The mitochondrial outer membrane is not a major diffusion barrier for ADP in mouse heart skinned fibre bundles

Area of Science:

• Biochemistry
• Mitochondrial Physiology
• Cardiac Muscle Metabolism

Background:

• Mitochondrial oxygen consumption is crucial for cellular energy production.
• The apparent affinity (Km) of mitochondria for ADP in skinned cardiac fibres is significantly higher than in isolated mitochondria.
• Creatine kinase (CK) plays a role in cellular energy buffering and mitochondrial function.

Purpose of the Study:

• To investigate the reasons for the high apparent Km of ADP in skinned cardiac fibre bundles.
• To determine the true affinity of cardiac mitochondria for ADP.
• To assess the role of creatine kinase and diffusion gradients in regulating mitochondrial ADP response.

Main Methods:

• Utilized saponin-skinned cardiac fibre bundles from wild-type and double-knockout mice (lacking both cytosolic and mitochondrial CK).
• Incubated fibre bundles with ADP-producing systems: creatine kinase/creatine or yeast hexokinase/glucose.
• Measured mitochondrial oxygen consumption rates in response to varying ADP concentrations.

Main Results:

• Incubation with extramitochondrial ADP-producing systems significantly decreased the apparent Km for ADP in skinned cardiac fibre bundles.
• The reduction in apparent Km suggests that diffusion gradients, not intrinsic mitochondrial properties, caused the initial high Km.
• Mitochondria from knockout mice also showed improved ADP response when provided with functional CK systems.

Conclusions:

• Cardiac mitochondria possess an intrinsic affinity for ADP comparable to isolated mitochondria.
• The high apparent Km observed in skinned cardiac fibres is primarily an artifact of diffusion limitations outside the mitochondria.
• Cellular energy buffering systems, like creatine kinase, are critical for efficient substrate supply to mitochondria in intact tissues.