Multiple cardiac contractile protein abnormalities in myopathic syrian hamsters (BIO 53:58)

j Mol Cell Cardiol 17, 95-107 (1985)

M u l t i p l e C a r d i a c C o n t r a c t i l e P r o t e i n A b n o r m a l i t i e s in M y o p a t h i c Syr ian H a m s t e r s (BIO 53 : 58)*~

Ashwani Malhotra, Marc Karell and James Scheuer

Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, N Y 10467, USA

(Received 15 November 1983, accepted in revised form 13 April 1984)

A. MALI-IOTRA, M. KARELL AND J. SCnEUER. Multiple Cardiac Contractile Protein Abnormalities in Myopathic Syrian Hamsters (BIO 53:58)�9 Journal of Molecular and Cellular Cardiology (1985) 17, 95 107. Hearts of genetically myopathic male hamsters (BIO 53 : 58) were studied at 1 month, 2 months, 3 months, 4 to 5 months and 7 months of age. The time course of alterations in the cardiac myofibrillar ATPase activity, the relationship of myofibrillar ATPase activity to free [Ca2+], myosin ATPase activity and the distribution of heavy chain myosin isoenzymes were evaluated. Mg 2§ Ca 2+ ATPase activity of cardiac myofibrils in myopathics was increased in 4 month and 7-month-old hamsters. Elevated Mg 2+ ATPase activity was found as early as in 2-month-old hamster. However, there was no loss in the regulation of the myopathic myofibrillar assembly as measured by the Pc, response (10 .7 M to 10 -4 M Ca2+). Scans of SDS electrophoresis slab gels of cardiac myofibrillar proteins from control (C) and myopathic animals (M) did not show any differences at any age group (1, 4 and 7 months). There was a significant decrease in myosin Ca 2+ ATPase activity and actin activated Mg 2 +-ATPase activity at 4 to 5 months and 7 months of age in the myopathic hearts. At all ages in normal and myopathic animals cardiac myosin consisted of three isoenzymes, V1, V 2 and V 3. At all ages in controls and at 1 to 3 months in myopathics, V 1 predominated and the isoenzyme distribution was V 1 > V 2 > V s . However, in myopathics at 4 to 5 months, the distribution was V 1 = V 3 > V 2 and at 7 months was V s > V 2 > V 1. Our experiments suggest alterations in different components of the contractile protein system that occur at different stages ofmyopathy. /

KEy WORDS: Cardiac muscle; Cardiomyopathy; Contractile proteins; Proteolytic enzyme inhibitors; Myofib- rils; Myosin ; Isoenzymes ; Pyrophosphate gels; Myosin ATPase ; Polyacrylamide gels.

Introduction I t h a s b e e n p r e v i o u s l y r e p o r t e d t h a t the m y o s i n a d e n o s i n e t r i p h o s p h a t a s e ( A T P a s e ) a c t i v i t y is dep re s sed in the h e a r t s o f myo- p a t h i c a n i m a l s a t a s tage w h e n t he h e a r t s a re in f a i lu re [-2, 18]. P a n g a n d Weg l i ck i [14] r e p o r t e d t h a t m y o f i b r i l l a r A T P a s e ac t iv i t i e s f r o m s i m i l a r a n i m a l s v a r i e d w i t h age a n d sex b u t w e r e u n i f o r m l y d e p r e s s e d a f t e r 50 d a y s of age. I n r e c e n t years , i t has b e e n s h o w n t h a t c a r d i a c m y o s i n in some species exists in t h r e e i so+nzymic fo rms a n d these m a y v a r y d e p e n d - i ng o n the a n i m a l ' s age, species, a n d phys io l - ogic or p a t h o l o g i c s t a t e [16]. I s o e n z y m i c c h a n g e s in myos in , c a n a c c o u n t for t he c h a n g e

* Supported by US Public Health Service Research Institute, Bethesda, Maryland.

in A T P a s e ac t iv i ty o b s e r v e d in a v a r i e t y o f m o d e l s o f c a r d i a c h y p e r t r o p h y [13].

