HuR-dependent expression of Wisp1 is necessary for TGFß-induced cardiac myofibroblast activity.

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.

Calcium cycling as a mediator of thermogenic metabolism in adipose tissue.

Canonical non-shivering thermogenesis (NST) in brown and beige fat relies on uncoupling protein 1 (UCP1)-mediated heat generation, although alternative mechanisms of NST have been identified, including sarcoplasmic reticulum (SR)-calcium cycling. Intracellular calcium is a crucial cell signaling molecule for which compartmentalization is tightly regulated, and the sarco-endoplasmic calcium ATPase (SERCA) actively pumps calcium from the cytosol into the SR. In this review, we discuss the capacity of SERCA-mediated calcium cycling as a significant mediator of thermogenesis in both brown and beige adipocytes. Here, we suggest two primary mechanisms of SR calcium mediated thermogenesis. The first mechanism is through direct uncoupling of the ATPase and calcium pump activity of SERCA, resulting in the energy of ATP catalysis being expended as heat in the absence of calcium transport. Regulins, a class of SR membrane proteins, act to decrease the calcium affinity of SERCA and uncouple the calcium transport function from ATPase activity, but remain largely unexplored in adipose tissue thermogenesis. A second mechanism is through futile cycling of SR calcium whereby SERCA-mediated SR calcium influx is equally offset by SR calcium efflux, resulting in ATP consumption without a net change in calcium compartmentalization. A fuller understanding of the functional and mechanistic role of calcium cycling as a mediator of adipose tissue thermogenesis and how manipulation of these pathways can be harnessed for therapeutic gain remains unexplored. Significance Statement Enhancing thermogenic metabolism in brown or beige adipose tissue may be of broad therapeutic utility to reduce obesity and metabolic syndrome. Canonical BAT-mediated thermogenesis occurs via uncoupling protein 1 (UCP1). However, UCP1-independent pathways of thermogenesis, such as sarcoplasmic (SR) calcium cycling, have also been identified, but the regulatory mechanisms and functional significance of these pathways remain largely unexplored. Thus, this mini-review discusses the state of the field with regard to calcium cycling as a thermogenic mediator in adipose tissue.

Adipocyte-specific deletion of HuR induces spontaneous cardiac hypertrophy and fibrosis.

Adipose tissue homeostasis plays a central role in cardiovascular physiology, and the presence of thermogenically active brown adipose tissue (BAT) has recently been associated with cardiometabolic health. We have previously shown that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) reduces BAT-mediated adaptive thermogenesis, and the goal of this work was to identify the cardiovascular impacts of Adipo-HuR-/-. We found that Adipo-HuR-/- mice exhibit a hypercontractile phenotype that is accompanied by increased left ventricle wall thickness and hypertrophic gene expression. Furthermore, hearts from Adipo-HuR-/- mice display increased fibrosis via picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene coexpression network analysis (WGCNA) across both cardiac and adipose tissue to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and cardiac fibrosis. RNA-seq results demonstrated a significant increase in proinflammatory gene expression in both cardiac and subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF-α and IL-6. In addition to inflammation-related genes, WGCNA identified a significant enrichment in extracellular vesicle-mediated transport and exosome-associated genes in scWAT, whose expression most significantly associated with the degree of cardiac fibrosis observed in Adipo-HuR-/- mice, implicating these processes as a likely adipose-to-cardiac paracrine mechanism. These results are significant in that they demonstrate the spontaneous onset of cardiovascular pathology in an adipose tissue-specific gene deletion model and contribute to our understanding of how disruptions in adipose tissue homeostasis may mediate cardiovascular disease.NEW & NOTEWORTHY The presence of functional brown adipose tissue in humans is known to be associated with cardiovascular health. Here, we show that adipocyte-specific deletion of the RNA binding protein HuR, which we have previously shown to reduce BAT-mediated thermogenesis, is sufficient to mediate a spontaneous development of cardiac hypertrophy and fibrosis. These results may have implications on the mechanisms by which BAT function and adipose tissue homeostasis directly mediate cardiovascular disease.

Mechanisms linking adipose tissue inflammation to cardiac hypertrophy and fibrosis.

Adipose tissue is classically recognized as the primary site of lipid storage, but in recent years has garnered appreciation for its broad role as an endocrine organ comprising multiple cell types whose collective secretome, termed as adipokines, is highly interdependent on metabolic homeostasis and inflammatory state. Anatomical location (e.g. visceral, subcutaneous, epicardial etc) and cellular composition of adipose tissue (e.g. white, beige, and brown adipocytes, macrophages etc.) also plays a critical role in determining its response to metabolic state, the resulting secretome, and its potential impact on remote tissues. Compared with other tissues, the heart has an extremely high and constant demand for energy generation, of which most is derived from oxidation of fatty acids. Availability of this fatty acid fuel source is dependent on adipose tissue, but evidence is mounting that adipose tissue plays a much broader role in cardiovascular physiology. In this review, we discuss the impact of the brown, subcutaneous, and visceral white, perivascular (PVAT), and epicardial adipose tissue (EAT) secretome on the development and progression of cardiovascular disease (CVD), with a particular focus on cardiac hypertrophy and fibrosis.

HuR-dependent expression of RyR2 contributes to calcium-mediated thermogenesis in brown adipocytes.

Several uncoupling protein 1 (UCP1)-independent thermogenic pathways have been described in thermogenic adipose tissue, including calcium-mediated thermogenesis in beige adipocytes via sarco/endoplasmic reticulum ATPase (SERCA). We have previously shown that adipocyte-specific deletion of the RNA binding protein human antigen R (HuR) results in thermogenic dysfunction independent of UCP1 expression. RNA sequencing revealed the downregulation of several genes involved in calcium ion transport upon HuR deletion. The goal of this work was to define the HuR-dependent mechanisms of calcium driven thermogenesis in brown adipocytes. We generated (BAT)-specific HuR-deletion (BAT-HuR -/- ) mice and show that their body weight, glucose tolerance, brown and white adipose tissue weights, and total lipid droplet size were not significantly different compared to wild-type. Similar to our initial findings in Adipo-HuR -/- mice, mice with BAT-specific HuR deletion are cold intolerant following acute thermal challenge at 4°C, demonstrating specificity of acute HuR-dependent thermogenesis to BAT. We also found decreased expression of ryanodine receptor 2 (RyR2), but no changes in RyR2, SERCA1, SERCA2, or UCP1 expression, in BAT from BAT-HuR -/- mice. Next, we used Fluo-4 calcium indicator dye to show that genetic deletion or pharmacological inhibition of HuR blunts the increase in cytosolic calcium concentration in SVF-derived primary brown adipocytes. Moreover, we saw a similar blunting in ß-adrenergic-mediated heat generation, as assessed by ERtherm AC fluorescence, in SVF-derived brown adipocytes following HuR inhibition or deletion. Mechanistically, we show that HuR directly binds and reduces the decay rate of RyR2 mRNA in brown adipocytes, and stabilization of RyR2 via S107 rescues ß-adrenergic-mediated cytosolic calcium increase and heat generation in HuR deficient brown adipocytes. In conclusion, our results suggest that HuR-dependent control of RyR2 expression plays a significant role in the thermogenic function of brown adipose tissue through modulation of SR calcium cycling.