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 DOI: 10.1258/002367795780740032

1995 29: 420Lab AnimL. P. Soldati, B. R. Barber, S. Salardi, I. Molinari and G. Bianchi

A new rat model for studying the calpain-calpastatin system  

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A new rat model for studying the calpain-calpastatin system

L. P. SoldatP, B. R. Barber2, s. Salardi2, I. Molinari2 & G. BianchP'Nephrology Unit, Department of Sciences and Biomedical Technologies, University of Milan, ViaOlgettina 60, 20132, Milano and 2prassis,Sigma-Tau Research Institute, via Forlanini 3, 20019 SettimoMilanese, Milano, Italy

SummaryA recombinant rat strain-Milan low-calpastatin strain (MLCS)-was derived from Milanhypertensive (MHS/Gib) and Milan normotensive (MNS/Gib) strains. The MLCS ratshave normal blood pressure and low calpastatin activity, and this strain is proposed as amodel for studies of the calpain-calpastatin system, which is involved in importantcellular mechanisms. Calpastatin polymorphism was observed in 10 different strains oflaboratory rats and a single locus hypothesis is suggested as the mode of inheritance.

Keywords Ratj calpastatinj calpainj blood pressure

Calpains (CaNP) are cytosolic neutralcalcium-dependent proteinases thatcatalyse the proteolysis of several plasmamembrane and cytoskeletal-associatedproteinsj their activity is naturallyregulated by a specific inhibitor proteintermed calpastain fCAST): (Murachi 1983j

Suzuki & Onho 1990, Croall & De Martino1991). A difference was observed betweencalpain activity in erythrocytes and kidneycells of the inbred Milan hypertensivestrain [MHS/Gib) of rats and those of itsnormotensive control strain (MNS/Gib)(pontremoli et a1. 1986a, 1986b, 1987). Itwas further shown that this difference wasdue to a lower inhibition of calpain bycalpastain in MHS/Gib cells. Thereduction of calpastatin activity leads to animbalance of the calpain:calpastatin ratioand is associated with an increase ofresidual proteolytic activity (Soldati L,unpublished data). Calpain activity hasbeen considered of importance in variouscellular mechanisms that may influenceactivation of platelet function (Fox et a1.1990a, 1990b, Puri et a1. 1990), control of

Correspondence to: Dr L P Soldati

Accepted 1 February 1995

cell cycle (Watanabe 1989/ Schoellmeyer1988/ March et a1. 1991), synaptic memoryand cytoskeleton remodelling (Siman &Noszek 1988, Kennedy 1989, Siman 1990).

MHS/Gib rats were originally selected toattain elevated blood pressure [meanvalue±1 SEM=169±0.3, range160-180 mmHg) after 2 months of age,and the normotensive MNS/Gib tomaintain normal levels (mean value ± ISEM= 131±0.3, range 125-140mmHg)throughout adult life (Bianchi et a1. 1975,1990). Subsequently, several differenceshave been revealed in other phenotypic andgenotypic characters (Bianchi et a1. 1984)and some of these have shown correlationswith blood pressure levels (Bianchi et a1.1975/ 1994, Salvati et a1. 1987).

In previous studies, we examined an F2population of these rats, derived by inter-crossing Fl (MHS/Gib X MNS/Gib) hybrids,and found no correlation between arterialpressure and calpastatin activity, thusconcluding that these inherited traits arenot related (Soldati et a1. 1991a, 1991b).This was corroborated by the observationthat an inbred, recombinant strainMH.NE/Brb, also derived from MHS/Gib and

laboratory Animals (1995) 29. 420-426

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low-calpastatin strain of rats

MNS/Gib crosses, demonstratedindependent segregation of these traits;hypertension being present together withnormal levels of CAST activity.

This study reports the selection of a newrecombinant strain possessing reciprocalsegregation of 2 characteristics-normalsystolic blood pressure as in MNS/Gib, andlow CAST activity as in MHS/Gibj thisnew strain has been designated Milan lowcalpastatin strain (MLCS)and is nowproposed as a model for investigationsinvolving calpain-calpastatin mechanisms.

Data obtained with classic geneticcrosses and 10 other rat strains are alsopresented to support the hypothesis thatvariation in calpastatin activity of rats isdue to polymorphism at a single Mendelianlocus.

Materials and methods

Animals and husbandryAll rats of the Milan hypertensive strain(MHS/Gib), Milan normotensive strain(MNS/Gib), their various crosses andinbred recombinants MH.NE/Brb andMH.NA/Brb used for these studies werebred in our own closed colonies underbarriered conditions at the Prassis Sigma-Tau Research Institute, Milan. Groups of 4other inbred (WKY/Cri, BN/Cri, SHR/Cri,F344/Cri) and 2 outbred (CR:CD-Sprague-Dawley origin and Cri:WI-Wistar originlstrains were obtained from Charles RiverItalia SpA, Calco/CO, Italy for comparisonof strain distribution patterns.