T h e p r e s e n t s t u d y was u n d e r t a k e n in a n a t t e m p t to d e t e r m i n e : (1) a t w h i c h s tage d u r i n g the course of c a r d i o m y o p a t h y t he A T P a s e ac t iv i ty o f m y o f i b r i l l a r p r o t e i n s b e c o m e s a l t e r e d a n d r e l a t i o n s h i p o f A T P a s e ac t i v i t y to free c a l c i u m c o n c e n t r a t i o n ; (2) w h e t h e r c h a n g e s in t he d i s t r i b u t i o n o f m y o s i n i s o e n z y m e s o c c u r as t he c a r d i o m y o p a t h y d e v e l o p s ; a n d (3) h o w the c h a n g e s in myof ib~ r i l l a r p r o t e i n s r e l a t e to c h a n g e s in m y o s i n A T P a s e ac t i v i t y d u r i n g t h e course o f c a rd io - m y o p a t h y .

Grant HL18824 from National Heart, Lung and Blood

~" This work was presented in preliminary form at the 66th Annual meeting of Federation of American Societies for Experimental Biology, April 15-23, 1982 held at New Orleans, Louisiana. (Malhotra et al., 41, 1526, 1982).

0022-2828/85/020095 + 13 $03.00/0 �9 1985 Academic Press Inc. (London) Limited

96 A. Malhotra et aL

M a t e r i a l s and M e t h o d s

Adenosine triphosphate (ATP), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), ethylene glycel bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and proteloytic enzyme inhibitors [-phenylmethyl sulfonyl flouride (PMSF), Leupeptin, N-c~-p- Tosyl-L-lysine chloromethyl ketone (TLCK) Pepstatin] were purchased from Sigma, St Louis, MO. Genetically myopathic male hamsters (Bio 53 :58) ages 1 month to 7 months were obtained from Telaco Co., Maine. The time points selected for study were 1 month, 2 months, 3 months, 4 to 5 months and 7 months after birth. For controls, random-bred animals of the same age were used. For each age group control and myopathic animals were sacrificed at the same time and their hearts processed and analyzed simultaneously. Animals of different age groups were received and processed at different times. Therefore statistical comparisons are made.only between studies of control and myopathic samples from animals of the same age. Animals were anesthetized with ether and their hearts removed. After washing the hearts free of blood, the atria and the connect- ing vessels were removed and the ventricular portions were homogenized with a Tekmar homogenizer for 60 s. Six to eight hearts were pooled in each group. In some studies, hearts were immediately prepared for extraction of contractile proteins. Other hearts were stored at --70~ in 50% glycerol containing 30 mM KC1, 10 mM KPO4 (pH 7.0), 2 mM D T T and 0.2 mM PMSF prior to preparation of the extracts. There was no difference in values for ATPase when hearts were prepared immediately or after a period of storage.

Myofibrils were isolated and purified with Triton X-100 by the technique of Solaro et al. [,17]. In some of the myofibrillar preparations from control and myopathic cardiac tissue, various proteolytic enzyme inhibitors like PMSF (0.2 m~), leupeptin (1 #g/ml) pepstatin (1 #g/ml) and T L C K (0.1 mM) were used. To check the purity of the myofibrils, the modified method of Maizel [10] was used. The samples were run on sodium dodecyl sulfate (SDS) gradient (5% to 16.5%) slab gel electrophoresis in Tris-glycine buffer system. Contaminant proteins or evidence of proteo-

lytic breakdown were not detected in these purified myofibrillar preparations. Crude tissue extracts for isoenzyme studies were obtained by a slightly modified procedure of Mercadier et al. [,,131. Approximately 100 mg of tissue was extracted for 20 min at 4~ with 4 to 5 vol. of a solution of 0.3 M KC1, 0.1 M KH2PO4, 0.05 M K2HPO4, 0.001 M MgC12, 0.01 M Na4P2OT, 3 mM NAN3, 1% (vol/vol) 2-mercaptoethanol (fl-MSH) and 0.2 mM PMSF. The homogenate was centrifuged at 3000 x g f o r 20 min at 4~ The supernatant was dialyzed against 40 mM Na4P20 7 (sodium pyrophosphate) 0.2% fl-mercaptoethanol (fl- MSH), mixed with an equal volume of cold glycerol and then stored at - 70~ until ready for use.

