Tnni3k Modifies Disease Progression in Murine Models of Cardiomyopathy

The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.


Introduction
Heart failure is the common final outcome for many forms of acute and chronic heart disease. The prognosis of heart failure is highly variable between patients, and the differences in phenotypic expression (symptoms, disease progression and course, and final outcome) create difficulties in the construction of predictive models [1]. Previous research has suggested that genetic factors can considerably modify the progression and outcome of heart failure [2]. However, these factors are difficult to identify directly in the human population because of wide genetic variability, uncontrollable environmental factors, and the intervention of medical therapy.
We have employed a disease-sensitized mouse model to map genetic factors that modify the progression and outcome of heart disease. In the Calsequestrin transgenic mouse, cardiac-specific overexpression of the calcium binding protein Calsequestrin (CSQ) leads to dilated cardiomyopathy [3]. This murine model recapitulates many of the hallmarks of human dilated cardiomyopathy including cardiac enlargement, depressed contractile function, abnormal beta-adrenergic receptor signaling and premature death [4]. Although all mice that overexpress CSQ develop dilated cardiomyopathy, disease progression and outcome varies greatly depending on the genetic background (inbred mouse strain) harboring the Csq transgene (Csq tg ) [5,6]. These differences are due to modifier genes whose multiple alleles differentially modulate the phenotype.
We have exploited these strain-specific phenotypic differences to map seven different loci (Heart failure modifier or Hrtfm) that modify the progression of cardiac dysfunction and the outcome of heart failure [5][6][7]. Hrtfm2, mapping to mouse chromosome 3, was identified in a cross between inbred strain DBA/2J (DBA), which harbors the original Csq tg , and C57BL/6 (B6). In this cross, the B6 allele at Hrtfm2 imparted a dominant, disease-accelerating effect on both cardiac dysfunction (as measured by echocardiography) and reduced survival [5]. Subsequently, Hrtfm2 was re-identified in a second cross between DBA/Csq tg animals and inbred strain AKR/J (AKR) [7]. In this cross, the AKR allele of Hrtfm2 also imparted a dominant, disease-accelerating effect on cardiac dysfunction and survival. The phenotypic effects of Hrtfm2 were robust, accounting for 30% of the genetic variability for survival and 22% for cardiac dysfunction. Capitalizing on the ancestral nature of the Hrtfm2 allele (ie, mapping within a murine haplotype block that has been retained throughout evolution and now found in multiple inbred strains), we employed haplotype-sharing analysis to effectively narrow the candidate interval from 16.5 Mb to 2 Mb, a region containing only 7 known genes [7]. We had previously suggested the Tnni3k gene as an attractive candidate based on its location within the shared haplotype interval and its biological significance as a cardiac-specific kinase that reportedly interacts with cardiac Troponin I (cTnI) [8].
Here we report the molecular characterization of allelic variation at the murine Tnni3k gene, and present in vivo functional evidence showing that Tnni3k underlies the heart failure modifier locus, Hrtfm2.

Results
As part of an effort to identify candidate genes for the Hrtfm loci, we performed microarray expression analysis of normal heart tissue from the inbred strains used in our mapping crosses to identify genes showing innate differences in transcript levels. Of the genes mapping within the Hrtfm2 linkage peak, only one gene exhibited significantly different transcript levels between the less susceptible strain DBA and the more susceptible strains B6 and AKR. Transcript levels of Tnni3k were elevated 12-fold in B6 and AKR compared with DBA, whereas levels of other transcripts mapping within the interval were not significantly different ( Figure 1A). These expression differences were validated by more-sensitive qRT-PCR analysis, where Tnni3k message levels were found to be 25-fold higher in B6 and AKR strains than those in DBA ( Figure 1B). In parallel, we genetically isolated the Hrtfm2 locus by creating a congenic line that carried AKR alleles across Hrtfm2 (an approximately 20 Mb region between rs13477425 and rs13477504) and DBA alleles throughout the rest of the genome. Quantitative RT-PCR showed that Tnni3k transcript levels in hearts from DBA.AKR-Hrtfm2 congenic mice were comparable to levels observed in B6 and AKR (AKR being the source of the Hrtfm2 locus), and not that seen in DBA (the genomic background), suggesting that the Tnni3k expression differences were driven by cisacting sequence elements within the Hrtfm2 locus, rather than transacting factors mapping elsewhere in the genome.
