CCMED-D-11-01204
ONLINE SUPPLEMENT
Crosstalk between calpain and caspase-3 during mechanical ventilationinduced diaphragmatic atrophy
W. Bradley Nelson, Ashley J. Smuder, Matthew B. Hudson, Erin E. Talbert and
Scott K. Powers
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METHODS and RESULTS
In Vitro Protease Inhibition Assay. Often, studies using pharmacological
inhibitors are criticized due to concerns about off-target effects of the
pharmacological agents. To determine if our agents could successfully inhibit
their target proteases while also not inhibiting other proteases believed to be
important in mechanical ventilation (MV)-induced diaphragm weakness, we
designed a series of in vitro experiments adapted from Pereira et al (1). Using
purchased purified active enzymes (calpain-1, caspase-3 and caspase-9), we
measured
the
cleavage
of
fluorogenic
substrates
when
exposed
to
concentrations of inhibitor similar to our calculated in vivo concentrations, based
on 70% body water. Sample fluorescence, an indicator of substrate cleavage,
was measured after 20 minutes of incubating substrate, active enzyme, and the
inhibitor. The calpain inhibitor (SJA-6017) prevented the majority of cleavage of
the calpain substrate by active calpain-1, while our caspase-3 inhibitor (ACDEVD-CHO) did not prevent cleavage of the substrate (Figure 1). Similarly, after
20 minutes of incubation, the caspase-3 inhibitor (AC-DEVD-CHO) prevented
cleavage of the caspase-3 substrate by active caspase-3, while the calpain
inhibitor did not prevent substrate cleavage by caspase-3 (Figure 2). Incubation
of the calpain inhibitor with active caspase-9 did not prevent substrate cleavage,
while the caspase-3 inhibitor did in fact inhibit the activity of caspase-9 (Figure 3).
Results from the in vitro assays are represented in Figures 1-3.
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Figure 1. Calpain-1 activity in the presence of the experimental inhibitors. Bars
represent end-point fluorescence after 20 minute incubation. Sub = substrate,
Calp-1 = purified calpain-1 enzyme, Calp Inhib = calpain inhibitor (SJA-6017),
Casp-3 Inhib = caspase-3 inhibitor (AC-DEVD-CHO). Values are means ± SEM,
groups with different letters are statistically different p<0.01, n=4.
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Figure 2. Caspase-3 activity in the presence of the experimental inhibitors. Bars represent end point
fluorescence after 20 min incubation. Sub = substrate, Casp-3 = purified caspase-3 enzyme, Casp-3
Inhib = caspase-3 inhibitor (AC-DEVD-CHO), Calp Inhib = calpain inhibitor (SJA-6017). Values are
means ± SEM, groups with different letters are statistically different p<0.01, n=4.
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Figure 3. Caspase-9 activity in the presence of the experimental inhibitors. Bars represent end point
fluorescence after 20 min incubation. Sub = substrate, Casp-9 = purified caspase-9 enzyme, Casp-3
Inhib = caspase-3 inhibitor (AC-DEVD-CHO), Calp Inhib = calpain inhibitor (SJA-6017). Values are
means ± SEM, groups with different letters are statistically different p<0.01, n=4.
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Standardization of protein loading for Western blotting. Ponceau-S stained
membranes were used to control for equal protein loading and transfer
differences. The membrane was scanned, and lanes were quantified to
normalize Western blots to protein loading. Figure 4 provides a representative
membrane that has been stained with Ponceau S.
Figure 4. Representative Ponceau S stained membrane. 1, control; 2, 12 hr MV; 3, 12 hr MV +
Calpain Inhibitor; 4, 12 hr MV + Caspase-3 Inhibitor. MW, molecular weight.
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Arterial blood gas and arterial blood pressure measurements.
To ensure that our MV protocol was successful in maintaining
homeostasis, we measured arterial pH, arterial blood pressures, heart rate,
arterial PO2, arterial PCO2 and in all MV animals at the beginning of the
experiments and at various time intervals during MV (see figure 5). The carotid
artery was cannulated to allow for blood gas sampling and direct blood pressure
monitoring via pressure transducer (Kent Scientific, Torrington, CT). Arterial
blood gases and arterial blood pH were made with ~ 150 μl sample and
measured via a clinical blood gas analyzer (GEM Premier 3000, Instrumentation
Laboratory, Lexington, MA). Heart rates were monitored using a lead II
electrocardiograph (model 2522B, BK Precision, Yorba Linda, CA).
Figure 5. Hemodynamic results from mechanically ventilated animals. pH values represent range of
values, all other values represent means ± SEM.
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References
1.
Pereira NA, Song Z. Some commonly used caspase substrates and inhibitors
lack the specificity required to monitor individual caspase activity. Biochem Biophys
Res Commun 2008;377(3):873-877.