cardiomyopathies are difficult and complicated diseases of heart muscle associated with heart failure and/or sudden cardiac death , which are classified by the world health organization ( who ) in 1995 into four main forms ; hypertrophic cardiomyopathy ( hcm ) , dilated cardiomyopathy ( dcm ) , restrictive cardiomyopathy ( rcm ) , and arrhythmogenic right ventricular cardiomyopathy ( arvc ) . hcm is characterized by ventricular muscle hypertrophy , which especially involves the increased thickness of interventricular septum , leading to a marked decrease in left ventricular ( lv ) chamber volume . hcm has impaired lv diastolic function probably because of hypertrophy itself , interstitial fibrosis and/or myocyte disarray , while it usually has normal , or slightly attenuated lv systolic function . , the prevalence of hcm is about 1/500 , in which more than 70% are familial cases . dcm is characterized by abnormal enlargement of lv or both ventricular chambers with impaired systolic function , affecting about 36.5 in 100,000 people , of whom about 25%30% are familial cases . its prognosis is poor due to high frequency of arrhythmias and sudden death , and there are no effective therapeutic drugs at end stage and heart transplantation is the most effective treatment for survival . rcm , which is sometimes familial , is a rare form of cardiomyopathy , characterized by impaired ventricular filling with normal or decreased diastolic volume of either , or both ventricles and normal , or near normal , systolic function and wall thickness . , arvc is characterized by progressive fibrofatty replacement of right ventricular myocardium , with palpitations , syncope , and sudden death . its prevalence is between 1/1000 and 1/5000 , with 10% of deaths occurring before age 19 and 50% before age 35 . reported the first hcm - causing mutation of -myosin heavy chain ( -myhc ) gene in 1990 , a large number of mutations in sarcomeric protein genes that encode -myhc , cardiac troponin t ( ctnt ) , cardiac troponin i ( ctni ) , cardiac troponin c ( ctnc ) , -tropomyosin ( -tm ) , cardiac myosin binding protein c ( mybp - c ) , ventricular myosin light chains 1 and 2 ( lc1 , lc2 ) , actin and titin / connection , etc.,, as well as cytoskeletal and nuclear membrane protein genes that encode dystrophin , desmin , tafazzin , -sarcoglycan , lamin a / c , etc., have been identified as a cause of hcm , dcm and rcm . in contrast , no sarcomeric protein genes have been shown to be responsible for arvc . molecular genetic testing has so far indicated that arvc is associated with more than eight genes which encode the transforming growth factor -3 gene ( tgfb3 ) , ryanodine receptor 2 , desmoplakin , transmembrane protein 43 , junction plakoglobin ( also known as gamma catenin ) , etc . , many studies have been made on the structural and/or functional consequences of mutations , but the molecular mechanisms by which mutations found in the above genes lead to the pathogenesis of various forms of cardiomyopathy are not entirely clear . sarcomere , a structural unit of the contractile apparatus myofibril in cardiac muscle , has thick and thin filaments , which are composed of myosin and actin / tropomyosin / troponin complex , respectively . the molecular mechanism of ca regulation of muscle contraction has extensively been investigated since dr . , it is now common knowledge that troponin consists of three subunits with distinct function and structure , in which tnt anchors tn complex to tm , tni inhibits the actin - myosin contractile interaction , and tnc removes the inhibitory action of tni upon binding ca . until now , there have been over 100 mutations found in three ctn subunits causing hcm , dcm or rcm . this article focuses on the molecular mechanisms underlying the pathogenesis of cardiomyopathies caused by these mutations . cardiac muscle contraction is generated by the interaction between myosin head and thin filament , upon activated actomyosin mg - atpase . actin molecules polymerize into a double helical filament , which forms the backbone of the thin filament . tm is an -helical coiled - coil fibrous protein interacting with adjacent tm molecules in a head to tail manner and is located along polymerized actin together with tn complex at a actin : tm : tn ratio of 7 : 1 : 1 . , tn consists of three subunits : tnt , which anchors tn complex to tm , also interacts directly with actin ; tni , which is involved in the inhibition of actomyosin mg - atpase and inhibits actin - myosin interactions at diastolic levels of intracellular ca ; tnc , which is a ca - binding subunit that removes tni inhibition upon ca binding . tn plays a key role in the transition from diastole to systole of cardiac muscle . when the cytoplasmic ca concentration of cardiac myocytes is low around 2  10 mol / l , the contractile interaction of actin with myosin head is inhibited by tn - tm . when