Jorge Espinoza-Derout

Electronic cigarettes (E-Cig) are a new way of delivering nicotine, gaining popularity among adolescents and young adults, who often do not realize their harmful effects. Although the adverse effects of E-Cigs on the liver and heart have been demonstrated, their effects on the skeletal muscle have not been well studied. In this study, we evaluated the skeletal muscle effects of E-Cig aerosol, delivered in a manner similar to human vaping, in a mouse model of obesity induced by a high-fat diet (HFD). C57BL/6 mice, fed either a normal chow diet (NCD) or HFD, were exposed to either saline aerosol control or aerosol generated from Blu PLUS^TM containing 0% or 2.4% nicotine for 12 weeks. Mice fed an NCD were included to distinguish whether E-Cig effects on the skeletal muscle required the presence of obesity induced by an HFD. The soleus muscle, an oxidative muscle rich in mitochondria, was assessed by Western blotting, electron microscopy, and biochemical assays. An NCD group was included to assess the baseline effects of HFD-induced obesity, on the skeletal muscle. The skeletal muscle from HFD-fed mice exposed to E-Cig 2.4% had reduced levels of phospho-AMPK compared with saline and E-Cig 0% groups, while E-Cigs had no effect on NCD-fed mice. Levels of phospho-adipose triglyceride lipase were also reduced in both E-Cig 2.4% and 0% compared with the saline group. These metabolic protein impairments were accompanied by increased levels of oxidative stress and phospho-p38 MAPK. Deregulation of the autophagy markers, microtubule-associated protein 1A/1B-light chain 3 (LC3-I; inactive form) and LC3-II (active form), was also observed, evidenced by decreased levels of LC3-II, ratio LC3-II/LC3-I, and increased levels of p62. Transmission electron microscopy analysis showed that E-Cig 2.4% induced damage to mitochondrial structure compared with the saline or E-Cig 0% groups. These findings suggest that E-Cig exposure on HFD impairs the skeletal muscle, adding to the growing list of affected organs for ongoing regulatory efforts concerning nicotine-containing substances.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by ubiquitous deficiency in the SMN protein. The identification of disease modifiers is key to understanding pathogenic mechanisms and broadening the range of targets for developing SMA therapies that complement SMN upregulation. Here, we report a cell-based screen that identified inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) as suppressors of proliferation defects induced by SMN deficiency in mouse fibroblasts. We further show that SMN deficiency induces p38 MAPK activation and that pharmacological inhibition of this pathway improves motor function in SMA mice through SMN-independent neuroprotective effects. Using a highly optimized p38 MAPK inhibitor (MW150) and combinatorial treatment in SMA mice, we observed synergistic enhancement of the phenotypic benefit induced by either MW150 or an SMN-inducing drug alone. By promoting motor neuron survival, pharmacological inhibition of p38 MAPK synergizes with SMN induction and enables enhanced synaptic rewiring of motor neurons within sensory-motor spinal circuits. These studies identify the p38 MAPK pathway as a therapeutic target and MW150 as a neuroprotective drug for combination therapy in SMA.

Electronic cigarettes (e-cigarettes) are a prevalent alternative to conventional nicotine cigarettes among smokers and people who have never smoked. Increased concentrations of serum free fatty acids (FFAs) are crucial in generating lipotoxicity. We studied the effects of acipimox, an antilipolytic drug, on e-cigarette-induced cardiac dysfunction. C57BL/6J wild-type mice on high fat diet were treated with saline, e-cigarette with 2.4% nicotine [e-cigarette (2.4%)], and e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction were diminished in mice exposed to e-cigarettes (2.4%) compared with saline and acipimox-treated mice. Mice exposed to e-cigarette (2.4%) had increased circulating levels of inflammatory cytokines and FFAs, which were diminished by acipimox. Gene Set Enrichment Analysis revealed that e-cigarette (2.4%)-treated mice had gene expression changes in the G2/M DNA damage checkpoint pathway that was normalized by acipimox. Accordingly, we showed that acipimox suppressed the nuclear localization of phospho-p53 induced by e-cigarette (2.4%). Additionally, e-cigarette (2.4%) increased the apurinic/apyrimidinic sites, a marker of oxidative DNA damage which was normalized by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1 protein levels and 4-hydroxynonenal (4-HNE). These markers of oxidative stress were decreased by acipimox. Therefore, inhibiting lipolysis with acipimox normalizes the physiological changes induced by e-cigarettes and the associated increase in inflammatory cytokines, oxidative stress, and DNA damage.

