Dysregulation of endoplasmic reticulum and mitochondrial (ER:Mit) contacts and mitochondrial calcium (mitCa2+) homeostasis are found in several neurodegenerative disorders, including Ataxia Telangiectasia (A-T). However, the cellular basis of these defects remains unclear. Previously, we demonstrated that the aberrantly elevated Interferon-Stimulated Gene 15 (ISG15) pathway inhibits protein polyubiquitylation, its dependent protein turnover, and mitophagy pathways in A-T. Literature indicates that silencing of mitochondrial ubiquitin ligase 1 (MUL1) stabilizes mitofusin2 (MFN2) and attenuates mitCa2+ uptake from ER to Mit (mitCa2+influx) in primary neurons. We have replicated these findings in apparently healthy fibroblasts. We hypothesized that elevated ISG15 may inhibit ubiquitin-dependent MUL1-mediated degradation of MFN2 and dysregulate ER:Mit contacts and mitCa2+ homeostasis in A-T fibroblasts. Concurrently, MFN2 is stabilized in A-T, MUL1-silenced A-T, MUL1/ISG15-silenced A-T vs ISG15-silenced A-T fibroblasts. Moreover, the number of ER:Mit contacts is increased in A-T vs ISG15-silenced A-T fibroblasts. Notably, mitCa2+efflux is significantly attenuated in A-T vs ISG15-silenced A-T fibroblasts in which mitCa2+efflux is restored to levels comparable to those observed in normal fibroblasts. The mitCa2+efflux remains attenuated in MUL1 and MUL1/ISG15-silenced A-T fibroblasts. We conclude that ISG15 impairs MUL1/MFN2-mediated regulation of ER:Mit contacts and attenuates mitCa2+efflux, which may, in turn, cause Ca2+ overload-mediated mitochondrial damage in A-T. These findings suggest that ISG15 silencers may correct mitochondrial abnormalities and improve mitochondrial health in A-T patients and in those with other neurodegenerative disorders in which ISG15 is elevated, such as ALS.

Traumatic brain injury (TBI) and alcohol misuse are inextricably linked and can increase the risk for development of neurodegenerative diseases, particularly in military veterans and contact sport athletes. Proteinopathy (defects in protein degradation) is considered an underlying factor in neurodegenerative diseases. Whether it contributes to TBI/alcohol-mediated neurodegeneration is unexplored, however. Our recent studies have identified ISGylation, a conjugated form of ISG15 (Interferon-Stimulated Gene 15) and inducer of proteinopathy, as a potential mechanistic link underlying TBI-mediated neurodegeneration and proteinopathy in veterans. In the current study, a rat model of combined TBI and alcohol use was utilized to investigate the same relationship. Here, we report sustained induction of Interferon β (IFNβ), changes in TAR DNA Binding 43 (TDP-43) ISGylation levels, TDP-43 proteinopathy (C-terminal fragmentation [CTF]), and neurodegeneration in the ventral horns of the lumbar spinal cords (LSCs) and/or motor cortices (MCs) of female rats post-TBI in a time-dependent manner. In males, these findings mostly remained non-significant, although moderate alcohol use appears to decrease neurodegeneration in males (but not females) post-TBI. We, however, do not claim that moderate alcohol consumption is beneficial for preventing TBI-mediated neurodegeneration. We have previously demonstrated that ISGylation is increased in the LSCs of veterans with TBI/ALS (amyotrophic lateral sclerosis). Here, we show increased ISGylation of TDP-43 in the LSCs of TBI/ALS-afflicted female veterans compared with male veterans. Knowing that ISGylation induces proteinopathy, we suggest targeting ISGylation may prevent proteinopathy-mediated neurodegeneration post-TBI, particularly in women; however, causal studies are required to confirm this claim.

Cytokine-driven hyper inflammation has been identified as a critical factor behind poor outcomes in patients severely infected with SARS-CoV-2 virus. Notably, protein ISGylation, a protein conjugated form of Type 1 IFN-inducible ubiquitin-like protein ISG15 (Interferon-Stimulated Gene 15), induces cytokine storm (CS) and augments colonic inflammation in colitis-associated colon cancers in mouse models. However, whether ISGylation is increased and causally responsible for CS and hyper inflammation in symptomatic COVID-19 patients is unknown. Here, we measured ISGylation levels in peripheral blood mononuclear cells (PBMCs) from 10 symptomatic (SARS-CoV-2-positive with symptoms) and asymptomatic (SARS-CoV-2-positive with no symptoms) COVID-19 patients, and 4 uninfected individuals (SARS-CoV-2-negative), using WesTm assay. Strikingly, we note significant increases in protein ISGylation and MX-1 (myxovirus-resistance protein-1) protein levels, both induced by type-I IFN, in symptomatic but not in asymptomatic patients and uninfected individuals. Knowing that ISGylation augments CS and intestinal inflammation in colon cancers, we propose that increased ISGylation may be an underlying cause of CS and inflammation in symptomatic patients.