Those rats used for selection of MLCSand genetic crosses were maintained in thebarriered facility, while those used forstrain comparison were housed inunbarriered rooms with identicalenvironmental conditions for 10 days priorto sampling.

A total of 312 rats were screened forCAST activity over the 11 generations ofselection reported and a further 115 Fl andbackcross hybrids and 85 parental strainrats were produced and sampled by thesame techniques for genetic studies. All ofthe rats were aged 2-3 months andweighed 250 to 350g body weight.

421

All requirements of EEC Directive609/1986 and the relevant Italian Law no.116, Jan. 1992 regarding housing,husbandry and procedures were observed.Breeding was carried out in transparentpolycarbonate cages 15x 45 xiS h cm withstainless steel wire covers 5 cm high(Tecniplast Gazzada S.r.!, Buguuggiate/VA,Italy). Weaned animals were maintained insimilar cages with a height of 20 cm. Therats were fed Altromin MT IA.RieperS.p.A, Vandoies/BZ, Italy) ad libitum withdrinking water provided ad libitum inbottles. The rooms were ventilated throughHEPA filters with 16 changes per hour offully conditioned air at 21± 2°C and60± 10% relative humidity.

Blood samplingFor between-strain comparison ofcalpastatin and calpain-I assays, 4 male ratsof each of the 10 strains were anaesthetizedby halothane inhalation, and blood waswithdrawn via the dorsal aorta withoutrecovery.

For selection of breeding animals, 2ml ofblood was withdrawn from the retro-orbitalplexus under halothane inhalationanaesthesia.

Blood pressure measurementsSystolic blood pressure was measured bythe indirect tail-cuff method on a W+ WBP Recorder (Ugo Basile, Via Borghi 43,Comerio/VA, Italy) after warming at 35°Cfor IS min in thermostated and ventilatedcabinets. Readings obtained on 3 separatedays were averaged for each animal.

Calpain-I and calpastatin (CAST) activityassayThe cytosolic fractions of calpastatin andcalpain-I were obtained by extraction fromerythrocytes and separated by hydrophobicchromatography and ion exchangechromatography and assayed for CASTactivity according to methods described byPontremoli et aI. 1986b.

The proteolytic activity of extractedcalpain-I was measured with the substrateacid-denatured human globin in presence of1mM calcium chloride at pH 7.5. The

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422 Soldati et al.

amino-groups liberated by the proteolyticenzyme were measured by the fluorescenceof added fluorescamine and compared toa standard solution of glycine. Oneunit was defined as the amount of enzymethat releases 1/!mol/h of free amino-groups.

The inhibitor activity of extractedcalpastatin, heated at 90°C for 3 min, wasmeasured under the same conditions withthe addition of 50 units of extractedcalpain-I from human erythrocytes. Oneunit of inhibitor was defined as the amountof calpastatin that inhibited one unit ofstandardized proteinase activity. Theactivities of both enzymes were correctedfor the haemoglobin content of packed redcells (U/mgHb). Due to inherentlimitations of these assays, the precisionexpressed as the coefficient of variationfor these methods were estimated to bewithin 15%.

Genetic analysisThe one-gene hypothesis, on whichselection was based, was further tested bymeasuring calpastatin activity levels inlarger cohorts of F1 offspring from crossesbetween MHS/Gib and MNS/Gib and fromreciprocal backcrosses of Fl x MNS/Giband Fl x MHS/Gib. One or 2 rats of eachparental strain were assayed ascontemporaneous controls with eachgroup sampled for selection. The rangesgiven for the parental populationscompared to the genetic crosses werederived from the pooled values of thesecontrol animals.

StatisticsComparisons between groups were made by2-way analysis of variance, and regressionanalysis by the least squares method.Regression of mean offspring values on

Fig 1 Percentual proportions of rats classifiedaccording to three pheno-genotypes in eachgeneration of selection for low CAST activity.BC=backcross of Fl to MNS; P=parental strain;S=selection generation; n=number of samples

7

14

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24

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56

53

51

55

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52

SelectionGenerition

looIrMHSl ~ PI50~PZ

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~CAST Activity U/mgHb

Recombinant strain (MLCS) selectionData collected from our previousinvestigations (Soldati et ai. 1991a, 1991b)indicated that the inheritance of CASTactivity in rat erythrocytes was controlledby a simple monogenic Mendelianmechanism. Rats of the homozygousMHS/Gib genotype had CAST valueswithin a range of 0 to <3 U/mgHb (n = 10),those of MNS/Gib within 6 to 8U/mgHb(n = 5) and the values of Fl hybrids(MNS/Gib X MHS/Gib) were within3 to 5U/mgHb (n=36). These 3ranges were then taken to represent the 3corresponding genotypes on which tobase the following selection breedingprogramme.