We analyzed heart tissue prepared from six inbred mouse strains to determine if these differences in levels of Tnni3k transcript would be observed at the protein level. We chose three additional strains that shared either the DBA or B6 haplotype at Tnni3k (Table S1). As predicted by the transcript levels, robust levels of TNNI3K protein were detected in B6, AKR, 12961/SvJ (129) and the DBA.AKR-Hrtfm2 congenic, which share the B6 haplotype. Surprisingly, no apparent protein was detected for DBA, C3H/HeJ and BALB/cByJ (BALB/c) strains, which share the DBA haplotype ( Figure 1C). Therefore, within the limits of detection of the antiserum, TNNI3K protein was apparently absent from hearts of strains sharing the DBA haplotype at the Tnni3k locus. The latter strains effectively represent Tnni3k null or extreme hypomorphic genotypes with no apparent effect on development or survival, and with no obvious pathological consequence.
The Tnni3k coding region of the mapping strains differed by a single, relatively conservative, non-synonymous coding SNP (rs30712233, T659I). By sequencing Tnni3k cDNA from the strains, we noted another, more consequential, strain-specific sequence difference. All strains sharing the B6 haplotype showed a single major transcript identical to the published cDNA. By contrast, all strains sharing the DBA haplotype exhibited a mixture of two transcripts consisting of the published transcript and a second transcript containing a 4-nucleotide insertion between exons 19 and 20 ( Figure 2A). This insertion was not present in the genomic DNA, but instead represented the addition of the first 4 nucleotides from intron 19 into the Tnni3k transcript. The insertion created a frameshift resulting in a premature termination codon immediately downstream ( Figure 2B). We determined that the frameshifted transcript accounted for approximately 70% of the message in DBA heart mRNA, but was not present in B6 or AKR ( Figure 2C). This transcript was not found in any of the EST databases for mouse or any other species, suggesting that it represented an aberrant message created by defective splicing, possibly caused by the use of a second 'gt' splice donor site 4 nucleotides downstream of the normal donor site.
The genomic region surrounding exons 19 and 20 harbors over 50 SNPs. Although in principle any of these could have caused the aberrant splicing, we focused on the SNP nearest to the splice donor junction. B6 and related strains (AKR, 129, MRL) possess an 'a' nucleotide at rs49812611, whereas DBA and related strains (A/J, C3H/HeJ (C3H), BALB/c) possess a 'g'. This SNP lies at the +9 position for the normal splice site, but importantly, this SNP lies at the +5 position with reference to the aberrant splice site. Thus, DBA and related strains harbor the consensus 'g' nucleotide at the +5 position for the aberrant site. During mRNA processing, the 'g' at the +5 splice donor position pairs with a 'c' in the U1 or U6 snRNA, resulting in a preference for 'g' at this position. Weight matrix scores for splice donor strength [9,10] for each possible splice donor site confirmed that the second (aberrant) splice site was the strongest splice site in the region only when the 'g' nucleotide is present at rs49812611 ( Figure 2D).
We tested the hypothesis that rs49812611 is the cause of aberrant splicing using an in vitro splicing system. Genomic DNA spanning exons 18 through 20 from both B6 and DBA were subcloned and transfected into 293T cells ( Figure 3A). These in vitro constructs recapitulated the splicing pattern observed in vivo, confirming that the splicing defect was caused by cis-acting sequences residing within the cloned 4 kb genomic fragment ( Figure 3B). Site-directed mutagenesis was used to investigate the role of rs49812611 in aberrant splicing. A single change at this SNP completely reversed the splicing pattern. DBA genomic DNA altered to carry the 'a' allele at rs49812611 generated no aberrant splice product, whereas the B6 DNA carrying the 'g' allele exhibited the aberrant product ( Figure 3B). These results showed that rs49812611 was responsible for the presence or absence of the aberrantly spliced message, although the extent of aberrant splicing may be modulated by other flanking sequence variation.
