Functional activity and morphology of isolated rat cardiac mitochondria under calcium overload. Effect of naringin.

The function of mitochondria as a regulator of myocyte calcium homeostasis has been extensively discussed. The aim of the present work was further clarification of the details of modulation of the functional activity of rat cardiac mitochondria by exogenous Ca2+ ions either in the absence or in the presence of the plant flavonoid naringin. Low free Ca2+ concentrations (40-250 nM) effectively inhibited the respiratory activity of heart mitochondria, remaining unaffected the efficacy of oxygen consumption. In the presence of high exogenous Ca2+ ion concentrations (Ca2+ free was 550 µM), we observed a dramatic increase in mitochondrial heterogeneity in size and electron density, which was related to calcium-induced opening of the mitochondrial permeability transition pores (MPTP) and membrane depolarization (Ca2+free ions were from 150 to 750 µM). Naringin partially prevented Ca2+-induced cardiac mitochondrial morphological transformations (200 µM) and dose-dependently inhibited the respiratory activity of mitochondria (10-75 µM) in the absence or in the presence of calcium ions. Our data suggest that naringin (75 µM) promoted membrane potential dissipation, diminishing the potential-dependent accumulation of calcium ions by mitochondria and inhibiting calcium-induced MPTP formation. The modulating effect of the flavonoid on Ca2+-induced mitochondria alterations may be attributed to the weak-acidic nature of the flavonoid and its protonophoric/ionophoric properties. Our results show that the sensitivity of rat heart mitochondria to Ca2+ ions was much lower in the case of MPTP opening and much higher in the case of respiration inhibition as compared to liver mitochondria.

Isolation and reconstruction of cardiac mitochondria from SBEM images using a deep learning-based method.

Mitochondrial morphological defects are a common feature of diseased cardiac myocytes. However, quantitative assessment of mitochondrial morphology is limited by the time-consuming manual segmentation of electron micrograph (EM) images. To advance understanding of the relation between morphological defects and dysfunction, an efficient morphological reconstruction method is desired to enable isolation and reconstruction of mitochondria from EM images. We propose a new method for isolating and reconstructing single mitochondria from serial block-face scanning EM (SBEM) images. CDeep3M, a cloud-based deep learning network for EM images, was used to segment mitochondrial interior volumes and boundaries. Post-processing was performed using both the predicted interior volume and exterior boundary to isolate and reconstruct individual mitochondria. Series of SBEM images from two separate cardiac myocytes were processed. The highest F1-score was 95% using 50 training datasets, greater than that for previously reported automated methods and comparable to manual segmentations. Accuracy of separation of individual mitochondria was 80% on a pixel basis. A total of 2315 mitochondria in the two series of SBEM images were evaluated with a mean volume of 0.78 µm3. The volume distribution was very broad and skewed; the most frequent mitochondria were 0.04-0.06 µm3, but mitochondria larger than 2.0 µm3 accounted for more than 10% of the total number. The average short-axis length was 0.47 µm. Primarily longitudinal mitochondria (0-30 degrees) were dominant (54%). This new automated segmentation and separation method can help quantitate mitochondrial morphology and improve understanding of myocyte structure-function relationships.

Analysis of Mitochondrial Function, Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles.

Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.

Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide.

NEW FINDINGS: What is the central question of this study? In adult rat hearts, exposure to hypobaric hypoxia increases tolerance to hypoxia-reoxygenation, termed endogenous cardioprotection. The mechanism involves the nitric oxide system and modulation of mitochondrial oxygen consumption. What is the cardiac energetic response in prepubertal rats exposed to hypobaric hypoxia? What is the main finding and its importance? Prepubertal rats, unlike adult rats, did not increase tolerance to hypoxia-reoxygenation in response acute exposure to hypobaric hypoxia, which impaired cardiac contractile economy. This finding could be related to a failure to increase nitric oxide synthase expression, hence modulation of mitochondrial oxygen consumption and ATP production. ABSTRACT: Studies in our laboratory showed that exposure of rats to hypobaric hypoxia (HH) increased the tolerance of the heart to hypoxia-reoxygenation (H/R), involving mitochondrial and cytosolic nitric oxide synthase (NOS) systems. The objective of the present study was to evaluate how the degree of somatic maturation could alter this healthy response. Prepubertal male rats were exposed for 48 h to a simulated altitude of 4400 m in a hypobaric chamber. The mechanical energetic activity in perfused hearts and the contractile functional capacity of NOS in isolated left ventricular papillary muscles were evaluated during H/R. Cytosolic nitric oxide (NO), production of nitrites/nitrates (Nx), expression of NOS isoforms, mitochondrial O2 consumption and ATP production were also evaluated. The left ventricular pressure during H/R was not improved by HH. However, the energetic activity was increased. Thus, the contractile economy (left ventricular pressure/energetic activity) decreased in HH. Nitric oxide did not modify papillary muscle contractility after H/R. Cytosolic p-eNOS-Ser1177 and inducible NOS expression were decreased by HH, but no changes were observed in NO production. Interestingly, HH increased Nx levels, but O2  consumption and ATP production in mitochondria were not affected by HH. Prepubertal rats exposed to HH preserved cardiac contractile function, but with a high energetic cost, modifying contractile economy. Although this could be related to the decreased NOS expression detected, cytosolic NO production was preserved, maybe through the Nx metabolic pathway, without modification of mitochondrial ATP production and O2  consumption. In this scenario, the treatment was unable to increase tolerance to H/R as observed in adult animals.

