Mitochondrial Dysfunction In Cardiomyocytes Research Articles

Evidence for enduring cardiac and multiorgan toxicity after repeated exposure to the synthetic cannabinoid JWH-018 in male rats

The use of synthetic cannabinoid receptor agonists (SCRAs) represents a public health concern. Besides abuse liability and cognitive impairments, SCRA consumption is associated with serious medical consequences in humans, including cardiotoxicity. The precise mechanisms underlying cardiac or other toxicities induced by SCRAs are not well understood. Here, we used in silico, in vivo, and ex vivo approaches to investigate the toxicological consequences induced by exposure to the SCRA JWH-018. Along with in silico predictive toxicological screening of 36 SCRAs by MC4PC software, adult male Sprague-Dawley rats were repeatedly exposed to JWH-018 (0.25mg/kg ip) for 14 consecutive days, with body temperature and cardiovascular parameters measured over the course of treatment. At 1 and 7 days after JWH-018 discontinuation, multiorgan tissue pathologies and heart mitochondria bioenergetics were assessed. The in silico findings predicted risk of cardiac adverse effects specifically for JWH-018 and other aminoalkylindole SCRAs (i.e., electrocardiogram abnormality and QT prolongation). The results from rats revealed that repeated, but not single, JWH-018 exposure induced hypothermia and cardiovascular stimulation (e.g., increased blood pressure and heart rate) which persisted throughout treatment. Post-mortem findings demonstrated cardiac lesions (i.e., vacuolization, waving, edema) 1 day after JWH-018 discontinuation, which may contribute to lungs, kidneys, and liver tissue degeneration observed 7 days later. Importantly, repeated JWH-018 exposure induced mitochondrial dysfunction in cardiomyocytes, i.e., defective lipid OXPHOS, which may represent one mechanism of JWH-018-induced toxicity. Our results demonstrate that repeated administration of even a relatively low dose of JWH-018 is sufficient to affect cardiovascular function and induce enduring toxicological consequences, pointing to risks associated with SCRA consumption.

LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

A novel function of claudin-5 in maintaining the structural integrity of the heart and its implications in cardiac pathology

This study aims to investigate the role of claudin-5 (Cldn5) in cardiac structural integrity. Proteomic analysis was performed to screen the protein profiles in enlarged left atrium from atrial fibrillation (AF) patients. Cldn5 shRNA adeno-associated virus (AAV) or siRNA was injected into the mouse left ventricle or added into HL1 cells respectively to knockdown Cldn5 in cardiomyocytes to observe whether the change of Cldn5 influences cardiac morphology and function, and affects those protein expressions stem from the proteomic analysis. Mitochondrial density and membrane potential were also measured by Mitotracker staining and JC-1 staining under the confocal microscope in HL1 cells. Cldn5 was reduced in cardiomyocytes from the left atrial appendage of AF patients compared to non-AF donors. Proteomic analysis showed 83 proteins were less abundant and 102 proteins were more abundant in AF patients. KEGG pathway analysis showed less abundant CACNA2D2, CACNB2, MYL2 and MAP6 were highly associated with dilated cardiomyopathy. Cldn5 shRNA AAV injection caused severe cardiac atrophy, dilation and myocardial dysfunction in mice. The decreases in mitochondrial numbers and mitochondrial membrane potentials in HL1 cells were observed after Cldn5 knockdown. We demonstrated for the first time the mechanism of Cldn5 downregulation-induced myocyte atrophy and myocardial dysfunction might be associated with the downregulation of CACNA2D2, CACNB2, MYL2 and MAP6, and mitochondrial dysfunction in cardiomyocytes.

GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.

Dexmedetomidine alleviates Hypoxia/reoxygenation-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway.