Since Tnni3k was originally identified as a positional candidate for Hrtfm2 due to differences in transcript levels between the mapping strains, we hypothesized that nonsense-mediated decay (NMD) was responsible for the drastically reduced levels of the frameshifted message seen in DBA. We investigated this in the mouse cardiomyocyte cell line, HL-1 [11], which shares the DBA haplotype at Tnni3k. We first confirmed that HL-1 cells expressed both aberrant and normal Tnni3k at levels comparable to wild-type DBA hearts, with the majority of the message being the aberrant variant that includes the 4-nucleotide insertion. HL-1 cardiomyocyte cells were then treated with either cycloheximide and emetine, two drugs commonly used to block NMD [12]. Treatment with either drug increased the level of aberrantly spliced transcript relative to the normally spliced message ( Figure 4A). As predicted, these treatments increased levels of total Tnni3k mRNA 16-fold

Author Summary
Heart failure is the common final outcome of many forms of acute and chronic heart disease. The prognosis of heart disease is highly variable between patients, and these differences in the phenotypic expression (symptoms, course, and final outcome) are in part due to genetic factors that have proven difficult to directly identify in the human population. To overcome this limitation, we employed a disease-sensitized mouse model of dilated cardiomyopathy to identify genes that modify the progression and outcome of the phenotype. Here we report the identification of a novel heart disease modifier gene, Tnni3k, that accelerates disease progression in two distinct mouse models of cardiomyopathy. This gene appears to play a critical role in modulating heart disease phenotypes and may provide a novel target for therapeutic intervention.
( Figure 4B), supporting a major role for NMD in the observed differences in transcript levels between strains.
Although these experiments determined the molecular mechanism underlying the observed differences in Tnni3k transcript levels, they did not address the in vivo role of Tnni3k in the progression of cardiomyopathy. We next investigated whether Tnni3k was the gene underlying the Hrtfm2 locus. We created transgenic mouse lines that expressed human TNNI3K protein in the heart. TNNI3K protein is highly conserved between human and mouse (91% identity), and transgenic expression of the human transcript enabled discrimination between the endogenous murine transcript and that derived from the transgene. Three independent founder lines were created, and qRT-PCR indicated that the human transgene was expressed at levels ranging from 5 to 20-fold above the endogenous B6 mouse transcript, depending on the founder. F1 generation mice from all three lines survived over a year, and cardiac function in 12 and 21-week transgenic animals were indistinguishable from wild-type animals. Consequently, TNNI3K expression alone did not result in overt cardiomyopathy or decreased survival due to heart failure. This was not unexpected, since in the absence of the Csq transgenic diseasesensitizer, there were no measurable differences in heart function between B6 and DBA animals, even though B6 express robust levels of TNNI3K whereas DBA shows no detectable protein.
By repeated backcrosses to DBA, the TNNI3K transgenes were introgressed into the DBA background that shows no detectable endogenous murine TNNI3K protein to test the hypothesis that in the presence of the Csq transgenic sensitizer, increased expression of TNNI3K would accelerate disease progression. The backcrossed transgenic lines continued to express robust levels of the human TNNI3K protein ( Figure S1). Two lines were chosen for all subsequent experimental crosses, and phenotypic data from N5 (or N6) animals from both lines were combined, as there was no discernable difference in the data derived from either transgenic line.
In addition to an apparently normal lifespan, TNNI3K transgenic animals did not show any signs of cardiac pathology by echocardiography. By contrast, expression of TNNI3K in the context of the Csq transgenic sensitizer resulted in profoundly premature death ( Figure 5). Of the four possible genotypes from a cross between Csq (sensitizer) and TNNI3K (modifier) transgenic lines, only the double transgenic mice showed a decrease in survival (p,0.00001). The observed survival differences were profound. All other genotypes survived on average at least 150 days, but all animals expressing both Csq and TNNI3K died within 21 days. This extreme premature death phenotype resembled that which we had previously observed when attempting to introgress the Csq transgene into the B6 background, which exhibits robust levels of endogenous mouse TNNI3K protein [5]. Starting with the sensitizer in the DBA background [4], we were unable to move the Csq transgene beyond the second generation, as N2 animals died within 40 days, precluding further backcrosses with B6 mice [5].