Effects of Different Exercise Interventions on Cardiac Function in Rats With Myocardial Infarction.

BACKGROUND: High-intensity interval training (HIIT) and aerobic training (AT) both improve cardiac function; however, their effects on cardiac function after myocardial infarction (MI) and the molecular mechanisms are unclear. In this study, HIIT, AT and sedentary (SED) interventions were performed for 4 weeks to compare the effects on cardiac function after MI and explore a more suitable approach for clinical application and the potential mechanisms. METHODS: Twenty-four (24) male rats were randomly divided into a control group (CON), MI-sedentary group (MI-SED), MI-aerobic training group (MI-AT), and MI-high-intensity interval training group (MI-HIIT). After 4 weeks of intervention the exercise capacity, heart rate (HR), left ventricular end-diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF), AMP-activated protein kinase α1 (AMPKα1), cardiomyocyte morphology, and cardiac mitochondria were assessed. RESULTS: After intervention: 1) exercise capacity in the MI-AT (49.08±3.141 m; p<0.001) and MI-HIIT (51.70±7.572 m; p<0.001) groups was significantly more increased than the MI-SED group; there was no significant difference between the MI-AT and MI-HIIT group (p=0.33). 2) LVEDD and LVESD in the MI-SED (p<0.01) and MI-HIIT (p<0.01) groups was significantly more increased than the CON group; the MI-AT group showed no significant difference in LVEDD and LVESD compared with the CON group; LVEF in the MI-AT (53.47±7.913%; p=0.03) and MI-HIIT (56.20±7.224%; p=0.006) groups was significantly more increased than the MI-SED group, and there was no statistical difference between the MI-AT and MI-HIIT groups. 3) AMPKα1 expression was significantly increased in the MI-AT (1.15±0.264; p=0.001) and MI-HIIT (1.04±0.238; p=0.003) groups and decreased in the MI-SED group (0.71±0.257; p<0.001) when compared with the CON group. 4) The MI-SED group exhibited sarcoplasmic dissolution and fibrous hyperplasia in the myocardium, cardiac mitochondrial damage and reduced mitochondrial numbers; the MI-HIIT group displayed swollen and vacuolated cardiac mitochondria with disrupted cristae; the MI-AT and MI-HIIT groups had significantly increased cardiac mitochondrial numbers than the MI-SED group; there was no statistical difference between the MI-AT and MI-HIIT groups. CONCLUSIONS: Aerobic training and HIIT for 4 weeks had similar cardioprotection and were superior to SED intervention. Both AT and HIIT improved cardiac function and exercise capacity by upregulating AMPKα1 expression. However, 4 weeks of intervention resulted in left ventricular dilation and cardiac myocardial mitochondrial injury in the MI-HIIT group.

Testosterone, myocardial function, and mortality.

The cardiovascular system is particularly sensitive to androgens, but some controversies exist regarding the effect of testosterone on the heart. While among anabolic abusers, cases of sudden cardiac death have been described, recently it was reported that low serum level of testosterone was correlated with increased risk of cardiovascular diseases (CVD) and mortality rate. This review aims to evaluate the effect of testosterone on myocardial tissue function, coronary artery disease (CAD), and death. Low testosterone level is associated with increased incidence of CAD and mortality. Testosterone administration in hypogonadal elderly men and women has a positive effect on cardiovascular function and improved clinical outcomes and survival time. Although at supraphysiologic doses, androgen may have a toxic effect, and at physiological levels, testosterone is safe and exerts a beneficial effect on myocardial function including mechanisms at cellular and mitochondrial level. The interaction with free testosterone and estradiol should be considered. Further studies are necessary to better understand the interaction mechanisms for an optimal androgen therapy in CVD.