MHS rats were crossed with MNS toproduce an Fl generation. These animalswere then backcrossed to the high-calpastatin strain MNS/Gib, and theoffspring selected by their systolic bloodpressure and CAST values into possibleheterozygotes or homozygotes for thecalpastatin gene. The presumed CASTheterozygotes with lowest levels of bothblood pressure and calpastatin were inter-crossed and selection applied to subsequentgenerations in the same manner.

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Low calpastatin strain of rats

cumulative selection differentials and meanvalues for parents was taken on selectiongenerations Sl to S7, as earlier matingswere obtained by different methods. Theregression of mean offspring on mid-parentvalues was calculated on all familiessampled whether selected or not formating, but backcrosses to inbred rats ofthe parental strains were excluded.Regression data were weighted for thenumbers of offspring in the respectivefamilies or generations. Inbreedingcoefficients (F)were calculated by pathwayrelationships of coancestry [f}(Falconer1966).

All calculations are based on raw datawithout transformations, other than wherepercentual proportions are indicated(Fig. I), and means are given with standarderrors of the mean (SEM),and number ofanimals (n) or degrees of freedom, asindicated in the text and figures.

Results

Recombinant strain selectionThe first backcross of F1 to MNS/Gibindividuals (parents not sampled for eitherparameter) produced 2 males and 5 femaleswith mean blood pressure of 135± 2.1 SEMmmHg and a mean CAST level of6.33 ± 0.4 SEM U/mgHb. Each of thesemales was mated to 2 females with theCAST values most similar to his own. Themeasurements of CAST in the offspring offollowing generations confirmed the generalworking hypothesis of association betweenphenotype classes and genotype.

Starting from the F1x MNSIGibbackcross population} response to selectionfor low calpastatin activity was rapid.Although some single animalsdemonstrated unexpectedly high values[from 3 to < 5 U/mgHb) in the third and fifthgenerations, for all selection generationsafter S2 the means corresponded to theMHS/Gib phenotypic class and from S6onwards all families produced only animalswith values lower than 3 U/mgHb. Theprogress of this selection is shown in Fig.I, as the percentual proportion of rats

423

in each generation of the 3 predefinedclasses.

The regression slope of generation meanscompared to mean values of their parentswas 0.84± 0.1 SEM (P= <0.001 withdegrees of freedom = 6, R2=0.93), and thatof response to cumulated selectiondifferential was 0.91 ± 0.09 SEM, [degreesof freedom = 6). These regressions comparedfavourably to that of offspring means onmid-parent values taken on all familiesbred (0.97±0.17 SEM, with 27 degrees offreedom), giving a general estimate ofheritability close to unity as would beexpected for a single locus trait (Falconer1966].

Blood pressureThe simultaneous selection for low bloodpressure closely followed a patternpreviously reported (Barber et al. 1988),and stable, uniform values within the range130-140 mmHg were attained within 2generations. There was, however, atransient return to intermediate values atthe S3 to S5 generations-presumably dueto recombination of recessive alleles atunknown loci for this polymeric character

mmHg•.••18'";;;)~ 17 HSIGib - - - - - - - - - - - - - - -w'""'16 -----------------c815...J

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Fig 2 Response to simultaneous selection fornormal blood pressure levels in MLCS rats selectedfor low CASTactivity. BC=backcross of Fl to MNS;P=parental strain; S=selection generation. Circles=generation means; bars= ± 1 standard deviation.Shaded areas represent ± 1 standard deviationaround the pooled means of contemporarygenerations of indicated parental strains MHS/Giband MNS/Gib. n = number of samples (includesanimals related to those in Fig. 1 which were notscreened for CASTactivity)

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424 Soldati et al.

3.53± 0.3

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(WKYICri). Such isolated segregations havebeen reported in other inbred strains(Tripodi et 01. 1991, Dubay et 01. 1993,Festing 1984) and underlines the need forcaution when considering isogenicity oftraits in any inbred strains for which thatstrain is not normally monitored. Some highCAST values were found in both WKYICriand SHR/Cri rats. But as these were closeto the limits Qfthe CAST values for ourlarger sample of MNS/Gib, the existence ofa third allele need not be envisaged.

Calpain-I activity, in the absence ofcalpastatin, was also measured in theerythrocytes of these 10 strains. Only thecalpain-I isoform is present in erythrocytesand no differences were found; the valuesfor all strains falling within the range0.31-1.62 (mean± 1 SEM values were0.85 ± 0.08) U/100 mg haemoglobin.

Genetic crossesThe distribution frequencies and statisticalresults of the genetic crosses are shown inFig. 4 and corresponded well to the ranges

ZO1 S10

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012345678910CAST Activity U/mgHb.

