Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac α-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen the mutations and and the mutations and the of depletion/reconstitution the functional consequences of mutations were ATPase The of depletion of of Tm-Tn from myofibrils in a loss of the ATPase and a loss in the ATPase reconstitution of wild-type Tm-Tn in regain in the regulation and in the ATPase and Tm mutations the of ATPase and ATPase activity. the mutations the of ATPase and the of ATPase activity. the mutations and in the cardiomyopathic To study the functional consequences of various cardiomyopathic mutations in human cardiac α-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen the mutations and and the mutations and the of depletion/reconstitution the functional consequences of mutations were ATPase The of depletion of of Tm-Tn from myofibrils in a loss of the ATPase and a loss in the ATPase reconstitution of wild-type Tm-Tn in regain in the regulation and in the ATPase and Tm mutations the of ATPase and ATPase activity. the mutations the of ATPase and the of ATPase activity. the mutations and in the cardiomyopathic by in the troponin troponin troponin troponin cardiac troponin troponin troponin cardiac of the of in the of (Tm), in the of Tm and a the of of the of in of the cardiac in from and a of the by cardiac and a of the by of mutations in the cardiac mutations in been in the of and The was in and the of a Tm in the of The was by of the of a of the the of the of Tm mutations of the in the Tm mutations in the of the in the Tm the in the Tm in the of the Tm or a of the by and of the or the and of mutations been in the cardiac the and Tm mutations been and The was in a was with of a The was in a with a of was with and a and of a in of cardiomyopathic mutations in the of of from in the troponin the in The and mutations mutations and the and the of native Tm from myofibrils and or from the of with the of the and the of Tm from The of method endogenous Tm and the complex (Tm-Tn) from myofibrils has been the the depletion of Tm-Tn from the reconstitution of and the of ATPase the functional consequences of and Tm mutations mutant and wild-type in study were with the the Tm with the was the wild-type Tm the and reconstitution in study of the study depletion of of Tm-Tn from myofibrils in loss of the ATPase and a loss in the ATPase activity. The reconstitution of Tm-Tn the and the ATPase The of the ATPase were by and ATPase The Tm mutations and the of ATPase activity. the the the of ATPase activity. in of the the loss of in Tm method ATPase the Tm and the of cardiomyopathic and of and cardiac was by of human cardiac Tm was the were by a and was by were a method with and were a Tm was with a and the Tm was a Tm was with a steps were the The of the was by cardiac troponin and troponin were and by in cardiac of recombinant and were by in of in the ATPase of Tm-Tn from cardiac myofibrils were from by by preparations in in were the by in a were and the and was and in a myofibril myofibrils were of endogenous Tm and by in depletion and in depletion were and the depletion of the in the and in the with of myofibrils and by the in myofibrils in the a of the of myofibril depletion and reconstitution myofibrils were of endogenous Tm-Tn in depletion was by of the myofibrils in depletion in a myofibrils were in reconstitution depletion and with of myofibrils were with Tm-Tn in Tm-Tn was removed from the myofibrils by in and myofibrils were ATPase of Tm-Tn myofibrils were in reconstitution and and in reconstitution of the depletion been and been reconstitution were reconstitution by in the reconstitution Five of myofibrils were with Tm-Tn by with Tm-Tn in and The myofibril and of in were in a of the myofibrils were and was removed and in a in the in ATPase were by the with a the ATPase ATPase were with the of of myofibrils myofibril and various the of were in a and the was with of The ATPase was and of was The was with of depletion and reconstitution were in or and or The of ATPase were in various with from was the of of myofibril were the were with and was of the Tm-Tn the of of endogenous Tm-Tn from myofibrils and the myofibrils were in a depletion and the of the and were the ATPase of from in the or of the ATPase in the of the ATPase in the of in the of removed from the The ATPase of myofibrils been and myofibrils been The was and in the of of the myofibrils in method the depletion of Tm and in the depletion the the the in the of and the were and was chosen the of depletion of myofibrils ATPase of been of endogenous were in depletion the endogenous and the of depletion was with myofibrils depletion of endogenous were by the of ATPase and and the of the depletion The the of been The were the of in Tm-Tn the of reconstitution of Tm-Tn the myofibrils and the the were with various of recombinant been with of regain or regulation the of been with Tm-Tn in the of and the ATPase in the of of and ATPase the of reconstitution with the in the in the reconstitution Tm-Tn the ATPase in the of was and the in the of was the myofibrils were with or the ATPase in the of were and the ATPase in the of were and the of and was from the Tm-Tn was the of and myofibrils with the in the of was chosen ATPase ATPase of myofibrils been and with recombinant The of myofibrils with of been with of recombinant human cardiac Five of were with Tm-Tn in The of Tm-Tn reconstitution was by the regain