A Mouse Model for Monitoring Calpain Activity under Physiological and Pathological Conditions

Marc Bartoli,Nathalie Bourg,Daniel Stockholm,Fabrice Raynaud,Antony Delevacque,Yang Han,Perrine Borel,Kenza Seddik,Nasser Armande,Isabelle Richard

doi:10.1074/jbc.m608803200

Abstract

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.

Highlights

Inappropriate calpain activation has been implicated in various disease states including traumatic spinal cord and brain injuries, cataract formation, cerebral and heart ischemia, hypertension, arthritis, Duchenne muscular dystrophy, and neurodegenerative disorders such as Alzheimer disease [2,3,4,5,6]
It is noted that when mice died during the experiment, a postmortem cleavage of the reporter was seen associated with a high decrease of FRETeff, stressing the importance of in vivo analysis when looking for physiological calpain activity
We demonstrated by the investigation that the fluorescent signals could readily be exploited in physiological and pathological studies in living mice

Summary

EXPERIMENTAL PROCEDURES

Generation of CAFI and SARCAFI Models—The construction of the reporter cassette was previously described [14]. Validation of the Reporter Functionality—After sampling, pulverized powder of different tissues was incubated in vitro in the presence of Ca2ϩ for 30 min at 37 °C in the presence or absence of MDL28170, a potent inhibitor of the ubiquitous calpains Subsequent analysis of these samples by anti-GFP Western blotting allowed the visualization of the extent of the reporter cleavage. It is noted that when mice died during the experiment, a postmortem cleavage of the reporter was seen associated with a high decrease of FRETeff, stressing the importance of in vivo analysis when looking for physiological calpain activity. A change of 4.3 and 6.1% in FRETeff was observed at days 4 and 14, respectively (Table 1 and Fig. 5, panel b) Another protocol was performed where the mice were forced to run on a treadmill for 1 h. This loss of FRETeff was very homogeneous among the studied muscle with no apparent damage seen in the myofibers

Crossing of CAFI Mice with a

Findings

DISCUSSION