CARF (CDKN2AIP) regulates cellular fate in response to various stresses. However, its role in metabolic stress is unknown. We found that fatty livers from mice exhibit low CARF expression. Similarly, overloaded palmitate inhibited CARF expression in HepG2 cells, suggesting that excess fat-induced stress downregulates hepatic CARF. In agreement with this, silencing and overexpressing CARF resulted in higher and lower fat accumulation in HepG2 cells, respectively. Furthermore, CARF overexpression lowered the ectopic palmitate accumulation in HepG2 cells. We were interested in understanding the role of hepatic CARF and underlying mechanisms in the development of NAFLD. Mechanistically, transcriptome analysis revealed that endoplasmic reticulum (ER) stress and oxidative stress pathway genes significantly altered in the absence of CARF. IRE1α, GRP78, and CHOP, markers of ER stress, were increased, and the treatment with TUDCA, an ER stress inhibitor, attenuated fat accumulation in CARF-deficient cells. Moreover, silencing CARF caused a reduction of GPX3 and TRXND3, leading to oxidative stress and apoptotic cell death. Intriguingly, CARF overexpression in HFD-fed mice significantly decreased hepatic steatosis. Furthermore, overexpression of CARF ameliorated the aberrant ER function and oxidative stress caused by fat accumulation. Our results further demonstrated that overexpression of CARF alleviates HFD-induced insulin resistance assessed with ITT and GTT assay. Altogether, we conclude that excess fat-induced reduction of CARF dysregulates ER functions and lipid metabolism leading to hepatic steatosis.

Electronic cigarettes or e-cigarettes are the most frequently used tobacco product among adolescents. Despite the widespread use of e-cigarettes and the known detrimental cardiac consequences of nicotine, the effects of e-cigarettes on the cardiovascular system are not well-known. Severalin vitroandin vivostudies delineating the mechanisms of the impact of e-cigarettes on the cardiovascular system have been published. These include mechanisms associated with nicotine or other components of the aerosol or thermal degradation products of e-cigarettes. The increased hyperlipidemia, sympathetic dominance, endothelial dysfunction, DNA damage, and macrophage activation are prominent effects of e-cigarettes. Additionally, oxidative stress and inflammation are unifying mechanisms at many levels of the cardiovascular impairment induced by e-cigarette exposure. This review outlines the contribution of e-cigarettes in the development of cardiovascular diseases and their molecular underpinnings.

Electronic cigarettes (e-cigarettes), also known as electronic nicotine delivery systems, are a popular alternative to conventional nicotine cigarettes, both among smokers and those who have never smoked. In spite of the widespread use of e-cigarettes and the proposed detrimental cardiac and atherosclerotic effects of nicotine, the effects of e-cigarettes on these systems are not known. In this study, we investigated the cardiovascular and cardiac effects of e-cigarettes with and without nicotine in apolipoprotein-E knockout (ApoE^−/−) mice. We developed an e-cigarette exposure model that delivers nicotine in a manner similar to that of human e-cigarettes users. Using commercially available e-cigarettes, bluCig PLUS, ApoE^−/− mice were exposed to saline, e-cigarette without nicotine [e-cigarette (0%)], and e-cigarette with 2.4% nicotine [e-cigarette (2.4%)] aerosol for 12 wk. Echocardiographic data show that mice treated with e-cigarette (2.4%) had decreased left ventricular fractional shortening and ejection fraction compared with e-cigarette (0%) and saline. Ventricular transcriptomic analysis revealed changes in genes associated with metabolism, circadian rhythm, and inflammation in e-cigarette (2.4%)-treated ApoE^−/− mice. Transmission electron microscopy revealed that cardiomyocytes of mice treated with e-cigarette (2.4%) exhibited ultrastructural abnormalities indicative of cardiomyopathy. Additionally, we observed increased oxidative stress and mitochondrial DNA mutations in mice treated with e-cigarette (2.4%). ApoE^−/− mice on e-cigarette (2.4%) had also increased atherosclerotic lesions compared with saline aerosol-treated mice. These results demonstrate adverse effects of e-cigarettes on cardiac function in mice. NEW & NOTEWORTHY The present study is the first to show that mice exposed to nicotine electronic cigarettes (e-cigarettes) have decreased cardiac fractional shortening and ejection fraction in comparison with controls. RNA-seq analysis reveals a proinflammatory phenotype induced by e-cigarettes with nicotine. We also found increased atherosclerosis in the aortic root of mice treated with e-cigarettes with nicotine. Our results show that e-cigarettes with nicotine lead to detrimental effects on the heart that should serve as a warning to e-cigarette users and agencies that regulate them.