Myocardial ischemia/reperfusion injury (MIRI) seriously threatens the health of people. The mitochondrial dysfunction in cardiomyocytes can promote the progression of MIRI. Dexmedetomidine (Dex) could alleviate the myocardial injury, which was known to reverse mitochondrial dysfunction in lung injury. However, the function of Dex in mitochondrial dysfunction during MIRI remains unclear. To assess the function of Dex in mitochondrial dysfunction during MIRI. To investigate the function of Dex in MIRI, H9C2 cells were placed in condition of hypoxia/reoxygenation (H/R). CCK8 assay was performed to test the cell viability, and the mitochondrial membrane potential was evaluated by JC-1 staining. In addition, the binding relationship between Sirt3 and Prdx3 was explored by Co-IP assay. Furthermore, the protein expressions were examined using western blot. Dex could abolish H/R-induced mitochondrial dysfunction in H9C2 cells. In addition, H/R treatment significantly inhibited the expression of Sirt3, while Dex partially restored this phenomenon. Knockdown of Sirt3 or Prdx3 obviously reduced the protective effect of Dex on H/R-induced mitochondrial injury. Meanwhile, Sirt3 could enhance the function of Prdx3 via deacetylation of Prdx3. Dex was found to attenuate H/R-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway. Thus, this study might shed new lights on exploring new strategies for the treatment of MIRI.

SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy.

Sirtuin 5 (SIRT5), localized in the mitochondria, has been identified as a protein desuccinylase and demalonylase in the mitochondria since the depletion of SIRT5 boosted the global succinylation and malonylation of mitochondrial proteins. We investigated the role of SIRT5 in diabetic cardiomyopathy (DCM) and identified the mechanism regarding lysine demalonylation in this process. Wild-type and SIRT5 knockout mice were induced with DCM, and primary cardiomyocytes and cardiac fibroblasts extracted from wild-type and SIRT5 knockout mice were subjected to high glucose (HG). SIRT5 deficiency exacerbated myocardial injury in DCM mice, aggravated HG-induced oxidative stress and mitochondrial dysfunction in cardiomyocytes, and intensified cardiomyocyte senescence, pyroptosis, and DNA damage. DCM-induced SIRT5 loss diminished glutathione S-transferase P (GSTP1) protein stability, represented by significantly increased lysine malonylation (Mal-Lys) modification of GSTP1. SIRT5 overexpression alleviated DCM-related myocardial injury, which was reversed by GSTP1 knockdown. Reduced SIRT5 transcription in DCM resulted from the downregulation of SPI1. SPI1 promoted the transcription of SIRT5, thereby ameliorating DCM-associated myocardial injury. However, SIRT5 deletion resulted in a significant reversal of the protective effect of SPI1. These observations suggest that SPI1 activates SIRT5 transcriptionally to mediate GSTP1 Mal-Lys modification and protein stability, thus ameliorating DCM-associated myocardial injury.

Abstract 12043: Neutrophil Extracellular Traps in Heart Tissue Drive Cardiac Dysfunction and Adverse Outcomes in Dilated Cardiomyopathy

Background: Neutrophil extracellular traps (NETs) have emerged as crucial contributors to cardiovascular diseases, but the significance of NETs in heart failure remains unclear. Aims: We aimed to clarify the relevance of NETs in myocardial tissue in heart failure. Methods and Results: We studied consecutive 62 patients with idiopathic dilated cardiomyopathy (DCM) who underwent endomyocardial biopsy. Using immunohistochemistry, NETs were identified in heart tissue by detecting the colocalization of citrullinated histone H3, neutrophil elastase, and DAPI. DCM patients had significantly higher numbers of NETs per tissue area and per neutrophil compared to control subjects without heart failure (n=11, Figure A ) (P < 0.05 and P < 0.05, respectively). When categorizing DCM patients based on the presence or absence of NETs, patients with NETs (n=32) had lower left ventricular ejection fraction and higher B-type natriuretic peptide levels than those without NETs (P < 0.05 and P < 0.05, respectively). Kaplan-Meier analysis revealed that NETs-positive DCM patients had lower event-free survival rates from the composite of cardiac events including cardiac deaths, decompensated heart failure, and left ventricular assist device implantation over a median 768-day follow-up ( Figure B ). In a multivariable Cox proportional hazard model, the presence of NETs independently associated with the risk of adverse cardiac events (hazard ratio, 5.96; P < 0.05). Ex vivo analysis revealed that NETs-containing conditioned medium from wild-type neutrophils impaired mitochondrial oxygen consumption in mouse adult cardiomyocytes (P < 0.05), while genetic ablation of peptidyl arginine deiminase 4, a molecule essential for NETs formation, abolished this effect. Conclusion: NETs in myocardial tissue contribute to cardiac dysfunction and adverse outcomes in DCM patients, potentially through mitochondrial dysfunction of cardiomyocytes.