cytoplasmic ca concentration is raised by electrochemical coupling involving a membrane controlled release of ca into sarcoplasm and ca mobilization known as calcium - induced calcium release , ca binds to tnc and triggers a series of conformational changes of protein - protein interactions in the thin filament , which relieves the inhibition by tn - tm and thus promotes the contractile interaction between actin and myosin. tnt has two functionally and structurally distinct domains named t1 and t2 . , t1 ( n - terminal region of tnt ) anchors tn complex to the thin filament through its strongly binding to tm . on the other hand , t2 ( c - terminal region of tnt ) interacts with other tn subunits ( tni and tnc ) and tm / actin. the molecular weight of tnt is 3136 ku , with 250300 amino acid residues . ctnt is a peptide of 288 amino acid residues with approximate 35 ku of molecular weight . in human heart , there are four ctnt isoforms ( ctnt1-ctnt4 ) produced by alternative splicing of ctnt transcripts at exon 4 and exon 5 . , it is suggested that the alternative splicing of ctnt may contribute to altered myofilament response to ca in cardiac muscle contraction . ctnt is a substrate for protein kinase c ( pkc ) containing up to four potential phosphorylation sites . sumandea , et al . reported that phosphorylation at thr-206 significantly reduces maximum force generation , ca - sensitivity and cross - bridge cycling rate , suggesting that pkc- mediated phosphorylation of ctnt may play a role in the regulation of cardiac contraction . studies on inherited cardiomyopathies caused by ctnt mutations have revealed an important function of this protein in the ca regulation of muscle contraction . since thierfelder , et al . found two missense mutations ( ile79asn and arg92gln ) and a splice donor site ( intron 16g1a ) mutation in ctnt in inherited hcm patients in 1994 and until only recently , a new mutation p80s was reported in a japanese family , 36 hcm - linked mutations , 13 dcm - linked mutations and 4 rcm - linked mutations dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. mutations in ctnt account for approximately 15%30% of all hcm case . however , they usually lead to poor prognosis and high incidence of sudden cardiac death ; e.g. , i79n , r92w , r92q and e160 , etc . , induce a malignant clinical phenotype with the average life of patients being no more than middle age . , , , the functional consequences of these mutations have been studied by many groups . ,  we studied the functional effects of these hcm mutations using rabbit left ventricular skinned fibers and myofibrils with a ctnt - exchange technique . , we found most of these hcm - linked ctnt mutations , such as i79n , r92q , r92l , r92w , r94l , a104v , r130c , e160 , e163r , e163k and e244d , etc . , increased the ca sensitivity of force generation in skinned fibers and shifted the force - pca relationship leftwards , but did not significantly affect the maximum force generating capability or atpase activity and ca - cooperativity . on the other hand , r278c , r278p , tnt28(+7 ) and tnt14 decreased ca - cooperativity in force generation in skinned fibers in addition to a ca - sensitizing effect. almost all hcm - causing mutations occur in the tm - anchoring region ( residues 79 - 170 and 272 - 288 ) in ctnt , strongly suggesting that these mutations impair the interaction of ctnt and tm leading to an increase in the ca - sensitivity by decreasing the inhibitory action of ctni on the thin filament . recently , manning , et al . suggested that the ctnt hcm - related mutations in , or flanking , the n - tail t1 domain ( residues 79 - 170 ) directly interacting with overlapping tm , may alter the flexibility of t1 , which is inversely proportional to ca - cooperativity . in 2000 , a deletion mutation k210 of ctnt was reported as the first dcm - causing mutation of sarcomeric proteins . since then , this mutation has extensively been studied both in vitro and in vivo . with the ctnt - exchange technique , we have shown that this mutation confers a lower ca - sensitivity on the force generation of skinned cardiac muscle fibers and atpase activity of cardiac myofibrils compared with wild - type , while having no effects on the maximum force , or atpase activity , and ca - cooperativity . other groups also reported similar results. almost all dcm - causing mutations reported in ctnt to date , e.g. , r131w , r141w , r205 , k210 , r205l , and d270n , etc . , have demonstrated a ca - desensitizing effect on skinned fiber force generation and myofibrillar or actomyosin atpase activity., these results strongly suggest that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by ctnt mutation , in contrast to hcm where ca sensitization is a primary mechanism for the pathogenesis . however , it is still not completely clarified how one amino acid mutation can increase or decrease the ca - sensitivity