We next determined whether natural levels of the murine TNNI3K protein would also exhibit disease-accelerating effects. This was investigated by crossing the congenic mice harboring the AKR allele at Hrtfm2 with mice containing the Csq transgene, both held for many generations in the DBA background. The progeny from this cross would harbor a only single AKR allele of Tnni3k, the appropriate genotype for Hrtfm2 which exhibited dominant effects in the original mapping crosses [5,7]. When the congenic mice DBA.AKR-Hrtfm2, were crossed with the Csq sensitized mice, the Csq tg offspring with even only a single AKR Hrtfm2 allele showed a decrease in survival (on average 107 days) compared to TaqMan qRT-PCR of Tnni3k from 5 wild-type mouse hearts from each strain confirms that transcript levels are higher (approximately 25-fold) in B6 and AKR compared to DBA (**p.0.0001 and *p.0.001). Three hearts from the Hrtfm2 congenic line harboring AKR alleles at the Tnni3k locus on a DBA genetic background (DBA.AKR-Hrtfm2) shows transcript levels similar to B6 and AKR hearts, which is significantly higher than observed in DBA hearts (**p.0.0001). Actb served as an endogenous control. Error bars indicate standard error of the mean (SEM). (C) Western blot analysis shows that three strains that share the DBA haplotype at Tnni3k show no detectable TNNI3K protein, while three strains with the B6 haplotype show robust expression. The DBA.AKR-Hrtfm2 congenic mouse shows high expression as predicted based on RNA expression. Receptor tyrosine kinase TEK was used as a protein loading control. doi:10.1371/journal.pgen.1000647.g001 those with two DBA Hrtfm2 alleles (more than 150 days, Figure 5). The Csq-sensitized Hrtfm2 congenic line also survived longer than Csq-sensitized F1(DBA/B6) animals, which survived on average to only 50 days [5]. In the original mapping cross, Hrtfm2 contributed approximately 30% of the genetic variance towards the survival trait [5]. Thus, as expected, the isolated B6/AKR Hrtfm2 allele contributed a robust but only partial effect on reduced survival when compared to the F1 animals, as other modifier loci had been crossed out of the congenic line.
To determine whether the premature death was related to cardiac dysfunction, we performed echocardiography on animals with all four possible genotypes resulting from cross between the Csq and TNNI3K transgenic lines. Echocardiography was performed at 14 days, the earliest possible age for reproducible echocardiographic data. Due to the extremely accelerated disease course and profound reduction in survival, only six of fourteen double transgenic mice survived to their scheduled 14-day echocardiogram. Fractional shortening in these TNNI3K tg /Csq tg mice was significantly decreased compared to the other three genotypes of animals (P,0.0232), demonstrating severely abnormal heart function of the double transgenic animal ( Figure 6A and 6B, Table S2). Even by 14 days, hearts from the TNNI3K tg /Csq tg mice were larger than those of the other genotypes, and by histological staining showed obvious chamber dilation ( Figure 6C). Thus, the double transgenic animals developed dilated cardiomyopathy by 14 days (or earlier) and all mice of this genotype died before this or shortly thereafter due to heart failure. Many of the double transgenic animals displayed bradycardia (a severe slowing of the heart rate), clearly evident in the echocardiograms. This phenotype, while a feature of the natural disease progression in the Csq transgenic model, is normally observed only in adult animals just prior to heart failure [5]. Thus, this hallmark of the natural progression of the Csq transgenic model is also greatly accelerated with overexpression of TNNI3K.
We also investigated the echocardiographic parameters of the DBA.AKR-Hrtfm2/Csq tg (see Figure 5). Echocardiography performed on these mice at 4 and 8 weeks of age showed decreased fractional shortening in DBA.AKR-Hrtfm2/Csq tg mice compared to the DBA/Csq tg littermates, indicating more severe level of cardiomyopathy ( Figure 7, Table S3). Furthermore, from age 4 to 8 weeks, percent fractional shortening decreased more rapidly in DBA.AKR-Hrtfm2/Csq tg mice (36%, p = 0.0004) than the littermates (18%, p = 0.186), suggesting that the presence of even a single AKR-Hrtfm2 locus (essentially half the normal AKR/B6 level of TNNI3K expression) can accelerate the progression of the Csq-induced cardiomyopathy.