TRAP1 inhibits MARCH5-mediated MIC60 degradation to alleviate mitochondrial dysfunction and apoptosis of cardiomyocytes under diabetic conditions.

Mitochondrial dysfunction and cell death play important roles in diabetic cardiomyopathy, but the underlying mechanisms remain unclear. Here, we report that mitochondrial dysfunction and cell apoptosis are prominent features of primary cardiomyocytes after exposure to high glucose/palmitate conditions. The protein level of MIC60, a core component of mitochondrial cristae, is decreased via ubiquitination and degradation under these conditions. Exogenous expression of MIC60 alleviates cristae disruption, mitochondrial dysfunction and apoptosis. Moreover, we identified MARCH5 as an E3 ubiquitin ligase that specifically targets MIC60 in this process. Indeed, MARCH5 mediates K48-linked ubiquitination of MIC60 at Lys285 to promote its degradation. Mutation of the ubiquitination site in MIC60 or the MIC60-interacting motifs in MARCH5 abrogates MARCH5-mediated MIC60 ubiquitination and degradation. Silencing MARCH5 significantly alleviates high glucose/palmitate-induced mitochondrial dysfunction and apoptosis in primary cardiomyocytes. In addition to E3 ubiquitin ligases, molecular chaperones also play important roles in protein stability. We previously reported that the mitochondrial chaperone TRAP1 inhibits the ubiquitination of MIC60, but the detailed mechanism is unknown. Here, we find that TRAP1 performs this function by competing with MARCH5 for binding to MIC60. Our findings provide new insights into the mechanism underlying mitochondrial dysfunction in cardiomyocytes in diabetic cardiomyopathy. MARCH5 promotes ubiquitination of MIC60 to induce MIC60 degradation, mitochondrial dysfunction and apoptosis in cardiomyocytes under diabetic conditions. TRAP1 inhibits MARCH5-mediated ubiquitination by competitively interacting with MIC60.

Mitochondria and NLRP3 inflammasome in cardiac hypertrophy.

Cardiac hypertrophy is the main adaptive response of the heart to chronic loads; however, prolonged or excessive hypertrophy promotes myocardial interstitial fibrosis, systolic dysfunction, and cardiomyocyte death, especially aseptic inflammation mediated by NLRP3 inflammasome, which can aggravate ventricular remodeling and myocardial damage, which is an important mechanism for the progression of heart failure. Various cardiac overloads can cause mitochondrial damage. In recent years, the mitochondria have been demonstrated to be involved in the inflammatory response during the development of cardiac hypertrophy in vitro and in vivo. As the NLRP3 inflammasome and mitochondria are regulators of inflammation and cardiac hypertrophy, we explored the potential functions of the NLRP3 inflammasome and mitochondrial dysfunction in cardiac hypertrophy. In particular, we proposed that the induction of mitochondrial dysfunction in cardiomyocytes may promote NLRP3-dependent inflammation during myocardial hypertrophy. Further in-depth studies could prompt valuable discoveries regarding the underlying molecular mechanisms of cardiac hypertrophy, reveal novel anti-inflammatory therapies for cardiac hypertrophy, and provide more desirable therapeutic outcomes for patients with cardiac hypertrophy.