The prevalence of electronic cigarette (e-cigarettes) use has rapidly increased in the world. Use of tobacco products has been associated with DNA damage and metabolic syndrome. Using Apolipoprotein E knockout (ApoE-/-) mice on a western diet (WD), a mouse model of nonalcoholic fatty liver disease (NAFLD), we recently demonstrated that nicotine in e-cigarettes activates hepatocyte apoptosis, and causes hepatic steatosis. This study examines the harmful effects of e-cigarettes on the liver with a special emphasis on DNA damage and mitochondrial dysfunction. ApoE-/- mice were exposed to saline, e-cigarettes without nicotine or e-cigarettes with 2.4% nicotine for 12 weeks using our newly developed mouse e-cigarette exposure model system that delivers nicotine to mice leading to equivalent serum cotinine levels found in human cigarette users. Mice exposed to e-cigarette (2.4%) had increased apurinic/apyrimidinic (AP) sites, a manifestation of DNA damage. Additionally, e-cigarette (2.4%) produced a decrease in NAD+/NADH ratio in hepatic cells, in comparison with saline and e-cigarette (0%). Western blot analysis showed that mice treated with e-cigarette (2.4%) had increased poly (ADP ribose) polymerase (PARP1) activity associated with reduced levels of Sirtuin 1 (SIRT1). Furthermore, hepatic oxidative stress and mitochondrial DNA mutations, as well as hepatic PTEN-induced kinase 1 (PINK1) were increased in mice treated with e-cigarette (2.4%). Transmission electron microscopy revealed that hepatocytes of mice treated with e-cigarette (2.4%) exhibited increased vacuolization of the mitochondria and a reduction in cellular organelles. These results demonstrate the adverse effects of e-cigarettes exposure leading to NAD+ deficiency which may suggest a mechanistic link between e-cigarette-induced hepatic DNA damage and mitochondrial dysfunction.

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by homozygous inactivation of the SMN1 gene and reduced levels of the survival motor neuron (SMN) protein. Since higher copy numbers of the nearly identical SMN2 gene reduce disease severity, to date most efforts to develop a therapy for SMA have focused on enhancing SMN expression. Identification of alternative therapeutic approaches has partly been hindered by limited knowledge of potential targets and the lack of cell-based screening assays that serve as readouts of SMN function. Here, we established a cell system in which proliferation of cultured mouse fibroblasts is dependent on functional SMN produced from the SMN2 gene. To do so, we introduced the entire human SMN2 gene into NIH3T3 cell lines in which regulated knockdown of endogenous mouse Smn severely decreases cell proliferation. We found that low SMN2 copy number has modest effects on the cell proliferation phenotype induced by Smn depletion, while high SMN2 copy number is strongly protective. Additionally, cell proliferation correlates with the level of SMN activity in small nuclear ribonucleoprotein assembly. Following miniaturization into a high-throughput format, our cell-based phenotypic assay accurately measures the beneficial effects of both pharmacological and genetic treatments leading to SMN upregulation. This cell model provides a novel platform for phenotypic screening of modifiers of SMN2 gene expression and function that act through multiple mechanisms, and a powerful new tool for studies of SMN biology and SMA therapeutic development.