For preparation and purification of myosin, either glycerinated myofibrils (frozen at - 7 0 ~ remaining after myofibrillar studies were used or 6 to 8 hearts were pooled for the preparation of myofibrils. Subsequent extraction of myosin was done using a KC1- pyrophosphate buffer (pH 7.0) and fractionation with a saturated solution of ammonium sulfate containing 10 m~ EDTA--1 mM D T T (pH 7.0) [11]. To inhibit proteolysis, PMSF (0.2 raM) was used throughout the different stages of the preparation. The myosin precipitated with 35% to 47% saturated ammonium sulfate was used. The myosin thus purified was shown by sodium dodecyl sulfate (SDS) gradient (5% to 16.5%) slab gel electrophoresis in Tris-glycine buffer system to be free of actin, troponin and tropomyosin and without evidence of proteolytic degradation. Polyacrylamide gel electrophoresis of myofibrils and myosin was performed on SDS tube gels as described by Weber and Osborn [19]. Similar electrophoretic profiles of myofibrils were obtained by SDS-gradient slabs. Scans of slab gels of myofibrillar and myosin preparations were performed on a Quick Scan Jr., from Helena Laboratories.

Cardiac myofibrillar ATPase activities were assayed in final volume of 1 ml at 30~ in a medium containing 50 mM KC1, 2 mM ATP, 2.5 mM MgC12, 20 mM Imidazole, (pH 7.0), 10 m~ NAN3, 0.1 m~ CaC12 or 2 mr~ EGTA and protein concentration 0.1 to 0.2 mg/ml. Mg 2+-Ca 2 § ATPase activity will be defined as Ca2+-stimulated

Contracti le Proteins in Cardiomyopathy 97

ATPase measured in the presence of M g 2+ (2.5 mM) and different concentrat ions of Ca 2 § E G T A ATPase act ivi ty will be defined as basal ATPase (Mg 2 § measured in the absence of free Ca 2 + but in the presence of 2.5 mM M g 2§ and 2 mM E G T A . For s tudying the free CaZ+-dependence of act ivat ion of myof ibr i l la r ATPase , C a 2 + / E G T A buffers were p repa red according to the procedure of H a t h a w a y and coworkers [6]. The react ion was s tar ted by the addi t ion of the substrate. For these studies, the prote in content of the enzyme was de te rmined by the b iure t method with bovine serum a lbumin as a s tandard . Es t imat ion of inorganic phospha te (Pi) was de te rmined by the micro method of Zak and coworkers [20].

Ca 2+ and K + - E D T A ATPase activities of myosin were assayed as described previously [11]. The final volume of incuba t ion medium was 1 ml of 0.3 M KC1, 50 mM Tris-C1 (pH 7.6), 10 mM CaC12 or 1 mM E D T A , and 5 mM ATP. The react ion was carr ied out at 30~ and was s tar ted by the add i t ion of the substrate. T h e amoun t of the enzyme added (60 to 75 #g) was adjusted so tha t only 15% to 20% of A T P was hydrolyzed. Results are

expressed as #mol Pi l ibe ra ted /mg prote in / min at 30~

Act in was ext rac ted from the rabb i t skeletal muscle as descr ibed in our ear l ier publ icat ions [11]. Actin ac t iva ted MgZ+-ATPase act ivi ty was measured in 50 mM KC1, 20 mM Imid- azole (pH 7.0), 2.5 mM MgC12, 2.0 mM ATP, 100 #g of myosin and vary ing concentrat ions of actin at 25~ Incubat ions were carr ied out as repor ted previously [11]. The micro- phospha te method of es t imat ing Pi was used [2o].

