Overactive mitochondrial DNA replication disrupts perinatal cardiac maturation.

High mitochondrial DNA (mtDNA) amount has been reported to be beneficial for resistance and recovery of metabolic stress, while increased mtDNA synthesis activity can drive aging signs. The intriguing contrast of these two mtDNA boosting outcomes prompted us to jointly elevate mtDNA amount and frequency of replication in mice. We report that high activity of mtDNA synthesis inhibits perinatal metabolic maturation of the heart. The offspring of the asymptomatic parental lines are born healthy but manifest dilated cardiomyopathy and cardiac collapse during the first days of life. The pathogenesis, further enhanced by mtDNA mutagenesis, involves prenatal upregulation of mitochondrial integrated stress response and the ferroptosis-inducer MESH1, leading to cardiac fibrosis and cardiomyocyte death after birth. Our evidence indicates that the tight control of mtDNA replication is critical for early cardiac homeostasis. Importantly, ferroptosis sensitivity is a potential targetable mechanism for infantile-onset cardiomyopathy, a common manifestation of mitochondrial diseases.

Nuclear receptor subfamily 4 group A member 1 promotes myocardial ischemia/reperfusion injury through inducing mitochondrial fission factor-mediated mitochondrial fragmentation and inhibiting FUN14 domain containing 1-depedent mitophagy.

This study investigated the mechanism by which NR4A1 regulates mitochondrial fission factor (Mff)-related mitochondrial fission and FUN14 domain 1 (FUNDC1)-mediated mitophagy following cardiac ischemia-reperfusion injury(I/R). Our findings showed that the damage regulation was positively correlated with the pathological fission and pan-apoptosis of myocardial cell mitochondria. Compared with wild-type mice (WT), NR4A1-knockout mice exhibited resistance to myocardial ischemia-reperfusion injury and mitochondrial pathological fission, characterized by mitophagy activation. Results showed that ischemia-reperfusion injury increased NR4A1 expression level, activating mitochondrial fission mediated by Mff and restoring the mitophagy phenotype mediated by FUNDC1. The inactivation of FUNDC1 phosphorylation could not mediate the normalization of mitophagy in a timely manner, leading to an excessive stress response of unfolded mitochondrial proteins and an imbalance in mitochondrial homeostasis. This process disrupted the normalization of the mitochondrial quality control network, leading to accumulation of damaged mitochondria and the activation of pan-apoptotic programs. Our data indicate that NR4A1 is a novel and critical target in myocardial I/R injury that exertsand negative regulatory effects by activating Mff-mediated mito-fission and inhibiting FUNDC1-mediated mitophagy. Targeting the crosstalk balance between NR4A1-Mff-FUNDC1 is a potential approach for treating I/R.

PKM2 regulates metabolic flux and oxidative stress in the murine heart.

Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.

3-N-Butylphthalide Confers Antiarrhythmic Features in Ischemia/Reperfusion Injury of Diabetic Heart by Targeting Mitochondria-Endoplasmic Reticulum Network and Inhibiting Oxidative Stress and Inflammation.

While 3-N-butylphthalide (NBP) has demonstrated notable cardioprotective effects, its precise role in mitigating myocardial arrhythmia following ischemia/reperfusion (IR) injury in diabetes remains unclear. This study aimed to explore the potential mechanisms through which NBP mitigates reperfusion-induced myocardial arrhythmia in diabetic rats, with a particular focus on mitochondrial function and biogenesis, endoplasmic reticulum (ER) stress, and oxidative/inflammatory responses. Sixty Sprague-Dawley rats were divided into non-diabetic and diabetic groups, subjected to in-vivo myocardial IR injury, and treated with NBP (100 mg/kg, intraperitoneally) through different modalities: preconditioning, postconditioning, or a combination of both. Electrocardiography (ECG) was employed to assess the incidence and severity of arrhythmia. Fluorometric, Western blotting and ELISA analyses were utilized to measure the mitochondrial, ER stress, and cellular outcomes. Treatment of non-diabetic rats with NBP in preconditioned, postconditioned, and combined approaches significantly reduced cardiotroponin-I and the frequency and severity of arrhythmias induced by IR injury. However, only the combined preconditioning plus postconditioning approach of NBP had protective and antiarrhythmic effects in diabetic rats, in an additive manner. Moreover, the NBP combined approach improved mitochondrial function and upregulated the expression of PGC-1?, Sirt1, and glutathione while concurrently downregulating ER stress and oxidative and pro-inflammatory-related proteins in diabetic rats. In conclusion, the combined approach of NBP treatment was effective in mitigating myocardial arrhythmia in diabetic rats. This approach coordinates interactions within the mitochondria-endoplasmic reticulum network and inhibits oxidative and inflammatory mediators, offering a promising strategy for managing myocardial arrhythmia in diabetic patients. Key words: Myocardial Infarction, Mitochondria, Arrhythmia, Reperfusion, Diabetes, Ischemia.