Fig 4 Distribution frequencies of CASTvalues ofrats from classic genetic crosses of MHS/Gib andMNS/Gib strains. BC=backcross of F1 to indicatedinbred strain. n = number of samples

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(Schlager et 01. 1986). After this period allgeneration means were not significantlydifferent (P= <0.001) in blood pressurelevels from the normotensive MNS/Gibinbred foundation colony (Fig. 2).

Sister by brother matings were adoptedexclusively from the fifth generation ofselection (S3)onwards and a coefficient ofinbreeding value of 0.93 was obtained forall families in the tenth generation (S8).

Sterility was observed in the 5th [S3)and6th [S4)generations and affected 61% ofmatings (14/23b this effect fell to 7%(2/30) for the next 3 generations. Theaverage litter size at birth (9.4± 0.3 SEM)and pre-weaning mortality [1.8%) in thestrain under selection has remainedcomparable to those of the 2 parentalstrains.

Strain distribution patternsPolymorphism of CAST activity levelsappeared to be highly distributed amongthe 10 strains of laboratory rats screened(Fig. 3). Apparent segregation was seen asexpected in one outbred strain (Cri:WIl,but occurred within an inbred strain

CAST Activity U/mgHb

STRAIN <1 I 1 I 2 3 I 4 I 5 6 I 7 1>8MHS/Glb • •• •MNS/Glb • • ••

MH.NE/Brb • •• •MH.NA/Brb • • ••

BN/Crl :: •SHR/Cri • ••WKY/Cri • •• •F344/Cri • •• ••Crl:WI • ••

Crl:CD •• ••Fig 3 Distribution of CAST polymorphism insamples of rats (n=4) from 10 different rat strains.Each data point represents one animal. MH.NE/Brband MH.NA/Brb are recombinants of MHS/Gib andMNS/Gib; all other strains are unrelated

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Low-calpastatin strain of rats 425

Fig 5 Relationship between means of CASTactivity in classic genetic crosses of MHS/Gib andMNS/Gib rats. Circles indicate means of populationsshown on base line: open circle = segregatingpopulation; BC= Backcross of F1 to indicated inbredstrain. Dashed line indicates extrapolation of MHSmean on the hypothesis of no dominance.Numbers of samples as in Fig. 4

DiscussionThe class values chosen as selectioncriteria for this trait correspondedextremely well to Mendelian expectationsfor a single locus throughout theexperiments with the MNS/Gib parentalpopulation showing greater variation thanthe MHS/Gib population (Fig. 4).Furthermore, the Fl population meantended towards the MHS/Gib range whichsuggested some phenotypic dominance inthis direction and complete selection was

apparently not reached within 2generations, which would be expected for asimple monogenic character withoutdominance.

However, the apparent difficulty inattaining complete selection in earlygenerations and the unexpecteddistributions could be due to a scale effector the limitations of the assay whichappears to be more variable with highervalues (e.g. MNS/Gib) and limited for thelower values (e.g. MHS/Gib). This couldhave led to possible misclassification ofsome phenotypes used for selectionmatings and to the apparent artefact ofdominance in the genetic crosses (see Figs4 and 5), but when 3 overall phenotypicclasses were defined in the crossexperiment (as used in Fig. 1 for theselection), at least 80% of each cohort wasclassified as expected with very littleoverflow into adjacent classes and thesecorresponded well in a Chi square test(P=O.55) to Mendelian proportions.

While this study highlights thedifficulties and limitations of classifyinggenotypes of biologically metric traits onthe basis of their phenotypes, the resultsdo give strong support to the hypothesisthat the genotypes of this polymorphism inrats may be adequately defined by theirphenotypic values, at least for the purposesof selection and monitoring its isogenicityin inbred colonies.

Considering the extensive list ofendogenous proteins susceptible todegradation by calpain and its suggestedinvolvement in research fields as diverse ascytology, neurology, cardiology andenzymology, we propose the new MLCSstrain as a further useful model to beutilized in conjunction with MNSIGib,MHS/Gib and their other derivedrecombinant strains, such as MH.NE/Brb.

Acknowledgments 1. Soldati was supported bygrants from the Italian Ministry of Public Educationand San Raffaele Hospital, Milan.

This work was partially supportcd by theEURHYPGEN Concerted Action Programme.

The authors are grateful to Pietro Dioguardi andMario Franco for skilled technology in the breedingand maintenance of the animals.

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used for selection. Both backcrossescontained an excess of animals with valuesin the lower of the 2 classes into whichthey were expected to fall, and all cohortsexcept MHS/Gib included animals withvalues that lay outside those of the smallerdata sets from previous studies (Soldati eta1. 1991a, 1991b).

There were no significant differencesbetween sexes.

The relationships among the means ofthese populations are plotted in Fig. 5and indicate about 15% dominancedeviation towards MHS/Gib values in allcross populations.

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426

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