titin is the third most abundant protein ( after myosin and actin ) in vertebrate striated muscle , with an average adult human containing ~0.5  kg . this extremely large protein , which is also known as connectin , spans each half sarcomere from the z - line to the m - line or center of the sarcomere [ 4 , 5 ] ( figure 1 ) . the c - terminal a - band segment of titin is attached to the thick filament via multiple binding sites for myosin and c - protein   and two c - terminal titin regions from adjacent half - sarcomeres overlap in the m - line region of the sarcomere . similarly titin 's n - terminal segment is anchored in the z - disk and overlaps another titin n - terminus from the adjacent sarcomere . titin is believed to function as a template in sarcomere assembly and for maintenance of sarcomere integrity [ 9 , 10 ] ( figure 1 ) . these concepts have been confirmed by recent work showing that titin loss in long - term disuse of skeletal muscle results in the disorganization of the ordered sarcomeric structure [ 11 , 12 ] . each end of the thick filament is linked to the nearest z - disk by titin . this provides axial continuity for the production of resting tension and maintains the thick filament in the center of sarcomere during generation of active force . titin 's several extensible elements establish titin as a critical , multifunctional sarcomeric component . these extensible elements are composed of ( 1 ) tandem ig segments ( consisting of serially linked immunoglobulin - like domains ) , ( 2 ) the pevk region ( so called for its high content of proline ( p ) , glutamate ( e ) , valine ( v ) , and lysine ( k ) residues ) , and ( 3 ) the cardiac - specific n2b unique sequence ( n2b - us ) ( figure 1 ) . in slack sarcomeres the tandem ig and pevk segments are collapsed . upon initial stretch , the collapsed tandem ig segments are straightened while their individual ig domains remained folded . with further stretch the pevk region extends [ 14 , 15 ] . modeling tandem ig and pevk segments as entropic springs with different bending rigidities indicated that in the physiological sl range ( a ) the ig - like domains of the tandem ig segments remain folded and ( b ) the pevk segment behaves as a permanently unfolded polypeptide [ 1619 ] . the cardiac - specific n2b unique sequence ( n2b - us ) forms a third spring element in cardiac titin and provides extensibility at the upper range of physiological sarcomere lengths in the heart [ 17 , 2023 ] . the original description of titin isoforms suggested that the n2b unique sequence occurred in cardiac muscle and a different unique region called n2a was found only in skeletal muscles . names of the full isoforms in these tissues were then n2b and n2a , respectively . it was later found that the myocardium expresses two major classes of titin isoforms : a smaller n2b and a larger n2ba that contained both the n2b and n2a unique sequences [ 24 , 25 ] ( figure 2 ) . all these known titin isoforms contain pevk and tandem ig segments [ 5 , 25 , 26 ] . the n2b isoform has fewer ig domains and a short pevk region ; the n2a class of isoforms contains more ig domains and a somewhat longer pevk region . nearly all z - disk and a - band / m - band titin domains are constitutively expressed in titin isoforms of human cardiac muscle . the main titin differential splicing occurs in the middle ig and pevk domains of the i - band - titin segment [ 24 , 27 ] . the current review will discuss the isoforms of titin and the alternative splicing patterns that lead to the different forms . a number of excellent reviews should be consulted for further details on the structure and function of titin [ 6 , 19 , 2833 ] . titin is the biggest protein in the human body , and there are a number of size variants . the earliest reports indicated that there were two electrophoretic bands : a larger t1 ( which was the full length version ) and t2 ( a proteolytic fragment extending from the pevk region through the carboxyl terminal m line end ) . because of titin 's extremely large size , migration is minimal in typical sds polyacrylamide gels , and it has been difficult to develop a reliable and quantitative gel procedure . an earlier study used 3.312% gradient polyacrylamide gels to detect and quantify titin and nebulin from short segments of single muscle fibers . this system separated titin t1 ( intact titin , mr ~3300kda ) and t2 ( breakdown product of titin , mr ~2000kda ) . another group employed 2% polyacrylamide slab gels strengthened with agarose to resolve two t1 bands plus the t2 . more recently 29.5% acrylamide gradient gels have been used to separate the large titin isoforms and fragments . these gels showed that the t1 mobility varied greatly between muscle sources , reflecting the difference in molecular mass of the 3.7-mda soleus titin and the 2.97-mda rat cardiac titin isoform . the two major t1 bands were ascribed to the titin isoforms n2b and n2ba in cardiac muscle . however , the gels mentioned above are physically difficult to work with , are more complex to pour , and often undergo distortion or tearing during fixing and staining . it is also very difficult to transfer large proteins out of acrylamide gels for western blotting . more recently our group has developed a different more reliable and reproducible system called vertical sds - agarose gel electrophoresis ( vage ) . this method employs sea kem gold agarose and allows high - resolution separation of titin isoforms . this system can also be