Preeti Tripathi

Cardiac inflammation after local irradiation is influenced by the kallikrein-kinin system

Radiotherapy of intrathoracic and chest wall tumors may lead to exposure of the heart to ionizing radiation, resulting in radiation-induced heart diseases (RIHD). The main manifestations of RIHD become apparent many years after treatment and include cardiomyopathy and accelerated atherosclerosis. This study examines the role of the kallikrein-kinin system (KKS) in RIHD by investigating the cardiac radiation response in a kininogen-deficient Brown Norway Katholiek (BN/Ka) rat model. BN/Ka rats and wild-type Brown Norway (BN) rats were exposed to local heart irradiation with a single dose of 18 Gy or 24 Gy and were observed for 3 to 6 months. Examinations included in vivo and ex vivo cardiac function, histopathology, gene and protein expression measurements, and mitochondrial swelling assays. Upon local heart irradiation, changes in in vivo cardiac function were significantly less in BN/Ka rats. For instance, a single dose of 24 Gy caused a 35% increase in fractional shortening in BN rats compared with a 16% increase in BN/Ka rats. BN rats, but not BN/Ka rats, showed a 56% reduction in cardiac numbers of CD2-positive cells, and a 57% increase in CD68-positive cells, together with a 52% increase in phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). Local heart irradiation had similar effects on histopathology, mitochondrial changes, and left ventricular mRNA levels of NADPH oxidases in the two genotypes. These results suggest that the KKS plays a role in the effects of radiation on cardiac function and recruitment of inflammatory cells. The KKS may have these effects at least in part by altering Erk1/2 signaling.

Radiation-Induced Alterations in Mitochondria of the Rat Heart

Radiation therapy for the treatment of thoracic cancers may be associated with radiation-induced heart disease (RIHD), especially in long-term cancer survivors. Mechanisms by which radiation causes heart disease are largely unknown. To identify potential long-term contributions of mitochondria in the development of radiation-induced heart disease, we examined the time course of effects of irradiation on cardiac mitochondria. In this study, Sprague-Dawley male rats received image-guided local X irradiation of the heart with a single dose ranging from 3–21 Gy. Two weeks after irradiation, left ventricular mitochondria were isolated to assess the dose-dependency of the mitochondrial permeability transition pore (mPTP) opening in a mitochondrial swelling assay. At time points from 6 h to 9 months after a cardiac dose of 21 Gy, the following analyses were performed: left ventricular Bax and Bcl-2 protein levels; apoptosis; mitochondrial inner membrane potential and mPTP opening; mitochondrial mass and expression of mitophagy mediators Parkin and PTEN induced putative kinase-1 (PINK-1); mitochondrial respiration and protein levels of succinate dehydrogenase A (SDHA); and the 70 kDa subunit of complex II. Local heart irradiation caused a prolonged increase in Bax/Bcl-2 ratio and induced apoptosis between 6 h and 2 weeks. The mitochondrial membrane potential was reduced until 2 weeks, and the calcium-induced mPTP opening was increased from 6 h up to 9 months. An increased mitochondrial mass together with unaltered levels of Parkin suggested that mitophagy did not occur. Lastly, we detected a significant decrease in succinate-driven state 2 respiration in isolated mitochondria from 2 weeks up to 9 months after irradiation, coinciding with reduced mitochondrial levels of succinate dehydrogenase A. Our results suggest that local heart irradiation induces long-term changes in cardiac mitochondrial membrane functions, levels of SDH and state 2 respiration. At any time after exposure to radiation, cardiac mitochondria are more prone to mPTP opening. Future studies will determine whether this makes the heart more susceptible to secondary stressors such as calcium overload or ischemia/reperfusion.

Effects of Late Administration of Pentoxifylline and Tocotrienols in an Image-Guided Rat Model of Localized Heart Irradiation

Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.