Chronic treatment with prebiotics, probiotics and synbiotics attenuated cardiac dysfunction by improving cardiac mitochondrial dysfunction in male obese insulin-resistant rats.

PURPOSE: In metabolic syndrome, the composition of gut microbiota has been disrupted, and is associated with left ventricular (LV) dysfunction. Several types of prebiotics, probiotics, and synbiotics have been shown to exert cardioprotection by restoring gut microbiota from dysbiosis and reducing systemic inflammation. However, the effects of prebiotics such as xylooligosaccharides (XOS); probiotics such as Lactobacillus paracasei STII01 HP4, and synbiotics on metabolic and LV function in obese insulin-resistant rats have not been investigated. In this study, we hypothesized that prebiotics and probiotics improve metabolic parameters, heart rate variability (HRV), blood pressure (BP), and LV function by attenuating cardiac mitochondrial dysfunction, systemic inflammation, and oxidative stress, and that synbiotics provide greater efficacy than a single regimen in obese insulin resistance. METHODS: Rats were fed with either normal diet or high-fat diet (HFD) for 12 weeks and then rats in each dietary group were randomly subdivided into four subgroups to receive either a vehicle, prebiotics, probiotics, or synbiotics for another 12 weeks. Metabolic parameters, BP, HRV, LV function, cardiac mitochondrial function, systemic inflammation, and oxidative stress were determined. RESULTS: HFD-fed rats had obese insulin resistance with markedly increased systemic inflammatory marker [Serum LPS; ND; 0.6 ± 0.1 EU/ml vs. HFD; 5.7 ± 1.2 EU/ml (p < 0.05)], depressed HRV, and increased BP and LV dysfunction [%ejection fraction; ND; 93 ± 2% vs. HFD; 83 ± 2% (p < 0.05)]. Prebiotics, probiotics, and synbiotics attenuated insulin resistance by improving insulin sensitivity and lipid profiles. All interventions also improved HRV, BP, LV function [%ejection fraction; HFV; 81 ± 2% vs. HFPE; 93 ± 3%, HFPO; 92 ± 1%, HFC; 92 ± 2% (p < 0.05)] by attenuating mitochondrial dysfunction, oxidative stress, and systemic inflammation in obese insulin-resistant rats. CONCLUSION: Prebiotics, probiotics, and synbiotics shared similar efficacy in reducing insulin resistance and LV dysfunction in obese insulin-resistant rats.

Identity and function of a cardiac mitochondrial small conductance Ca2+-activated K+ channel splice variant.

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload.

α-MHC MitoTimer mouse: In vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart.

In order to maintain an efficient, energy-producing network in the heart, dysfunctional mitochondria are cleared through the mechanism of autophagy, which is closely linked with mitochondrial biogenesis; these, together with fusion and fission comprise a crucial process known as mitochondrial turnover. Until recently, the lack of molecular tools and methods available to researchers has impeded in vivo investigations of turnover. To investigate the process at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein characterized by transition from green fluorescence to a more stable red conformation over 48 h, and its rate of maturation is stable under physiological conditions. We fused the Timer cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of a cardiac-restricted promoter. This construct was used to create the alpha-MHC-MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice demonstrated a high degree of heterogeneity in the ratio of red-to-green fluorescence of MitoTimer in cardiac tissue. Further, scattered solitary mitochondria within cardiomyocytes display a much higher red-to-green fluorescence (red-shifted) relative to other mitochondria in the cell, implying a block in import of newly synthesized MitoTimer likely due to lower membrane potential. These red-shifted mitochondria may represent older, senescent mitochondria. Concurrently, the cardiomyocytes also contain a subpopulation of mitochondria that display a lower red-to-green fluorescence (green-shifted) relative to other mitochondria, indicative of germinal mitochondria that are actively engaged in import of newly-synthesized mito-targeted proteins. These mitochondria can be isolated and sorted from the heart by flow cytometry for further analysis. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.

Temperature acclimation of mitochondria function from the hearts of a temperate wrasse (Notolabrus celidotus).

