CABG Patients Develop Global DNA Hypermethylation, That Negatively Affect the Mitochondrial Function and Promote Post-Surgical Cognitive Decline: A Proof of Concept in Small Cohort.

Global DNA hypermethylation and mitochondrial dysfunction are reported to be associated with the development of mild cognitive decline (MCI). The present study aims to generate preliminary data that connect the above association with post-surgical coronary artery bypass grafting (CABG) cognitive decline in patients. Data were collected from 70 CABG patients and 25 age-matched controls. Cognitive function was assessed using the Montreal Cognitive Assessment (MOCA) test on day 1 (before surgery) and on the day of discharge. Similarly, blood was collected before and one day after the CABG procedure for mitochondrial functional analysis and expression of DNA methylation genes. Test analysis score suggested 31 (44%) patients had MCI before discharge. These patients showed a significant decrease in complex I activity and an increase in malondialdehyde levels (p < 0.001) from the control blood samples. Post-surgical samples showed a significant reduction in blood MT-ND1 mRNA expression from control and from pre-surgical samples (p < 0.005), along with elevated DNMT1 gene expression (p < 0.047), with an insignificant increase in TET1 and TET3 gene expression. Correlation analysis showed a significant positive relation between cognitive decline and elevated blood DNMT1 and declined blood complex I activity, signifying that cognitive decline experienced by post-surgical CABG patients is associated with increased DNMT1 expression and declined complex I activity. Based on the data, we conclude that both DNA hypermethylation and mitochondrial dysfunction are associated with post-CABG MCI, where the former is negatively correlated, and the latter is positively correlated with post-surgical MCI in CABG cases. Additionally, a multimarker approach that comprises MOCA, DNA methylation, DNMT, and NQR activities can be utilized to stratify the population that is sensitive to developing post-CABG MCI.

Recent advances in potential of Fisetin in the management of myocardial ischemia-reperfusion injury-A systematic review.

BACKGROUND: The primary therapeutic strategy in managing ischemic heart diseases is to restore the perfusion of the myocardial ischemic area by surgical methods that often result in an unavoidable injury called ischemia-reperfusion injury (IR). Fisetin is an effective flavonoid with antioxidant and anti-inflammatory properties, proven to be cardioprotective against IR injury in both in-vitro and invivo models, apart from its promising health benefits against cancer, diabetes, and neurodegenerative ailments. PURPOSE: The potential of fisetin in attenuating myocardial IR is inconclusive as the effectiveness of fisetin needs more understanding in terms of its possible target sites and underlying different mechanisms. Considering the surge in recent scientific interests in fisetin as a pharmacological agent, this review not only updates the existing preclinical and clinical studies with fisetin and its underlying mechanisms but also summarizes its possible targets during IR protection. METHODS: We performed a literature survey using search engines Pubmed, PMC, Science direct, Google, and research gate published across the years 2006-2021. The relevant studies were extracted from the databases with the combinations of the following keywords and summarized: myocardial ischemia-reperfusion injury, natural products, flavonoid, fisetin, PI3K, JAK-STAT, Nrf2, PKC, JNK, autophagy. RESULTS: Fisetin is reported to be effective in attenuating IR injury by delaying the clotting time, preserving the mitochondrial function, reducing oxidative stress, and inhibiting GSK 3ß. But it failed to protect diseased cardiomyocytes challenged to IR. As discussed in the current review, fisetin not only acts as a conventional antioxidant and anti-inflammatory agent to exert its biological effect but may also exert modulatory action on the cellular metabolism and adaptation via direct action on various signalling pathways that comprise PI3K, JAK-STAT, Nrf2, PKC, JNK, and autophagy. Moreover, the dosage of fisetin and co-morbidities like diabetes and obesity are found to be detrimental factors for cardioprotection. CONCLUSION: For further evaluation and smooth clinical translation of the fisetin molecule in IR treatment, researchers should pay close attention to the potential of fisetin to possibly alter the key cardioprotective pathways and dosage, as the efficacy of fisetin is tissue and cell type-specific and varies with different doses.

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria.

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.

Long-term administration of fisetin was not as effective as short term in ameliorating IR injury in isolated rat heart.