Robust Mitochondrial Isolation from Rodent Cardiac Tissue.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Cross-sex hormonal replacement: Some effects over mitochondria.

Transgender is a term for people whose gender identity or expression differs from their natal sex. These individuals often seek cross-hormonal therapy to simulate the individual´s desired gender. However, the use of estrogens and testosterone has side effects such as a higher propensity to cancer, weight changes and cardiovascular diseases. Testosterone has also been linked with hypertension. Still, little is known about the outcomes and prevalence of metabolic perturbations in the trans community. Here we aim to analyze if cross-administering sexual hormones affects heart mitochondrial function. Mitochondria produces the ATP needed for heart function. In fact, different studies show that mitochondrial dysfunction precedes cardiac damage. In this work we used either female rats castrated and injected with testosterone or male rats castrated and injected with estrogens for 4 months. We performed an electrocardiogram, and then we isolated heart mitochondria to measure the rate of oxygen consumption, calcium fluxes, membrane potential, superoxide dismutase activity, lipoperoxidation and cytokines. We detected wide modifications in all parameters associated to cross-hormonal administration.

Characterization of a novel mitophagy-related 5-genes signature for diagnosis of acute myocardial infarction.

Myocardial infarction (MI) and the ensuing heart failure (HF) remain the main cause of morbidity and mortality worldwide. One of the strategies to combat MI and HF lies in the ability to accurately predict the onset of these disorders. Alterations in mitochondrial homeostasis have been reported to be involved in the pathogenesis of various cardiovascular diseases (CVDs). In this regard, perturbations to mitochondrial dynamics leading to impaired clearance of dysfunctional mitochondria have been previously established to be a crucial trigger for MI/HF. In this study, we found that MI patients could be classified into three clusters based on the expression levels of mitophagy-related genes and consensus clustering. We identified a mitophagy-related diagnostic 5-genes signature for MI using support vector machines-Recursive Feature Elimination (SVM-RFE) and random forest, with the area under the ROC curve (AUC) value of the predictive model at 0.813. Additionally, the single-cell transcriptome and pseudo-time analyses showed that the mitoscore was significantly upregulated in macrophages, endothelial cells, pericytes, fibroblasts and monocytes in patients with ischemic cardiomyopathy, while sequestosome 1 (SQSTM1) exhibited remarkable increase in the infarcted (ICM) and non-infarcted (ICMN) myocardium samples dissected from the left ventricle compared with control samples. Lastly, through analysis of peripheral blood from MI patients, we found that the expression of SQSTM1 is positively correlated with troponin-T (P < 0.0001, R = 0.4195, R2 = 0.1759). Therefore, this study provides the rationale for a cell-specific mitophagy-related gene signature as an additional supporting diagnostic for CVDs.