Understanding how mitochondrial function alters with acclimation may provide insight to the limits these organelles place on temperate fish hearts facing seasonal temperature fluctuations. This investigation determined if compromised cardiac mitochondrial function contributed to heart failure (HF) in the New Zealand wrasse Notolabrus celidotus acclimated at their mean summer and winter ocean temperatures. To test this hypothesis, fish were acclimated to cold (CA, 15°C) and warm (WA, 21°C) temperatures. The temperature of HF was determined by Doppler sonography and mitochondrial function in permeabilised cardiac fibres was tested using high resolution respirometry. Heat stress mediated HF occurred at a THF of 26.7±0.4°C for CA fish, and at 28.2±0.6°C for WA fish. Biochemical analyses also revealed that WA fish had elevated resting plasma lactate indicating an increased dependence on anaerobic pathways. When cardiac fibres were tested with increasing temperatures, apparent breakpoints in the respiratory control ratio (RCR-I) with substrates supporting complex I (CI) oxygen flux occurred below the THF for both acclimated groups. While WA cardiac mitochondria were less sensitive to increasing temperature for respirational flux supported by CI, Complex II, and chemically uncoupled flux, CA fish maintained higher RCRs at higher temperatures. We conclude that while acclimation to summer temperatures does alter cardiac mitochondrial function in N. celidotus, these changes need not be beneficial in terms of oxidative phosphorylation efficiency and may come at an energetic cost, which would be detrimental in the face of further habitat warming.

Megamitochondria in Cardiomyocytes of a Knockout (Klf15-/-) Mouse.

The Kruppel-like factors (KLF) family of zinc-finger transcriptional regulators control many aspects of cardiomyocyte structure and function. Deletion of Klf15 from the nuclear genome in mice affects cardiac mitochondria. Some become grossly enlarged, extending many sarcomeres in length. These display many sites of incipient pinching, but there is little attenuation of the megamitochondria at these sites; there are no examples of organelles that clearly have reached the point where further membrane encroachment will cause separation into smaller daughter mitochondria. It is clear that deletion of Klf15 interferes with nuclear control of mitochondrial fission, whereas fusion appears to be unaffected.

Could thermal sensitivity of mitochondria determine species distribution in a changing climate?

For many aquatic species, the upper thermal limit (Tmax) and the heart failure temperature (THF) are only a few degrees away from the species' current environmental temperatures. While the mechanisms mediating temperature-induced heart failure (HF) remain unresolved, energy flow and/or oxygen supply disruptions to cardiac mitochondria may be impacted by heat stress. Recent work using a New Zealand wrasse (Notolabrus celidotus) found that ATP synthesis capacity of cardiac mitochondria collapses prior to T(HF). However, whether this effect is limited to one species from one thermal habitat remains unknown. The present study confirmed that cardiac mitochondrial dysfunction contributes to heat stress-induced HF in two additional wrasses that occupy cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats. With exposure to heat stress, T. lunare had the least scope to maintain heart function with increasing temperature. Heat-exposed fish of all species showed elevated plasma succinate, and the heart mitochondria from the cold temperate N. fucicola showed decreased phosphorylation efficiencies (depressed respiratory control ratio, RCR), cytochrome c oxidase (CCO) flux and electron transport system (ETS) flux. In situ assays conducted across a range of temperatures using naive tissues showed depressed complex II (CII) and CCO capacity, limited ETS reserve capacities and lowered efficiencies of pyruvate uptake in T. lunare and N. celidotus. Notably, alterations of mitochondrial function were detectable at saturating oxygen levels, indicating that cardiac mitochondrial insufficiency can occur prior to HF without oxygen limitation. Our data support the view that species distribution may be related to the thermal limits of mitochondrial stability and function, which will be important as oceans continue to warm.

Multiple mitochondria-targeted components screened from Sini decoction improved cardiac energetics and mitochondrial dysfunction to attenuate doxorubicin-induced cardiomyopathy.