of regulation of ATPase and the in ATPase The the of ATPase of The were the of in of and of and the of depletion and of myofibrils were by and with of Tm was the of Tm and in the of Tm in and the of Tm in myofibrils with To the of the depletion and reconstitution myofibril were and a the of the and were removed with the depletion of the of the the of The mutant and the of ATPase ATPase of Tm in in a the of depletion and reconstitution of and mutant in cardiac To and of myofibril was by cardiac myofibrils myofibrils been and with or mutant of the and from and and of the of myofibril the of Tm and removed with the depletion and of Tm-Tn and with or ATPase of and myofibrils been with recombinant or were the in the of Tm-Tn reconstitution the ATPase activity. myofibrils were of endogenous the a loss of by the of the the various and a of ATPase with the ATPase of the myofibrils were with or the the of and ATPase was chosen with mutant of and Tm mutations the of The ATPase a of the of native myofibrils the loss in in myofibrils been with mutant with the of and with The the of in the of of Tm ATPase the of the mutations the of ATPase myofibrils were myofibrils were with a of various mutant and wild-type The ATPase were with the myofibrils and the of the mutations the and the of ATPase was mutations in in the ATPase and a in ATPase The and of Tm mutations the Ca2+-dependent ATPase The ATPase of myofibrils been and with or of the mutant the steps in of and Tm ATPase the were the and were from the with The the of in The of of Tm ATPase been and with mutant ATPase from of the mutations a in the and in the ATPase the of ATPase activity. The and of Tm mutations Ca2+-dependent The ATPase of myofibrils been and with mutant with of of and mutations a in the were and were from the The the of in The of of and with Tm-Tn the of ATPase of the of myofibrils has a in the mutations in of the myofibrils the of of and the of the of ATPase the of mutations in a depletion/reconstitution The method depletion of endogenous Tm-Tn the method from cardiac myofibrils the loss of proteins. by and of the of the of of in the depletion method of reconstitution of Tm-Tn the myofibrils in reconstitution of activity. the of the of the endogenous Tm-Tn removed and replaced with Tm-Tn method the of mutations in the of the of the of by cardiac myofibrils the of the ATPase the has been the with has been by The of ATPase by from the and the of the functional of Tm-Tn depletion and reconstitution with and the the method and with the of the in Tm of Tm has the and of functional by and the ATPase in ATPase the the myofibrils were with were in ATPase and the of Tm in the of ATPase myofibrils been with ATPase in a in Tm and with or the the a with in in the depletion the of myofibrils been in depletion were with the of of ATPase was by a in the regulation of the of of ATPase in has with the or the ATPase the of and the in the of the functional of the Tm mutations the of a in Tm was in the in cardiac troponin The Tm was chosen a the method in and in by a in by a loss in the of the Tm was by and a in by in of a of and and with and the of the a the of the loss in of the reconstitution of the of the reconstitution the loss of the the the was the and the the loss of the in of Tm and the of a in the of ATPase activity. 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The reconstitution of Tm-Tn the and the ATPase The of the ATPase were by and ATPase The Tm mutations and the of ATPase activity. the the the of ATPase activity. in of the the loss of in Tm method ATPase the Tm and the of cardiomyopathic and of and cardiac was by of human cardiac Tm was the were by a and was by were a method with and were a Tm was with a and the Tm was a Tm was with a steps were the The of the was by cardiac troponin and troponin were and by in cardiac of recombinant and were by in of in the ATPase of Tm-Tn from cardiac myofibrils were from by by preparations in in were the by in a were and the and was and in a myofibril myofibrils were of endogenous Tm and by in depletion and in depletion were and the depletion of the in the and in the with of myofibrils and by the in myofibrils in the a of the of Tm-Tn myofibrils were in reconstitution and and in reconstitution of the depletion been and been reconstitution were reconstitution by in the reconstitution Five of myofibrils were with Tm-Tn by with Tm-Tn in and The myofibril and of in were in a of the myofibrils were and was removed and in a in the in ATPase were by the with a the ATPase ATPase were with the of of myofibrils myofibril and various the of were in a and the was with of The ATPase was and of was The was with of depletion and reconstitution were in or and or The of ATPase were in various with from was the of of myofibril were the were with and was and of and cardiac was by of human cardiac Tm was the were by a and was by were a method with and were a Tm was with a and the Tm was a Tm was with a steps were the The of the was by cardiac troponin and troponin were and by in cardiac of recombinant and were by in of in the cardiac was by of human cardiac Tm was the were by a and was by were a method with and were a Tm was with a and the Tm was a Tm was with a steps were the The of the was by cardiac troponin and troponin were and by in cardiac of recombinant and were by in of in the ATPase of Tm-Tn from cardiac myofibrils were from by by preparations in in were the by in a were and the and