Chronic alcohol consumption is well known to contribute to an array of health complications and increases the incidence and severity of acute respiratory distress syndrome (ARDS), an acute lung injury marked by severe pulmonary edema and impairment of normal gas exchange. Despite advancements in understanding its pathogenesis, the mortality rate for ARDS remains over 40% and there are currently no effective pharmacological treatments for ARDS. Chronic alcohol consumption contributes to the pathogenesis of ARDS by altering the tight junction composition of the pulmonary epithelium which is essential to maintain normal barrier function. In this study, we investigate the use of a pharmacological peptide, aCT1, which mimics the cytoplasmic carboxy-terminal PDZ binding motif of the gap junction protein Connexin43 (Cx43) to disrupt endogenous Cx43 from binding scaffold proteins, including the tight junction protein Zonula Occludens-1 (ZO-1). By inhibiting ZO-1 binding to Cx43, we hypothesize that more ZO-1 is available to associate with tight junction proteins, including claudins, to enhance epithelial barrier function. We have previously shown that chronic alcohol use exacerbates acute endotoxin-mediated lung injury by compromising both the alveolar and pulmonary capillary epithelial barriers. Here, we tested whether treatment with aCT1 strengthens alveolar barrier function and mitigates the effects of alcohol-mediated impairment of tight junctions. Male C57BL/6J mice were pair-fed 20% ethanol or water for 14 weeks to establish a chronic alcohol model in vivo. Mice were sedated and given an intratracheal instillation of 5mg/kg of the bacterial endotoxin lipopolysaccharide (LPS) derived from E. coli O55:B5 to induce a second, direct insult in the alcohol-primed lung to impact the lung epithelium. Mice were administered via nebulizer 1h post-LPS instillation either 5mg/kg of the aCT1 peptide or a scrambled control. Evans Blue (EB) dye was injected into mouse tail veins 24h after LPS instillation. To measure alveolar flooding, bronchoalveolar lavage fluid (BAL) was collected 1h later and EB in BAL was measured and normalized to serum EB. We found that administration of aCT1 decreased the leakage of EB dye from the blood into the alveolar air space in both water-fed and alcohol-fed mice, thus improving lung barrier function. This improvement in pulmonary leak suggests that aCT1 has the capacity to mitigate ARDS. Further investigation will determine whether aCT1 has a positive impact on short-term mortality rates due to endotoxemia and whether it can improve pulmonary barrier dysfunction due to other causes of acute lung injury.

Mitophagy is defective in several neurodegenerative diseases, including Ataxia Telangiectasia (A-T). However, the molecular mechanism underlying defective mitophagy in A-T is unknown. Literature indicates that damaged mitochondria are transported to the perinuclear region prior to their removal via mitophagy. Our previous work has indicated that conjugation of SUMO2 (Small Ubiquitin-like Modifier 2) to mitofusins (Mfns) may be necessary for congression of mitochondria into SUMO2-/ubiquitin-/LC3-positive compact structures resembling mito-aggresomes at the perinuclear region in CCCP-treated HEK293 cells. Here, we demonstrate that Mfns are SUMOylated, and mitochondria are transported to the perinuclear region; however, mitochondria fail to congress into mito-aggresome-like structures in CCCP-treated A-T cells. Defect in mitochondrial congression is causally related to constitutively elevated ISG15 (Interferon-Stimulated Gene 15), an antagonist of the ubiquitin pathway, in A-T cells. Suppression of the ISG15 pathway restores mitochondrial congression, reduce oxidative stress, and level of unhealthy mitochondria, which is suggestive of restoration of mitophagy in A-T cells. ISG15 is also constitutively elevated and mitophagy is defective in Amytrophic Lateral Sclerosis (ALS). The constitutively elevated ISG15 pathway therefore appears to be a common unifying biochemical mechanism underlying defective mitophagy in neurodegenerative disorders thus, implying the broader significance of our findings, and suggest the potential role of ISG15 inhibitors in their treatment.

