Mitochondrial biogenesis protects metabolism from mitochondrial dysfunction produced by activation of innate immunity by lipopolysaccharide (LPS) or other bacterial products. Here we tested the hypothesis in mouse heart that activation of toll-like receptor-4 (TLR4), which induces early-phase genes that damage mitochondria, also activates mitochondrial biogenesis through induction of nitric oxide synthase (NOS2). We compared three strains of mice: wild type (Wt) C57BL/6J, TLR4(-/-), and NOS2(-/-)for cardiac mitochondrial damage and mitochondrial biogenesis by real-time RT-PCR, Western analysis, immunochemistry, and isoform analysis of myosin heavy chain (MHC) after sublethal heat-killed Escherichia coli (HkEC). After HkEC, Wt mice displayed significant myocardial mtDNA depletion along with enhanced TLR4 and NOS2 gene and protein expression that normalized in 72 h. HkEC generated less cytokine stress in TLR4(-/-)and NOS2(-/-)than Wt mice, NOS2(-/-)mice had mtDNA damage comparable to Wt, and both knockout strains failed to restore mtDNA copy number because of mitochondrial transcriptosome dysfunction. Wt mice also showed the largest beta-MHC isoform switch, but MHC recovery lagged in the NOS2(-/-)and TLR4(-/-)strains. The NOS2(-/-)mice also unexpectedly revealed the codependency of TLR4 expression on NOS2. These findings demonstrate the decisive participation of NOS2 induction by TLR4 in optimization of mitochondrial biogenesis and MHC expression after gram-negative challenge.