Rationale: Sini decoction (SND) is an efficient formula against DOX-induced cardiomyopathy (DCM), but the active ingredient combination (AIC) and mechanisms of SND remain unclear. Therefore, the present study aimed to identify the AIC and elucidate the underlying mechanism of AIC on DCM. Methods: The AIC were screened by a novel comprehensive two-dimensional cardiac mitochondrial membrane chromatography (CMMC)-TOFMS analysis system and further validated by cell viability, reactive oxygen species (ROS) generation, ATP level, and mitochondrial membrane potential in DOX-induced H9c2 cell injury model. Then, an integrated model of cardiac mitochondrial metabolomics and proteomics were applied to clarify the underlying mechanism in vitro. Results: The CMMC column lifespan was significantly improved to more than 10 days. Songorine (S), neoline, talatizamine, 8-gingerol (G) and isoliquiritigenin (I), exhibiting stronger retention on the first-dimension CMMC column, were screened to have protective effects against DOX cardiotoxicity in the H9c2 cell model. S, G and I were selected as an AIC from SND according to the bioactivity evaluation and the compatibility theory of SND. The combined in vitro use of S, G and I produced more profound therapeutic effects than any component used individually on increasing ATP levels and mitochondrial membrane potential and suppressing intracellular ROS production. Moreover, SGI attenuated DCM might via regulating mitochondrial energy metabolism and mitochondrial dysfunction. Conclusions: The provided scientific evidence to support that SGI combination from SND could be used as a prebiotic agent for DCM. Importantly, the proposed two-dimensional CMMC-TOFMS analytical system provides a high-throughput screening strategy for mitochondria-targeted compounds from natural products, which could be applied to other subcellular organelle models for drug discovery.

Long COVID-19 and the Heart: Is Cardiac Mitochondria the Missing Link?

Significance: Although corona virus disease 2019 (COVID-19) has now gradually been categorized as an endemic, the long-term effect of COVID-19 in causing multiorgan disorders, including a perturbed cardiovascular system, is beginning to gain attention. Nonetheless, the underlying mechanism triggering post-COVID-19 cardiovascular dysfunction remains enigmatic. Are cardiac mitochondria the key to mediating cardiac dysfunction post-severe acute respiratory syndrome coronavirus 2 (post-SARS-CoV-2) infection? Recent Advances: Cardiovascular complications post-SARS-CoV-2 infection include myocarditis, myocardial injury, microvascular injury, pericarditis, acute coronary syndrome, and arrhythmias (fast or slow). Different types of myocardial damage or reduced heart function can occur after a lung infection or lung injury. Myocardial/coronary injury or decreased cardiac function is directly associated with increased mortality after hospital discharge in patients with COVID-19. The incidence of adverse cardiovascular events increases even in recovered COVID-19 patients. Disrupted cardiac mitochondria postinfection have been postulated to lead to cardiovascular dysfunction in the COVID-19 patients. Further studies are crucial to unravel the association between SARS-CoV-2 infection, mitochondrial dysfunction, and ensuing cardiovascular disorders (CVD). Critical Issues: The relationship between COVID-19 and myocardial injury or cardiovascular dysfunction has not been elucidated. In particular, the role of the cardiac mitochondria in this association remains to be determined. Future Directions: Elucidating the cause of cardiac mitochondrial dysfunction post-SARS-CoV-2 infection may allow a deeper understanding of long COVID-19 and resulting CVD, thus providing a potential therapeutic target. Antioxid. Redox Signal. 38, 599-618.

Long-term administration of Western diet induced metabolic syndrome in mice and causes cardiac microvascular dysfunction, cardiomyocyte mitochondrial damage, and cardiac remodeling involving caveolae and caveolin-1 expression.

BACKGROUND: Long-term consumption of an excessive fat and sucrose diet (Western diet, WD) has been considered a risk factor for metabolic syndrome (MS) and cardiovascular disease. Caveolae and caveolin-1 (CAV-1) proteins are involved in lipid transport and metabolism. However, studies investigating CAV-1 expression, cardiac remodeling, and dysfunction caused by MS, are limited. This study aimed to investigate the correlation between the expression of CAV-1 and abnormal lipid accumulation in the endothelium and myocardium in WD-induced MS, and the occurrence of myocardial microvascular endothelial cell dysfunction, myocardial mitochondrial remodeling, and damage effects on cardiac remodeling and cardiac function. METHODS: We employed a long-term (7 months) WD feeding mouse model to measure the effect of MS on caveolae/vesiculo-vacuolar organelle (VVO) formation, lipid deposition, and endothelial cell dysfunction in cardiac microvascular using a transmission electron microscopy (TEM) assay. CAV-1 and endothelial nitric oxide synthase (eNOS) expression and interaction were evaluated using real-time polymerase chain reaction, Western blot, and immunostaining. Cardiac mitochondrial shape transition and damage, mitochondria-associated endoplasmic reticulum membrane (MAM) disruption, cardiac function change, caspase-mediated apoptosis pathway activation, and cardiac remodeling were examined using TEM, echocardiography, immunohistochemistry, and Western blot assay. RESULTS: Our study demonstrated that long-term WD feeding caused obesity and MS in mice. In mice, MS increased caveolae and VVO formation in the microvascular system and enhanced CAV-1 and lipid droplet binding affinity. In addition, MS caused a significant decrease in eNOS expression, vascular endothelial cadherin, and ß-catenin interactions in cardiac microvascular endothelial cells, accompanied by impaired vascular integrity. MS-induced endothelial dysfunction caused massive lipid accumulation in the cardiomyocytes, leading to MAM disruption, mitochondrial shape transition, and damage. MS promoted brain natriuretic peptide expression and activated the caspase-dependent apoptosis pathway, leading to cardiac dysfunction in mice. CONCLUSION: MS resulted in cardiac dysfunction, remodeling by regulating caveolae and CAV-1 expression, and endothelial dysfunction. Lipid accumulation and lipotoxicity caused MAM disruption and mitochondrial remodeling in cardiomyocytes, leading to cardiomyocyte apoptosis and cardiac dysfunction and remodeling.

Association of an Increased Abnormal Mitochondria Ratio in Cardiomyocytes with a Prolonged Oxygen Uptake Time Constant during Cardiopulmonary Exercise Testing of Patients with Non-ischemic Cardiomyopathy.

Objective The cardiac function, blood distribution, and oxygen extraction in the muscles as well as the pulmonary function determine the oxygen uptake (VO2) kinetics at the onset of exercise. This factor is called the VO2 time constant, and its prolongation is associated with an unfavorable prognosis for heart failure (HF). The mitochondrial function of skeletal muscle is known to reflect exercise tolerance. Morphological changes and dysfunction in cardiac mitochondria are closely related to HF severity and its prognosis. Although mitochondria play an important role in generating energy in cardiomyocytes, the relationship between cardiac mitochondria and the VO2 time constant has not been elucidated. Methods We calculated the ratio of abnormal cardiac mitochondria in human myocardial biopsy samples using an electron microscope and measured the VO2 time constant during cardiopulmonary exercise testing. The VO2 time constant was normalized by the fat-free mass index (FFMI). Patients Fifteen patients with non-ischemic cardiomyopathy (NICM) were included. Patients were divided into two groups according to their median VO2 time constant/FFMI value. Results Patients with a low VO2 time constant/FFMI value had a lower abnormal mitochondria ratio than those with a high VO2 time constant/FFMI value. A multiple linear regression analysis revealed that the ratio of abnormal cardiac mitochondria was independently associated with a high VO2 time constant/FFMI. Conclusion An increased abnormal cardiac mitochondria ratio might be associated with a high VO2 time constant/FFMI value in patients with NICM.

Protective Effect of Curcumin, Chrysin and Thymoquinone Injection on Trastuzumab-Induced Cardiotoxicity via Mitochondrial Protection.

Mitochondrial dysfunction may lead to cardiomyocyte death in trastuzumab (TZM)-induced cardiotoxicity. Accordingly, this study was designed to evaluate the mitochondrial protective effects of curcumin, chrysin and thymoquinone alone in TZM-induced cardiotoxicity in the rats. Forty-eight male adult Wistar rats were divided into eight groups: control group (normal saline), TZM group (2.5 mg/kg I.P. injection, daily), TZM + curcumin group (10 mg/kg, I.P. injection, daily), TZM + chrysin (10 mg/kg, I.P. injection, daily), TZM + thymoquinone (0.5 mg/kg, I.P. injection, daily), curcumin group (10 mg/kg, I.P. injection, daily), chrysin group (10 mg/kg, I.P. injection, daily) and thymoquinone group (10 mg/kg, I.P. injection, daily). Blood and tissue were collected on day 11 and used for assessment of creatine phosphokinase, lactate dehydrogenase (LDH), troponin, malondialdehyde (MDA) amount, glutathione levels and mitochondrial toxicity parameters. TZM increased mitochondrial impairments (reactive oxygen species formation, mitochondrial swelling, mitochondrial membrane potential collapse and decline in succinate dehydrogenase activity) and histopathological alterations (hypertrophy, enlarged cell, disarrangement, myocytes degeneration, infiltration of fat in some areas, hemorrhage and focal vascular thrombosis) in rat heart. As well as TZM produced a significant increase in the level of CK, LDH, troponin, MDA, glutathione disulfide. In most experiments, the co-injection of curcumin, chrysin and thymoquinone with TZM restored the level of CK, LDH, troponin, MDA, GSH, mitochondrial impairments and histopathological alterations. The study revealed the cardioprotective effects of curcumin, chrysin and thymoquinone against TZM-induced cardiotoxicity which could be attributed to their antioxidant and mitochondrial protection activities.