Depolarized/damaged mitochondria aggregate at the perinuclear region prior to mitophagy in cells treated with mitochondrial stressors. However, the cellular mechanism(s) by which damaged mitochondria are transported and remain aggregated at the perinuclear region is unknown. Here, we demonstrate that mitofusins (Mfn1/2) are post-translationally modified by SUMO2 (Small Ubiquitin-related Modifier 2) in Human embryonic kidney 293 (Hek293) cells treated with protonophore CCCP and proteasome inhibitor MG132, both known mitochondrial stressors. SUMOylation of Mfn1/2 is not for their proteasomal degradation but facilitate mitochondrial congression at the perinuclear region in CCCP- and MG132-treated cells. Additionally, congressed mitochondria (mito-aggresomes) colocalize with LC3, ubiquitin, and SUMO2 in CCCP-treated cells. Knowing that SUMO functions as a "molecular glue" to facilitate protein-protein interactions, we propose that SUMOylation of Mfn1/2 may congress, glues, and confines damaged mitochondria to the perinuclear region thereby, protectively quarantining them from the heathy mitochondrial network until their removal via mitophagy in cells.

Veterans who have served in the military are at a nearly 60% greater risk of being diagnosed with amyotrophic lateral sclerosis (ALS). Literature reports suggest that a history of traumatic brain injury (TBI) may be a risk factor for ALS in veterans. However, no diagnostic biomarkers are available for identifying ALS risk/development in TBI-exposed veterans. Here, using a Wes assay, we show that ISGylation, a conjugated form of interferon-stimulated gene 15 protein, is significantly elevated in the lumbar spinal cords (SC-Ls) of TBI-ALS compared with ALS veterans without a previous history of TBI (nonTBI-ALS). Although not as striking as in TBI-ALS veterans, ISGylation is also increased in nonTBI-ALS compared with normal veterans. Notably, no changes in ISGylation were seen in occipital lobe samples obtained from the same patients, suggesting that elevated ISGylation is distinct to ALS disease-specific SC-Ls. Moreover, we detected increased ISGylation in cerebral spinal fluid samples of TBI-ALS veterans. Other results using cultured lymphocyte cell lines show a similar trend of increased ISGylation in ALS patients from the general population. Together, these data suggest that ISGylation could serve as a diagnostic biomarker for TBI-ALS veterans, nonTBI-ALS veterans, and nonveterans affected by ALS.

Interferon-Stimulated Gene 15 (ISG15), an antagonist of the canonical ubiquitin pathway, is frequently overexpressed in various cancers. In cancer cells, ISG15 is detected as free (intracellular) and conjugated to cellular proteins (ISGylation). Free ISG15 is also secreted into the extracellular milieu. ISGylation has protumor functions and extracellular free ISG15 has immunomodulatory properties in vitro. Therefore, whether ISG15 is a tumor suppressor or tumor promoter in vivo remains controversial. The current study aimed to clarify the role of free ISG15 in tumorigenesis. Breast cancer cells stably expressing control, ISG15, and UbcH8 (ISG15-specific E2 ligase) shRNAs were used to assess the immunoregulatory and antitumor function of free ISG15 in cell culture ( in vitro) and in nude mice (in vivo). We show that extracellular free ISG15 suppresses breast tumor growth and increases NK cell infiltration into xenografted breast tumors in nude mice, and intracellular free ISG15 enhances major histocompatibility complex (MHC) class I surface expression in breast cancer cells. We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo. These findings provides the basis for developing strategies to increase systemic levels of free ISG15 to treat cancer patients overexpressing the ISG15 pathway.

Aberrant expression of the oncogenic Kirsten-Ras (Ki-Ras) and interferon-stimulated gene 15 (ISG15) pathways is common in breast and other cancers. However, whether these dysregulated pathways cooperate to promote malignancy is not known. This study links Ki-Ras and ISG15 in a previously unidentified regulatory loop that may underlie malignant transformation of mammary cells. We show that oncogenic Ki-Ras regulates the expression of the ISG15 pathway (free ISG15 and ISG15 conjugates), and ISG15, in turn, stabilizes Ki-Ras protein by inhibiting its targeted degradation via lysosomes in breast cancer cells. Disruption of this loop by silencing either Ki-Ras or the ISG15 pathway restored the disrupted cellular architecture, a hallmark feature of most cancer cells. We also demonstrate that ISG15 and UbcH8 (ISG15-specific conjugating enzyme) shRNAs reversed Ki-Ras mutation-associated phenotypes of cancer cells, such as increased cell proliferation, colony formation, anchorage-independent growth in soft agar, cell migration, and epithelial–mesenchymal transition. As UbcH8-silenced breast cancer cells are devoid of ISG15 conjugates but have free ISG15, our results using UbcH8-silenced cells suggest that ISG15 conjugates, and not free ISG15, contributes to oncogenic Ki-Ras transformation. We have thus identified the conjugated form of ISG15 as a critical downstream mediator of oncogenic Ki-Ras, providing a potential mechanistic link between ISG15 and Ki-Ras-mediated breast tumorigenesis. Our findings, which show that inhibition of the ISGylation reverses the malignant phenotypes of breast cancer cells expressing oncogenic Ki-Ras, support the development of ISG15 conjugation inhibitors for treating breast and also other cancers expressing oncogenic Ki-Ras.

