Age-dependent regulation of skeletal muscle mitochondria by the thrombospondin-1 receptor CD47

Abstract

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

Figure 1. Tissue survey of mitochondrial mRNA expression

RNA was prepared from the indicated tissues with the RNA Later^c method and levels of mRNA for cytochrome c (cyt c), cytochrome b (cyt b), nuclear respiratory factor-1 (NRF-1) and PGC1a were determined by Q-PCR using the Sybr Green method. Data is plotted as the ratio of the CD47-null value to the WT value, both of which were first normalized to the level of mRNA for the ribosomal protein 36B4. Tissue from 5 to 16 mice was processed independently for each value shown.

Figure 2. Electron microscopy of soleus muscle from CD47-null and WT mice

Soleus was rapidly harvested and fixed in a mixture of paraformaldehyde and glutaraldehyde and prepared for thin section TEM. WT muscle is shown in A and CD47-null in B. White arrows indicate mitochondria.The final magnification is 10,000X for both. In C, per cent mitochondrial area in ten randomly selected fields were measured for each of 3 animals for each genotype. The CD47 null data (top panel) is for soleus, a type I slow twitch muscle and the TSP1 null data (bottom panel) is for gastrocnemius, a type II fast twitch muscle. Comparable increases were seen in both muscle types in both knockouts.

Figure 3. Age-dependent decrease in expression of mitochondrial marker RNAs in CD47 null mice

RNA prepared as in figure 1 was amplified using the Sybr Green method. Data is normalized to values for 36B4 mRNA and expressed as the ratio of CD47 null to WT values. At 12 months of age, values for all three RNAs were the same in CD47 null and WT muscle.

Figure 4. Young CD47-null mice have improved endurance and muscle fiber type switching

(A) Running endurance of 3 month old CD47-nulls is significantly superior to WTs (n=7–8). (B) CD47-null gastrocnemius muscle, normally a fast twitch muscle, expresses high levels of markers of slow twitch, mitochondria-rich fibers compared to WT. Q-PCR data expressed as the ratio of CD47-null to WT value for each mRNA as in figure 1 (n=8).

Figure 5. Body composition of CD47 null and WT mice

(A) CD47-null mice weigh significantly less than WTs at both 3 and 12 months of age. Lean (B) and fat (C) body mass was determined by DEXA at both ages. CD47-nulls have significantly less body fat than WTs, accounting for the difference in total body weight (n=4–6).

Figure 6. CD47-null aortic smooth muscle cells produce much less ROS that WTs

Early passage aortic smooth muscle cells from male 6 week old null (solid bar) and WT mice (empty bar) were seeded on black 96 well tissue culture plates, grown to confluence, then treated with dihydroethidine which reacts with superoxide to produce fluorescent ethidium (HE), dichlorofluorescein (DCF) which becomes fluorescent after reaction with several types of ROS or Mitosox which detects primarily mitochondrial superoxide. The increase of fluorescence over time (slope) was normalized to cell number determined as the DAPI (nuclear) fluorescence of the same cells. The figure is representative of 3 separate experiments with different batches of cells. Aortic smooth muscle cells were isolated from 6–8 weeks old mice.