Jin-Xin-Kang alleviates heart failure by mitigating mitochondrial dysfunction through the Calcineurin/Dynamin-Related Protein 1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic heart failure (CHF) is a severe consequence of cardiovascular disease, marked by cardiac dysfunction. Jin-Xin-Kang (JXK) is a traditional Chinese herbal formula used for the treatment of CHF. This formula consists of seven medicinal herbs, including Ginseng (Ginseng quinquefolium (L.) Alph.Wood), Astragali Radix (Astragalus membranaceus (Fisch.) Bunge), Salvia miltiorrhiza (Salvia miltiorrhiza Bunge), Descurainiae Semen Lepidii Semen (Descurainia sophia (L.) Webb ex Prantl), Leonuri Herba (Leonurus japonicus Houtt.), Cinnamomi Ramulus (Cinnamomum cassia (L.) J.Presl), and Ilex pubescens (Ilex pubescens Hook. & Arn.). Its clinical efficacy has been validated through prospective randomized controlled studies. However, the specific mechanisms of action for this formula have yet to be elucidated. AIM OF THE STUDY: This study aimed to investigate the effect of JXK on mitochondrial function and its mechanism in the treatment of CHF. METHODS: JXK components were qualitatively analyzed using UPLC-Q-Orbitrap-MS. HF was induced in mice via transverse aortic constriction (TAC). After successful model establishment, lyophilized JXK-L (4.38 g/kg) and JXK-H (13.14 g/kg) were administered for 8 weeks. In vitro, hypertrophic myocardium was induced using angiotensin II (Ang II) for 48 h, followed by JXK-L and JXK-H treatment. Network pharmacology and molecular docking techniques were used to predict the relevant targets of JXK. Cardiac function, serum markers, and histopathological changes were evaluated to assess cardiac function. Immunofluorescence of Tomm20, mitochondrial membrane potential, and ROS were measured to assess mitochondrial dysfunction. Protein expression of calcineurin (CaN) and Drp1 in the myocardium was assessed by Western blot analysis. RESULTS: We detected that the active components of JXK include terpenes, glycosides, flavonoids, amino acids, and alkaloids, among others. In mice with CHF, JXK improved cardiac function and reversed ventricular remodeling. Network pharmacology indicated that JXK can inhibit the calcium signaling pathway. The molecular docking results demonstrated that the active components of JXK effectively bind with CaN. Both in vitro and in vivo experiments confirmed that JXK regulated the CaN/Drp1 pathway and alleviated mitochondrial dysfunction. CONCLUSION: JXK can inhibit the CaN/Drp1 pathway to improve mitochondrial function, and consequently treat CHF.

MICU1 and MICU2 control mitochondrial calcium signaling in the mammalian heart.

Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.

Myocardial ultrastructure of human heart failure with preserved ejection fraction.

Over half of patients with heart failure have a preserved ejection fraction (>50%, called HFpEF), a syndrome with substantial morbidity/mortality and few effective therapies1. Its dominant comorbidity is now obesity, which worsens disease and prognosis1-3. Myocardial data from patients with morbid obesity and HFpEF show depressed myocyte calcium-stimulated tension4 and disrupted gene expression of mitochondrial and lipid metabolic pathways5,6, abnormalities shared by human HF with a reduced EF but less so in HFpEF without severe obesity. The impact of severe obesity on human HFpEF myocardial ultrastructure remains unexplored. Here we assessed the myocardial ultrastructure in septal biopsies from patients with HFpEF using transmission electron microscopy. We observed sarcomere disruption and sarcolysis, mitochondrial swelling with cristae separation and dissolution and lipid droplet accumulation that was more prominent in the most obese patients with HFpEF and not dependent on comorbid diabetes. Myocardial proteomics revealed associated reduction in fatty acid uptake, processing and oxidation and mitochondrial respiration proteins, particularly in very obese patients with HFpEF.

Cardiac troponin I directly binds and inhibits mitochondrial ATP synthase with a noncanonical role in the post-ischemic heart.

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. However, its role in mitochondria is unknown. Here we show that cTnI localized to mitochondria in the heart, inhibited mitochondrial functions when stably expressed in noncardiac cells and increased the opening of the mitochondrial permeability transition pore under oxidative stress. Direct, specific and saturable binding of cTnI to F1FO-ATP synthase was demonstrated in vitro using immune-captured ATP synthase and in cells using proximity ligation assay. cTnI binding doubled ATPase activity, whereas skeletal troponin I and several human pathogenic cTnI variants associated with familial hypertrophic cardiomyopathy did not. A rationally designed peptide, P888, inhibited cTnI binding to ATP synthase, inhibited cTnI-induced increase in ATPase activity in vitro and reduced cardiac injury following transient ischemia in vivo. We suggest that cTnI-bound ATP synthase results in lower ATP levels, and releasing this interaction during cardiac ischemia-reperfusion may increase the reservoir of functional mitochondria to reduce cardiac injury.