Ataxia-telangiectasia (A-T) is a cerebellar neurodegenerative disorder; however, the basis for the neurodegeneration in A-T is not well established. Lesions in the ubiquitin and autophagy pathways are speculated to contribute to the neurodegeneration in other neurological diseases and may have a role in A-T neurodegeneration. Our recent studies revealed that the constitutively elevated ISG15 pathway impairs targeted proteasome-mediated protein degradation in A-T cells. Here, we demonstrate that the basal autophagy pathway is activated in the ubiquitin pathway-compromised A-T cells. We also show that genotoxic stress triggers aberrant degradation of the proteasome and autophagy substrates (autophagic flux) in A-T cells. Inhibition of autophagy at an early stage using 3-methyladenine blocked UV-induced autophagic flux in A-T cells. On the other hand, bafilomycin A1, which inhibits autophagy at a late stage, failed to block UV-induced autophagic flux, suggesting that overinduction of autophagy may underlie aberrant autophagic flux in A-T cells. The ISG15-specific shRNA that restored proteasome function restores autophagic function in A-T cells. These findings suggest that autophagy compensates for the ISG15-dependent ablation of proteasome-mediated protein degradation in A-T cells. Genotoxic stress overactivates this compensatory mechanism, triggering aberrant autophagic flux in A-T cells. Supporting the model, we show that autophagy is activated in the brain tissues of human A-T patients. This highlights a plausible causal contribution of a novel "ISG15 proteinopathy" in A-T neuronal cell death.

The interferon-stimulated gene 15 (ISG15) pathway is highly elevated in breast cancer; however, very little is known about how the ISG15 pathway contributes to breast tumorigenesis. In the current study, using the gene disruption approach, we demonstrate that both ISG15 and UbcH8 (ISG15-specific conjugating enzyme) disrupt F-actin architecture and formation of focal adhesions in ZR-75-1 breast cancer cells. In addition, ISG15 and UbcH8 promote breast cancer cell migration. We also demonstrate that ISG15 inhibits ubiquitin/26S proteasome-mediated turnover of proteins implicated in tumor cell motility, invasion and metastasis. Together, our results suggest that the aberrant activation of the ISG15 pathway confers a motile phenotype to breast cancer cells by disrupting cell architecture and stabilizing proteins involved in cell motility, invasion and metastasis. Because the cellular architecture is conserved and the ISG15 pathway is constitutively activated in tumor cells of different lineages, it is reasonable to assume that our observations in breast cancer must hold true for many other tumors.

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

We have recently shown that the inability of repetitive ischemia (RI) to activate p38 MAPK (p38) and Akt in metabolic syndrome [JCR:LA-cp (JCR)] rats was associated with impaired coronary collateral growth (CCG). Furthermore, Akt and p38 activation correlated with optimal O(2)(-). levels and were altered in JCR rats, and redox-sensitive p38 activation was required for CCG. Here, we determined whether the activation of Src, a possible upstream regulator, was altered in JCR rats and whether redox-dependent Src and Akt activation were required for CCG. CCG was assessed by myocardial blood flow (microspheres) and kinase activation was assessed by Western blot analysis in the normal zone and collateral-dependent zone (CZ). RI induced Src activation (approximately 3-fold) in healthy [Wistar-Kyoto (WKY)] animals but not in JCR animals. Akt inhibition decreased (approximately 50%), and Src inhibition blocked RI-induced CCG in WKY rats. Src inhibition decreased p38 and Akt activation. Myocardial oxidative stress (O(2)(-). and oxidized/reduced thiols) was measured quantitatively (X-band electron paramagnetic resonance). An antioxidant, apocynin, reduced RI-induced oxidative stress in JCR rats to levels induced by RI in WKY rats versus the reduction in WKY rats to very low levels. This resulted in a significant restoration of p38 (approximately 80%), Akt (approximately 65%), and Src (approximately 90%) activation in JCR rats but decreased the activation in WKY rats (p38: approximately 45%, Akt: approximately 65%, and Src: approximately 100%), correlating with reduced CZ flow in WKY rats (approximately 70%), but significantly restored CZ flow in JCR rats (approximately 75%). We conclude that 1) Akt and Src are required for CCG, 2) Src is a redox-sensitive upstream regulator of RI-induced p38 and Akt activation, and 3) optimal oxidative stress levels are required for RI-induced p38, Akt, and Src activation and CCG.