easily adapted for the characterization of other very large proteins from a variety of sources . two rat embryonic / neonatal forms ( n2ba - n1 ; n2ba - n2 ) with apparent sizes of approximately 3710 and 3590kda , respectively , are found during late embryonic and immediately after birth . these are gradually replaced by two adult forms ( n2ba - a1 ; n2ba - a2 ) with sizes of 3390 and 3220 . it has been postulated that these shorter versions are due to deletion of large numbers of exons from the exon 50 to 71 and 50 to 90 middle ig regions ( see below ) . early cdna sequencing of titin from human cardiac and soleus indicated that both isoforms were derived from a single gene and obtained by alternative splicing [ 5 , 24 ] . the complete cdna and genomic sequence determination of human cardiac titin have provided a template for later study of titin splicing variants . kolmerer and coworkers   searched for alternatively expressed exons in the m - line region of titin and found that six exons ( called mex1 to 6 ) coded for the most carboxyl terminus of titin . message for human cardiac titin always included exon mex5 while rabbit cardiac titin message contained small amounts with the mex5 missing and mouse hearts had approximately equal proportions of mex5 ( + ) and mex5 (  ) . rabbit skeletal muscles contained varying ratios of mex5 ( + ) and mex5 (  ) . the proportion of mex5 ( + ) containing message ( and presumably the titin segment it codes ) appears to correlate with the m line fine structure differences that occur between tissues [ 4042 ] , developmental stage , and species . the z - disk region of titin consists of 4 ig - domains ( z1 to z4 ) with large interdomain insertions between z2 and z3 and between z3 and z4 [ 5 , 45 ] . the latter insertion contains a series of 45 amino acid residue repeats ( z repeats ) . alternative splicing results in the expression of 6 or 7 repeats in human and rabbit heart , 4 to 6 in slow skeletal muscle , and 2 to 4 in fast skeletal muscle . the number of repeats correlates with the z line thickness [ 45 , 46 ] . the i - band region of titin is believed to be responsible for the myofibrillar passive tension response to stretch . this splicing pattern has been verified in human , rabbit , rat , and pig hearts . pcr amplification with primers from i27 to i84 of rabbit , pig , and rat ventricle is consistent with an expected size of 760  bp ( based on the human sequence ) . dog heart yielded two fragments ( a major 615  bp and a minor 760  bp ) and the amino acid sequence deduced from the nucleotide sequences revealed that the dog ventricle short pevk peptide contains a deletion of 53 amino acid residues from the amino terminal end of the n2b pevk region ( corresponding to exons 219 and 220 ) . the absence of exon 224 also occurs in some clones from human heart ( see figure 3 and discussion below ) . variable numbers of titin exons between 50 and 219 , including the n2a unique region , are expressed in cardiac and skeletal muscle . this splicing pattern gives rise to the n2a protein isoforms in skeletal muscle and the n2ba isoforms in cardiac muscle [ 24 , 27 ] . n2ba isoforms ( containing both n2a and n2b unique elements ) have multiple splicing pathways in the i27 to i68 segment ( figure 2 ) . splice pathways have been identified between i27 ( exon 50 ) and exons 51 , 70 , 71 , 75 , 77 , 80 , and 90 [ 24 , 38 ] . the expression of more middle ig domains in fetal rat ventricle is consistent with the larger size of fetal titin n2ba isoforms observed by electrophoresis . the n2b unique exon 49 is excluded in skeletal muscles by splicing together i15 to i27 , and only human soleus muscle expresses consecutive exons between 50 and 102 ( i27 to i79 ) . altogether , skeletal titin transcripts always include the n2a segment and exclude the n2b exon ; cardiac titins always include the n2b exon . the n2ba pevk region has an exceptionally varied pattern of splicing ( figure 3 ) . initial studies with dog cardiac muscle resulted in six clones ( a46 a - f ) , all from the same pcr amplification , having different exon patterns and pevk lengths ( 703 , 788 , 894 , 900 , 703 , and 819 amino acids respectively ) . subsequent work with the human pevk revealed that 10 different clones from a single individual had 10 different patterns of splicing . in addition a completely new pattern of n2ba splicing has been recently obtained in which exon 108 , found only in n2ba isoforms , was directly linked to the constitutively expressed pevk cluster in the 219 to 225 exon region ( figure 3 ) . this so - called  short pevk n2ba  class of isoforms has been observed in clones from both healthy and diseased hearts . such a pattern would result in titin molecules with apparently less potential pevk extension during stretch and thus leads to a stiffer sarcomere . it is not clear whether such message types are expressed into protein and whether there are a significant proportion of such messages transcribed to affect the passive tension . if an increased proportion of these splicing products occurred with aging , for example , it might partially explain the increased cardiac stiffness that often occurs during the later period of life . a mutant rat model has been recently described that results in significant alterations in titin isoform expression [ 26 , 47 ] . homozyogous mutants express a giant titin with apparent size on sds agarose gels of about 3.9  mda . this is larger than the size of titin from human soleus ( 3.7  mda ) . since the