We next investigated whether TNNI3K expression would exhibit a disease accelerating effect in a model of cardiomyopathy that was unrelated to Calsequestrin over-expression. Transverse aortic constriction (TAC) induces left ventricular hypertrophy in response to pressure overload [13]. We performed TAC on TNNI3K transgenic animals and wild-type littermate controls.  Table S4). This confirmed that TNNI3K expression has a detrimental effect on heart function outside the context of the transgenic Csq sensitizer.

Discussion
We had previously mapped 7 loci (Hrtfm1-7) that modify heart disease progression using the Calsequestrin (Csq) transgenic mouse model [5][6][7]. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. We have shown that the murine Tnni3k locus harbors an ancestral SNP in intron 19 that activates a cryptic splice site, generating an aberrant transcript that undergoes NMD, leading to drastically reduced message levels and  an apparent absence of TNNI3K protein. In DBA and other inbred mouse strains sharing the same haplotype at Tnni3k, drastically reduced levels of TNNI3K protein have no obvious effect on normal development or physiology, suggesting that any trace amounts of protein that remain are sufficient for its normal function, or that the lack of protein is compensated by functional redundancy of another gene. In vivo transgenic and congenic mouse lines confirm that TNNI3K levels are a significant determinant of the rate of disease progression and outcome, since expression of this protein accelerates disease progression in two independent and unrelated models of cardiomyopathy. However, we did not observe a simple, linear relationship between the level of TNNI3K transgenic overexpression and the strength of the modifying effect. In these models, the levels of overexpression in the transgenic lines may have crossed the threshold required for maximal phenotypic effects.
The modifying effects of TNNI3K expression were not dependent on the allele at the nonsynonymous coding SNP (rs30712233, T659I). DBA and most inbred mouse strains encode Threonine at this position. Most other species also encode Threonine at the homologous position. By contrast, B6 and AKR inbred strains encode the Isoleucine variant. The human TNNI3K transgene employed in the validation experiments coincidentally encodes the highly conserved Threonine variant. The modifying effects of the human transgene carrying the conserved variant (695T) directly parallel, and are even stronger, than those observed using the congenic line containing the B6/AKR variant (695I). Thus, robust phenotype modifying effects were observed independent of the murine Tnni3k coding variant. Nonetheless, these experiments did not address whether the 695I polymorphism also alters TNNI3K protein function.
TNNI3K was identified as a cardiac-specific protein kinase that interacted with cardiac Troponin I (cTnI) in the yeast-two hybrid interaction assay [8], however, cTnI has not been established as a phosphorylation target. TNNI3K protein contains seven ankyrin repeats in the N-terminus followed by a dual-specificity protein kinase domain and a short C-terminal serine-rich domain. The overall domain structure of TNNI3K resembles that of Integrinlinked Kinase (ILK). ILK mediates communication from the cellular matrix to intracellular signaling molecules such as PKB and GSK3b, and plays important roles in cardiac growth, contractility and repair [14,15]. Sequence and structural homology might imply similar functions for TNNI3K. A yeast twohybrid interaction screen with a C-terminal fragment of TNNI3K identified several additional sarcomeric proteins as putative binding partners such as cardiac a-actin and myosin binding protein C [8]. These studies suggest that TNNI3K might modulate sarcomere function through interactions with key components of the sarcomeric complex. However, to date, none of these proteins has been validated as a phosphorylation target of TNNI3K in cardiomyocytes, and the in vivo function of TNNI3K remains unknown.