Mitochondrial calcium uniporter channel gatekeeping in cardiovascular disease.

The mitochondrial calcium (mCa2+) uniporter channel (mtCU) resides at the inner mitochondrial membrane and is required for Ca2+ to enter the mitochondrial matrix. The mtCU is essential for cellular function, as mCa2+ regulates metabolism, bioenergetics, signaling pathways and cell death. mCa2+ uptake is primarily regulated by the MICU family (MICU1, MICU2, MICU3), EF-hand-containing Ca2+-sensing proteins, which respond to cytosolic Ca2+ concentrations to modulate mtCU activity. Considering that mitochondrial function and Ca2+ signaling are ubiquitously disrupted in cardiovascular disease, mtCU function has been a hot area of investigation for the last decade. Here we provide an in-depth review of MICU-mediated regulation of mtCU structure and function, as well as potential mtCU-independent functions of these proteins. We detail their role in cardiac physiology and cardiovascular disease by highlighting the phenotypes of different mutant animal models, with an emphasis on therapeutic potential and targets of interest in this pathway.

Caprylic Acid Inhibits High Mobility Group Box-1-Induced Mitochondrial Damage in Myocardial Tubes.

Myocardial damage significantly impacts the prognosis of patients with cancer; however, the mechanisms of myocardial damage induced by cancer and its treatment remain unknown. We previously reported that medium-chain fatty acids (MCFAs) improve cancer-induced myocardial damage but did not evaluate the differences in effect according to MCFA type. Therefore, this study investigated the role of inflammatory cytokines in cancer-induced myocardial damage and the effects of three types of MCFAs (caprylic acid [C8], capric acid [C10], and lauric acid [C12]). In a mouse model, the C8 diet showed a greater effect on improving myocardial damage compared with C10 and C12 diets. Myocardial tubes differentiated from H9C2 cardiomyoblasts demonstrated increased mitochondrial oxidative stress, decreased membrane potential and mitochondrial volume, and inhibited myocardial tube differentiation following treatment with high-mobility group box-1 (HMGB1) but not interleukin-6 and tumor necrosis factor-α cytokines. However, HMGB1 treatment combined with C8 improved HMGB1-induced mitochondrial damage, enhanced autophagy, and increased mitochondrial biogenesis and maturation. However, these effects were only partial when combined with beta-hydroxybutyrate, a C8 metabolite. Thus, HMGB1 may play an important role in cancer-related myocardial damage. C8 counteracts HMGB1's effects and improves cancer-related myocardial damage. Further clinical studies are required to investigate the effects of C8.

Astaxanthin ameliorated isoproterenol induced myocardial infarction via improving the mitochondrial function and antioxidant activity in rats.

The present study evaluated the cardioprotective effect of astaxanthin (ASX) against isoproterenol (ISO) induced myocardial infarction in rats via the pathway of mitochondrial biogenesis as the possible molecular target of astaxanthin. The control group was injected with normal physiological saline subcutaneously for 2 days. The second group was injected with ISO at a dose of 85 mg/kg bwt subcutaneously for 2 days. The third, fourth and fifth groups were supplemented with ASX at doses of 10, 20, 30 mg/kg bwt, respectively daily by oral gavage for 21 days then injected with ISO dose of 85 mg/kg bwt subcutaneously for 2 successive days. Isoproterenol administration in rats elevated the activities of Creatine kinase-MB (CK-MB), aspartate transaminase (AST), lactate dehydrogenase (LDH), and other serum cardiac biomarkers Troponin-I activities, oxidative stress biomarkers, malondialdehyde(MDA), Nuclear factor-kappa B (NF-KB), while it decreased Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), Nuclear factor erythroid-2-related factor 2 (Nfe212), mitochondrial transcriptional factor A (mt TFA), mitochondrial DNA copy number and glutathione system parameters. However, Astaxanthin decreased the activities of serum AST, LDH, CK-MB, and Troponin I that elevated by ISO. In addition, it increased glutathione peroxidase and reductase activities, total glutathione and reduced GSH content, and GSH/GSSG ratio, mtDNA copy number, PGC-1α expression and Tfam expression that improved mitochondrial biogenesis while it decreased GSSG and MDA contents and NF-KB level in the cardiac tissues. This study indicated that astaxanthin relieved isoproterenol induced myocardial infarction via scavenging free radicals and reducing oxidative damage and apoptosis in cardiac tissue.