Analyses of myosin isoenzymes from crude myosin extracts and from purif ied myosin prepara t ions were done by po lyacry lamide gel electrophoresis using non-dissociat ing condi- tions at 2~ in a Pha rmac ia appa ra tus (GE-2/ 4) as described by Hoh et al. [7] and d 'Albis et al. [1]. The runn ing buffer conta ined 0.02 M Na4P2OT, 10% glycerol and in 0.001 M E D T A , 0.01% (v/v) f l -MSH, p H 8.5 [1, 7]. Cycl indr ical 4% po lyacry lamide gels (60 m m x 6 mm) were p repared with ac ry lamide and N,N 'me thy lene b isacrylamide (30 : 0.8). Approx ima te ly 2 to 5 /~g of pure myosin or crude myosin extracts were layered on each gel and run at a constant vol tage gradient of

TABLE 1. Weight relationships in control and myopathic (BIO 53 : 58) hamsters

HW/BW ratio Male Age (month) Body weight (g) Heart weight (g) (mg/g)

Control (15) 1 51.8 __ 1.2 0.135 __+ 0.037 2.664 __ 0.054 Myopathic (15) 55.9 __+ 1.2 0.145 _+ 0.035 2.599 _+ 0.043

P < 0.05 N.S. N.S.

Control (7) 2 87.6 + 2.6 0.183 __ 0.005 2.093 __+ 0.045 Myopathic (7) 86.0 __ 1.2 0.192 _+ 0.004 2.232 + 0.061

N.S. N.S. N.S.

Control (15) 3 94.3 ___ 2.0 0.346 __ 0.060 3.669 ___ 0.089 Myopathic (15) 91.4 __ 2.2 0.337 + 0.049 3.687 __ 0.167

N . S . N , S . N .S .

Control (18) 4 114.9 __+ 2.4 0.359 _+ 0.009 3.125 + 0.077 Myopathic (19) 109.1 ___ 1.8 0.326 + 0.014 2.988 __ 0.067

P < 0.05 N.S. N.S.

Control (20) 5 96.0 _+ 1.4 0.299 _+ 0.003 3.122 + 0.041 Myopathic (18) 107.4 _+ 1.4 0.313 __ 0.005 2.915 _+ 0.041

P < 0.001 P < 0.05 P < 0.001

Control (10) 6 123.5 _ 3.4 0.387 _ 0.010 3.133 _+ 0.089 Myopathic (15) 118.0 _+ 1.6 0.372 _+ 0.008 3.152 __ 0.040

N .S . N .S N .S .

Control (24) 7 114.4 __ 1.9 0.324 __ 0.006 2.832 __ 0.047 Myopathic (21) i i i . 5 + 1.0 0.363 +_ 0.005 3.313 _+ 0.052

N.S. P < 0.001 P < 0.001

Results are mean _ s.E. Numbers in parentheses indicate the number of animals in each group.

98 A. Malhotra et aL

14 V/cm for 20 to 22 h. The gels were stained and destained [7] and densitometric scans were recorded at 550 nm on a Beckman Acta M-V1 spectrophotometer. The relative quali- tative estimate of each isoenzyme was calcu- lated from the area under the peak height. Statistical differences between mean values were evaluated by an unpaired t-test.

MHC

R e s u l t s

Table 1 shows the body weight (BW), heart weight (HW) and heart to body weight ratios (HW/BW) of male myopathic and control animals. There were gradual increases in the body and heart weights from 1 month to 4 months of age in controls as well as myopathic animals. At 5 months of age, body and hear t weights were higher but H W / B W ratios were lower in myopathics as compared to the controls. At 7 months body weights were similar in both groups but heart weights and H W / B W ratios were higher in myopathics than in controls.

A representative SDS gradient slab gel electrophoresis pattern (SDS) of myofibrils and myosin in control (C) and myopathic (M) group (4 months old) is shown in Figure 1. Electrophoretic analysis on SDS slab or disc gels did not reveal any loss or breakdown in the myofibrillar proteins in any of C and M groups. Also the scans of the slabs (Fig. 2) for different hamster age groups did not demon- strate any differences between any of the myofibrillar proteins.