Recently, expression of TNNI3K was shown to be protective in a different cardiomyopathic disease context [16,17]. In a murine model of cardiac ischemia, intramyocardial transplantation of Tnni3k-overexpressing P19CL6 cells promoted cardiomyogenesis and improved cardiac function. We note that P19CL6 cells were originally derived from the C3H lineage of mice [18], which share with DBA, the ''null'' haplotype for Tnni3k (see Figure 1). Thus, the resulting phenotype may have been due to the restoration of Tnni3k expression in otherwise null cells, rather than to overt overexpression of the gene. A locus for susceptibility to coxsackievirus B3-induced myocarditis maps to a locus on distal mouse chromosome 3 (Vms1, viral myocarditis susceptibility locus) that virtually overlaps Hrtfm2 and which includes Tnni3k in its confidence interval [17]. In this disease model, inbred strain C57BL/10 provides the protective allele at the locus, and strain A/J provides the susceptibility allele. Assuming that Tnni3k also underlies the Vms1 locus, viralinduced myocarditis might represent another disease context where expression of TNNI3K is protective. These combined data suggest that expression of TNNI3K may be detrimental in certain pathological conditions such as pressure overload or aberrant sarcomeric calcium regulation, but protective in other disease contexts. In either scenario, TNNI3K appears to play a critical role in modulating disease progression and outcome in heart disease.
Since protein kinases are critical cell cycle regulators, kinase inhibitors have become a major avenue for the development of novel cancer therapeutics. TNNI3K may be an ideal candidate for the development of small molecule kinase inhibitors for categories of heart disease where TNNI3K expression is detrimental. In these cases, selective inhibition of TNNI3K would be particularly useful as it might slow disease progression, which may prove beneficial in treating individuals with rapidly progressing disease. In other scenarios of disease, augmentation of TNNI3K activity or protein levels may instead prove beneficial. Further investigation of TNNI3K function in these and other cardiomyopathic mouse  Tnni3k, a Genetic Modifier of Cardiomyopathy models will lead to increased understanding of its role in both normal and pathological contexts, and may provide a novel target for therapy for heart disease.

Animal care and handling
All mice were handled according to approved protocol and animal welfare regulations of the Institutional Review Board at Duke University Medical Center. All inbred mouse strains used in the course of this study were obtained from Jackson Laboratory (Bar Harbor, ME). Transgenic mice overexpressing Csq [3] were maintained on a DBA/2J genetic background.

DBA.AKR-Hrtfm2 congenic mouse
Through repeated backcrossing to DBA/2J, a congenic mouse was created which retains AKR genomic DNA at the Hrtfm2 locus in the DBA genetic background. At generation N2, breeders were selected which were heterozygous at Hrtfm2 and homozygous DBA at the other mapped modifier loci [7]. Genome-wide SNP genotyping was carried out using the Mouse MD linkage panel with 1449 SNPs (Illumina, San Diego, CA). By generation N6, the animals were homozygous for DBA alleles throughout the genome and only showed heterozygosity for an approximately 20 Mb interval on chromosome 3, the region containing Hrtfm2. Once we had reached the generation N10 backcross, the DBA.AKR-Hrtfm2 mouse was maintained by intercross.
Mouse RNA isolation, microarray analysis, and qRT-PCR Whole hearts removed from age-and sex-matched wild type animals from each of the three primary strains (B6, DBA, AKR) were used to examine RNA transcript levels. Total RNA was isolated using the RNeasy Kit (Qiagen, Valencia, CA). Microarray analysis was done on an Affymetrix Mouse probe set (Mouse 430 2.0 Array, Affymetrix, Santa Clara, CA). Analysis was done using GeneSpring GX 7.3 Expression Analysis (Agilent Technologies, Santa Clara, CA). For the TaqMan expression analysis, total RNA was extracted from whole mouse hearts using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized from 1 mg total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) and used as the template for qRT-PCR. Tnni3k cDNA was amplified using the predesigned gene expression assay (TaqMan, ABI, assay ID: Mm01318633_m1). Beta-actin (Actb) was used as the endogenous control (TaqMan, ABI, catalogue number 4352341E). All amplifications were carried out in triplicate on an ABI Prism 7000 Real Time PCR system and analyzed with ABI software. All statistical analyses were done using an unpaired, two-tailed T-test. Analysis of TNNI3K protein expression Whole heart protein lysates were prepared using flash-frozen heart tissue resuspended in lysis buffer with protease inhibitors. Lysates were analyzed by SDS-PAGE and Western blot performed with standard methods. A polyclonal peptide antiserum was developed to the C-terminal 14 amino acids of mouse TNNI3K protein (LHSRRNSGSFEDGN). Antiserum from 2 rabbits was purified on a Protein A column (GenScript, Piscataway, NJ). TNNI3K antibody was used at a 1:1000 dilution in TBST with 5% dry milk. Secondary anti-rabbit antibody conjugated to HRP followed by incubation with Pierce SuperSignal West Pico Chemiluminescant Substrate (Thermo Fisher Scientific, Rockford, IL) and exposure to X-OMAT film (Kodak) to visualize protein bands. Western blot analysis was used to confirm specificity of the antibody. As predicted, the mTNNI3K antibody detects a 90 kDa protein from lysates prepared from 293T cells transiently transfected with a full length Tnni3k expression vector and in protein lysates from wild-type mouse hearts.
