Histone deacetylase 4 (HDAC4) regulates numerous gene expression programs through its signal-dependent repression of myocyte enhancer factor 2 (MEF2) and serum response factor (SRF) transcription factors. In cardiomyocytes, calcium/calmodulin-dependent protein kinase II (CaMKII) signaling promotes hypertrophy and pathological remodeling, at least in part by phosphorylating HDAC4, with consequent stimulation of MEF2 activity. In this paper, we describe a novel mechanism whereby protein kinase A (PKA) overcomes CaMKII-mediated activation of MEF2 by regulated proteolysis of HDAC4. PKA induces the generation of an N-terminal HDAC4 cleavage product (HDAC4-NT). HDAC4-NT selectively inhibits activity of MEF2 but not SRF, thereby antagonizing the prohypertrophic actions of CaMKII signaling without affecting cardiomyocyte survival. Thus, HDAC4 functions as a molecular nexus for the antagonistic actions of the CaMKII and PKA pathways. These findings have implications for understanding the molecular basis of cardioprotection and other cellular processes in which CaMKII and PKA exert opposing effects.

Thum et al. conclude that microRNA-21 (miR-21) is essential for cardiac hypertrophy and fibrosis in response to pressure overload (1). They also claim that our failure to observe a blockade to these processes in mice treated with an 8-mer locked nucleic acid–modified oligonucleo­tide against miR-21 (called Anti-21) (2) is due to the ineffectiveness of such inhibitors. We wish to point out several caveats to their study regarding the role of miR-21 in cardiac hypertrophy and their conclusions regarding the efficacy of the Anti-21 oligonucleotide. First, we find that Anti-21 inhibits miR-21 with a half-maximal inhibitory concentration of 0.9 nM, indicating the efficacy of Anti-21. Second, Thum et al. do not state the method they used to measure miR-21 inhibition, though we assume it to be quantitative PCR (qPCR). In our hands, qPCR alone is unreliable for measuring miRNA inhibition, especially for 8-mer inhibitors, since they may be displaced during qPCR and thereby give an underrepresentation of miRNA inhibition. To demonstrate functional inhibition of a miRNA, it is important to show data from multiple assays, such as small RNA Northern blots, luciferase reporter assays, and target derepression, as shown in our study (2). Such data are lacking in the Thum et al. rebuttal, which makes comparison of the different chemistries impossible. Thum et al. also state that we measured miR-21 inhibition on day 2 after dosing with Anti-21, when in fact we measured inhibition 3 weeks after dosing. At this time point, we observed inhibition of miR-21 in pressure-overloaded hearts at a level significantly below that of control mice. Thus, their approach for inhibition and/or measurement of miR-21 by their 8-mer inhibitors differs markedly from ours, since we observed robust miR-21 inhibition 3 weeks after injection, as demonstrated by multiple readouts (2). Using mismatched oligonucleotide controls is also important for interpreting miRNA inhibition studies in vivo, as described in our paper, rather than using PBS as a control, as reported by Thum et al. (1). Finally, Thum et al. postulate that constitutive genetic deletion of miR-21 in mice may not reveal the functions of miR-21 in cardiac disease because of compensatory events that mask such functions. If such compensation occurs, it must be specific for the cardiac functions of miR-21, since miR-21 null mice are resistant to lung tumorigenesis (3), consistent with the documented pro-oncogenic functions of miR-21. To further address the possibility of genetic compensation, we have deleted a floxed miR-21 allele immediately prior to thoracic aortic constriction in mice using a ubiquitously expressed tamoxifen-regulated Cre transgene. These animals show cardiac hypertrophy and fibrosis comparable to that of their Cre-negative littermates. Genetic compensation therefore cannot account for the normal pathological cardiac remodeling response in miR-21 null mice. Moreover, functions of other miRNAs in heart disease can be revealed by genetic deletion in mice, as shown for miR-208 (4). Thus, while 22-mer oligonucleotide inhibitors against miR-21 are efficacious in inhibiting cardiac hypertrophy (1), other loss-of-function approaches appear ineffective. We remain enthusiastic about miRNAs as therapeutic targets and welcome such dialog.

MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3' untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent cardiac hypertrophy and fibrosis in rodents in response to pressure overload. In contrast, we have shown here that miR-21-null mice are normal and, in response to a variety of cardiac stresses, display cardiac hypertrophy, fibrosis, upregulation of stress-responsive cardiac genes, and loss of cardiac contractility comparable to wild-type littermates. Similarly, inhibition of miR-21 through intravenous delivery of a locked nucleic acid-modified (LNA-modified) antimiR oligonucleotide also failed to block the remodeling response of the heart to stress. We therefore conclude that miR-21 is not essential for pathological cardiac remodeling.

Erythrocyte formation occurs throughout life in response to cytokine signaling. We show that microRNA-451 (miR-451) regulates erythropoiesis in vivo. Mice lacking miR-451 display a reduction in hematrocrit, an erythroid differentiation defect, and ineffective erythropoiesis in response to oxidative stress. 14-3-3zeta, an intracellular regulator of cytokine signaling that is repressed by miR-451, is up-regulated in miR-451(-/-) erythroblasts, and inhibition of 14-3-3zeta rescues their differentiation defect. These findings reveal an essential role of 14-3-3zeta as a mediator of the proerythroid differentiation actions of miR-451, and highlight the therapeutic potential of miR-451 inhibitors.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small-cell lung cancer (NSCLC) accounts for 80% of cases. MicroRNA-21 (miR-21) expression is increased and predicts poor survival in NSCLC. Although miR-21 function has been studied in vitro with cancer cell lines, the role of miR-21 in tumor development in vivo is unknown. We utilize transgenic mice with loss-of-function and gain-of-function miR-21 alleles combined with a model of NSCLC to determine the role of miR-21 in lung cancer. We show that overexpression of miR-21 enhances tumorigenesis and that genetic deletion of miR-21 partially protects against tumor formation. MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.

Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKII delta, the major CaMKII isoform expressed in the heart, we generated CaMKII delta-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKII delta-null mice showed diminished kinase activity toward histone deacetylase 4 ( HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKII delta-null mice, underscoring the specificity of the CaMKII delta signaling pathway for HDAC4 phosphorylation. We conclude that CaMKII delta functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKII delta as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload.

Abstract 281 Johannes Backs, David Patrick, Thea Backs, Svetlana Bezprozvannaya, Xiaoxia Qi, Joseph A Hill, Eric N Olson, UT Southwestern Medical Center at Dallas, Dallas, TX Johannes Backs, 2007 Finalist and Presenting Author

Injection drug use is inextricably linked to commercial sex work and the transmission of sexually transmitted disease (STD). In many communities prevention efforts have been stalled owing to the marginal existence of this community. This study describes the sexual activities, condom use, reported STDs, and commercial sex work in a large cohort of injection drug users. Seventy two per cent of male and 92% of female subjects in the cohort were sexually active. Among female subjects, 57% reported more than 100 lifetime partners. Condoms were generally not used with regular partners, used about half the time with casual partners, and used about 80% of the time with paying partners. Female sex workers were more likely to have unstable housing and to report incarceration in the previous six months. Reducing the transmission of STDs and HIV in drug using communities is a public health priority. While existing prevention programmes should be strengthened, innovative approaches to STD surveillance, diagnosis, and prevention are needed.