of force generation of cardiac muscle in the respective form of inherited cardiomyopathy . we demonstrated that the dcm - causing mutation of ctnt r141w increased the affinity of ctnt for tm , by using a quartz - crystal microbalance . this result strongly suggests that r141w mutation in the strong tm - binding region in ctnt strengthens the integrity of ctni in the thin filament by stabilizing the interaction between ctnt and tm , which might allow ctni to inhibit the thin filament more effectively , leading to ca desensitization . rcm - causing mutations , unlike hcm- and dcm - causing mutations , are rare and recently found in ctnt ( i79n , e96 , and e136k ) . , pinto , et al . , reported that e96 showed a large increase in the ca - sensitivity and impaired abilities to inhibit atpase and to relax skinned fibers , which could contribute to the severe diastolic dysfunction in rcm . more recently , pinto , et al . , found a novel double deletion in ctnt of two highly conserved amino acids ( n100 and e101 ) in a rcm pediatric patient , which also conferred a large increase in the ca - sensitivity . in addition , this double deletion mutation decreased the ca - cooperativity of force development , suggesting alterations in intra - filament protein - protein interactions . ctni binds to a specific region of each tm molecule with a 38 nm periodicity along the tm / actin filament . , ctni is the inhibitory subunit of ctn complex , which is responsible for inhibition of actomyosin atpase activity . in the absence of ca , ctni inhibits contraction through interacting with actin ; the inhibitory effect of ctni is relieved during muscle contraction through interacting with ctnc upon ca binding to ctnc . hence , ctni is a key regulatory protein in cardiac muscle contraction and relaxation cycle . ctni consists of several functional domains , a cardiac specific n - terminal extension region ( residues 1 - 32 ) , an it - arm region , the inhibitory region ( residues 128 - 147 ) , the switch region ( residues 147 - 163 ) , and the c - terminal mobile domain . , phosphorylation of ctni by several different kinases plays important roles in the regulation of cardiac muscle contraction under physiological or pathological conditions . protein kinase a ( pka)-mediated phosphorylation of ctni at ser-23/ser-24 reduces myofilament ca - sensitivity , increases the rate of ca dissociation from ctnc , increases crossbridge cycling rate , and enhances unloaded shortening velocity. pkc may phosphorylate ctni at ser-23/ser-24 , ser-43/ser-45 and thr-144 ; phosphorylation at ser-43/ser-45 decreases maximum ca - activated force generation in skinned fibers and maximal sliding velocity in motility assays . , we have recently found that ctni is phosphorylated by pkc at ser-23/ser-24 and thr-144 , which leads to the depressed cooperative activation of the thin filament . phosphorylation of ctni at different sites by different protein kinases may have distinct effects in vivo , although they are poorly understood. the effects of ctni phosphorylation could be affected by some mutations that cause inherited cardiomyopathies . thirty , four , and nine mutations of ctni have so far been found in hcm , dcm , and rcm , respectively , , , most of which are located in the c - terminal region ( figure 2 ) . in 1997 , kimura , et al . reported six mutations in ctni ( r145 g , r145q , r162w , k183 , g203s , and k206q ) associated with hcm in an autosomal dominant manner . studies on the functional consequences of these mutations at the level of skinned cardiac muscle force generation and myofibrillar atpase activity revealed that they have a ca - sensitizing effect on cardiac muscle contraction as in the hcm - linked mutations in ctnt . studies of the ca affinity of reconstituted cardiac thin filament with a fluorescence ( iaans ) labeling technique revealed that r145 g , which is located in the inhibitory region of ctni , increases the basal level of atpase activity and increases the ca binding affinity of the regulatory site of ctnc in the thin filament . mutations of r145 g , r145q , r162w , and g203s show increased minimum force in skinned cardiac muscle fibers with no significant changes in the maximum force . a deletion mutation k183 increases the ca - sensitivity with no effects on either maximum or minimum force generation of skinned cardiac muscle . dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. until 2009 , only one mutation a2v was reported to cause dcm in an autosomal recessive mannar , which impairs the interaction of ctni with ctnt . in 2009 , carballo , et al . reported that the mutations of k36q and n185k of ctni cause dcm in an autosomal dominant manner . k36q has been shown to mediate the movement of the n - terminal region in ctni upon phosphorylation of s22/23 by camp- dependent protein kinase . functional studies of k36q and n185k have revealed that these dcm