was and in a myofibril myofibrils were of endogenous Tm and by in depletion and in depletion were and the depletion of the in the and in the with of myofibrils and by the in myofibrils in the a of the of Tm-Tn myofibrils were in reconstitution and and in reconstitution of the depletion been and been reconstitution were reconstitution by in the reconstitution Five of myofibrils were with Tm-Tn by with Tm-Tn in and The myofibril and of in were in a of the myofibrils were and was removed and in a in the in ATPase were by the with a the of Tm-Tn from cardiac myofibrils were from by by preparations in in were the by in a were and the and was and in a myofibril myofibrils were of endogenous Tm and by in depletion and in depletion were and the depletion of the in the and in the with of myofibrils and by the in myofibrils in the a of the of Tm-Tn myofibrils were in reconstitution and and in reconstitution of the depletion been and been reconstitution were reconstitution by in the reconstitution Five of myofibrils were with Tm-Tn by with Tm-Tn in and The myofibril and of in were in a of the myofibrils were and was removed and in a in the in ATPase were by the with a the ATPase ATPase were with the of of myofibrils myofibril and various the of were in a and the was with of The ATPase was and of was The was with of depletion and reconstitution were in or and or The of ATPase were in various with from ATPase were with the of of myofibrils myofibril and various the of were in a and the was with of The ATPase was and of was The was with of depletion and reconstitution were in or and or The of ATPase were in various with from was the of of myofibril were the were with and was was the of of myofibril were the were with and was of the Tm-Tn the of of endogenous Tm-Tn from myofibrils and the myofibrils were in a depletion and the of the and were the ATPase of from in the or of the ATPase in the of the ATPase in the of in the of removed from the The ATPase of myofibrils been and myofibrils been The was and in the of of the myofibrils in method the depletion of Tm and in the depletion the the the in the of and the were and was chosen the of depletion of myofibrils Tm-Tn the of reconstitution of Tm-Tn the myofibrils and the the were with various of recombinant been with of regain or regulation the of been with Tm-Tn in the of and the ATPase in the of of and ATPase the of reconstitution with the in the in the reconstitution Tm-Tn the ATPase in the of was and the in the of was the myofibrils were with or the ATPase in the of were and the ATPase in the of were and the of and was from the Tm-Tn was the of and myofibrils with the in the of was chosen ATPase ATPase of myofibrils been and with recombinant The of myofibrils with of been with of recombinant human cardiac Five of were with Tm-Tn in The of Tm-Tn reconstitution was by the regain of regulation of ATPase and the in ATPase The the of ATPase of The were the of in of and of and the of depletion and of myofibrils were by and with of Tm was the of Tm and in the of Tm in and the of Tm in myofibrils with To the of the depletion and reconstitution myofibril were and a the of the and were removed with the depletion of the of the the of The mutant and the of ATPase ATPase of Tm in in a the of depletion and reconstitution of and mutant in cardiac To and of myofibril was by cardiac myofibrils myofibrils been and with or mutant of the and from and and of the of myofibril the of Tm and removed with the depletion and of Tm-Tn and with or ATPase of and myofibrils been with recombinant or were the in the of Tm-Tn reconstitution the ATPase activity. myofibrils were of endogenous the a loss of by the of the the various and a of ATPase with the ATPase of the myofibrils were with or the the of and ATPase was chosen with mutant of and Tm mutations the of The ATPase a of the of native myofibrils the loss in in myofibrils been with mutant with the of and with The the of in the of of Tm ATPase the of the mutations the of ATPase myofibrils were myofibrils were with a of various mutant and wild-type The ATPase were with the myofibrils and the of the mutations the and the of ATPase was mutations in in the ATPase and a in ATPase The and of Tm mutations the Ca2+-dependent ATPase The ATPase of myofibrils been and with or of the mutant the steps in of and Tm ATPase the were the and were from the with The the of in The of of Tm ATPase been and with mutant ATPase from of the mutations a in the and in the ATPase the of ATPase activity. The and of Tm mutations Ca2+-dependent The ATPase of myofibrils been and with mutant with of of and mutations a in the were and were from the The the of in The of of and with Tm-Tn the of ATPase of the of myofibrils has a in the mutations in of the myofibrils the of of and of the Tm-Tn the of of endogenous Tm-Tn from myofibrils and the myofibrils were in a depletion and the of the and were the ATPase of from in the or of the ATPase in the of the ATPase in the of in the of removed from the The ATPase of myofibrils been and myofibrils been The was and in the of of the myofibrils in method the depletion of Tm and in the depletion the the the in the of and the were and was chosen the of depletion of myofibrils Tm-Tn the of reconstitution of Tm-Tn the myofibrils and the the were with various of recombinant been with of regain or regulation the of been with Tm-Tn in the of and the ATPase in the of of and ATPase the of reconstitution with the in the