Fluorescent RT-PCR assay cDNAs were subjected to qRT-PCR using primers designed to detect either a 116 bp or a 120 bp cDNA PCR product. The forward primer was targeted 25 bp upstream of the predicted 4 base insertion and was fluorescently labeled: 59-6FAM-AGATTTCTGCAGTCCCTGGAT-39 while the unlabeled reverse primer was targeted 48 bp downstream of the predicted 4 base insertion with the sequence: 59-AAGACATCAGCCTT-GATGGTG-39. Accumulation of both fragments was quantified using the GeneMapper analysis program on the ABI Prism 3730 DNA Sequencer (Applied Biosystems). Ratios of properly spliced and mis-spliced products were calculated based on relative amplification of both cDNA products.

Cloning of mTnni3k splicing constructs, cell culture, and transfection
To create the Tnni3k genomic splicing constructs, DBA genomic DNA and B6 BAC clone RP23-180023 were used as templates to generate genomic 4 kb fragments that included part of intron 17, exon 18, intron 18, exon 19, intron 19, exon 20 and part of intron 20. The sequence of the forward PCR primer was 59-ACTTACTTATGTGCTTCTCTTAGTTATGTGC-39; the reverse primer was 59-GGATTTAAACATAGGTGTGTACC-TAATTGT-39. PCR products were sub-cloned into pSPL3 (Invitrogen). Clones were verified by direct sequencing. Human embryonic kidney HEK293T (293T) cells (ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum at 37uC in 5% CO 2 . Cells were grown on 35 mm 2 plates and transfected with 1 mg plasmid DNA using FuGene reagent (Roche, Indianapolis, IN) according to the manufacturer's protocol. RNA was extracted with TRIzol (Invitrogen) 24 hr post-transfection and RT-PCR was carried out using standard methods.
In vitro splicing assay HEK293T cells were grown to approximately 80% confluence in 6-well plates, then transfected using with 1 mg of DBA-or B6-pSPL3 plasmid mixed with FuGene reagent. All transfections were performed in triplicate. Total RNA was extracted with TRIzol 20 hr post-transfection. RT-PCR was carried out using standard methods. Ratios of properly spliced and aberrantly spliced products for the Tnni3k construct were determined by the fluorescent RT-PCR assay described above.

Site-directed mutagenesis
A single base was changed at rs49812611 (IVS19+9), in the DBA-pSPL3 construct (GRA) and the B6-pSPL3 construct (ARG) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA) with PfuTurbo proofreading DNA polymerase. All clones were sequenced to verify proper incorporation of the SNP.

Culture of cardiomyocytes and NMD blocking experiments
HL-1 cardiomyocytes [11] were cultured in Claycomb Medium (SAFC Laboratories, Lenexa, KS) supplemented with Fetal Bovine Serum at 10%, 2 mM L-Glutamine, 100 mg/ml Penicillin/ Streptomycin, and 100 mM fungizone. Cells were cultured at 37uC with 5% CO 2 . Although the HL-1 cardiomyocytes were derived from a heart isolated from a mixed B6-DBA mouse [11] direct sequencing of genomic DNA from the cell line showed that it is homozygous for DBA alleles at the Tnni3k locus. HL-1 cells were treated with 5.7610 22 mM cycloheximide or 3.3610 22 mM emetine. Each treatment was performed in triplicate and RNA was isolated from cells 24 hours post treatment. RT-PCR was performed on RNA isolated from cells treated with NMD blocking drugs and untreated controls. Ratios of properly spliced and aberrantly spliced products were measured using the fluorescent RT-PCR splicing assay as described above. Total transcript levels were determined using the Tnni3k TaqMan assay described above.