mutations of ctni decrease the maximum activity and ca - sensitivity of actin - myosin s1 atpase and significantly reduce the ca binding affinity of the regulatory site of ctnc in the thin filament . more recently , murakami , et al . reported a new missense mutation p16 t in dcm patients . in 2003 , published the first report of ctni mutations in rcm patients ( l144q , r145w , a171 t , k178e , d190 g , and r192h ) . we examined the functional and structural consequences of these six mutations by using in vitro assays of skinned cardiac muscle fibers and nmr and revealed that they cause dramatic ca - sensitization of force generation in cardiac muscle with a subtle structural perturbation within ctni molecule . ca - sensitizing effects of these rcm mutations are much greater than those of hcm mutations in ctni . however , d190 g mutation with the smallest ca - sensitizing effect was reported to cause both rcm and hcm in a single large family , suggesting that rcm and hcm are caused by the same mechanism through ca - sensitization with greater sensitization leading to rcm . in addition , all six mutations elevate the resting force of skinned cardiac muscle fibers at low ca concentrations , and four mutations , l144q , r145w , a171 t , and r192h decrease the maximum level of force generation at high ca concentrations . troponin c is a member of the ef - hand family of ca binding proteins and consists of two globular lobes at n- and c - terminus , which are connected with an -helix . each globular lobe contains a pair of ca - binding sites , hence there are four ca - binding sites named i through iv from the n - terminal of vertebrate skeletal tnc ( stnc ) . in contrast to stnc , site i of ctnc dose not bind ca under physiological conditions . analysis of equilibrium ca binding of ctnc demonstrates that the dissociation constant ( kd ) of site ii is approximate 1.2  10 m , and plays an important ca - regulatory role in cardiac muscle contraction . on the other hand , the dissociation constant ( kd ) of sites iii and iv are approximate 7.4 x 10 m. sites iii and iv also bind mg with kd at 0.9  10 m , play a structural role to keep the c lobe tightly bound to ctni and to stabilize the interaction between ctnc and ctnt . compared with ctnt and ctni , much fewer mutations have so far been found in ctnc , with only six mutations both in hcm and dcm . c84y and d145e increase the ca - sensitivity of force generation in skinned cardiac muscle fibers . a8v and d145e also increase the force recovery . in contrast to these mutations , e134d shows no changes in ca - sensitivity and force recovery . fluorescence studies using iaans - labeled ctnc suggested that l29q increases the ca binding affinity of site ii of ctnc . functional studies indicated that this mutation increased the ca - sensitivity with no effect on the maximal force generation , and increased the ca affinity of ctnc isolated or incorporated into troponin complex and thin filament . in contrast to the dcm - linked mutations found in ctnt and ctni , g159d mutation in ctnc does not significantly decrease the ca - sensitivity of force generation of skinned fibers , but it induces a decrease in atpase activity of reconstituted myofilaments as well as filament sliding velocity . interestingly , biesiadecki , et al . provide evidence that g159d may alter myocardial functional responses to -adrenergic stimulation by blunting the ca - desensitizing effect of ctni phosphorylation at ser-23/ser-24 . afterward , two novel missense mutations localized in the regulatory ca - binding site ii of ctnc , e59d and d75y , were identified in a dcm patient . these mutations showed a decrease in the myofilament ca - sensitivity and ca binding affinity in the force - pca relationship measurements and fluorescence spectroscopy , respectively . d75y disrupts molecular motions critical for ca binding and cardiomyocyte contractility , whereas functional defect caused by e59d is benign . most recently , pinto , et al . reported y5h , m103i , and i148v mutations in ctnc in dcm patients . functional studies showed that y5h and m103i decrease the ca2-sensitivity of force generation and that the effects of pka phosphorylation of ctni on the myofilament ca - sensitivity is abolished by m103i but diminished by y5h and i148v . t1 ( n - terminal region of tnt ) anchors tn complex to the thin filament through its strongly binding to tm . on the other hand , t2 ( c - terminal region of tnt ) interacts with other tn subunits ( tni and tnc ) and tm / actin. the molecular weight of tnt is 3136 ku , with 250300 amino acid residues . ctnt is a peptide of 288 amino acid residues with approximate 35 ku of molecular weight . in human heart , there are four ctnt isoforms ( ctnt1-ctnt4 ) produced by alternative splicing of ctnt transcripts at exon 4 and exon 5 . , it is suggested that the alternative splicing of ctnt may contribute to altered myofilament response to ca in cardiac muscle contraction . ctnt is a substrate for protein kinase c ( pkc ) containing up to four potential phosphorylation sites . sumandea , et al . reported that phosphorylation at thr-206 significantly reduces maximum force generation , ca - sensitivity and cross - bridge cycling rate , suggesting that pkc- mediated phosphorylation of ctnt may play a role in the regulation of cardiac contraction . studies on inherited cardiomyopathies caused by ctnt mutations have revealed an important function of this protein in the ca regulation of muscle contraction . since thierfelder , et al . found two missense mutations ( ile79asn and arg92gln ) and a splice donor site ( intron 16g1a ) mutation in ctnt in inherited hcm patients in 1994 and until only recently , a new mutation p80s was reported in a japanese family , 36 hcm - linked mutations , 13 dcm - linked mutations and 4 rcm - linked mutations dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. mutations in ctnt account for approximately 15%30% of all hcm case . however , they usually lead to poor prognosis and high incidence of sudden cardiac death ; e.g. , i79n , r92w , r92q and e160 , etc . , induce a malignant clinical phenotype with the average life of patients being no more than middle age . , , , the functional consequences of these mutations have been studied by many groups . ,  we studied the functional effects of these hcm mutations using rabbit left ventricular skinned fibers and myofibrils with a ctnt - exchange technique . , we found most of these hcm - linked ctnt mutations , such as i79n , r92q , r92l , r92w , r94l , a104v , r130c , e160 , e163r , e163k and e244d , etc . , increased the ca sensitivity of force generation in skinned fibers and shifted the force - pca relationship leftwards , but did not significantly affect the maximum force generating capability or atpase activity and ca - cooperativity . on the other hand , r278c , r278p , tnt28(+7 ) and tnt14 decreased ca - cooperativity in force generation in skinned fibers in addition to a ca - sensitizing effect. almost all hcm - causing mutations occur in the tm - anchoring region ( residues 79 - 170 and 272 - 288 ) in ctnt , strongly suggesting that these mutations impair the interaction of ctnt and tm leading to an increase in the ca - sensitivity by decreasing the inhibitory action of ctni on the thin filament . suggested that the ctnt hcm - related mutations in , or flanking , the n - tail t1 domain ( residues 79 - 170 ) directly interacting with overlapping tm , may alter the flexibility of t1 , which is inversely proportional to ca - cooperativity . in 2000 , a deletion mutation k210 of ctnt was reported as the first dcm - causing mutation of sarcomeric proteins . since then , this mutation has extensively been studied both in vitro and in vivo . with the ctnt - exchange technique , we have shown that this mutation confers a lower ca - sensitivity on the force generation of skinned cardiac muscle fibers and atpase activity of cardiac myofibrils compared with wild - type , while having no effects on the maximum force , or atpase activity , and ca - cooperativity . other groups also reported similar results. almost all dcm - causing mutations reported in ctnt to date , e.g. , r131w , r141w , r205 , k210 , r205l , and d270n , etc . , have demonstrated a ca - desensitizing effect on skinned fiber force generation and myofibrillar or actomyosin atpase activity., these results strongly suggest that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by ctnt mutation , in contrast to hcm where ca sensitization is a primary mechanism for the pathogenesis . however , it is still not completely clarified how one amino acid mutation can increase or decrease the ca - sensitivity of force generation of cardiac muscle in the respective form of inherited cardiomyopathy . we demonstrated that the dcm - causing mutation of ctnt r141w increased the affinity of ctnt for tm , by using a quartz - crystal microbalance . this result strongly suggests that r141w mutation in the strong tm - binding region in ctnt strengthens the integrity of ctni in the thin filament by stabilizing the interaction between ctnt and tm , which might allow ctni to inhibit the thin filament more effectively , leading to ca desensitization . rcm - causing mutations , unlike hcm- and dcm - causing mutations , are rare and recently found in ctnt ( i79n , e96 , and e136k ) . , reported that e96 showed a large increase in the ca - sensitivity and impaired abilities to inhibit atpase and to relax skinned fibers , which could contribute to the severe diastolic dysfunction in rcm . more recently , pinto , et al . , found a novel double deletion in ctnt of two highly conserved amino acids ( n100 and e101 ) in a rcm pediatric patient , which also conferred a large increase in the ca - sensitivity . in addition , this double deletion mutation decreased the ca - cooperativity of force development , suggesting alterations in intra - filament protein - protein interactions . ctni binds to a specific region of each tm molecule with a 38 nm periodicity along the tm / actin filament . , ctni is the inhibitory subunit of ctn complex , which is responsible for inhibition of actomyosin atpase activity . in the absence of ca , ctni inhibits contraction through interacting with actin ; the inhibitory effect of ctni is relieved during muscle contraction through interacting with ctnc upon ca binding to ctnc . hence , ctni is a key regulatory protein in cardiac muscle contraction and relaxation cycle . ctni consists of several functional domains , a cardiac specific n - terminal extension region ( residues 1 - 32 ) , an it - arm region , the inhibitory region ( residues 128 - 147 ) , the switch region ( residues 147 - 163 ) , and the c - terminal mobile domain . , phosphorylation of ctni by several different kinases plays important roles in the regulation of cardiac muscle contraction under physiological or pathological conditions . protein kinase a ( pka)-mediated phosphorylation of ctni at ser-23/ser-24 reduces myofilament ca - sensitivity , increases the rate of ca dissociation from ctnc , increases crossbridge cycling rate , and enhances unloaded shortening velocity. pkc may phosphorylate ctni at ser-23/ser-24 , ser-43/ser-45 and thr-144 ; phosphorylation at ser-43/ser-45 decreases maximum ca - activated force generation in skinned fibers and maximal sliding velocity in motility assays . , we have recently found that ctni is phosphorylated by pkc at ser-23/ser-24 and thr-144 , which leads to the depressed cooperative activation of the thin filament . phosphorylation of ctni at different sites by different protein kinases may have distinct effects in vivo , although they are poorly understood. the effects of ctni phosphorylation could be affected by some mutations that cause inherited cardiomyopathies . thirty , four , and nine mutations of ctni have so far been found in hcm , dcm , and rcm , respectively , , , most of which are located in the c - terminal region ( figure 2 ) . in 1997 , kimura , et al . reported six mutations in ctni ( r145 g , r145q , r162w , k183 , g203s , and k206q ) associated with hcm in an autosomal dominant manner . studies on the functional consequences of these mutations at the level of skinned cardiac muscle force generation and myofibrillar atpase activity revealed that they have a ca - sensitizing effect on cardiac muscle contraction as in the hcm - linked mutations in ctnt . studies of the ca affinity of reconstituted cardiac thin filament with a fluorescence ( iaans ) labeling technique revealed that r145 g , which is located in the inhibitory region of ctni , increases the basal level of atpase activity and increases the ca binding affinity of the regulatory site of ctnc in the thin filament . mutations of r145 g , r145q , r162w , and g203s show increased minimum force in skinned cardiac muscle fibers with no significant changes in the maximum force . a deletion mutation k183 increases the ca - sensitivity with no effects on either maximum or minimum force generation of skinned cardiac muscle . dcm : dilated cardiomyopathy ; hcm : hypertrophic cardiomyopathy ; rcm : restrictive cardiomyopathy ; tm : tropomyosin ; tnc : troponin c ; tni : troponin i. until 2009 , only one mutation a2v was reported to cause dcm in an autosomal recessive mannar , which impairs the interaction of ctni with ctnt . in 2009 , carballo , et al . reported that the mutations of k36q and n185k of ctni cause dcm in an autosomal dominant manner . k36q has been shown to mediate the movement of the n - terminal region in ctni upon phosphorylation of s22/23 by camp- dependent protein kinase . functional studies of k36q and n185k have revealed that these dcm mutations of ctni decrease the maximum activity and ca - sensitivity of actin - myosin s1 atpase and significantly reduce the ca binding affinity of the regulatory site of ctnc in the thin filament . more recently , murakami , et al . reported a new missense mutation p16 t in dcm patients . in 2003 , published the first report of ctni mutations in rcm patients ( l144q , r145w , a171 t , k178e , d190 g , and r192h ) . we examined the functional and structural consequences of these six mutations by using in vitro assays of skinned cardiac muscle fibers and nmr and revealed that they cause dramatic ca - sensitization of force generation in cardiac muscle with a subtle structural perturbation within ctni molecule . ca - sensitizing effects of these rcm mutations are much greater than those of hcm mutations in ctni . however , d190 g mutation with the smallest ca - sensitizing effect was reported to cause both rcm and hcm in a single large family , suggesting that rcm and hcm are caused by the same mechanism through ca - sensitization with greater sensitization leading to rcm . in addition , all six mutations elevate the resting force of skinned cardiac muscle fibers at low ca concentrations , and four mutations , l144q , r145w , a171 t , and r192h decrease the maximum level of force generation at high ca concentrations . troponin c is a member of the ef - hand family of ca binding proteins and consists of two globular lobes at n- and c - terminus , which are connected with an -helix . each globular lobe contains a pair of ca - binding sites , hence there are four ca - binding sites named i through iv from the n - terminal of vertebrate skeletal tnc ( stnc ) . in contrast to stnc , site i of ctnc dose not bind ca under physiological conditions . analysis of equilibrium ca binding of ctnc demonstrates that the dissociation constant ( kd ) of site ii is approximate 1.2  10 m , and plays an important ca - regulatory role in cardiac muscle contraction . on the other hand , the dissociation constant ( kd ) of sites iii and iv are approximate 7.4 x 10 m. sites iii and iv also bind mg with kd at 0.9  10 m , play a structural role to keep the c lobe tightly bound to ctni and to stabilize the interaction between ctnc and ctnt . compared with ctnt and ctni , much fewer mutations have so far been found in ctnc , with only six mutations both in hcm and dcm . c84y and d145e increase the ca - sensitivity of force generation in skinned cardiac muscle fibers . a8v and d145e also increase the force recovery . in contrast to these mutations , e134d shows no changes in ca - sensitivity and force recovery . fluorescence studies using iaans - labeled ctnc suggested that l29q increases the ca binding affinity of site ii of ctnc . functional studies indicated that this mutation increased the ca - sensitivity with no effect on the maximal force generation , and increased the ca affinity of ctnc isolated or incorporated into troponin complex and thin filament . in contrast to the dcm - linked mutations found in ctnt and ctni , g159d mutation in ctnc does not significantly decrease the ca - sensitivity of force generation of skinned fibers , but it induces a decrease in atpase activity of reconstituted myofilaments as well as filament sliding velocity . provide evidence that g159d may alter myocardial functional responses to -adrenergic stimulation by blunting the ca - desensitizing effect of ctni phosphorylation at ser-23/ser-24 . afterward , two novel missense mutations localized in the regulatory ca - binding site ii of ctnc , e59d and d75y , were identified in a dcm patient . these mutations showed a decrease in the myofilament ca - sensitivity and ca binding affinity in the force - pca relationship measurements and fluorescence spectroscopy , respectively . d75y disrupts molecular motions critical for ca binding and cardiomyocyte contractility , whereas functional defect caused by e59d is benign . most recently , pinto , et al . reported y5h , m103i , and i148v mutations in ctnc in dcm patients . functional studies showed that y5h and m103i decrease the ca2-sensitivity of force generation and that the effects of pka phosphorylation of ctni on the myofilament ca - sensitivity is abolished by m103i but diminished by y5h and i148v . in order to determine the significance of the in vitro findings concerning inherited cardiomyopathies , especially to validate whether the shift in cardiac myofilament ca- sensitivity is the most important and a common cause of cardiomyopathies associated with tn mutations in vivo , it is necessary and important to produce animal models . we have succeeded to create a knock - in mouse model in which a deletion of three base pairs coding for k210 in ctnt found in dcm patients was introduced into mouse endogenous ctnt gene using gene - targeting technology . this mouse model showed a particularly high incidence of sudden death in their growth periods from one to three months old , and enlarged hearts and heart failure , closely recapitulating the phenotypes of human dcm patients . , surface ecg showed that mutant mice commonly had an electrophysiological abnormality in the heart with long qt , which might be involved in their frequent sudden death . telemetric ecg recordings showed that mice died by abruptly developing repetitive torsade de pointes several hours before death , which ultimately degenerated into ventricular fibrillation . consistent with the results of in vitro studies , skinned cardiac muscle fibers prepared from mutant mice showed significantly lower ca - sensitivity in force generation . ca transient analysis using fura-2 in cardiomyocytes showed a significant increase in the peak amplitude in mutant mice , suggesting that ca transient is augmented to compensate for decreased myofilament ca - sensitivity . an increased intracellular camp level might be responsible for the augmented ca transient in mutant mice . skinned fibers prepared from f110i - ctnt transgenic mice showed an increased ca - sensitivity of force and atpase activity , and a much increased energy cost . in contrast , no changes in force or the atpase- pca dependencies were observed in transgenic r278c- ctnt skinned fibers . the increased ca - sensitivity and higher energy cost in f110i - ctnt hearts may be responsible for the severe phenotype compared with r278c - ctnt . this result supports the hypothesis that a greater shift in ca - sensitivity of force development results in more severe clinical phenotype and prognosis . , a knock - in mouse model of hcm caused by ctni mutation of r21c also showed an increased ca - sensitivity of force development , consistent with the findings in mouse models of hcm caused by ctnt mutations . heterozygous and homozygous mutant mice both developed a remarkable degree of cardiac hypertrophy and fibrosis . r21c is the only mutation identified within the unique n - terminus of ctni and is located in the region close to -adrenergic- activated pka - mediated phosphorylation sites . heterozygous mice reduced and homozygous mice abolished the well - known decrease in the ca sensitivity of force generation by the phosphorylation of ctni at ser23/ser24 with pka , suggesting that the impaired regulation of myofilament ca sensitivity by pka phosphorylation in ctni may also play a role in pathogenesis of hcm caused by this mutation . although studies using knock - in and transgenic mouse models have provided much important and useful information , we have to note that mouse models are different from human in ca handling during contraction / relaxation and in alterations in ca flux during heart failure . , made a hcm - causing r146g - ctni transgenic rabbit model that reflects the human system more accurately . this rabbit model also showed an increased ca - sensitivity of skinned fibers prepared from ventricular papillary muscle , as well as cardiomyocyte disarray , fibrosis , and altered connexin43 organization , which recapitulate the human hcm phenotype . human patients should have different contractile dynamics in cardiac muscle compared with mouse models , because mouse heart mainly expresses the -myosin heavy chain ( mhc ) isoform , whereas human heart mainly expresses the -mhc isoform with much lower atpase activity . , recently created a mouse model expressing hcm - related mutation r92l ctnt and -mhc by crossing the transgenic mouse strain expressing r92l ctnt with the transgenic mouse strain predominantly expressing the -mhc isoform . they found that the length - dependence of contractile activation and myofilament ca - sensitivity was blunted in these mice , suggesting that the mhc isoform has an important effect on the outcome of ctnt mutations . many functional consequences of ctn mutations in inherited cariomyopathies have been revealed , such as changes in regulatory protein - protein interaction , alterations of crossbridge cycling rate or unloaded shortening velocity , and alterations of atpase activity or phosphorylation level of key regulatory proteins like ctni . , however , it should be noted that almost all mutations in ctn shift the ca - sensitivity of cardiac muscle contraction . recently , liu , et al . , demonstrated by fluorescence studies that cardiomyopathy - related cardiac tn mutations alter the ca association and dissociation rates of ctnc in the thin filament , also implying that the alteration of steady state ca - sensitivity of the thin filament by the mutations in ctn may be a primary cause for cardiomyopathies . changes in ca - sensitivity and subsequent alteration of ca homeostasis could be a common , and the most important underlying mechanisms that trigger the arrhythmias leading to cardiac sudden death or the development of congestive heart failure . , , , to fully understand the pathogenic mechanism of inherited cardiomyopathies and their therapies , it is important to generate animal models that closely recapitulate human pathological features . we attempted to treat the dcm - causing knock - in k210-ctnt mice with a ca sensitizer pimobendan , which was expected to be beneficial for the mice with decreased cardiac myofilament ca - sensitivity . this result strongly supports the hypothesis that ca desensitization of cardiac muscle contraction is a primary mechanism for the pathogenesis of dcm caused by this ctnt mutation , which has been led by our in vitro studies , and suggests that ca sensitizers might be beneficial for the treatment of dcm patients associated with the sarcomeric regulatory protein mutations that cause myofilament ca desensitization , including k210 ctnt mutation . on the other hand , ca desensitizers might be beneficial for the treatment of hcm , in which increased ca - sensitivity could be a primary pathogenic mechanism . further studies using animal models closely recapitulating human disease phenotypes are important to develop therapeutic drugs for inherited cardiomyopathy patients .