A Tocotrienol-Enriched Formulation Protects against Radiation-Induced Changes in Cardiac Mitochondria without Modifying Late Cardiac Function or Structure

Radiation-induced heart disease (RIHD) is a common and sometimes severe late side effect of radiation therapy for intrathoracic and chest wall tumors. We have previously shown that local heart irradiation in a rat model caused prolonged changes in mitochondrial respiration and increased susceptibility to mitochondrial permeability transition pore (mPTP) opening. Because tocotrienols are known to protect against oxidative stress-induced mitochondrial dysfunction, in this study, we examined the effects of tocotrienols on radiation-induced alterations in mitochondria, and structural and functional manifestations of RIHD. Male Sprague-Dawley rats received image-guided localized X irradiation to the heart to a total dose of 21 Gy. Twenty-four hours before irradiation, rats received a tocotrienol-enriched formulation or vehicle by oral gavage. Mitochondrial function and mitochondrial membrane parameters were studied at 2 weeks and 28 weeks after irradiation. In addition, cardiac function and histology were examined at 28 weeks. A single oral dose of the tocotrienol-enriched formulation preserved Bax/Bcl2 ratios and prevented mPTP opening and radiation-induced alterations in succinate-driven mitochondrial respiration. Nevertheless, the late effects of local heart irradiation pertaining to myocardial function and structure were not modified. Our studies suggest that a single dose of tocotrienols protects against radiation-induced mitochondrial changes, but these effects are not sufficient against long-term alterations in cardiac function or remodeling.

Effects of radiation on the epidermal growth factor receptor pathway in the heart

Abstract Purpose: Radiation-induced heart disease (RIHD) is a serious side-effect of thoracic radiotherapy. The epidermal growth factor receptor (EGFR) pathway is essential for the function and survival of cardiomyocytes. Hence, agents that target the EGFR pathway are cardiotoxic. Tocotrienols protect from radiation injury, but may also enhance the therapeutic effects of EGFR pathway inhibitors in cancer treatment. This study investigated the effects of local irradiation on the EGFR pathway in the heart and tests whether tocotrienols may modify radiation-induced changes in this pathway. Methods: Male Sprague-Dawley rats received image-guided localized heart irradiation with 21 Gy. Twenty four hours before irradiation, rats received a single dose of tocotrienol-enriched formulation or vehicle by oral gavage. At time points from 2 h to 9 months after irradiation, left ventricular expression of EGFR pathway mediators was studied. Results: Irradiation caused a decrease in the expression of epidermal growth factor (EGF) and neuregulin-1 (Nrg-1) mRNA from 6 h up to 10 weeks, followed by an upregulation of these ligands and the receptor erythroblastic leukemia viral oncogene homolog (ErbB)4 at 6 months. In addition, the upregulation of Nrg-1 was statistically significant up to 9 months after irradiation. A long-term upregulation of ErbB2 protein did not coincide with changes in transcription or post-translational interaction with the chaperone heat shock protein 90 (HSP90). Pretreatment with tocotrienols prevented radiation-induced changes at 2 weeks. Conclusions: Local heart irradiation causes long-term changes in the EGFR pathway. Studies have to address how radiation may interact with cardiotoxic effects of EGFR inhibitors.

Roles of sensory nerves in the regulation of radiation-induced structural and functional changes in the heart.

PURPOSE Radiation-induced heart disease (RIHD) is a chronic severe side effect of radiation therapy of intrathoracic and chest wall tumors. The heart contains a dense network of sensory neurons that not only are involved in monitoring of cardiac events such as ischemia and reperfusion but also play a role in cardiac tissue homeostasis, preconditioning, and repair. The purpose of this study was to examine the role of sensory nerves in RIHD. METHODS AND MATERIALS Male Sprague-Dawley rats were administered capsaicin to permanently ablate sensory nerves, 2 weeks before local image-guided heart x-ray irradiation with a single dose of 21 Gy. During the 6 months of follow-up, heart function was assessed with high-resolution echocardiography. At 6 months after irradiation, cardiac structural and molecular changes were examined with histology, immunohistochemistry, and Western blot analysis. RESULTS Capsaicin pretreatment blunted the effects of radiation on myocardial fibrosis and mast cell infiltration and activity. By contrast, capsaicin pretreatment caused a small but significant reduction in cardiac output 6 months after irradiation. Capsaicin did not alter the effects of radiation on cardiac macrophage number or indicators of autophagy and apoptosis. CONCLUSIONS These results suggest that sensory nerves, although they play a predominantly protective role in radiation-induced cardiac function changes, may eventually enhance radiation-induced myocardial fibrosis and mast cell activity.

Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway.