A large body of evidence suggests that hypoxia drives aggressive molecular features of malignant cells irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by frequent involvement of diverse hypoxic microenvironments. We studied the impact of long-term deep hypoxia (1% O2) on the biology of lymphoma cells. Only 2 out of 6 tested cell lines (Ramos, and HBL2) survived ≥ 4 weeks under hypoxia. The hypoxia-adapted (HA)b Ramos and HBL2 cells had a decreased proliferation rate accompanied by significant suppression of both oxidative phosphorylation and glycolytic pathways. Transcriptome and proteome analyses revealed marked downregulation of genes and proteins of the mitochondrial respiration complexes I and IV, and mitochondrial ribosomal proteins. Despite the observed suppression of glycolysis, the proteome analysis of both HA cell lines showed upregulation of several proteins involved in the regulation of glucose utilization including the active catalytic component of prolyl-4-hydroxylase P4HA1, an important druggable oncogene. HA cell lines demonstrated increased transcription of key regulators of auto-/mitophagy, e.g., neuritin, BCL2 interacting protein 3 (BNIP3), BNIP3-like protein, and BNIP3 pseudogene. Adaptation to hypoxia was further associated with deregulation of apoptosis, namely upregulation of BCL2L1/BCL-XL, overexpression of BCL2L11/BIM, increased binding of BIM to BCL-XL, and significantly increased sensitivity of both HA cell lines to A1155463, a BCL-XL inhibitor. Finally, in both HA cell lines AKT kinase was hyperphosphorylated and the cells showed increased sensitivity to copanlisib, a pan-PI3K inhibitor. In conclusion, our data report on several shared mechanisms of lymphoma cell adaptation to long-term hypoxia including: 1. Upregulation of proteins responsible for glucose utilization, 2. Degradation of mitochondrial proteins for potential mitochondrial recycling (by mitophagy), and 3. Increased dependence on BCL-XL and PI3K-AKT signaling for survival. In translation, inhibition of glycolysis, BCL-XL, or PI3K-AKT cascade may result in targeted elimination of HA lymphoma cells.

Hypertension, dyslipidemia, and insulin resistance in the spontaneously hypertensive rat (SHR) can be alleviated by rescuing CD36 fatty acid translocase. The present study investigated whether transgenic rescue of CD36 in SHR could affect mitochondrial function and activity of selected metabolic enzymes in the heart. These analyses were conducted on ventricular preparations derived from SHR and from transgenic strain SHR- Cd36 that expresses a functional wild-type CD36. Our respirometric measurements revealed that mitochondria isolated from the left ventricles exhibited two times higher respiratory activity than those isolated from the right ventricles. Whereas, we did not observe any significant changes in functioning of the mitochondrial respiratory system between both rat strains, enzyme activities of total hexokinase, and both mitochondrial and total malate dehydrogenase were markedly decreased in the left ventricles of transgenic rats, compared to SHR. We also detected downregulated expression of the succinate dehydrogenase subunit SdhB (complex II) and 70 kDa peroxisomal membrane protein in the left ventricles of SHR- Cd36 . These data indicate that CD36 may affect in a unique fashion metabolic substrate flexibility of the left and right ventricles.