The current study aims to determine the comparative efficacy of fisetin in reducing myocardial ischemia-reperfusion injury (IR) in isolated rat hearts when the drug was given either oral or intraperitoneal (ip) for short-term and long-term administration. Rats treated with fisetin (20 mg/kg-oral/ip) for short (30 min prior to surgery) and long (15 days prior to surgery followed by 1-day washout) duration were subjected to myocardial IR using Langendorf perfusion system. Hemodynamics, cardiac injury, mitochondrial functional assessment, and fisetin levels were estimated. Unlike the long-term administration of fisetin, the short-term treated-rat heart exhibited significant cardioprotection, measured via hemodynamic indices (RPP in mmHg × beats/min × 10 ^ 4: IR - 4 ± 0.1, FIPS - 2.49 ± 0.18, FIPL - 1.87 ± 0.14), reduced infarct size (in % area of infarct: IR - 38 ± 5, FIPS - 17 ± 1, FOS - 14 ± 2), improved mitochondrial ETC enzyme activity (NQR activity in IFM: FIPS - 0.25 ± 0.016, FIPL - 0.20 ± 0.02), and declined oxidative stress (GSH in IFM: FIPS - 1.52 ± 0.14, FIPL - 1.25 ± 0.22). However, no significant difference in the protection was observed between the animals treated with oral or intraperitoneally administered fisetin. Single dose of fisetin administration before IR protocol was more effective than 15 days of fisetin-treated drug followed by 1-day washout, thus may not be suitable for long-term dietary supplement for post-surgical cardiac rehabilitation.

Preconditioning the rat heart with 5-azacytidine attenuates myocardial ischemia/reperfusion injury via PI3K/GSK3ß and mitochondrial KATP signaling axis.

5-Azacytidine is well known for its clinical usage in cancer treatments. The present study investigates the role of 5-azacytidine as a cardioprotective agent to ameliorate ischemia/reperfusion (I/R) injury. The cardioprotective effect of 5-azacytidine was evaluated in three experimental models: in vitro, ex vivo, and in vivo. The cardioprotective effect was evaluated via cell viability, hemodynamic indices, infarct size measurement, and assessment of histopathology, oxidative stress, and mitochondrial function. The experiments were repeated in the presence of PI3K/GSK3ß and mitochondrial KATP (mtKATP ) cardioprotective signaling pathway inhibitors to understand the underlying mechanism. 5-Azacytidine improved the cell viability by 29% in I/R-challenged H9C2 cells. Both isolated rat heart and LAD ligation model confirmed the infarct sparing effect of 5-azacytidine against I/R. It also provided a beneficial effect by normalizing the altered hemodynamics, reducing the infarct size and cardiac injury markers, reversing the perturbation of mitochondria, reduced oxidative stress, and improved the pPI3K and pAKT protein expression from I/R. In addition, it also augmented the activation of PI3K/AKT and mtKATP signaling pathway, confirmed by using wortmannin (PI3K inhibitor), SB216763 (GSK3ß inhibitor), and glibenclamide (mtKATP channel closer). The effectiveness of 5-azacytidine as a cardioprotective agent is attributed to its activation of the PI3K/GSK3ß and mtKATP channel signaling axis, thereby preserving mitochondrial function and reducing oxidative stress.

Evaluating the effects of carbon monoxide releasing molecule-2 against myocardial ischemia-reperfusion injury in ovariectomized female rats.

PURPOSE: Cardioprotective effect of carbon monoxide, a gasotransmitter against myocardial ischemia-reperfusion injury (I/R), is well established in preclinical studies with male rats. However, its ischemic tolerance in post-menopausal animals has not been examined due to functional perturbations at the cellular level. METHODS: The protective role of carbon monoxide releasing molecule-2 (CORM-2) on myocardial I/R was studied in female Wistar rats using the Langendorff apparatus. The animals were randomly divided into normal and ovariectomized (Ovx) female rats and were maintained 2 months post-surgery. Each group was further divided into 4 subgroups (n = 6/subgroup): normal, I/R, CORM-2-control (20 µmol/L), and CORM-2-I/R. The cardiac injury was estimated via myocardial infarct size, lactate dehydrogenase, and creatine kinase levels in coronary effluent and cardiac hemodynamic indices. Mitochondrial functional activity was assessed by measuring mitochondrial electron transport chain enzyme activities, swelling behavior, mitochondrial membrane potential, and oxidative stress. RESULTS: Hemodynamic indices were significantly lower in ovariectomized rat hearts than in normal rat hearts. Sixty minutes of reperfusion of ischemic heart exhibited deteriorated cardiac physiological recovery in both ovariectomized and normal groups, where prominent decline was observed in ovariectomized rat. However, preconditioning the isolated heart with CORM-2 improved hemodynamics parameters significantly in both ovariectomized and normal rat hearts challenged with I/R, but with a limited degree of protection in ovariectomized rat hearts. The protective effect of CORM-2 was further confirmed via a reduction in cardiac injury, preservation of mitochondrial enzymes, and reduction in oxidative stress in all groups. CONCLUSION: CORM-2 administration significantly attenuated myocardial I/R injury in ovariectomized rat hearts by attenuating I/R-associated mitochondrial perturbations and reducing oxidative stress.