Table 2 summarizes the Mg a § z § ATPase activity of cardiac myofibrils isolated in the absence of proteolytic enzyme inhibitors from 1 month to 7-month-old control and myopathic hamster hearts. Cardiac myofibrillar ATPase ( M g 2 + - C a 2+) activity was not changed at 1 to 3 months of age in myopathic hamsters when compared to controls. However, Mg 2+-Ca 2 § ATPase activity was slightly increased in the hearts of myopathic hamsters as compared to the control animals in the age group 4 to 5 months of age but was lower in myopathics at 7 months. Myofibrillar ATPase activity in the presence of 2 mM E G T A (EGTA ATPase activity) was significantly higher in cardiac muscle from myopathic hamsters than controls and the ratio of

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FIGURE 1. Sodium dodecylsulfate polyacrylamide slab gradient gel electrophoresis (5% to 16.5%) of highly purified cardiac myofibrils (# 1, 2) and myosin ( # 3, 4) from control (C) and myopathic (M) animals (4 months old). MHC, LC 1 and LC 2 indicate myosin heavy chains, light chain 1 and 2 respectively; A, actin; Tn-T, troponin-T; TM, tropomyosin; Tn-I, Troponin-I. Troponin-C does not stain very well with Coomasie Blue R. Various proteins are identified according to purified standards.

Mg z+ Ca 2+ ATPase to E G T A ATPase was consistently lower at 2 months through 7 months of age.

Because of variability of results in control hearts in Table 2, the experiments were repeated with protease inhibitors being present during isolation and purification. Table 3 shows the results in the presence of various protease inhibitors (see caption for details). The overall absolute values for Mg 2 + - C a 2 + and Mg z +-ATPase were found to be higher when compared to the ATPase data in Table 2. The possible explanations could be (1) cardiac tissue in these experiments was used up within 1 to 2 weeks after freezing the fresh cardiac muscle (in buffered glycerol (pH 7.0) at --70~ or (2) various protease inhibitors helped preserve the higher

Contractile Proteins in Cardlomyopathy 99

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FIGURE 2. Scans of SDS-slab gels (5% to 16.5%) in representative cardiac myofibrillar preparations in 1 to 7-month-old hamsters. Top : control, Bottom : myopathic gel scans.

ATPase activities. However, the data are qualitatively similar to those observed in Table 2 except in 7-month-old hamster hearts. There were no differences in ATPase values in control and myopathic groups at 1 month of age. Ca 2 § stimulated Mg 2 § and Mg 2+ ATPase (basal) were found to be increased at 4 and 7 months. The ratio of Mg 2+ Ca 2+ ATPase to Mg 2 +-ATPase was found to be identical in control (C) and myopathic (M) group in 1-month-old hamsters, but were decreased in 4 and 7-month-old groups.

The Ca2+-dependent activities of cardiac myofibrillar ATPase activity with increasing free C a 2 + concentration (10 - s M to 10 -4 ~a) is shown in Figure 3. Panels (a), (b), and (c) show the sigmoid curves for control (C) and myopathic (M) hearts at l, 4 and 7 months. Panel (d) shows the net myofibrillar ATPase activation [ ( M g 2+ �9 C a 2 + AT Pa se -E GT A

ATPase)] for control and myopathic 4- month-old groups. The basal Mg 2+-ATPase (EGTA ATPase) and Ca = + stimulated Mg 2 +- ATPase activity of myofibrils at different free Ca /+ ion concentrations were significantly higher in myopathic than control hearts at 4 months and 7 months. There were no significant differences in net ATPase activation in control and myopathic groups at any calcium concentrat ion [Fig. 3 (d)]. Myofibrillar ATPase activities were also studied in mixed (C + M) myofibrils. The activities, including the E G T A ATPase (basal Mg 2+ ATPase), were an average of control and myopathic at all free Ca 2 + concentrations (not shown).