Fatty acid translocase (FAT/CD36) plays an important role in fatty acid uptake by different cell types and may also participate in regulation of calcium homeostasis and eicosanoid production. CD36 deficiency or polymorphisms in the CD36 gene are linked to some physiological irregularities. It is known that the expression of FAT/CD36 is aberrant in the spontaneously hypertensive rat (SHR), one of the most widely studied rat strains in cardiovascular research. In this work, we compared the cardiac proteome of SHR and transgenic SHR-Cd36 rats, who carry a copy of the wild type CD36 gene. Protein expression profiling was based on two-dimensional gel electrophoresis (2DE) coupled to tandem mass spectrometry and label-free LC/MS. These two complementary proteomic approaches allowed us to investigate proteome differences in the left and right heart ventricles of SHR and SHR-Cd36 rats. In total, we identified 26 differently expressed myocardial proteins, out of which 18 were found in the right ventricles and 8 in the left ventricles. Besides that, we determined a great number of proteins uniquely expressed either in the left or right ventricles. These data indicate a large qualitative disparity between the left and right ventricles. Genetic manipulations may affect different proteins in both heart ventricles. Biological significance: This is the first report revealing a relatively broad impact of transgenic expression of CD36 on the heart at the proteome level. Comparison of the protein profiles in both the left and right ventricles revealed differences in several proteins involved especially in energy metabolism. The observed downregulation of the respiratory chain enzymes in transgenic SHR-Cd36 rats may suggest a shift in regulation of energy metabolism due to expression of fatty acid translocase FAT/CD36. This study highlights the important role of cardiac tissue proteomic profiling for mapping of proteins which might be altered by targeted genetic manipulations.

Dysfunction or abnormalities in the regulation of fatty acid translocase Cd36, a multifunctional membrane protein participating in uptake of long-chain fatty acids, has been linked to the development of heart diseases both in animals and humans. We have previously shown that the Cd36 transgenic spontaneously hypertensive rat (SHR-Cd36), with a wild type Cd36, has higher susceptibility to ischemic ventricular arrhythmias when compared to spontaneously hypertensive rat (SHR) carrying a mutant Cd36 gene, which may have been related to increased β-adrenergic responsiveness of these animals (Neckar et al., 2012 Physiol. Genomics 44:173–182). The present study aimed to determine whether the insertion of the wild type Cd36 into SHR would affect the function of myocardial G protein-regulated adenylyl cyclase (AC) signaling. β-Adrenergic receptors (β-ARs) were characterized by radioligand-binding experiments and the expression of selected G protein subunits, AC, and protein kinase A (PKA) was determined by RT-PCR and Western blot analyses. There was no significant difference in the amount of trimeric G proteins, but the number of β-ARs was higher (by about 35 %) in myocardial preparations from SHR-Cd36 as compared to SHR. Besides that, transgenic rats expressed increased amount (by about 20 %) of the dominant myocardial isoforms AC5/6 and contained higher levels of both nonphosphorylated (by 11 %) and phosphorylated (by 45 %) PKA. Differently stimulated AC activity in SHR-Cd36 significantly exceeded (by about 18–30 %) the enzyme activity in SHR. Changes at the molecular level were reflected by higher contractile responses to stimulation by the adrenergic agonist dobutamine. In summary, it can be concluded that the increased susceptibility to ischemic arrhythmias of SHR-Cd36 is attributable to upregulation of some components of the β-AR signaling pathway, which leads to enhanced sensitization of AC and increased cardiac adrenergic responsiveness.

CD36 fatty acid translocase plays a key role in supplying heart with its major energy substrate, long‐chain fatty acids (FA). Spontaneously hypertensive rat (SHR) harbors a deletion variant of Cd36 gene. We analyzed the effects of mutant Cd36 on cardiac susceptibility to ischemia‐reperfusion injury in adult SHR‐Cd36 transgenic rats with wild type Cd36 compared to age‐matched SHR controls. Using an open‐chest model of coronary artery occlusion, we found that SHR‐Cd36 showed profound ischemic arrhythmogenesis resulting in significantly increased total number of premature ventricular complexes (2623±517 vs. 849±250) in SHR controls. On the other hand, transgenic SHR showed significantly reduced infarct size (52.6±4.3% vs. 72.4±2.9% of area at risk). Similar differences were observed in isolated perfused hearts and the increased incidence of arrhythmias in transgenic SHR was abolished by reserpine suggesting the involvement of catecholamines. Furthermore, forskolin‐stimulated activity of adenylyl cyclase was increased in the SHR‐Cd36. It can be concluded that Cd36 plays an important role in modulating cardiac susceptibility to ischemia‐reperfusion injury. The proarrhythmic effect of Cd36 transgene appears to be related to increased adrenergic stimulation. Supported by grants: GAAV IAAX01110901, GAUK 429611