Inhibition of PI3K/mTOR/KATP channel blunts sodium thiosulphate preconditioning mediated cardioprotection against ischemia-reperfusion injury.

Recent studies have shown that pre and postconditioning the heart with sodium thiosulfate (STS) attenuate ischemia-reperfusion (IR) injury. However, the underlying mechanism involved in the cardioprotective signaling pathway is not fully explored. This study examined the existing link of STS mediated protection (as pre and post-conditioning agents) with PI3K, mTOR, and mPTP signaling pathways using its respective inhibitors. STS was administered to the isolated perfused rat heart through Kreb's Heinselit buffer before ischemia (precondition: SIPC) and reperfusion (postcondition: SPOC) in the presence and absence of the PI3K, mTOR, and mPTP signaling pathway inhibitors (wortmannin, rapamycin, and glibenclamide respectively). SIPC failed to improve the IR injury-induced altered cardiac hemodynamics, increased infarct size, and the release of cardiac injury markers in the presence of these inhibitors. On the other hand, the SPOC protocol effectively rendered the cardioprotection even in the PI3K/mTOR/KATP inhibitors presence. Interestingly, the SIPC's identified mode of action viz reduction in oxidative stress and the preservation of mitochondrial function were lost in the inhibitors' presence. Based on the above results, we conclude that the underlying mechanism of SIPC mediated cardioprotection works via the PI3K/mTOR/KATP signaling pathway axis activation.

Attenuation of cardiac ischemia-reperfusion injury by sodium thiosulfate is partially dependent on the effect of cystathione beta synthase in the myocardium.

Our early studies have shown that sodium thiosulfate (STS) treatment attenuated the ischemia-reperfusion (IR)-induced injury in an isolated rat heart model by decreasing apoptosis, oxidative stress, and preserving mitochondrial function. Hydrogen sulfide, the precursor molecule is reported to have similar efficacy. This study aims to investigate the role of endogenous hydrogen sulfide in STS-mediated cardioprotection against IR in an isolated rat heart model. D, L-propargylglycine (PAG), an inhibitor of cystathionine γ-lyase was used as endogenous H2S blocker. In addition, we used the hypoxia-reoxygenation (HR) model to study the impact of STS in cardiomyocytes (H9C2) and fibroblast (3T3) cells. STS treatment to animal and cells prior to IR or HR decreased cell injury. The sensitivity of H9C2 and 3T3 cells towards HR (6 h hypoxia followed by 12 h reoxygenation) challenge varies, where, 3T3 cells exhibited higher cell death (54%). Cells treated with PAG prior to STS abrogate the protective effect in 3T3 (cell viability 61%) but not in H9C2 (cell viability 82%). Further evaluation in rat heart model showed partial recovery (46% RPP) of heart from those hearts pretreated with PAG prior to STS condition. In conclusion, we demonstrated that STS-mediated cardioprotection to IR-challenged rat heart is not fully dependent on endogenous H2S level and this dependency may be linked to higher fibroblast content in rat heart.

Sodium thiosulfate post-conditioning protects rat hearts against ischemia reperfusion injury via reduction of apoptosis and oxidative stress.

Pharmacological agents given at the time of reperfusion can protect the heart from ischemia reperfusion injury (IR). Being a calcium chelator, antioxidant and mitochondrial potassium channel modulator, sodium thiosulfate (STS) was chosen to treat myocardial IR injury. Isolated rat heart model was used to induce IR injury and the hemodynamic changes were monitored using PowerLab (AD Instruments, Australia). STS at a dose of 1 mM given at the early stage of reperfusion significantly reduced the infarct size and recovered the failing heart from reperfusion injury. Its action was based on reduction of apoptosis as evidenced from decreased activity of caspase-3 in the myocardium, lowered expression of casp-3 and PARP, which was supported by absence of significant DNA fragmentation and histological derangement of fibers compared to the injury control. An evaluation of the inter-dependency of H2S and STS biosynthesis in the STS treated groups showed no significant changes in the level of STS, H2S and rhodanese, except the cystathionine gamma lyase activity that improved upon treatment. The mechanism underlying the antiapoptotic, mitochondrial preservation and antioxidant effects of STS were related to the biosynthesis of H2S. The fact that inhibition of cystathionine gamma lyase limited the STS mediated cardio protection supports this observation.