Chi Fung Lee

Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

Normalization of NAD+ Redox Balance as a Therapy for Heart Failure.

Background: Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, but there is no therapy for mitochondrial dysfunction. Methods: We assessed the reduced/oxidized ratio of nicotinamide adenine dinucleotide (NADH/NAD+ ratio) and protein acetylation in the failing heart. Proteome and acetylome analyses were followed by docking calculation, mutagenesis, and mitochondrial calcium uptake assays to determine the functional role of specific acetylation sites. The therapeutic effects of normalizing mitochondrial protein acetylation by expanding the NAD+ pool also were tested. Results: Increased NADH/NAD+ and protein hyperacetylation, previously observed in genetic models of defective mitochondrial function, also are present in human failing hearts as well as in mouse hearts with pathologic hypertrophy. Elevation of NAD+ levels by stimulating the NAD+ salvage pathway suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses. Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD+ ratio. Hyperacetylation of mitochondrial malate-aspartate shuttle proteins impaired the transport and oxidation of cytosolic NADH in the mitochondria, resulting in altered cytosolic redox state and energy deficiency. Furthermore, acetylation of oligomycin-sensitive conferring protein at lysine-70 in adenosine triphosphate synthase complex promoted its interaction with cyclophilin D, and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD+ redox balance either genetically or pharmacologically. Conclusions: We show that mitochondrial protein hyperacetylation due to NAD+ redox imbalance contributes to the pathologic remodeling of the heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear benefit of normalizing NADH/NAD+ imbalance in the failing hearts. These findings have a high translational potential as the pharmacologic strategy of increasing NAD+ precursors are feasible in humans.

Elimination of NADPH Oxidase Activity Promotes Reductive Stress and Sensitizes the Heart to Ischemic Injury

Background The NADPH oxidase family (Nox) produces reactive oxygen species by adding the electron donated by NADPH to oxygen. Excessive reactive oxygen species production under a variety of pathological conditions has been attributed to increased Nox activity. Here, we aimed at investigating the role of Nox in cardiac ischemic injury through gain‐ and loss‐of‐function approaches. Methods and Results We modulated Nox activity in the heart by cardiac‐specific expression of Nox4 and dominant negative Nox4. Modulation of Nox activity drastically changes the cellular redox status. Increasing Nox activity by cardiac‐specific overexpression of Nox4 imposed oxidative stress on the myocardium [increased NAD(P)+/NAD(P)H and decreased glutathione/glutathione disulfide ratio] and worsened cardiac energetics and contractile function after ischemia‐reperfusion. Overexpression of the dominant negative Nox4 (DN), which abolished the Nox function, led to a markedly reduced state [decreased NAD(P)+/NAD(P)H and increased glutathione/glutathione disulfide ratio] at baseline and paradoxically promoted mitochondrial reactive oxygen species production during ischemia resulting in no recovery of heart function after reperfusion. Limiting the generation of reducing equivalent through modulating carbon substrates availability partially restored the NAD+/NADH ratio and protected dominant negative Nox4 hearts from ischemic injury. Conclusions This study reveals an important role of Nox in cardiac redox regulation and highlights the complexity of developing therapies that affect the intricately connected redox states.

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Significance Mitochondria meet the majority of living cells’ demand for ATP and, as important regulators of redox homeostasis, metabolite levels, and calcium buffering, are a critical link between cell energetics and signaling. Disruption of these processes can induce adaptive or pathological signaling responses to stress and under severe stress promote cell death. Mitochondria have a complex proteome with conformations and interactions that are not well understood. Mitochondrial dysfunction is a direct cause of rare inherited diseases and is implicated in common metabolic diseases and age-related pathology. This study illuminates protein interactions and conformational features of nearly one-third of the mitochondrial proteome. Network information on this scale will enable groundbreaking insights into mitochondrial function, dysfunction, and potential therapeutic targets for mitochondrial-based pathology. Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I–III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks

Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules). We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression.

