In Situ Real-Time Chemiluminescence Imaging of Reactive Oxygen Species Formation from Cardiomyocytes

We have applied the highly sensitive chemiluminescence (CL) imaging technique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubation of H9c2 cells in culture with luminol and horseradish peroxidase (HRP), suggesting constitutive formation of ROS by the cardiomyocytes. Addition of benzo(a)pyrene-1,6-quinone (BPQ) to cultured H9c2 cells resulted in a 4-5-fold increase in the formation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells were sustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase, suggesting the primary involvement of superoxide and hydrogen peroxide (H2O2). In contrast to BPQ-mediated redox cycling, blockage of mitochondrial electron transport chain by either antimycin A or rotenone exerted marginal effects on the ROS formation by cultured H9c2 cells. Upregulation of cellular antioxidants for detoxifying both superoxide and H2O2 by 3H-1,2-dithiole-3-thione resulted in marked inhibition of both constitutive and BPQ-augmented ROS formation in cultured H9c2 cells. Taken together, we demonstrate the sensitive detection of ROS by CL imaging in cultured cardiomyocytes

Doxorubicin-induced cytotoxicity in rat myocardial H9c2 cells: the roles of reactive oxygen species and redox balance

Doxorubicin (Dox) is one of the most potent anti-neoplastic agents approved by the Food and Drug Administration. Its efficacy, however, is limited due to its well-documented cardiotoxic side effect. Since the first observation of this dosage-dependent side effect, the mechanisms and events leading to cardiotoxicity following exposure to doxorubicin have received much attention. However, the exact pathogenesis of Dox-induced cardiotoxicity remains to be elucidated. Although increased production of reactive oxygen species (ROS) from the redox cycling of Dox has been recognized as the primary mechanism of Dox-induced cardiotoxicity, it must be noted that many of the studies supporting the oxidative stress-induced cardiotoxicity hypothesis were conducted with supraclinical drug concentrations. This study examined the effect of clinically-relevant concentrations of Dox on H9c2 rat cardiomyoblasts. Through MTT-reduction cell viability assay, it was determined that exposure of H9c2 cells to Dox concentrations above 0.5 µM for more than 12 hours resulted in significant reduction in cell viability. To verify the role of oxidative stress on the development of cytotoxicity, ROS levels after exposure to low concentrations of Dox (less than 2 µM) were measured. Quantitative measurements of both cellular and mitochondrial ROS levels revealed no significant changes to superoxide presence while exhibiting significant decrease in hydrogen peroxide presence. However, despite the decreased presence of two major types of ROS, the potency of antioxidant responses from the H9c2 cells were found to have significantly increased. Also, exposure to Dox at clinically relevant concentrations led to significant increase in the gene expressions of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1(ICAM-1), two key adhesion molecules that have been implicated in Dox-induced cardiotoxicity. These results suggest that lower concentrations of Dox can stimulate intracellular anti-oxidative response that may thwart intracellular ROS levels required for maintaining of proper cell functions, ultimately leading to redox imbalance and inflammation in cardiomyocytes. While attempting to further investigate into the specific mode of cell death induced by Dox treatment, it was found that the innate fluorescence of Dox may be potent enough to be recognized by various fluorescence-based detection methods. Dox was found to exhibit fluorescence spectra consisting of a maximum excitation wavelength of 493 nm and a maximum emission wavelength of 592 nm, which were similar to the fluorescence characteristics of common fluorescent markers such as FITC, PI, MitoSOX, and DCF-DA which are widely used to assess cell viability, as well as ROS production. Furthermore, nuclear accumulation of Dox was confirmed by fluorescence microscopy, and spectrofluorometric measurements which detected the cellular uptake of Dox. This suggests that the innate fluorescence of Dox can be a valid probe used for future investigations for the uptake, release and distribution of Dox both in vitro and in vivo. Altogether, this study demonstrated for the first time that exposure to Dox at clinically relevant plasma concentrations significantly decreased hydrogen peroxide levels below the basal levels in both intact H9c2 rat cardiomyocytes and in isolated mitochondria. Cells treated with Dox showed a significant increase in the expression of genes associated with anti-oxidative response and inflammation. Utilizing the intrinsic fluorescence of Dox, it was found that incubation of H9c2 cells with Dox resulted in time-dependent intracellular uptake of Dox. This study may contribute in advancing our understanding of mechanisms responsible for Dox-induced cardiotoxicity and thereby improving the efficacy of Dox, one of the most prominent components of many chemotherapy regimens

Fluorescent probes for the detection of disease-associated biomarkers

Fluorescent probes have emerged as indispensable chemical tools to the field of chemical biology and medicine. The ability to detect intracellular species and monitor physiological processes has not only advanced our knowledge in biology but has provided new approaches towards disease diagnosis. In this review, we detail the design criteria and strategies for some recently reported fluorescent probes that can detect a wide range of biologically important species in cells and in vivo. In doing so, we highlight the importance of each biological species and their role in biological systems and for disease progression. We then discuss the current problems and challenges of existing technologies and provide our perspective on the future directions of the research area. Overall, we hope this review will provide inspiration for researchers and prove as useful guide for the development of the next generation of fluorescent probes.</p

Cardiac Specific Overexpression of Mitochondrial Omi/HtrA2 Induces Myocardial Apoptosis and Cardiac Dysfunction.