Table 4 shows the Ca2+-ATPase activity and K + - E D T A ATPase activity of cardiac myosin from control and myopathic animals in three different age groups (I month, 4 to 5 months and 7 months). The CaZ+-myosin ATPase activities were similar in the hearts of

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T A B L E 4. M y o s i n A T P a s e activi ty" in hea r t s o f ma l e con t ro l and m y o p a t h i c h a m s t e r s (B IO 53 : 58)

C a 2 + - A T P a s e K + - E D T A A T P a s e Age N u m b e r o f N u m b e r o f

(mon ths ) S tud ies b C on t ro l M y o p a t h i c Studies b Con t ro l M y o p a t h i c

1 10 0.90 + 0.04 0.88 + 0.04 9 1.26 +_ 0.09 1.16 + 0.05 N.S. N.S.

4 - 5 13 0.92 • 0,03 0.75 • 0.03 8 1.41 __ 0.11 1.38 _. 0. I0 P < 0.02 N.S.

7 18 1.00 • 0.02 0.76 ___ 0.03 12 1.17 ___ 0.05 1.14 __ 0.05 P < 0.001 N.s.

" Results are #real Pi/mg . m i n - 1 a t 30~ mean •

b Each preparation is a pool of 6-8 hearts.

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K + - E D T A myosin ATPase activities were similar in all the groups. SDS gel electrophoresis on gradient polyacrylamide (5% to 16.5%) slab gels in the tris-glycine buffer system (pH 8.8) did not show any differences

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Contracti le Proteins in Cardiomyopathy 103

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FIGURE 4. Pyrophosphate gel patterns (insets) and scans of gel patterns in representative samples from hearts of control (top row) and myopathic (bottom row) animals at (a) 1 month, (b) 4 to 5 months and (c) 7 months. These are representative patterns of different myosin preparations (6-8) used for myosin ATPase measurements.

in the myosin prepara t ions in the different age go groups.

T a b l e 5 shows M g 2 + - A T P a s e act ivi ty of 40 purif ied myosins from control and myopa th ic hamster hearts at ages 1 month , 4 to 5 months 0 and 7 months in the absence and presence of s0

I increasing concentrat ions of actin. Act in- ~ v2 ac t iva ted MgZ+-myosin ATPase activities ~, 40 were s i m i l a r i n m y o s i n f r o m c o n t r o l a n d m y o - ~ ~ ~-~ ~ ~-~ ~ pathics at 1 month. However ac t in-ac t iva ted ~ 0 M g / + - A T P a s e was depressed 20% at 2.5 pM 80 and 5 p ~ actin in myosin from myopa th i c f v3 - , ~ hearts a t 4 to 5 months. Act in ac t iva ted 4o M g 2 + - A T P a s e was depressed even to a grea ter degree (26%) in hearts of myopa th ic o ~ ~ animals at age 7 months at both the act in ~ 2 s 4 6 7

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concentrat ions. FIGURE 5. Percent distribution of myosin isoenzymes F igure 4 shows representat ive pyrophosp- from control and myopathic hearts from 1 month to 7

ha te gel pa t te rns and densi tometr ic scans of months. Significant differences (P < 0.001) are present card iac myosin isoenzymes at various stages of for V 1 and for V 3 between control and myopathic at 4, 6 deve lopmen t in control (C) and ca rd iomyopa - and 7 months. Each preparation is a pool of 6 to 8 hearts. thic (M) hamsters. M e a n values for isoenzyme Numbers in parentheses (6), (4), (4), (7), (8) and (6)

indicate the number of studies for 1, 2, 3, 4, 6 and 7- peaks are shown in Figure 5. At all ages month-old hamster in control (C)and myopathic (M) myosin consisted of three isoenzyme bands groups.


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V3 (%)

FIGURE 6. Relationship between myosin lsoenzymes V 1 and V a (%) content and actin (5 #m) activated Mg 2+-ATPase activity of cardiac myosin from 1,4 and 7- month-old hamsters (figures in parentheses). Mean of 6 to 7 different myosin preparations in each C and M group is shown in the figure. �9 control; O, myopathic.