Cardiovascular disease is the major cause of worldwide morbidity and mortality. This is the first study to investigate the cardioprotective effects of folic acid (FA) on left ventricular remodeling in response to acute volume overload generated by aorto‐caval fistula (ACF) Adult male Sprague‐Dawley rats were subjected to ACF for 72 hr ± pretreatment with folic acid (30 mg/kg/day) along with sham operated controls. Echocardiography and in vivo pressure volume loops indicated left ventricular (LV) dilation in ACF vs. sham that was associated with oxidative stress (decreased GSH/GSSG ratio), extracellular matrix (ECM) degradation and apoptosis. Interestingly, TUNEL‐positive cells resided only in the microvascular component of the ACF LV. Endothelial cell apoptosis lead to decreased LV capillary density, whereas ECM degradation in response to ACF was associated with mast cell degranulation and increased MT1‐MMP expression compared to sham. Folic acid supplementation remarkably reversed LV dilation, prevented apoptosis, decreased oxidative stress, and increased both VEGF isoform expression and capillary density despite continued volume overload induced by ACF. The prominent cardioprotective effects of FA may be due to its potent antioxidant and proangiogenic activities.

Hyperhomocysteinemia is a risk factor for atherosclerosis. This study investigated molecular mechanisms by which homocysteine alters rat aortic vascular smooth muscle cell (VSMC) function. Low‐passage VSMC were treated for 24 hr with a pathophysiological level of homocysteine (150 μM). Paradoxically, homocysteine increased both VSMC proliferation and apoptosis. Proteomic analysis indicated that homocysteine induced a marked increased in cofilin and galectin‐1. Interestingly, homocysteine‐induced VSMC motility was associated with high expression of cofilin. On the other hand, immunocytochemistry results showed that the expression of galectin‐1 is co‐localized to apoptotic cells. The link between galectin‐1 and apoptosis was related to high expression of the proapoptotic Bax‐1. Conversely, the apoptotic effect of homocysteine involved activation of caspase‐3 and PARP cleavage. These results demonstrate for the first time that the proliferative and migratory responses to homocysteine were associated with high expression of cofilin, whereas the apoptotic pathway was associated with high levels of galectin‐1 and increased Bax‐1 expression, caspase‐3 activation and PARP cleavage to its active form. This novel finding may form a basis for new therapeutic strategies for treatment of atherosclerosis. Supported by HL056046 and P20RR18766 (PAL).

Coronary artery (CA) remodeling may lead to increased arterial stiffness and decreased coronary flow reserve. The molecular mechanisms that dictate diabetes‐induced CA remodeling are undefined. This study established a link between oxidative stress, angiotensin II (Ang II), CA remodeling and stiffness in Type 2 diabetic mice. CA (<100 μ) were isolated from 16 wk heterozygous (Db/db) and diabetic (db/db) mice ± treatment with the NADPH‐oxidase inhibitor apocynin (5mM) or the AT1R blocker candesartan (1mg/kg/day). Mean arterial pressure, measured by telemetry, was similar between groups. CA were mounted on a pressure myograph for measurement of structural and passive mechanical properties. Significant inward remodeling was detected in db/db vs. Db/db, defined by increased wall thickness, media:lumen ratio, remodeling index and decreased compliance. This inward remodeling in db/db CA was associated with increased oxidative stress and collagen accumulation as measured by DHE and picrosirius red staining respectively. Immunoblot analysis indicted that basal JNK, ERK, and AKT phosphorylation were increased in db/db CA tissue lysates vs. Db/db. In vivo treatment with apocynin or candesartan reduced inward CA remodeling. These data suggest that increased oxidative stress and Ang II is critical for altered structural and functional remodeling of CA in Type 2 diabetes. Supported by HL056046 and P20RR18766 (PAL).