Myocardial apoptosis is a significant problem underlying ischemic heart disease. We previously reported significantly elevated expression of cytoplasmic Omi/HtrA2, triggers cardiomyocytes apoptosis. However, whether increased Omi/HtrA2 within mitochondria itself influences myocardial survival in vivo is unknown. We aim to observe the effects of mitochondria-specific, not cytoplasmic, Omi/HtrA2 on myocardial apoptosis and cardiac function. Transgenic mice overexpressing cardiac-specific mitochondrial Omi/HtrA2 were generated and they had increased myocardial apoptosis, decreased systolic and diastolic function, and decreased left ventricular remodeling. Transiently or stably overexpression of mitochondria Omi/HtrA2 in H9C2 cells enhance apoptosis as evidenced by elevated caspase-3, -9 activity and TUNEL staining, which was completely blocked by Ucf-101, a specific Omi/HtrA2 inhibitor. Mechanistic studies revealed mitochondrial Omi/HtrA2 overexpression degraded the mitochondrial anti-apoptotic protein HAX-1, an effect attenuated by Ucf-101. Additionally, transfected cells overexpressing mitochondrial Omi/HtrA2 were more sensitive to hypoxia and reoxygenation (H/R) induced apoptosis. Cyclosporine A (CsA), a mitochondrial permeability transition inhibitor, blocked translocation of Omi/HtrA2 from mitochondrial to cytoplasm, and protected transfected cells incompletely against H/R-induced caspase-3 activation. We report in vitro and in vivo overexpression of mitochondrial Omi/HtrA2 induces cardiac apoptosis and dysfunction. Thus, strategies to directly inhibit Omi/HtrA2 or its cytosolic translocation from mitochondria may protect against heart injury

Role of mineralocorticoid receptor regulation during experimental myocardial infarction

Ischaemic heart disease remains the leading cause of death worldwide. Following an ischaemic event, the primary strategy is to restore blood flow (reperfusion). However, this triggers release of reactive oxygen species, activation of stress-related gene transcription, autophagy and cell death processes leading to further injury (reperfusion injury). Elevated plasma aldosterone levels produce adverse cardiac effects, while mineralocorticoid receptor (MR) antagonists (spironolactone or eplerenone) reduce mortality, although mechanisms have not been defined. The aim of this thesis was to determine the role of MR regulation during experimental myocardial infarction (MI). This was achieved by using an ex-vivo isolated rat heart model and occluding a branch of the left coronary artery (30min) followed by reperfusion (150min). Increased levels of oxidative stress with activated autophagy and apoptosis confirmed our model of MI. Since there are sex differences in cardiac damage during MI, our studies show that androgens downregulate anti-apoptotic protein Bcl-xL, which shifts the balance towards apoptosis leading to aggravated cardiac damage in males compared to females. Expression levels of MR have been reported to be upregulated during MI in males, which could contribute to the aggravated damage, we did not find any significant change in MR expression between male and female rats and hence male rats were used for subsequent studies. Activation of MR by aldosterone (10 nM) increased cardiomyocyte apoptosis and aggravated infarct size during MI; prevented by low-dose MR antagonists. Low-dose (10 nM) spironolactone alone maintained redox balance, prevented activation of stress-related gene transcription and degradation of anti-apoptotic protein ARC, which prevented initiation of apoptosis. These studies provide direct evidence that MR activation aggravates cardiac damage during MI and provide mechanisms for the cardioprotective action of low-dose MR antagonists clinically

Restoration of Mitochondrial Cardiolipin Attenuates Cardiac Damage in Swine Renovascular Hypertension