Analysis of the prognostic value of mitochondria-related genes in patients with acute myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI) is a leading cause of death worldwide. Mitochondrial dysfunction is a key determinant of cell death post-AMI. Preventing mitochondrial dysfunction is thus a key therapeutic strategy. This study aimed to explore key genes and target compounds related to mitochondrial dysfunction in AMI patients and their association with major adverse cardiovascular events (MACE). METHODS: Differentially expressed genes in AMI were identified from the Gene Expression Omnibus (GEO) datasets (GSE166780 and GSE24519), and mitochondria-related genes were obtained from MitoCarta3.0 database. By intersection of the two gene groups, mitochondria-related genes in AMI were identified. Next, the identified genes related to mitochondria were subject to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Protein-protein interaction (PPI) network was constructed, and key genes were screened. Then, targeted drug screening and molecular docking were performed. Blood samples from AMI patients and healthy volunteers were analyzed for the key genes expressions using quantitative real time polymerase chain reaction (qRT-PCR). Later, receiver operating characteristic (ROC) curves assessed the diagnostic value of key genes, and univariate and multivariate COX analyses identified risk factors and protective factors for MACE in AMI patients. RESULTS: After screening and identification, 138 mitochondria-related genes were identified, mainly enriched in the processes and pathways of cellular respiration, redox, mitochondrial metabolism, apoptosis, amino acid and fatty acid metabolism. According to the PPI network, 5 key mitochondria-related genes in AMI were obtained: translational activator of cytochrome c oxidase I (TACO1), cytochrome c oxidase subunit Va (COX5A), PTEN-induced putative kinase 1 (PINK1), SURF1, and NDUFA11. Molecular docking showed that Cholic Acid, N-Formylmethionine interacted with COX5A, nicotinamide adenine dinucleotide + hydrogen (NADH) and NDUFA11. Subsequent basic experiments revealed that COX5A and NDUFA11 expressions were significantly lower in the blood of patients with AMI than those in the corresponding healthy volunteers; also, AMI patients with MACE had lower COX5A and NDUFA11 expressions in the blood than those without MACE (P < 0.01). ROC analysis also showed high diagnostic value for COX5A and NDUFA11 [area under the curve (AUC) > 0.85]. In terms of COX results, COX5A, NDUFA11 and left ventricular ejection fraction (LVEF) were protective factors for MACE in AMI, while C-reactive protein (CRP) was a risk factor. CONCLUSION: COX5A and NDUFA11, key mitochondria-related genes in AMI, may be used as biomarkers to diagnose AMI and predict MACE.

COX-2 optimizes cardiac mitochondrial biogenesis and exerts a cardioprotective effect during sepsis.

BACKGROUND: Septic cardiomyopathy is a component of multiple organ dysfunction in sepsis. Mitochondrial dysfunction plays an important role in septic cardiomyopathy. Studies have shown that cyclooxygenase-2 (COX-2) had a protective effect on the heart, and prostaglandin E2 (PGE2), the downstream product of COX-2, was increasingly recognized to have a protective effect on mitochondrial function. OBJECTIVE: This study aims to demonstrate that COX-2/PGE2 can protect against septic cardiomyopathy by regulating mitochondrial function. METHODS: Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and RAW264.7 macrophages and H9C2 cells were used to simulate sepsis in vitro. The NS-398 and celecoxib were used to inhibit the activity of COX-2. ZLN005 and SR18292 were used to activate or inhibit the PGC-1α activity. The mitochondrial biogenesis was examined through the Mitotracker Red probe, mtDNA copy number, and ATP content detection. RESULTS: The experimental data suggested that COX-2 inhibition attenuated PGC-1α expression thus decreasing mitochondrial biogenesis, whereas increased PGE2 could promote mitochondrial biogenesis by activating PGC-1α. The results also showed that the effect of COX-2/PGE2 on PGC-1α was mediated by the activation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). Finally, the effect of COX-2/PGE2 on the heart was also verified in the septic mice. CONCLUSION: Collectively, these results suggested that COX-2/PGE2 pathway played a cardioprotective role in septic cardiomyopathy through improving mitochondrial biogenesis, which has changed the previous understanding that COX-2/PGE2 only acted as an inflammatory factor.

Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Metabolic cycles: A unifying concept for energy transfer in the heart.

It is still debated whether changes in metabolic flux are cause or consequence of contractile dysfunction in non-ischemic heart disease. We have previously proposed a model of cardiac metabolism grounded in a series of six moiety-conserved, interconnected cycles. In view of a recent interest to augment oxygen availability in heart failure through iron supplementation, we integrated this intervention in terms of moiety conservation. Examining published work from both human and murine models, we argue this strategy restores a mitochondrial cycle of energy transfer by enhancing mitochondrial pyruvate carrier (MPC) expression and providing pyruvate as a substrate for carboxylation and anaplerosis. Metabolomic data from failing heart muscle reveal elevated pyruvate levels with a concomitant decrease in the levels of Krebs cycle intermediates. Additionally, MPC is downregulated in the same failing hearts, as well as under hypoxic conditions. MPC expression increases upon mechanical unloading in the failing human heart, as does contractile function. We note that MPC deficiency also alters expression of enzymes involved in pyruvate carboxylation and decarboxylation, increases intermediates of biosynthetic pathways, and eventually leads to cardiac hypertrophy and dilated cardiomyopathy. Collectively, we propose that an unbroken chain of moiety-conserved cycles facilitates energy transfer in the heart. We refer to the transport and subsequent carboxylation of pyruvate in the mitochondrial matrix as an example and a proposed target for metabolic support to reverse impaired contractile function.

Salvianolic acid B improves mitochondrial dysfunction of septic cardiomyopathy via enhancing ATF5-mediated mitochondrial unfolded protein response.

AIMS: Septic cardiomyopathy is characterized by impaired contractile function and mitochondrial activity dysregulation. Salvianolic acid B (Sal B) is a potent therapeutic compound derived from the traditional Chinese medicine Salvia miltiorrhiza. This study explored the protective effects of Sal B on septic heart injury, emphasizing the mitochondrial unfolded protein response (UPRmt). MATERIALS AND METHODS: An in vivo mouse model of lipopolysaccharide (LPS)-induced heart injury was utilized to assess Sal B's protective role in septic cardiomyopathy. Additionally, cell models stimulated by LPS were developed to investigate the mechanisms of Sal B on UPRmt. Quantitative polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence were employed for molecular analysis. RESULTS: Sal B, administered at doses of 10, 30, and 60 mg/kg, demonstrated protective effects on cardiac contractile function, reduced heart inflammation, and mitigated cardiac injury in LPS-exposed mice. In cardiomyocytes, LPS induced apoptosis, elevated mitochondrial ROS levels, promoted mitochondrial fission, and decreased mitochondrial membrane potential, all of which were alleviated by Sal B. Mechanistically, Sal B was found to induce UPRmt both in vivo and in vitro. ATF5, identified as a UPRmt activator, was modulated by LPS and Sal B, resulting in increased ATF5 expression and its translocation from the cytosol to the nucleus. ATF5-siRNA delivery reversed UPRmt upregulation, exacerbating mitochondrial dysfunction in LPS-stimulated cardiomyocytes and counteracting the mitochondrial function enhancement in Sal B-treated cardiomyocytes. CONCLUSIONS: This study provides evidence that Sal B confers cardiac protection by enhancing UPRmt, highlighting its potential as a therapeutic approach for mitigating mitochondrial dysfunction in septic cardiomyopathy.