VDAC3 has differing mitochondrial functions in two types of striated muscles

Abstract

Voltage-dependent anion channel (VDAC) is an abundant mitochondrial outer membrane protein. In mammals, three VDAC isoforms have been characterized. We have previously reported alterations in the function of mitochondria when assessed in situ in different muscle types in VDAC1 deficient mice (Anflous et al., 2001). In the present report we extend the study to VDAC3 deficient muscles and measure the respiratory enzyme activity in both VDAC1 and VDAC3 deficient muscles. While in the heart the absence of VDAC3 causes a decrease in the apparent affinity of in situ mitochondria for ADP, in the gastrocnemius, a mixed glycolytic/oxidative muscle, the affinity of in situ mitochondria for ADP remains unchanged. The absence of VDAC1 causes multiple defects in respiratory complex activities in both types of muscle. However, in VDAC3 deficient mice the defect is restricted to the heart and only to complex IV. These functional alterations correlate with structural aberrations of mitochondria. These results demonstrate that, unlike VDAC1, there is muscle-type specificity for VDAC3 function and therefore in vivo these two isoforms may fulfill different physiologic functions.

Fig. 1

Functional properties of mitochondria. The in situ affinity of mitochondria for ADP (Km[ADP] in μM) was determined in saponin-skinned fibers prepared from wild type control (black bar) and vdac3^-/- (white bar) heart (A) and gastrocnemius (C) in the absence (w/o) and presence of 25 mM creatine. Calculated rate of respiration in the presence of the maximal ADP concentration (Vmax) was determined in fibers prepared from wild type control (black bar) and vdac3^-/- (white bar) heart (B) and gastrocnemius (D) in the absence (w/o) and presence of 25 mM creatine. Values are expressed in μmoles of oxygen/min/g dry weigh (gdw). The error bar represents the SEM. *** p<0.001 vs. wild type.

Fig. 2

Electron microscopy images of muscles. Heart and gastrocnemius muscles were dissected from wild type and vdac3^-/- mice and mounted for electron microscopy analysis. A) and B) show intermyofibrillar mitochondria from wild type and vdac3^-/- heart respectively. The head arrows indicate one mitochondrion that spans two sarcomeres units. The asterisks indicate an average of mitochondria within two sarcomeres units. C) and D) show intermyofibrillar mitochondria from wild type and vdac3^-/- gastrocnemius respectively. E) and F) show subsarcolemmal mitochondria from wild type and vdac3^-/- gastrocnemius respectively. Magnification × 2,000 (A) and (B), × 6,000 (C), (D), (E) and (F).

Fig. 3

Expression analysis of different VDAC isoforms. Western blots corresponding to the sample homogenates from wild type and vdac3^-/- muscles were probed with antibodies specifics for different VDAC isoforms. Actin antibody was used for loading control. The panel indicates the ratio of VDAC1 (1) and VDAC2 (2) band signal to actin band signal in wild type (black bar) and vdac3^-/- (white bar) heart (H) and gastrocnemius (G). The bands were quantified by a Densitometer. Each bar represents the mean of two values. Lower panel: H: heart; G: gastrocnemius. 1: data relative to VDAC1 antibody; 2: data relative to VDAC3 antibody. Black bar: Wild type sample; White bar: vdac3^-/- sample.

Fig. 4

Respiratory enzyme activities. The activities of complex I, II, I + III, II + III and IV were determined and normalized to citrate synthase activity for mitochondrial content. The normalized activity in wild type control was taken as 100% and the normalized activity in the mutant muscles was calculated as a percent of the wild type control. A) Normalized respiratory enzyme activity in the heart. B) Normalized respiratory enzyme activity in the gastrocnemius. The black bar represents wild type muscle, the white bar represents vdac1^-/- muscle and the gray bar represents vdac3^-/- muscle. The error bars refer to SEM. * p<0.05 vs. wild type, ** p<0.01 vs. wild type, *** p<0.001 vs. wild type.

Fig. 5

Expression analysis of COXIV subunit. Western blot corresponding to the sample homogenates from wild type (Wt), vdac1^-/- and vdac3^-/- heart was probed with an antibody specific for COXIV subunit. Cytochrome c was used for loading control. Wt= Wild type