BACKGROUND: Renovascular hypertension (RVH) impairs cardiac structure and left ventricular (LV) function, but whether mitochondrial injury is implicated in RVH-induced myocardial damage and dysfunction has not been defined. We hypothesized that cardiac remodeling in swine RVH is partly attributable to cardiac mitochondrial injury. METHODS AND RESULTS: After 12 weeks of hypercholesterolemic (HC)-RVH or control (n=14 each), pigs were treated for another 4 weeks with vehicle or with the mitochondrial-targeted peptide (MTP), Bendavia (0.1 mg/kg subcutaneously, 5 days/week), which stabilizes mitochondrial inner-membrane cardiolipin (n=7 each). Cardiac function was subsequently assessed by multidetector-computed tomography and oxygenation by blood-oxygen-level-dependent magnetic resonance imaging. Cardiolipin content, mitochondrial biogenesis, as well as sarcoplasmic-reticulum calcium cycling, myocardial tissue injury, and coronary endothelial function were assessed ex vivo. Additionally, mitochondrial cardiolipin content, oxidative stress, and bioenergetics were assessed in rat cardiomyocytes incubated with tert-butyl hydroperoxide (tBHP) untreated or treated with MTP. Chronic mitoprotection in vivo restored cardiolipin content and mitochondrial biogenesis. Thapsigargin-sensitive sarcoplasmic reticulum Ca(2+)-ATPase activity that declined in HC-RVH normalized in MTP-treated pigs. Mitoprotection also improved LV relaxation (E/A ratio) and ameliorated cardiac hypertrophy, without affecting blood pressure or systolic function. Myocardial remodeling and coronary endothelial function improved only in MTP-treated pigs. In tBHP-treated cardiomyocytes, mitochondrial targeting attenuated a fall in cardiolipin content and bioenergetics. CONCLUSIONS: Chronic mitoprotection blunted myocardial hypertrophy, improved LV relaxation, and attenuated myocardial cellular and microvascular remodeling, despite sustained HC-RVH, suggesting that mitochondrial injury partly contributes to hypertensive cardiomyopathy

Assessment of mitochondrial dysfunction and implications in cardiovascular disorders

Mitochondria play a pivotal role in cellular function, not only acting as the powerhouse of the cell, but also regulating ATP synthesis, reactive oxygen species (ROS) production, intracellular Ca2+ cycling, and apoptosis. During the past decade, extensive progress has been made in the technology to assess mitochondrial functions and accumulating evidences have shown that mitochondrial dysfunction is a key pathophysiological mechanism for many diseases including cardiovascular disorders, such as ischemic heart disease, cardiomyopathy, hypertension, atherosclerosis, and hemorrhagic shock. The advances in methodology have been accelerating our understanding of mitochondrial molecular structure and function, biogenesis and ROS and energy production, which facilitates new drug target identification and therapeutic strategy development for mitochondrial dysfunction-related disorders. This review will focus on the assessment of methodologies currently used for mitochondrial research and discuss their advantages, limitations and the implications of mitochondrial dysfunction in cardiovascular disorders

p47phox-dependent oxidant signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-induced cardiac hypertrophy and apoptosis

The p47phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47phox in the heart remains unclear. In this study, we used wild-type (WT) and p47phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis

HIF-1α in the Heart: Provision of Ischemic Cardioprotection and Remodeling of Nucleotide Metabolism

In our studies we found that stabilized expression of HIF-1α in heart led to better recovery of function and less tissue death after 30 minutes of global ischemia, via mechanisms that preserve the mitochondrial polarization. Our group previously showed that HIF-1α conferred ischemic tolerance by allowing cardiomyocytes to use fumarate as an alternative terminal electron acceptor to sustain anaerobic mitochondrial polarization. The source of fumarate was identified as the purine nucleotide cycle (PNC). Here we discovered that HIF-1α upregulates AMP deaminase 2 (AMPD2), the entry point to the PNC. The combination of glycolysis and the PNC may protect the heart\u27s nucleotide resources. We subsequently examined the effects that HIF-1α exerts on nucleotide metabolism in the ischemic heart. We found that HIF-1α expression reduces adenosine accumulation in the ischemic heart. As ATP is depleted during ischemia, AMP accumulates. Our results suggest that AMP metabolism is shunted towards AMPD2 rather than the adenosine producing 5\u27-nucleotidase pathway. Subsequently, we treated hearts with the PNC inhibitor hadacidin followed by 30 minutes of global ischemia. Inclusion of hadacidin reduced ATP and adenylate energy charge in the hearts. These findings allow us to propose that activity of the PNC prevents the F0F1 ATP synthase from consuming glycolytic ATP in order to maintain mitochondrial polarization during ischemia. Thus, the PNC provides ATP sparing effects and preserves the energy charge in the ischemic heart. The fact that ATP and adenylate energy charge is better preserved during the initial 20 minutes of ischemia in HIF-1α expressing hearts is supportive of our observation that HIF-1α upregulates the PNC. HIF-1α also upregulates adenosine deaminase, which degrades adenosine. The limitation of adenosine accumulation may help HIF-1α expressing hearts avoid toxicity due to chronic adenosine exposure. Finally, we found that HIF-1α induces the expression of the nucleotide salvage enzyme hypoxanthine phosphoribosyl transferase (HPRT). Upon reperfusion HPRT serves to reincorporate the nucleotide degradation product, hypoxanthine, into the adenylate pool and may prevent the production of reactive oxygen species. Collectively, HIF-1α robustly protects the heart from ischemic stress and it upregulates several pathways whose cardioprotective role may extend beyond the remodeling of nucleotide metabolism