soleus sequence includes all the middle ig domains and virtually all the ig domains from the rest of the gene , it was initially assumed that the mutant rat heart must then express the full pevk exon cohort plus the n2b unique region . sequences obtained from pcr clones bridging the exon 108 to 225 region were not much larger than those from wild type . comparisons of exon expression between wild type and mutants showed some differences , primarily in the exon region between 175 and 219 typically associated with fetal exons ( figure 2 ) [ 26 , 48 ] . the inclusion of the n2b unique region would add some mass , but the possible inclusion of exon 48 would add 243 kda . among the ten clones from the normal human 2 in figure 3 human clones listed , there are 20 exons that are not present in all clones . if all these splicing events were independent , this means that there are 2 combinations , a total of 1,048,576 possible pathways . this far exceeds the approximately 38,016 potential splice variants identified previously with the dscam ( down syndrome cell adhesion molecule ) gene [ 49 , 50 ] . if all the clones in this table from 5 different individuals are included , then there are 38 differential exons and the number of potential clones exceeds 274 billion . microarrays have also been used to compare tissue sources for exon expression [ 48 , 51 ] . the identification of exon 156 as skeletal muscle specific   is also consistent with the absence of this exon from every human ( figure 4 ) and rat cardiac clone we have sequenced . their disadvantages include the fact that almost no splicing information can be obtained and the results include pooled data from all titins in the sample , including the novex isoforms . additionally the duplication of three segments of the titin gene in the region of the pevk   means that some microarray probes are identical . the major disadvantages include the fact that only possible splice patterns are sampled and that identified clones may not adequately represent , either quantitatively or qualitatively , the actual tissue patterns . however , the cloning approach has verified that titin splicing is highly diverse , and this complicates interpretation of mechanical experiments . titin isoform splicing not only generates diversity of titin isoforms in skeletal muscle , but also results in changes of the ratio of titin isoforms ( n2ba / n2b ) during heart development . before birth , mammalian heart mainly expresses more compliant n2ba isoforms . during perinatal heart development the larger n2ba isoform is gradually replaced by a smaller isoform n2b , and the n2b isoform becomes the predominant titin isoform in the adult left ventricles ( lv ) of smaller mammalian species [ 37 , 53 , 54 ] . the n2ba titin isoform prevails over n2b in the adult hearts of larger mammals , including humans [ 25 , 55 ] . recent studies have shown that the ratio of titin isoforms is altered in some heart diseases . a canine tachycardia - induced model of dilated cardiomyopathy ( dcm ) used to understand titin response to a chronic mechanical challenge of the heart indicated that two weeks of pacing gives rise to an exaggerated transmural titin isoform ratio gradient   and four weeks of pacing results in a decrease of the n2ba / n2b titin ratio , accompanied by an increase in titin - based passive tension . another study of spontaneously hypertensive rat model ( shr ) also showed a reduced ratio of n2ba / n2b titin in response to pressure overload , consistent with elevated passive tension of heart . more recent analyses showed that the left ventricle biopsies from patients with diastolic heart failure ( hf ) had a reduced n2ba / n2b titin . chronically ischemic lvs of coronary - artery - disease ( cad ) patients with congestive heart failure ( hf ) had nearly 50% n2ba titin ( compared to n2ba+n2b ) while approximate 30% n2ba was found in the lvs of control donor patients . analysis of explanted nonischaemic human dcm hearts again demonstrated increased proportions of n2ba / n2b [ 61 , 62 ] . these results are similar to the recently described rat model with lower ejection fraction ( unpublished data ) which expresses the n2ba isoform almost exclusively [ 26 , 47 ] . long - term hypothyroidism ( which results in diastolic dysfunction ) changed the titin isoform ratios as well . propylthiouracil ( ptu ) treatment in rats induced the expression of additional cardiac pevk and i domain exons similar to those in the large titin isoform of the fetal heart . consequently , titin - based passive and restoring forces were found to be significantly reduced in cardiac muscle of ptu - treated rats [ 63 , 64 ] . the mechanisms controlling titin splicing remain unknown , but the ability to manipulate the splicing of this protein has great potential for affecting human health .
titin is an extremely large protein found in highest concentrations in heart and skeletal muscle . 
 the single mammalian gene is expressed in multiple isoforms as a result of alternative splicing . although titin isoform expression is controlled developmentally and in a tissue specific manner , the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene . 
 over 1 million human splice pathways for a single individual can be potentially derived from the pevk region alone . 
 a new splicing pattern for the human cardiac n2ba isoform type has been found in which the pevk region includes only the n2b type exons . 
 the alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein .