Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector

For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the <i>in vivo</i> mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism

<div><p>It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.</p></div

Citrate synthase and cytochrome c oxidase activities.

<p># Significant difference between control and BDK-mKO mice.</p

Plasma BCAA concentrations of control and BDK-mKO mice with and without the running exercise bout.

<p># Significant difference between control and BDK-mKO mice. * Significant difference in the same group of mice with and without the exercise bout.</p

Exercise performance of control and BDK-mKO mice.

<p><b>(A)</b> Running distance to exhaustion before and after 2 weeks of training, and (B) swimming time to exhaustion of untrained mice on each of 4 consecutive days. # Significant difference between control and BDK-mKO mice.</p

Metabolites in the glycolytic pathway.

<p>Changes in metabolite levels in skeletal muscle of BDK-mKO mice and control mice with and without the exercise bout are shown. # Significant difference between control and BDK-mKO mice. G6P, glucose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; 2PG, 2-phosphoglycerate; and PEP, phosphoenolpyruvate.</p

Acetyl-CoA and metabolites in the TCA cycle.

<p>Changes in metabolite levels in skeletal muscle of BDK-mKO mice and control mice with and without the exercise bout are shown. # Significant difference between control and BDK-mKO mice. * Significant difference in the same group of mice with and without exercise bout. αKG, α-ketoglutarate.</p

BCAAs and their metabolites.

<p>Changes in the metabolite levels in the skeletal muscle of control and BDK-mKO mice with and without the exercise bout are shown. # Significant difference between control and BDK-mKO mice. * Significant difference in the same group of mice with and without the exercise bout. PMP, pyridoxamine 5'-phosphate; KIC, α-ketoisocaproate; KMV, α-keto-ß-methylvalerate; KIV, α-ketoisovalerate; ßMB-CAR, ß-methylbutyryl-carnitine; αMB-CAR, α-methylbutyryl-carnitine; IB-CAR, isobutyryl-carnitine; and αKG, α-ketoglutarate.</p

NADH, NAD⁺, and high energy compounds.

<p>NADH, NAD⁺, and high energy compounds.</p

Additional file 1: of A Phase I clinical trial of EUS-guided intratumoral injection of the oncolytic virus, HF10 for unresectable locally advanced pancreatic cancer

Table S1. Criteria Defining Resectability Status. (DOCX 18 kb