ca 2+ storage in sarcoplasmic reticulum (SR) was depleted with caffeine, and SR Ca 2+ reuptake was blocked by thapsigargin at the same time, probenecid did not show any effects in either Ca 2+ transient or Ca 2+ sparks. Our patch clamp experiments indicate that probenecid treatment does not trigger any significant transmembrane Ca 2+ influx. These results point to the important role of TRPV2 in regulating SR Ca 2+ release. In conclusion, TRPV2 activation may contribute to increased SR Ca 2+ release, leading to the enhancement of myocyte contractility. Thus, TRPV2 plays a potentially important role in controlling the cellular function of heart.

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Using a new class of nontoxic and degradable glycopolymer-based vehicles termed poly(glycoamidoamine)s, we demonstrate virus-like delivery efficacy of oligodeoxynucleotide (ODN) decoys to cardiomyoblasts (H9c2), primary cardiomyocytes, and the mouse heart. These glycopolymers bind and compact ODN decoys into nanoparticle complexes that are internalized by the cell membrane and mediate nuclear uptake of DNA in 90+% of cultured primary cardiomyocytes and 87% of the mouse myocardium. Experimental results reveal that decoys delivered via these glycopolymers block the activation of the transcription factor NF-κB, a major contributor to ischemia/reperfusion injury. Decoy complexes formed with glycopolymer T4 significantly blocked downstream gene expression of Cox-2 and limited myocardial infarction in vivo, phenocopying a transgenic mouse model. These promising delivery vehicles may facilitate high-throughput genetic approaches in animal models. Additionally, the low toxicity, biodegradation, and outstanding delivery efficacy suggest that these nanomedicines may be clinically applicable for gene regulatory therapy.

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3′-UTR. This shortening of the 3′-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3′-UTR relieves translational suppression observed in the long 3′-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

Microgravity (micro-g) environments have been shown to elicit dysregulation of specific genes in a wide assay of cell types. It is known that the activation of transcription factors and molecular signaling pathways influence various physiological outcomes associated with stress and adaptive responses. Nuclear factor-kappa B (NF-kappaB) is one of the most prevailing oxidation-sensitive transcription factors. It is hypothesized that simulated microgravity would activate NF-kappaB and its downstream transcriptional networks, thus suggesting a role for NF-kappaB in microgravity induced muscle atrophy. To investigate the activation of NF-kappaB in a rat cardiac cell line (H9c2) under micro-g, rotating wall vessel bioreactors were used to simulate micro-g conditions. Western blotting revealed that mean nuclear translocation of NF-kappaB p65 subunit was 69% for micro-g and 46% for unit-g dynamic control as compared with a 30 min TNF-alpha positive control (p

The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell (EC) death. Treatment with IL-18 increased NF-κB activation, PTEN induction, suppressed Akt activation, and stimulated EC death. While ectopic expression of p65 enhanced PTEN transcription, adenoviral transduction of dnIκB-α, dnp65, or dnIKKβ was inhibitory. Furthermore, IL-18 suppressed HO-1 mRNA expression via enhanced mRNA degradation. Overexpression of HO-1, treatment with HO-1 inducer hemin or the CO donor Cobalt (III) protoporphyrin IX all reversed IL-18-mediated NF-κB activation, PTEN induction, Akt suppression, and EC death. Furthermore, hemin induced HO-1 expression, and HO-1 knockdown, HO-1 inhibition, or CO scavengers all reversed the pro-survival effects of hemin. In addition, the CO donors CORM-1 and CORM-3 and the heme metabolites biliverdin and bilirubin attenuated IL-18-induced EC death via a similar signaling pathway. IL-18 induced p38α MAPK activation, and suppressed p38β isoform expression. While p38α knockdown attenuated, p38β knockdown potentiated IL-18-mediated EC death. Hemin and HO-1 reversed IL-18-mediated p38α induction, and restored p38β levels. These results demonstrate that IL-18 suppresses HO-1 expression and induces EC death. HO-1 overexpression, HO-1 induction, or treatment with heme metabolites all reverse IL-18-mediated p38α MAPK and NF-κB activation, PTEN induction, Akt suppression, and EC death. Thus, HO-1 inducers and CO donors may have the therapeutic potential to effectively block IL-18 signaling and reduce IL-18-dependent vascular injury and inflammation.

This is a response to a letter by Christians (1) In response to the letter by Christians asserting viability of HSF-1 knock-out (KO) mice, we acknowledge that our statement that “the homozygous mice are lethal” was an oversimplification, and we apologize for this. To set the record straight, HSF-1 KO mice have been created, and a small percentage of HSF-1−/− mice are viable in a background-dependent manner. According to the Mouse Genome Informatics Database (MGI:96238), there are 11 gene-targeted or gene-trapped alleles for HSF-1; only two of these are available as live mice, and the issues with viability have been described for the strain from the Benjamin laboratory (2). Due to substantial prenatal lethality, HSF-1 KO mice on a 129 strain background are mostly nonviable, as these mice represented only 1.2% of the live births from the F1 HSF-1+/− heterozygous parents (2). HSF-1−/− mice on a BALB/c or ICR background displayed slightly higher live birth rates for HSF-1 null offspring at 11.8 and 15.0%, respectively, but these backgrounds would have been inappropriate for our work. The hearts of mice on a BALB/c background have been shown to be hyperdynamic due to abnormally high levels of α-skeletal actin in the heart (3), and the outbred nature of ICR mice would make them improperly controlled relative to our previous work on murine cardiac ischemic/reperfusion injury. In addition, HSF-1 null C57BL/6 mice that survive to adulthood express some severely altered phenotypes, including growth retardation, exaggerated expression of TNF-α (which would certainly affect infarction), and female infertility (2). Despite the phenotypic deficiencies and prenatal lethality issues with HSF-1 KO mice, we did not intend to ignore that these mice have been created and characterized. However, research employing the HSF-1 homozygous mice must be carefully interpreted due to potential confounding effects of the complex issues with viability and altered phenotypes cited above.