Summary 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 inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-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 C-I deficiency inhibited Sirt3 activity, leading to 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.

Rationale: Mitochondrial dysfunction plays a pivotal role in the development of heart failure. Animal studies suggest that impaired mitochondrial biogenesis attributable to downregulation of the peroxisome proliferator-activated receptor ␥ coactivator (PGC)-1 transcriptional pathway is integral of mitochondrial dysfunction in heart failure. Objective: The study sought to define mechanisms underlying the impaired mitochondrial biogenesis and function in human heart failure. Methods and Results: We collected left ventricular tissue from end-stage heart failure patients and from nonfailing hearts (n‫,32؍‬ and 19, respectively). The mitochondrial DNA (mtDNA) content was decreased by >40% in the failing hearts, after normalization for a moderate decrease in citrate synthase activity (P<0.05). This was accompanied by reductions in mtDNA-encoded proteins (by 25% to 80%) at both mRNA and protein level (P<0.05). The mRNA levels of PGC-1␣/␤ and PRC (PGC-1-related coactivator) were unchanged, whereas PGC-1␣ protein increased by 58% in the failing hearts. Among the PGC-1 coactivating targets, the expression of estrogen-related receptor ␣ and its downstream genes decreased by up to 50% (P<0.05), whereas peroxisome proliferator-activated receptor ␣ and its downstream gene expression were unchanged in the failing hearts. The formation of D-loop in the mtDNA was normal but D-loop extension, which dictates the replication process of mtDNA, was decreased by 75% in the failing hearts. Furthermore, DNA oxidative damage was increased by 50% in the failing hearts. Conclusions: Mitochondrial biogenesis is severely impaired as evidenced by reduced mtDNA replication and depletion of mtDNA in the human failing heart. These defects are independent of the downregulation of the PGC-1 expression suggesting novel mechanisms for mitochondrial dysfunction in heart failure. (Circ Res. 2010;106:1541-1548.)Key Words: mtDNA Ⅲ mitochondrial biogenesis Ⅲ human heart failure Ⅲ PGC-1 Ⅲ oxidative damage M itochondrial dysfunction has been observed in a variety of cardiac diseases, including myocardial ischemia, diabetic cardiomyopathy and heart failure. Accumulating evidence have suggested that mitochondrial dysfunction accounts for impaired myocardial energetics and increased cell death during myocardial injury and the development of heart failure. 1,2 However, the molecular mechanisms responsible for the mitochondrial dysfunction under these pathological conditions are poorly understood making it difficult to develop mitochondriatargeted therapy.Recent studies have revealed a central role of peroxisome proliferator-activated receptor ␥ coactivator (PGC)-1 family proteins in mitochondrial biogenesis and function in multiple organs including the heart. 3-5 Downregulation of PGC-1␣ and its target genes have been observed in a number of rodent models of heart failure raising the intriguing possibility that impaired mitochondrial biogenesis can be a causal mechanism for mitochondrial dysfunction in heart failure. 6,7 However, despite strong evidence sugges...

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

cAMP-dependent protein kinase induction of PPAR␥ coactivator-1␣ (PGC-1␣) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1␣ in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1␣ and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1␣ reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBP␤, but not C/EBP␣ and C/EBP␦, bound to the CRE on the PGC-1␣ promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBP␤ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBP␤ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBP␤ rescued cAMP-inducible PGC-1␣ and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBP␤ on the CRE in PGC-1␣ proximal promoter.