Creation and testing of a TNNI3K transgenic mouse A full-length 2.5 kb human TNNI3K cDNA was amplified from normal human heart RNA following RT-PCR and cloned into a vector downstream of the murine a-myosin heavy chain (aMHC) promoter. An artificial minx intron was inserted upstream of the TNNI3K start codon. The construct was linearized and an 8 kb fragment containing the aMHC promoter, cDNA and SV40 polyadenylation sequence was purified and used for microinjection. B6SJLF1/J blastocysts were injected with the linearized transgene and subsequently implanted into surrogate mice. The resulting founder animals were genotyped for presence of the TNNI3K transgene using a 59 primer in the aMHC promoter and a 39 primer in the TNNI3K transgene. Three transgenic lines were chosen for backcrossing to the DBA strain. Western blot analysis of heart lysates with a polyclonal antibody (Bethyl Laboratories, Montgomery, TX) raised against a human C-terminal TNNI3K peptide (FHSCRNSSSFEDSS) confirmed similar levels of expression of the TNNI3K transgene in each line ( Figure S1). This was repeated for several generations of backcrossing to DBA. Southern blot analysis of DNA from founder animals and subsequent generations (N2-N3) indicated that two founder lines carried 10-20 copies of the transgene while the third line appeared to have .100 copies. qRT-PCR with SYBRgreen (Invitrogen) was performed on heart cDNA from several transgenic mice to determine the relative expression difference between endogenous mouse Tnni3k and transgenic human TNNI3K expression.

M-mode echocardiography
Transthoracic two-dimensional M-mode echocardiography was performed between 12 and 18 weeks of age in conscious mice using either a Vevo 770 echocardiograph (Visual Sonics, Toronto, Canada) or an HDI 5000 echocardiograph with a 15-MHz frequency probe (Phillips Electronics, Bothell, WA). Measurements of cardiac function include heart rate, posterior and septal wall thickness, left-ventricular end diastolic diameter (LVEDD) and left-ventricular end systolic diameter (LVESD). Fractional short-ening (FS) was calculated with the formula: FS = (LVEDD2 LVESD)/LVEDD, as previously described [4].

Histology
Hearts were fixed in 10% neutral buffered formalin, dehydrated in 75%, 90% and 100% ethanol, and embedded in paraffin; sections 5 mm in thickness were cut and then stained with Masson's trichrome stain.

Transverse aortic constriction
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and transverse aortic constriction (TAC) was performed as previously described [13]. TAC was performed on 14 TNNI3K transgene-positive animals and 14 transgenenegative (wild-type) littermates at 10 weeks of age. One of the transgene-negative controls and three transgene-positive animals died following surgery, which is a normal complication of this procedure. The remaining 24 mice were then analyzed by echocardiography (as described above), at 4 and 8 weeks following the surgery. Figure S1 Calsequestrin and TNNI3K protein expression in transgenic lines. (A) Calsequestrin protein expression detected in Csq tg and Csq tg ; TNNI3K tg mice heart lysates using anti-CSQ polycolonal antibody. A representative result is shown. (B) Stable expression of human TNNI3K protein in two independent transgenic lines. TNNI3K expression was detected in mice heart lysates from both TNNI3K tg lines using anti-TNNI3K polycolonal antibody. A representative result is shown from N7 backcross animals for both transgenic lines.   Table S2 M-mode echocardiograms of 14-day-old mice from a cross between TNNI3K tg and Csq tg transgenic animals. Measure-ments of cardiac function included left-ventricular end diastolic diameter (LVEDD), left-ventricular end systolic diameter (LVESD), posterior (PW) and septal (IVSW) wall thickness, ejection time (ET), and heart rate (HR). Heart weight (HW) and body weight (BW) were measured and the ratio of heart weight to body weight was determined. Data is shown as mean6sd.