(Vt, V 2 and V3) as observed in the rat heart [3, 7, 11]. In controls at all ages and at 1 to 3 months in cardiomyopathic animals, V 1 predominated and isoenzyme distribution was V 1 > V 2 > V a. However, in myopathics at 4 to 5 months to 7 months there was a gradual shift to predominance of V 3 over V 2 and V1. V 2 was consistent (18% to 24% of total isoenzyme content) in both control and myopathic throughout the whole age range.

Figure 6 (a, b) shows the relationship between the actin activated MgZ+-myosin ATPase activity and cardiac myosin isoenzyme (V 1 or V3% ) content in C and M group at 1, 4 and 7 months respectively. The activity of Mge+-act ivated ATPase closely paralleled the proportions of V1 or V 3 present.

D i s c u s s i o n

In this paper we have explored the alterations in the myofibrillar ATPase in the absence and presence of calcium, myosin ATPase activities and heavy chain myosin isoenzyme distribution in the hearts of cardiomyopathic hamsters (BIO 53 : 58) during the course of their disease. Mg 2 + - C a 2 + ATPase activity o fmyo- fibrils appeared not to be altered up to three months of age. However, increased E G T A ATPase or Mg 2 +-ATPase activity was noted in myopathics as early as two months of age. Mg 2 + - C a 2 + ATPase activity was increased in myofibrils from myopathic hearts at four to seven months. These findings contrast with those of Pang et al. [14] who found diminished Mg 2 + - C a 2 + ATPase activity of myofibrils in hearts of female (BIO 53 : 58) hamsters as early as 24 days, but not in males. In their study, males showed enhanced Mg 2+ Ca 2+ ATPase and E G T A ATPase activities at 44 days, similar to our study at 4 and 7 months. They observed a depression in Mg 2+ Ca 2+ ATPase and a moderate increase in E G T A ATPase activity at 50 days, similar to our 7-month findings. In their study females showed no significant difference in E G T A ATPase activity. The present studies were of male hamsters. The reasons for the sex a n d timing differences are uncertain, but it seems that more careful studies of the regulatory proteins, troponin-tropomyosin, might clarify this situation. Both the findings of Pang et al. [I4] and ours suggest abnormalities in the basal (calcium-independent) ATPase activity of myofibrils. Although we had previously reported a myofibrillar protease extracted from hearts of myopathic hamsters that is active in vitro against myosin, troponin and tropomyosin [9], in the present study on cardiac myofibrils there was no loss or breakdown in any myofibrillar components in any age group. Also the findings were observed when protease inhibitors were employed. Therefore it is unlikely that the abnormalities in basal myofibrillar ATPase was due to in vitro protease effects.

In order to explore the loss in calcium sensitivity in myopathic hamsters, cardiac myofibrillar ATPase activities were examined at different free calcium ion concentrations. There did not appear to be alterations in the half maximal activation of myofibrillar


Mg z +-Ca 2+ by free Ca 2 +, and the shape of curves relating free Ca 2+ to normalized ATPase (Fig. 3d) are the same in C and M preparations. The similar types of Ca 2 +-dose response sigmoid curves showed that there was no loss in the regulation of myopathic myofibrillar assembly (Fig. 3), Thus, the differences observed in myopathic myofibrils seemed to be independent of calcium ion concentration. The possibility was considered that the higher ATPase activity of myofibrils from myopathic hearts could be attributed to the presence of an activator. But when myofibrils from C and M were mixed, the ATPase was an average of C and M, suggesting that no activator was present. This loss in Ca 2+ sensitivity could be due to an alteration in the myosin or the troponin-tropomyosin (Tn-Tm) system or both. Daniel and Hartshorne [4] have presented an evidence that the sulfhydryl groups important for the Ca 2+- sensitivity of natural actomyosin are located on the heavy subunits of myosin. Thus the conformation changes in myosin may be partially responsible for the observed increase in Ca 2 + independent myofibrillar ATPase activity in the hearts of myopathic animals. In order to evaluate the contribution of Tn-Tm to overall alterations in contractile proteins, direct studies of T n - T m in the reassociated actomyosin system may yield an answer. Changes in these proteins have not been explored in cardiomyopathic hearts.