Reactive oxygen species (ROS) are implicated in coronary collateral growth (CCG). We evaluated the requirement for ROS in human coronary artery endothelial cell (HCAEC) tube formation, CCG in vivo, and signaling (p38 MAP kinase) by which ROS may stimulate vascular growth. The flavin-containing oxidase inhibitor diphenyleneiodonium (DPI) or the superoxide dismutase inhibitor diethyldithiocarbamate (DETC) blocked vascular endothelial growth factor-induced HCAEC tube formation in Matrigel. We assessed the effect of DPI and DETC on CCG in a rat model of repetitive ischemia (RI) (40 s left anterior descending coronary artery occlusion every 20 min for 2 h 20 min, 3 times/day, 10 days). DPI or DETC was given intraperitoneally, or the NAD(P)H oxidase inhibitor apocynin was given in drinking water. Collateral-dependent flow (measured by using microspheres) was expressed as a ratio of normal and ischemic zone flows. In sham-operated rats, collateral flow in the ischemic zone was 18 +/- 6% of normal zone; in the RI group, collateral flow in the ischemic zone was 83 +/- 5% of normal zone. DPI prevented the increase in collateral flow after RI (25 +/- 4% of normal zone). Similar results were obtained with apocynin following RI (32 +/- 7% of that in the normal zone). DETC achieved similar results (collateral flow after RI was 21 +/- 2% of normal zone). DPI and DETC blocked RI-induced p38 MAP kinase activation in response to vascular endothelial growth factor and RI. These results demonstrate a requirement for optimal ROS concentration in HCAEC tube formation, CCG, and p38 MAP kinase activation. p38 MAP kinase inhibition prevented HCAEC tube formation and partially blocked RI-induced CCG (42 +/- 7% of normal zone flow), indicating that p38 MAP kinase is a critical signaling mediator of CCG.

AT1R blockers reportedly inhibit or promote angiogenesis. Since coronary collateral growth (CCG) is redox-sensitive, we hypothesized that controversial effects of angiotensin II (AII) are due to boundaries of redox signaling which regulate CCG. We stimulated CCG in normal (WKY) and metabolic syndrome (JCR) rats by transient, repetitive ischemia (RI) for 10 days. Blood flow (microspheres, ml/min/g) was measured in normal (NZ) and collateral-dependent (CZ) zones. In WKY, RI increased CZ flow (CZF) (0.13 to 0.84), but RI+sAII (subpressor dose of AII) increased CZF more (0.13 to 1.42). In contrast, a hypertensive dose of AII (hAII) decreased CZF compared to RI (0.12 to 0.69 [RI+hAII]). Candesartan also abrogated CZF in WKY (0.12 to 0.56). RI CZF in JCR was significantly decreased compared to WKY (0.09 to 0.12), and was associated with a large increase in superoxide (DHE). CZF in JCR was partially restored by candesartan (0.09 to 0.45), accompanied by reduction in superoxide comparable to WKY+RI. Activation of p38 was directly related to CCG; when non-existent, there was no CCG. Thus, AII regulates CCG via optimal amounts of ROS and activation of redox-sensitive signaling. With normal oxidative stress (WKY), AT1R blockade decreases p38 activation and RI-induced CCG; with elevated basal oxidative stress (JCR), AT1R blockade rescues RI-induced CCG, by normalizing oxidative stress and activating p38. Supported by P20RR18766 (PR).

Repeated administration of MDMA (ecstasy) produces cardiac toxicity including eccentric left ventricular (LV) dilation and systolic dysfunction. While the mechanism(s) underlying this toxicity are unknown; increased oxidative stress appears play an important role. MDMA metabolites undergo redox cycling to produce superoxide. In vivo, MDMA treated hearts display redox modification of complexes I and V of the mitochondrial electron transport chain and components of the mitochondrial permeability transition pore, potentially leading to apoptosis. We hypothesized that metabolites of MDMA produce cardiac toxicity, at least in part, by initiating apoptosis in cardiac myocytes. Freshly isolated rat ALVM were treated with the MDMA metabolites alpha-Me dopamine or N-Me-alpha-Me dopamine. Both metabolites induced a dose-dependent release of Cyt c from the mitochondria and the cleavage of caspase-3. Both metabolites also caused the cleavage (ie. activation) of nuclear poly (ADP-ribose) polymerase-1 (PARP-1) suggesting DNA breakage. Our results indicate that MDMA metabolites directly induce apoptosis in adult cardiac myocytes which may contribute to MDMA-associated cardiac toxicity. Support AHA 0615646B, NIH P20-RR018766. MDMA courtesy of NIDA.