Developing chicken cardiac muscle mitochondria are resistant to variations in incubation oxygen levels.

Background: Chronic exposure to hypoxia during vertebrate development can produce abnormal cardiovascular morphology and function. The aim of this study was to examine cardiac mitochondria function in an avian model, the chicken, in response to embryonic development under hypoxic (15% O2), normoxic (21% O2), or hyperoxic (40% O2) incubation conditions. Methods: Chicken embryos were incubated in hypoxia, normoxia, or hyperoxia beginning on day 5 of incubation through hatching. Cardiac mitochondria oxygen flux and reactive oxygen species production were measured in permeabilized cardiac fibers from externally pipped and 1-day post hatchlings. Results: Altering oxygen during development had a large effect on body and heart masses of externally pipped embryos and 1-day old hatchlings. Hypoxic animals had smaller body masses and absolute heart masses, but proportionally similar sized hearts compared to normoxic animals during external pipping. Hyperoxic animals were larger with larger hearts than normoxic animals during external pipping. Mitochondrial oxygen flux in permeabilized cardiac muscle fibers revealed limited effects of developing under altered oxygen conditions, with only oxygen flux through cytochrome oxidase being lower in hypoxic hearts compared with hyperoxic hearts. Oxygen flux in leak and oxidative phosphorylation states were not affected by developmental oxygen levels. Mitochondrial reactive oxygen species production under leak and oxidative phosphorylation states studied did not differ between any developmental oxygen treatment. Conclusions: These results suggest that cardiac mitochondria function of the developing chicken is not altered by developing in ovo under different oxygen levels.

Superiority of focused ion beam-scanning electron microscope tomography of cardiomyocytes over standard 2D analyses highlighted by unmasking mitochondrial heterogeneity.

BACKGROUND: Cardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two-dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three-dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology. METHODS: Spatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild-type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam-scanning electron microscopy (FIB-SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild-type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3; Bnip3-/- ) using FIB-SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis. RESULTS: The FIB-SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = -0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = -0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild-type 89% vs. Bnip3-/- 68%; complexity: P < 0.001) forming large myofibril-distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3-/- mice also show a higher respiration rate (P < 0.01). CONCLUSIONS: Here, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

Protective effects of quercetin against oxidative stress induced by bisphenol-A in rat cardiac mitochondria.

Research has shown a relationship between the exposures to a chemical agent called bisphenol-A (BPA), which is extensively used in the production of polycarbonate plastics and the incidence of cardiovascular diseases. This association is most likely caused by the BPA's ability to disrupt multiple cardiac mechanisms, including mitochondrial functions. Therefore, this study aimed to explore the ability of quercetin (QUER) to limit the cardiotoxic effect of BPA in the rat's cardiac mitochondria. The experiment was carried out on 32 male Wistar rats, which were randomly assigned to four groups. The negative control group received olive oil; the positive control group received olive oil plus BPA (250 mg/kg); the third group received olive oil, BPA, and QUER (75 mg/kg); and the fourth group received olive oil and QUER, all orally for 14 days. The rats were slaughtered 24 h after the last treatment. The measured parameters included creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) as the biomarkers of cardiotoxicity, triglyceride (TG), total cholesterol (TC), and low-density and high-density lipoprotein cholesterol (LDL-C and HDL-C) as the measures of dyslipidemia, glutathione (GSH) content, catalase activity (CAT), reactive oxygen species (ROS), lipid peroxidation (LPO), and the level of damage to the mitochondrial membranes as the indicators of the impact of QUER on the BPA cardiotoxic effect. Finally, the rats treated with QUER showed better results in terms of serum CK-MB, serum LDH, serum lipid profile, GSH level, CAT activity, mitochondrial membrane potential (ΔΨm), LPO, and ROS. According to the results, QUER could be used as a protective agent against BPA-induced mitochondrial toxicity.