Supplementary Materials B A 2 9 3 1 7 10 4 5 6 8 11 12 13 14 Color key for aligment scores 2500 500 1000 1500 2000 <40 40-50 50-80 80-200 >=200 Query: Sus Scrofa ankrd1 intron 8 20 40 60 120 Query: Sus Scrofa ankrd1 intron 7 80 100 DD36 DD159 Supplementary Fig. 1. Results of BLAST searches for the intron 7 and 8 of the pig ankrd1 gene against the NCBI EST database. Pig ankrd1 intron 8 (A) and intron 7 (B) sequences were used as queries. For each query, the alignment score graph (left) and summary table (right) are shown. A - From 14 ESTs with similarity to the pig ankrd1 intron 8 sequence, eight are expressed in heart and skeletal muscle-derived libraries. White boxes - DD159 and DD36 bands (see Fig. 1). B – All ESTs with similarity to the pig ankrd1 intron 7 sequence are expressed in heart and skeletal muscle tissues. The pig ankrd1 intron 6 sequence did not reveal a significant similarity with a minimum alignment score value of 50.

305 ANK1 ANK2 ANK3 e5 e6 e7 i7 e8 150 200 250 300 350 319 ANK4 e9 309 i8 339 i6 ANKRD1 ANKRD1-i8 ANKRD1-i7,8 ANKRD1-i6,7,8 FS A 1 MMVLKVEELV TGKKNGGGDA GEFLPEDFRD GEYEAAVTLE KQEDLKTLPA HFVSLGEQQW 61 KIEKEREAEL KKKKLEQRSK LENLEDLEII IQLKKRKKYR KTKVPVVKEP EPEVITEPVD 121 VPRFLKAALE NKLPVVEKFL SDKNNPDVCD EYKRTALHRA CLEGHLAIVE KLIEAGAQIE 181 FRDMLESTAI HWASRGGNLD VLKLLLNKGA KISARDKLLS TALHVAVRTG HYECAEHLIA 241 CEADLNAKDR EGDTPLHDAV RLNRYKMIRL LITYGADLNV KNCVSAQARG PSYRLSNEQA 301 WLTYLKIVF ANKRD1 - 36.1 kDa 241 CEADLNAKDR EGDTPLHDAV RLNRYKMIRL LITYGADLNV KNCAGKTPMD LVLNWQNGTK 301 AIFDSLKENS YKASRIATF ANKRD1-i8 - 35.1 kDa 241 CEADLNAKDR VSSLVSLGLL KLHSPFMWNP CPSRAPLQVP LAGLRPTLAP SLCLSRKEIP 301 PCTTP ANKRD1-i7,8 - 34.2 kDa i7-e8 e7-e8-e9 181 FRDMLESTAI HWASRGGNLD VLKLLLNKGA KISARDKVFL LLPPSFSSLP LRTCLGVWAG 241 LWGSAGSSTS QLLSTALHVA VRTGHYECAE HLIACEADLN AKDRVSSLVS LGLLKLHSPF 301 MWNPCPSRAP LQVPLAGLRP TLAPSLCLSR KEIPPCTTP ANKRD1-i6,7,8 - 37.7 kDa i6 e7-e8-i8 i6-e7-i7 e8 e7 e9 i8 B Supplementary Fig. 2. A - Predicted C-terminal structure of intronless and intron-retaining ANKRD1 protein isoforms. Exon/exon (e/e) and exon/intron (e/i) arrangements (vertical dotted lines, green) are shown. Regions coding by the exon- and intron-derived sequences are marked in grey and red, respectively. Ankyrin repeat (ANK) domains were mapped using the ScanProsite algorithm. Titin-binding (horizontal white rectangle) and calsequestrin-2-binding (horizontal black rectangles) sequences are shown (Mikhailov, Torrado, 2008). FS – frame shift position. In the ANKRD1-i7,8 and ANKRD1-i6,7,8, the retention of intron 7 would produce a frameshift reading (blue) of exon 8. In both ANKRD1-i7,8 and ANKRD1-i6,7,8 isoforms, a retention, respectively, of the intron 7 or intron 6 and 7 would result in modification of the C-terminus through removal of the ankyrin repeat domain 4 that, in turn, might affect these isoform’s interactions with cardiac calsequestrin-2. B - Predicted amino acid sequences of pig intron-retained ANKRD1 isoforms. Intron-derived sequences are shown in red, while adjacent exon 7, 8 and 9 sequences are shown in blue. Deduced MW values (kDa) are also indicated.

Supplementary Table 1: Primers used in this study P – pig; H – human; e – exon; i – intron; F/R – forward/reverse primers. Each anchor primer has T7 sequence on the 5´end, while each arbitrary primer has M13 sequence on the 5´end (boldface).