Mitochondrion as a Target for Heart Failure Therapy – Role of Protein Lysine Acetylation –

Heart failure is a leading cause of death worldwide. Despite medical advances, the dismal prognosis of heart failure has not been improved. The heart is a high energy-demanding organ. Impairments of cardiac energy metabolism and mitochondrial function are intricately linked to cardiac dysfunction. Mitochondrial dysfunction contributes to impaired myocardial energetics and increased oxidative stress in heart failure, and the opening of mitochondrial permeability transition pore triggers cell death and myocardial remodeling. Therefore, there has been growing interest in targeting mitochondria and metabolism for heart failure therapy. Recent developments suggest that mitochondrial protein lysine acetylation modulates the sensitivity of the heart to stress and hence the propensity to heart failure. This article reviews the role of mitochondrial dysfunction in heart failure, with a special emphasis on the regulation of the nicotinamide adenine dinucleotide (NAD(+)/NADH) ratio and sirtuin-dependent lysine acetylation by mitochondrial function. Strategies for targeting NAD(+)-sensitive mechanisms in order to intervene in protein lysine acetylation and, thereby, improve stress tolerance, are described, and their usefulness in heart failure therapy is discussed.

Promoting PGC-1α-driven mitochondrial biogenesis is detrimental in pressure-overloaded mouse hearts.

Mitochondrial dysfunction in animal models of heart failure is associated with downregulation of the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α pathway. To test whether PGC-1α is an appropriate therapeutic target for increasing mitochondrial biogenesis and improving function in heart failure, we used a transgenic (TG) mouse model of moderate overexpression of PGC-1α (∼3-fold) in the heart. TG mice had small increases in citrate synthase activity and mitochondria size in the heart without alterations in myocardial energetics or cardiac function at baseline. In vivo dobutamine stress increased fractional shortening in wild-type mice, but this increase was attenuated in TG mice, whereas ex vivo isolated perfused TG hearts demonstrated normal functional and energetic response to high workload challenge. When subjected to pressure overload by transverse aortic constriction (TAC), TG mice displayed a significantly greater acute mortality for both male and female mice; however, long-term survival up to 8 wk was similar between the two groups. TG mice also showed a greater decrease in fractional shortening and a greater increase in left ventricular chamber dimension in response to TAC. Mitochondrial gene expression and citrate synthase activity were mildly increased in TG mice compared with wild-type mice, and this difference was also maintained after TAC. Our data suggest that a moderate level of PGC-1α overexpression in the heart compromises acute survival and does not improve cardiac function during chronic pressure overload in mice.

Chemical Crosslinking Mass Spectrometry Analysis of Protein Conformations and Supercomplexes in Heart Tissue

While modern structural biology technologies have greatly expanded the size and type of protein complexes that can now be studied, the ability to derive large-scale structural information on proteins and complexes as they exist within tissues is practically nonexistent. Here, we demonstrate the application of crosslinking mass spectrometry to identify protein structural features and interactions in tissue samples, providing systems structural biology insight into protein complexes as they exist in the mouse heart. This includes insights into multiple conformational states of sarcomere proteins, as well as interactions among OXPHOS complexes indicative of supercomplex assembly. The extension of crosslinking mass spectrometry analysis into the realm of tissues opens the door to increasing our understanding of protein structures and interactions within the context of the greater biological system.

Failed Power Plant Turns Into Mass Murder: New Insight on Mitochondrial Cardiomyopathy

Mitochondria are known as the powerhouse of the cell. For a high-energy–consuming organ, such as the heart, continuous ATP production via oxidative metabolism in the mitochondria is essential. Apart from ATP generation, mitochondria are also key to the regulation of cellular metabolism, calcium homeostasis, and reactive oxygen species (ROS) generation.1 Mitochondrial dysfunction has been strongly implicated in a variety of cardiovascular diseases including ischemic heart disease and heart failure. Furthermore, a large portion of mitochondrial disease patients, a condition caused by mutation of genes for mitochondrial proteins, develop cardiomyopathy indicating a causal role of mitochondria in cardiac dysfunction. Given its significant role in the pathogenesis and the current lack of effective therapy for mitochondrial dysfunction, there is a clear need for discovery and innovation in mitochondrial medicine.2,3 Article, see p 74 It is well established that the fetal heart relies heavily on glycolysis for energy metabolism. A switch from glycolysis to oxidative metabolism in the early postnatal period is associated with explosive mitochondrial biogenesis.4,5 The switch is critical for the postnatal maturation of the heart. Loss of PGC-1α/β (peroxisome proliferator–activated receptor gamma coactivator), the powerful transcriptional regulators of mitochondrial biogenesis, in perinatal and postnatal periods, results in lethal cardiomyopathy.6,7 The role of mitochondria in the embryonic cardiomyocytes is, however, less explored. Recent studies using pluripotent cell–derived cardiomyocytes have suggested intriguing functions of mitochondria beyond energy provision in the regulation of cardiomyocytes maturation.6,8–10 …