Pgam5-mediated PHB2 dephosphorylation contributes to endotoxemia-induced myocardial dysfunction by inhibiting mitophagy and the mitochondrial unfolded protein response.

Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5) have been associated with altered mitochondrial homeostasis in several cardiovascular diseases; however, their role in endotoxemia-related myocardial dysfunction has not been explored. Our experiments were aimed to evaluate the potential contribution of Pgam5 and PHB2 to endotoxemia-induced mitochondrial dysfunction in cardiomyocytes, with a focus on two endogenous protective programs that sustain mitochondrial integrity, namely mitophagy and the mitochondrial unfolded protein response (UPRmt). We found that PHB2 transgenic mice are resistant to endotoxemia-mediated myocardial depression and mitochondrial damage. Our assays indicated that PHB2 overexpression activates mitophagy and the UPRmt, which maintains mitochondrial metabolism, prevents oxidative stress injury, and enhances cardiomyocyte viability. Molecular analyses further showed that Pgam5 binds to and dephosphorylates PHB2, resulting in cytosolic translocation of mitochondrial PHB2. Silencing of Pgam5 or transfection of a phosphorylated PHB2 mutant in mouse HL-1 cardiomyocytes prevented the loss of mitochondrially-localized PHB2 and activated mitophagy and UPRmt in the presence of LPS. Notably, cardiomyocyte-specific deletion of Pgam5 in vivo attenuated LPS-mediated myocardial dysfunction and preserved cardiomyocyte viability. These findings suggest that Pgam5/PHB2 signaling and mitophagy/UPRmt are potential targets for the treatment of endotoxemia-related cardiac dysfunction.

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation–transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

A ROS-Responsive Liposomal Composite Hydrogel Integrating Improved Mitochondrial Function and Pro-Angiogenesis for Efficient Treatment of Myocardial Infarction.

Mitochondrial dysfunction of cardiomyocytes (CMs) has been identified as a significant pathogenesis of early myocardial infarction (MI). However, only a few agents or strategies have been developed to improve mitochondrial dysfunction for the effective MI treatment. Herein, a reactive oxygen species (ROS)-responsive PAMB-G-TK/4-arm-PEG-SG hydrogel is developed for localized drug-loaded liposome delivery. Notably, the liposomes contain both elamipretide (SS-31) and sphingosine-1-phosphate (S1P), where SS-31 acts as an inhibitor of mitochondrial oxidative damage and S1P as a signaling molecule for activating angiogenesis. Liposome-encapsulated PAMB-G-TK/4-arm-PEG-SG hydrogels demonstrate myocardium-like mechanical strength and electrical conductivity, and ROS-sensitive release of SS-31 and S1P-loaded liposomes. Further liposomal release of SS-31, which can target cytochrome c in the mitochondrial inner membrane of damaged CMs, inhibits pathological ROS production, improving mitochondrial dysfunction. Meanwhile, S1P released from the liposome induces endothelial cell angiogenesis by activating the S1PR1/PI3K/Akt pathway. In a rat MI model, the resulting liposomal composite hydrogel improves cardiac function by scavenging excess ROS, improving mitochondrial dysfunction, and promoting angiogenesis. This study reports for the first time a liposomal composite hydrogel that can directly target mitochondria of damaged CMs for a feedback-regulated release of encapsulated liposomes to consume the overproduced pathological ROS for improved CM activity and enhanced MI treatment.

The Regulatory Effects of Magnolia Officinalis Extract on Aging-induced Mitochondrial Dysfunction in Cardiomyocytes

The Regulatory Effects of Magnolia Officinalis Extract on Aging-induced Mitochondrial Dysfunction in Cardiomyocytes

Functional remodeling of perinuclear mitochondria alters nucleoplasmic Ca2+signaling in heart failure

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure (HF) development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) vs. perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF vs. PN mitochondria in pressure overload-induced cardiac remodeling in mice, and in non-failing and failing human cardiomyocytes.