I t is also possible that alterations in myosin could contribute to the findings in myofibrils. Studies on the purified myosin indicated that cardiomyopathy may be characterized by a significant decrease in myosin Ca 2+-ATPase activity at 4 to 5 and 7 months of age but no change in enzymatic activity was noted at 1 month. Earlier reports from our laboratory and others [2, 18] also showed a depressed Ca/ +-myosin ATPase activity in the hyper- trophic and final stages of cardiomyopathy in another myopathic strain BIO 14 : 6.

Aetin activated MgZ+-ATPase of cardiac myosin was reported to be depressed in myopathic hamsters of the V - M X 7-1 strain at 90 days of age [15]. However, in that report a time study was not performed. In the present investigation a tendency toward depression in this physiologically important ATPase may have been present as early as 1 month, but

statistically significant decreases in ATPase activities were observed at 4 to 5 months and 7 months respectively. Results of the actin activation studies support the myosin Ca z+- ATPase activity data.

The relative distribution of isoenzymes of myosin has been suggested to be responsible for different ATPase activities [3, 12, 13, 16]. The present study shows that hamster cardiac myosin is made up of three isoenzymes similar to rat. The distribution of these isoenzymes changes during development and especially as the pathological process of myopathy prog- resses. The change of predominant V 1 (HC~ homodimer) to slow migrating V 3 isoenzyme (HCp homodimer) during myopathy parallels the changes in actin activated Mg 2+-myosin ATPase and myosin Ca2+-ATPase activities in a similar manner to that previously shown in pathologic states in rat heart [13-]. The distribution of myosin isoenzymes was identical in crude tissue extracts and pure myosins suggesting that the changes observed were not due to any alterations in the myosin during the extraction procedure [2].

The current finding suggests that the myopathic state is associated with altered balance of isoenzyme synthesis as has been reported in altered thyroid state [3, 12]. However, the mechanism of this change is not clear.

Our experiments suggest abnormalities in several different components of the contractile protein system. The myofibrillar data are sug- gestive of a change in the regulatory protein(s) that occurs quite early in the course of development. The isoenzyme shifts are compatible with an alteration in myosin isoenzyme synthesis or degradation. It is possible that changes in the contractile protein assembly are partially responsible for abnormalities in cardiac function in the hearts of myopathic hamsters. However, the relation of the contractile protein changes to myocardial contractile function and the development of congestive failure are unclear. At any given cytoplasmic calcium concentration, myofibrillar contractile function might be expected to be increased in myopathic hearts, and calcium levels in these hearts might be elevated. This might tend to counteract the depressed myosin ATPase activity. I t is prob- able that contractile protein changes are not the entire explanation, since there are indica-

Contract i le Prote ins in Card iomyopathy 107

tions of alterations in the cell membrane itself [5]. The interesting observation that myocardial damage may be partially prevented by treatment with verapamil at one time point without protection of the contractile proteins also suggests a sarcolemmal factor in the development ofmyocardiopathy [8, 15].

Acknowledgements The authors wish to acknowledge the excel- lent technical assistance of Mr John Katt Val- salan. We also thank Ms Janet Ellen Holwell and Mrs Lori Fields for their secretarial assistance.

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8 JASmN, G., BAjusz, E. Prevention of Myocardial Degeneration in Hamsters with Hereditary Cardiomyopathy. In: Fleckenstein, A. and Rona, F. (Eds) Recent Advances in Studies on Cardiac Structure and Metabolism, vol 6. 219 229 (1975).

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14 PANG, D. C., WEGLmKI, W. B. Alteration ofmyofibrillar ATPase activities in hearts ofcardiomyopathic hamsters (BIO 53 : 58).J Mol Cell Cardiol 12, 445-456 (1980).

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Multiple cardiac contractile protein abnormalities in myopathic syrian hamsters (BIO 53:58)