OBJECTIVE— Many of the effects of angiotensin (Ang) II are mediated through specific plasma membrane receptors. However, Ang II also elicits biological effects from the interior of the cell (intracrine), some of which are not inhibited by Ang receptor blockers (ARBs). Recent in vitro studies have identified high glucose as a potent stimulus for the intracellular synthesis of Ang II, the production of which is mainly chymase dependent. In the present study, we determined whether hyperglycemia activates the cardiac intracellular renin-Ang system (RAS) in vivo and whether ARBs, ACE, or renin inhibitors block synthesis and effects of intracellular Ang II (iAng II). RESEARCH DESIGN AND METHODS— Diabetes was induced in adult male rats by streptozotocin. Diabetic rats were treated with insulin, candesartan (ARB), benazepril (ACE inhibitor), or aliskiren (renin inhibitor). RESULTS— One week of diabetes significantly increased iAng II levels in cardiac myocytes, which were not normalized by candesartan, suggesting that Ang II was synthesized intracellularly, not internalized through AT 1 receptor. Increased intracellular levels of Ang II, angiotensinogen, and renin were observed by confocal microscopy. iAng II synthesis was blocked by aliskiren but not by benazepril. Diabetes-induced superoxide production and cardiac fibrosis were partially inhibited by candesartan and benazepril, whereas aliskiren produced complete inhibition. Myocyte apoptosis was partially inhibited by all three agents. CONCLUSIONS— Diabetes activates the cardiac intracellular RAS, which increases oxidative stress and cardiac fibrosis. Renin inhibition has a more pronounced effect than ARBs and ACE inhibitors on these diabetes complications and may be clinically more efficacious.

SummaryLong noncoding RNAs (lncRNAs) are commonly dysregulated in tumors, but only a handful are known to play pathophysiological roles in cancer. We inferred lncRNAs that dysregulate cancer pathways, oncogenes, and tumor suppressors (cancer genes) by modeling their effects on the activity of transcription factors, RNA-binding proteins, and microRNAs in 5,185 TCGA tumors and 1,019 ENCODE assays. Our predictions included hundreds of candidate onco- and tumor-suppressor lncRNAs (cancer lncRNAs) whose somatic alterations account for the dysregulation of dozens of cancer genes and pathways in each of 14 tumor contexts. To demonstrate proof of concept, we showed that perturbations targeting OIP5-AS1 (an inferred tumor suppressor) and TUG1 and WT1-AS (inferred onco-lncRNAs) dysregulated cancer genes and altered proliferation of breast and gynecologic cancer cells. Our analysis indicates that, although most lncRNAs are dysregulated in a tumor-specific manner, some, including OIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergistically dysregulate cancer pathways in multiple tumor contexts.

The prevailing paradigm is that cardiac ANG II is synthesized in the extracellular space from components of the circulating and/or local renin-angiotensin system. The recent discovery of intracrine effects of ANG II led us to determine whether ANG II is synthesized intracellularly in neonatal rat ventricular myocytes (NRVM). NRVM, incubated in serum-free medium, were exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent angiotensin type 1 (AT(1)) receptor-mediated internalization of ANG II. ANG II was measured in cell lysates and the culture medium, which represented intra- and extracellularly synthesized ANG II, respectively. Isoproterenol increased ANG II concentration in cell lysates and medium of NRVM in the absence or presence of candesartan. High glucose markedly increased ANG II synthesis only in cell lysates in the absence and presence of candesartan. Western analysis showed increased intracellular levels of angiotensinogen, renin, and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of ANG II in the cytoplasm and nucleus of high-glucose-exposed NRVM and along the actin filaments in isoproterenol-exposed cells. ANG II synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and angiotensin-converting enzyme in isoproterenol-exposed cells. In summary, the site of ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are stimulus dependent. Significantly, NRVM synthesized and retained ANG II intracellularly, which redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.

The occurrence of a functional intracellular renin-angiotensin system (RAS) has emerged as a new paradigm. Recently, we and others demonstrated intracellular synthesis of ANG II in cardiac myocytes and vascular smooth muscle cells that was dramatically stimulated in high glucose conditions. Cardiac fibroblasts significantly contribute to diabetes-induced diastolic dysfunction. The objective of the present study was to determine the existence of the intracellular RAS in cardiac fibroblasts and its role in extracellular matrix deposition. Neonatal rat ventricular fibroblasts were serum starved and exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent receptor-mediated uptake of ANG II. Under these conditions, an increase in ANG II levels in the cell lysate represented intracellular synthesis. Both isoproterenol and high glucose significantly increased intracellular ANG II levels. Confocal microscopy revealed perinuclear and nuclear distribution of intracellular ANG II. Consistent with intracellular synthesis, Western analysis showed increased intracellular levels of renin following stimulation with isoproterenol and high glucose. ANG II synthesis was catalyzed by renin and angiotensin-converting enzyme (ACE), but not chymase, as determined using specific inhibitors. High glucose resulted in increased transforming growth factor-beta and collagen-1 synthesis by cardiac fibroblasts that was partially inhibited by candesartan but completely prevented by renin and ACE inhibitors. In conclusion, cardiac fibroblasts contain a functional intracellular RAS that participates in extracellular matrix formation in high glucose conditions, an observation that may be helpful in developing an appropriate therapeutic strategy in diabetic conditions.

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Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

These studies suggest that the intracellular renin-angiotensin system is an important component of the local system. Alternative mechanisms of angiotensin II synthesis and action suggest a need for novel therapeutic agents to block the intracellular renin-angiotensin system.

RATIONALE Heat shock proteins (Hsps) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. OBJECTIVE To determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in Ca-cycling. METHODS and RESULTS Hsp20 overexpression resulted in significant enhancement of cardiac function in intact animals, coupled with augmented Ca-cycling and sarcoplasmic reticulum (SR) Ca-load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca-affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing non-phosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20-overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1/PLN axis. This hypothesis was supported by cell-fractionation, co-immunoprecipitation and co-immunolocalization studies, which revealed an association between Hsp20, PP1 and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73–160 in Hsp20 and AA 163–330 in PP1. CONCLUSIONS Hsp20 is a novel regulator of SR Ca-cycling by targeting the PP1/PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.