FNDC5/irisin reduces ferroptosis and improves mitochondrial dysfunction in hypoxic cardiomyocytes by Nrf2/HO-1 axis.

Myocardial infarction is characterized by cardiomyocyte death and mitochondrial dysfunction induced by ischemia. Ferroptosis, a novel form of cell death, has been found to play critical roles under ischemic conditions. Recently, several studies have shown that fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, protect the heart against injury. However, its protective effect on ferroptosis and mitochondrial impairments is still unclear. Thus, our aim was to investigate the role of irisin in ferroptosis and mitochondrial dysfunction in cardiomyocytes under hypoxic conditions. Cardiomyocytes were treated with FNDC5 overexpression and/or irisin under normoxic and hypoxic conditions. Cell viability was assessed by Cell Counting Kit-8 assay. Reactive oxygen species production was evaluated by dihydroethidium staining. In addition, the intracellular ferrous iron level (Fe2+ ) and the relative concentration of malondialdehyde and ATP content were determined using an Iron Assay Kit, Lipid Peroxidation Assay Kit, and ATP Bioluminescent Assay Kit. The superoxide dismutase level in cells was measured using an Enzyme-Linked ImmunosorbentAssay Kit. Furthermore, an immunoblotting assay was used to determine ferroptosis-related mitochondrial proteins. Hypoxia promoted cell death, increased ferroptosis, and caused mitochondrial dysfunction in cardiomyocytes. Interestingly, FNDC5 overexpression and/or irisin administration elevated cell viability, decreased ferroptosis, and reversed mitochondrial impairments induced by hypoxia. Mechanistically, FNDC5/irisin reduced ferroptosis and reversed mitochondrial impairments by Nrf2/HO-1 axis in hypoxic cardiomyocytes. Thus, we have demonstrated that FNDC5/irisin plays a protective role in ferroptosis and mitochondrial dysfunction in hypoxia-induced cardiomyocytes.

A case study of a patient-centered reverse translational systems-based approach to understand adverse event profiles in drug development.

Adverse drug reactions (ADRs) of targeted therapy drugs (TTDs) are frequently unexpected and long‐term toxicities detract from exceptional efficacy of new TTDs. In this proof‐of‐concept study, we explored how molecular causation involved in trastuzumab‐induced cardiotoxicity changes when trastuzumab was given in combination with doxorubicin, tamoxifen, paroxetine, or lapatinib. The data analytical platform Molecular Health Effect was utilized to map population ADR data from the US Food and Drug Administration (FDA) Adverse Event Reporting System to chemical and biological databases (such as UniProt and Reactome), for hypothesis generation regarding the underlying molecular mechanisms causing cardiotoxicity. Disproportionality analysis was used to assess the statistical relevance between adverse events of interest and molecular causation. Literature search was performed to compare the established hypotheses to published experimental findings. We found that the combination therapy of trastuzumab and doxorubicin may affect mitochondrial dysfunction in cardiomyocytes through different molecular pathways such as BCL‐X and PGC‐1α proteins, leading to a synergistic effect of cardiotoxicity. We found, on the other hand, that trastuzumab‐induced cardiotoxicity would be diminished by concomitant use of tamoxifen, paroxetine, and/or lapatinib. Tamoxifen and paroxetine may cause less cardiotoxicity through an increase in antioxidant activities, such as glutathione conjugation. Lapatinib may decrease the apoptotic effects in cardiomyocytes by altering the effects of trastuzumab on BCL‐X proteins. This patient‐centered systems‐based approach provides, based on the trastuzumab‐induced ADR cardiotoxicity, an example of how to apply reverse translation to investigate ADRs at the molecular pathway and target level to understand the causality and prevalence during drug development of novel therapeutics.

Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity

Cyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the β-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca2+/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.

