Cardiac and skeletal muscle critically depend on mitochondrial energy metabolism for their normal function. Recently, we showed that apoptosis-inducing factor (AIF), a mitochondrial protein implicated in programmed cell death, plays a role in mitochondrial respiration. However, the in vivo consequences of AIF-regulated mitochondrial respiration resulting from a loss-of-function mutation in Aif are not known. Here, we report tissue-specific deletion of Aif in the mouse. Mice in which Aif has been inactivated specifically in cardiac and skeletal muscle exhibit impaired activity and protein expression of respiratory chain complex I. Mutant animals develop severe dilated cardiomyopathy, heart failure, and skeletal muscle atrophy accompanied by lactic acidemia consistent with defects in the mitochondrial respiratory chain. Isolated hearts from mutant animals exhibit poor contractile performance in response to a respiratory chain-dependent energy substrate, but not in response to glucose, supporting the notion that impaired heart function in mutant animals results from defective mitochondrial energy metabolism. These data provide genetic proof that the previously defined cell death promoter AIF has a second essential function in mitochondrial respiration and aerobic energy metabolism required for normal heart function and skeletal muscle homeostasis.

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Δ mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Δ mitochondria have increased cation contents, particularly K⁺ and low-membrane-potential Δψ. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K⁺/H⁺ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K⁺ homeostasis and organelle volume control.

Ca²⁺ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1—an IMM protein with a crucial role in mitochondrial K⁺/H⁺ exchange and volume homeostasis—also acts as a Ca²⁺/H⁺ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K⁺/H⁺ and Na⁺/H⁺ exchange. Decreased exchange activity resulted in matrix K⁺ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na⁺/Ca²⁺ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca²⁺ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca²⁺ fluxes may reflect those on Na⁺/H⁺ exchange activity.