Mitochondria are indispensable for bioenergetics and for the regulation of physiological/signaling events in cellular life. Although TNF-alpha-induced oxidative stress and mitochondrial dysfunction are evident in several pathophysiological states, the molecular mechanisms coupled with impaired cardiac function and its potential reversal by drugs such as Tempol or apocyanin have not yet been explored. Here, we hypothesize that TNF-alpha-induced oxidative stress compromises cardiac function by altering the mitochondrial redox state and the membrane permeability transition pore (MPTP) opening, thereby causing mitochondrial dysfunction. We measured the redox states in the cytosol and mitochondria of the heart to understand the mechanisms related to the MPTP and the antioxidant defense system. Our studies demonstrate that TNF-alpha-induced oxidative stress alters redox homeostasis by impairing the MPTP proteins adenine nucleotide translocator and voltage-dependent anion channel, thereby resulting in the pore opening, causing uncontrolled transport of substances to alter mitochondrial pH, and subsequently leading to dysfunction of mitochondria and attenuated cardiac function. Interestingly, we show that the supplementation of Tempol along with TNF-alpha restores mitochondrial and cardiac function.

AimsMutant protein aggregation (PA) cardiomyopathy (MPAC) is characterized by reductive stress (RS), PA (of chaperones and cytoskeletal components), and ventricular dysfunction in transgenic mice expressing human mutant CryAB (hmCryAB). Sustained activation of nuclear erythroid-2 like factor-2 (Nrf2) causes RS, which contributes to proteotoxic cardiac disease. The goals of this pre-clinical study were to (i) investigate whether disrupting Nrf2-antioxidant signalling prevents RS and rescues redox homeostasis in hearts expressing the mutant chaperone and (ii) elucidate mechanisms that could delay proteotoxic cardiac disease. Methods and resultsNon-transgenic (NTG), transgenic (TG) with MPAC and MPAC-TG:Nrf2-deficient (Nrf2-def) mice were used in this study. The effects of Nrf2 diminution (Nrf2+) on RS mediated MPAC in TG mice were assessed at 6 -7 and 10 months of age. The diminution of Nrf2 prevented RS and prolonged the survival of TG mice ( 50 weeks) by an additional 20-25 weeks. The TG:Nrf2-def mice did not exhibit cardiac hypertrophy at even 60 weeks, while the MPAC-TG mice developed pathological hypertrophy and heart failure starting at 24 -28 weeks of age. Aggregation of cardiac proteins was significantly reduced in TG:Nrf2-def when compared with TG mice at 7 months. Preventing RS and maintaining redox homeostasis in the TG:Nrf2-def mice ameliorated PA, leading to decreased ubiquitination of proteins. ConclusionNrf2 deficiency rescues redox homeostasis, which reduces aggregation of mutant proteins, thereby delaying the proteotoxic pathological cardiac remodelling caused by RS and toxic protein aggregates.--

Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.

Background: Radical forms of oxygen and nitrogen species (ROS/RNS) are highly reactive with nucleic acids, proteins and lipids and promote their oxidation. Normally, cellular ROS/RNS concentrations are tightly controlled by the inducible antioxidant system, which is predominantly regulated by the transcription factor Nrf2 (nuclear erythroid-2 like factor-2) and its cytosolic repressor protein, Keap1. We hypothesized that a decrease or an abrogation of Nrf2 impairs cardiac function and induce hypertrophy upon endurance stress in aging heart. Methods: Age-matched wild-type (WT) and Nrf2-/- (KO) mice (n=12/gp) at 2 and >20 months were subjected to endurance exercise stress (EES; 20 meter/min, 12% grade) and assessed the activation of Nrf2/ARE-dependent transcriptional mechanisms in the heart. Cardiac hypertrophy was determined by echocardiography, heart/body weight ratio and biochemical/molecular marker analyses. Results: Interestingly, both old-WT and Nrf2-/- mice exhibited oxidative stress on EES due to significant decrease or abrogation of Nrf2 nuclear levels, respectively, suggesting that aged-WT is equally susceptible to stress as Nrf2-/- mice. Age-dependent loss of Nrf2 decreased the transcription of Nrf2-dependent antioxidants and thereby elevated ROS levels to cause a more oxidized intracellular environment. Importantly, the loss of Nrf2 induced cardiac hypertrophy upon endurance stress in the aged (>20 mon) mice. At the end of 2-weeks of endurance stress, both the old-WT and Nrf2-/- mice had developed cardiac hypertrophy. Also, qPCR analysis showed significant (p<0.05) upregulation of hypertrophy markers (ANF and BNF) in the old-WT or Nrf2-/- when compared to sedentary WT mice confirmed cardiac hypertrophy due to loss of Nrf2. These results indicate that either a decrease or an abrogation of Nrf2 can increase susceptibility to stress induced hypertrophy in an aging heart. Conclusions: We conclude that enhancing protein levels and stability of nuclear Nrf2 could activate the transcription of major antioxidant enzymes and biosynthesis of key antioxidants. Enhancing protective mechanisms against oxidative stress in the elderly is expected to prevent or delay the onset of age-associated cardiac hypertrophy and cardiomyopathy.

Cardiomyopathy and heart failure (HF) is a growing cause of human morbidity and mortality across the globe. Antioxidant‐based treatments to detoxify ROS have been largely unsuccessful to protect humans from various cardiovascular diseases. Although transcriptional activation of antioxidants appear to be beneficial, their chronic effects remain elusive. Here, we tested a hypothesis that the sustained activation of Nrf2/antioxidant signaling will promote reductive stress (RS) and lead to hypertrophic cardiomyopathy. Using novel transgenic mouse models expressing Nrf2 in the heart (α‐MHC‐Nrf2‐TG) and their non‐transgenic (NTg) littermates, we studied the chronic effects of a hyper‐reductive condition on cardiac structure and function (echocardiography – Vevo2100 Imager). Furthermore, myocardial redox potential for glutathione (GSH/GSSG), transcript (qPCR), and protein (immunoblotting) levels for Nrf2‐related antioxidants in the myocardium of NTg and TG mice (n=6–12/gp.) were performed at different ages. Results from this novel study demonstrated ~40% mortality in TG mice compared to NTg by 60 weeks of age (Kaplan‐Meier survival plots). An abnormally higher GSH redox ratio was maintained along with a significantly diminished ROS in TG vs. NTg mice (p<0.05) indicated a hyper‐reductive state. This was strongly associated with a significant increase in Nrf2 function (promoter activity) with augmented levels of antioxidant genes and proteins (p<0.05) in TG vs. NTg mice. Subsequent analyses revealed that progressive cardiac remodeling and impaired cardiac output in TG relative to NTg mice. Thus, diminution of the obligatory oxidative signaling with chronic activation of Nrf2‐antioxidants could shift the redox equilibrium to the “reductive” side and cause cardiac disease. Recent clinical observations revealed a sub‐set of heart failure (HF) patients exhibiting a hyper‐reductive (i.e. RS) condition, suggesting a role for RS in the development of HF. Ongoing studies are focused on understanding the molecular mechanisms for RS‐pathogenesis in the myocardium.Support or Funding InformationThis work is supported by an R01 grant from NIH/NHLBI (HL118067).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.