Calcitriol attenuates the cytotoxicity induced by aluminium phosphide via inhibiting mitochondrial dysfunction and oxidative stress in rat isolated cardiomyocytes

These days, poisoning with aluminium phosphide (AlP), is one of the main health threats in human societies. Previous studies have been reported that cardiotoxicity induced by AlP, via mitochondrial dysfunction and oxidative stress is the main cause of death in victims. On the other, collectively, multiple lines of evidence strongly suggest that calcitriol has mitochondrial protective and antioxidant effects. Therefore, we assumed that calcitriol could presumably ameliorate AlP-induced oxidative stress and mitochondrial dysfunction in cardiomyocytes. Mitochondria and cardiomyocytes were isolated by differential centrifugation and collagenase perfusion respectively from rat heart. The isolated cardiomyocytes and mitochondria were cotreated with different concentrations of calcitriol (0.2, 0.4 and 1 μg/ml) and AlP (20 μg/ml) for 3 h. The parameters of cellular toxicity including; cytotoxicity, reactive oxygen species (ROS) formation, malondialdehyde (MDA) level, mitochondria membrane potential (ΔΨm) collapse, lysosomal membrane integrity, the level of oxidized and reduced glutathione (GSH and GSSG), and mitochondrial toxicity parameters including; succinate dehydrogenase (SDH) activity and mitochondrial swelling were analyzed using biochemical and flow cytometric evaluations. Administration of AlP significantly increased cytotoxicity, GSH depletion, cellular ROS formation, MDA level, mitochondrial and lysosomal dysfunction in isolated cardiomyocytes. In isolated mitochondria, AlP decreased SDH activity and mitochondrial swelling. The cotreatment of isolated cardiomyocytes and mitochondria with calcitriol (0.4 and 1 μg/ml) and AlP (20 μg/ml) showed the ability to reduce the toxic effects of AlP. These findings suggest a potential therapeutic role of calcitriol in protecting cardiomyocytes and cardiac mitochondria from oxidative damage induced by AlP. According to the results, calcitriol exerted ameliorative effects against AlP-induced cytotoxicity and mitochondrial toxicity, and the effect was attributed to the antioxidant properties.

Serum from pediatric dilated cardiomyopathy patients promotes dysregulation of cardiolipin biosynthesis and mitochondrial dysfunction

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondria function are dysregulated in pediatric hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in failing adult hearts. We investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using Seahorse assay, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mtDNA copy number determined by qPCR. Mass spectroscopy was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and copy number were not significantly altered, we showed that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and gene expression of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by PPARα. Pediatric DCM serum circulating factors can promote CL remodeling resulting in mitochondrial dysfunction in cardiomyocytes. These findings suggest that CL could be a therapeutic target for this particular population.

Bevacizumab-Induced Mitochondrial Dysfunction, Endoplasmic Reticulum Stress, and ERK Inactivation Contribute to Cardiotoxicity.

The molecular mechanisms underlying the cardiotoxicity associated with bevacizumab, a first-line immunotherapeutic agent used to treat lung cancer, are not fully understood. Here, we examined intracellular signal transduction in cardiomyocytes after exposure to different doses of bevacizumab in vitro. Our results demonstrated that bevacizumab significantly and dose-dependently reduces cardiomyocyte viability and increases cell apoptosis. Bevacizumab treatment also led to mitochondrial dysfunction in cardiomyocytes, as evidenced by the decreased ATP production, increased ROS production, attenuated antioxidative enzyme levels, and reduced respiratory complex function. In addition, bevacizumab induced intracellular calcium overload, ER stress, and caspase-12 activation. Finally, bevacizumab treatment inhibited the ERK signaling pathway, which, in turn, significantly reduced cardiomyocyte viability and contributed to mitochondrial dysfunction. Together, our results demonstrate that bevacizumab-mediated cardiotoxicity is associated with mitochondrial dysfunction, ER stress, and ERK pathway inactivation. These findings may provide potential treatment targets to attenuate myocardial injury during lung cancer immunotherapy.