in the reconstitution Tm-Tn the ATPase in the of was and the in the of was the myofibrils were with or the ATPase in the of were and the ATPase in the of were and the of and was from the Tm-Tn was the of and myofibrils with the in the of was chosen ATPase of and of and the of depletion and of myofibrils were by and with of Tm was the of Tm and in the of Tm in and the of Tm in myofibrils with To the of the depletion and reconstitution myofibril were and a the of the and were removed with the depletion of the of the the of The mutant and the of Tm-Tn and with or ATPase of and myofibrils been with recombinant or were the in the of Tm-Tn reconstitution the ATPase activity. myofibrils were of endogenous the a loss of by the of the the various and a of ATPase with the ATPase of the myofibrils were with or the the of and ATPase was chosen with mutant of Tm ATPase the of the mutations the of ATPase myofibrils were myofibrils were with a of various mutant and wild-type The ATPase were with the myofibrils and the of the mutations the and the of ATPase was mutations in in the ATPase and a in ATPase The and of Tm ATPase been and with mutant ATPase from of the mutations a in the and in the ATPase the of ATPase activity. The and of and with Tm-Tn the of ATPase of the of myofibrils has a in the mutations in of the myofibrils the of of and the of the of ATPase the of mutations in a depletion/reconstitution The method depletion of endogenous Tm-Tn the method from cardiac myofibrils the loss of proteins. by and of the of the of of in the depletion method of reconstitution of Tm-Tn the myofibrils in reconstitution of activity. the of the of the endogenous Tm-Tn removed and replaced with Tm-Tn method the of mutations in the of the of the of by cardiac myofibrils the of the ATPase the has been the with has been by The of ATPase by from the and the of the functional of Tm-Tn depletion and reconstitution with and the the method and with the of the in Tm of Tm has the and of functional by and the ATPase in ATPase the the myofibrils were with were in ATPase and the of Tm in the of ATPase myofibrils been with ATPase in a in Tm and with or the the a with in in the depletion the of myofibrils been in depletion were with the of of ATPase was by a in the regulation of the of of ATPase in has with the or the ATPase the of and the in the of the functional of the Tm mutations the of a in Tm was in the in cardiac troponin The Tm was chosen a the method in and in by a in by a loss in the of the Tm was by and a in by in of a of and and with and the of the a the of the loss in of the reconstitution of the of the reconstitution the loss of the the the was the and the the loss of the in of Tm and the of a in the of ATPase activity. The ATPase of the the in of ATPase the of ATPase of mutations the of The of of Tm in the of the mutations of the or of the ATPase in the of the complex in the of the the with Tm the of a in the and the cardiac the of ATPase the of with the loss in in the was a the in the functional of the Tm mutations mutations in the of mutations in in the study of the mutations in the ATPase activity. a in in with a in the the of ATPase ATPase in the the in the ATPase the and Tm mutations with the the from of a of the of mutations in the the the consequences in with cardiac the and functional consequences of mutations in of the To of and the and mutations in the in The the of the of ATPase the of mutations in a depletion/reconstitution The method depletion of endogenous Tm-Tn the method from cardiac myofibrils the loss of proteins. by and of the of the of of in the depletion method of reconstitution of Tm-Tn the myofibrils in reconstitution of activity. the of the of the endogenous Tm-Tn removed and replaced with Tm-Tn method the of mutations in the of the of the of by cardiac myofibrils the of the ATPase the has been the with has been by The of ATPase by from the and the of the functional of Tm-Tn depletion and reconstitution with and the the method and with the of the in Tm of Tm has the and of functional by and the ATPase in ATPase the the myofibrils were with were in ATPase and the of Tm in the of ATPase myofibrils been with ATPase in a in Tm and with or the the a with in in the depletion the of myofibrils been in depletion were with the of of ATPase was by a in the regulation of the of of ATPase in has with the or the ATPase the of and the in The the of the functional of the Tm mutations the of a in Tm was in the in cardiac troponin The Tm was chosen a the method in and in by a in by a loss in the of the Tm was by and a in by in of a of and and with and the of the a the of the loss in of the reconstitution of the of the reconstitution the loss of the the the was the and the the loss of the in of Tm and the of a in the of ATPase activity. The ATPase of the the in of ATPase the of ATPase of mutations the of The of of Tm in the of the mutations of the or of the ATPase in the of the complex in the of the the with Tm the of a in the and the cardiac the of ATPase the of with the loss in in the was a the in the functional of the Tm mutations mutations in the of mutations in in the study of the mutations in the ATPase activity. a in in with a in the the of ATPase ATPase in the the in the ATPase activity. the and Tm mutations with the the from of a of the of mutations in the the the consequences in with cardiac the and functional consequences of mutations in of the To of and the and mutations in the in