Background Chronic stress-induced cardiac pathology exhibits both a wide range in severity and a high degree of heterogeneity in clinical manifestation in human patients. This variability is contributed to by complex genetic and environmental etiologies within the human population. Genetic approaches to elucidate the genetics underlying the acquired forms of cardiomyopathies, including genome-wide association studies (GWAS), have been largely unsuccessful, resulting in limited knowledge as to the contribution of genetic variations for this important disease. Methods and Results Using the β-adrenergic agonist isoproterenol as a specific pathological stressor to circumvent the problem of etiological heterogeneity, we performed a GWAS for genes influencing cardiac hypertrophy and fibrosis in a large panel of inbred mice. Our analyses revealed 7 significant loci and 17 suggestive loci, containing an average of 14 genes, affecting cardiac hypertrophy, fibrosis and surrogate traits relevant to HF. Several loci contained candidate genes which are known to contribute to Mendelian cardiomyopathies in humans or have established roles in cardiac pathology based on molecular or genetic studies in mouse models. In particular, we identify Abcc6 as the gene underlying a fibrosis locus by validating that an allele with a splice mutation of Abcc6 dramatically and rapidly promotes isoproterenol induced cardiac fibrosis. Conclusions Genetic variants significantly contribute to the phenotypic heterogeneity of stress induced cardiomyopathy. Systems genetics is an effective approach to identify genes and pathways underlying the specific pathological features of cardiomyopathies. Abcc6 is a previously unrecognized player in the development of stress-induced cardiac fibrosis.

We aimed to understand the genetic control of cardiac remodeling using an isoproterenol-induced heart failure model in mice, which allowed control of confounding factors in an experimental setting. We characterized the changes in cardiac structure and function in response to chronic isoproterenol infusion using echocardiography in a panel of 104 inbred mouse strains. We showed that cardiac structure and function, whether under normal or stress conditions, has a strong genetic component, with heritability estimates of left ventricular mass between 61% and 81%. Association analyses of cardiac remodeling traits, corrected for population structure, body size and heart rate, revealed 17 genome-wide significant loci, including several loci containing previously implicated genes. Cardiac tissue gene expression profiling, expression quantitative trait loci, expression-phenotype correlation, and coding sequence variation analyses were performed to prioritize candidate genes and to generate hypotheses for downstream mechanistic studies. Using this approach, we have validated a novel gene, Myh14, as a negative regulator of ISO-induced left ventricular mass hypertrophy in an in vivo mouse model and demonstrated the up-regulation of immediate early gene Myc, fetal gene Nppb, and fibrosis gene Lgals3 in ISO-treated Myh14 deficient hearts compared to controls.

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The chemotherapeutic effect of doxorubicin (Dox) is limited by cumulative dose-dependent cardiotoxicity in cancer survivors. Dexrazoxane (DRZ) is approved to prevent Dox-induced cardiotoxicity. Humanin and its synthetic analog HNG have a cytoprotective effect on the heart. To investigate the cardioprotective efficacy of HNG alone or in combination with DRZ against Dox-induced cardiotoxicity, 80 adult male mice were randomly divided into 8 groups to receive the following treatments via intraperitoneal injection: saline dailym HNG (5 mg/kg) daily, DRZ (60 mg/kg) weekly, Dox (3 mg/kg) weekly, DRZ + HNG, Dox + HNG, Dox + DRZ, and Dox + HNG + DRZ. Echocardiograms were performed before and at 4, 8, and 9.5 wk after the beginning of treatment. All mice were euthanized at 10 wk. In the absence of Dox, HNG, DRZ, or DRZ + HNG had no adverse effect on the heart. Dox treatment caused decreases in ejection fraction and cardiac mass and increases in cardiomyocyte apoptosis and intracardiac fibrosis. HNG or DRZ alone blunted the Dox-induced decrease in left ventricle posterior wall thickness and modestly ameliorated the Dox-induced decrease in ejection fraction. HNG + DRZ significantly ameliorated Dox-induced decreases in ejection function, cardiac fibrosis, and cardiac mass. Using a targeted analysis for the mitochondrial gene array and protein expression in heart tissues, we demonstrated that HNG + DRZ reversed DOX-induced altered transcripts that were biomarkers of cardiac damage and uncoupling protein-2. We conclude that HNG enhances the cardiac protective effect of DRZ against Dox-induced cardiotoxicity. HNG + DRZ protects mitochondria from Dox-induced cardiac damage and blunts the onset of cardiac dysfunction. Thus, HNG may be an adjuvant to DRZ in preventing Dox-induced cardiotoxicity. NEW & NOTEWORTHY Doxorubicin (Dox) is commonly used for treating a wide range of human cancers. However, cumulative dosage-dependent carditoxicity often limits its clinical applications. We demonstrated in this study that treating young adult male mice with synthetic humanin analog enhanced the cardiac protective effect of dexrazoxane against chemotherapeutic agent Dox-induced cardiac dysfunction. Thus, humanin analog can potentially serve as an adjuvant to dexrazoxane in more effectively preventing Dox-induced cardiac dysfunction and cardiomyopathy.

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Background: Heart failure (HF) is characterized by complex transcriptional networks that direct the heart from a healthy to diseased state. Although some contributing genes have been identified through molecular biology and GWAS, heritability studies suggest that many genes have resisted discovery through these approaches. Identifying connective regulatory loci, especially master transcriptional regulators that affect the expression of many genes, offers a promising means of discovering novel relevant genes that have not been detected with other methods. Methods and Results: Transcriptional regulators of HF were identified using nine-week-old female mice from 93 lines of the Hybrid Mouse Diversity Panel. Mice received 30 ug/g/day of isoproterenol (ISO) for 3 weeks to induce cardiac dysfunction. Transcriptomes were generated from left ventricles of these mice along with age-matched controls. Expression Quantitative Trait Loci (eQTLs) for 13,156 transcripts were identified using a mixed model in three conditions (control, treated, delta). Suggestive (P<1E-4) eQTLs were sorted into 500kb bins tiled across the genome to identify loci that regulate a significant number of transcripts. Ten hotspot loci that regulate over 5% (658 of 13156) of expressed genes were identified, several of which contain genes with known roles in HF, including Drosha , Akap5 and Dicer1 . Several novel regulators were also identified, including the Serine Proteinase Inhibitor, Serpina3n , which resides in a locus that regulates the change in expression of 9.7% (1276 of 13156) of all genes and is strongly correlated with changes in heart weight after ISO treatment. In subsequent in vitro work, Serpina3n knockdown resulted in reduced cellular hypertrophy, changes to hypertrophy-related gene expression, and modulation of the expression of several genes linked to its locus. Conclusion: GWAS performed on over 20,000 transcripts in control and ISO-treated hearts identified 10 genomic loci that regulate over 5% of the expressed genes in the heart. Serpina3n is a novel master regulator of HF. Further analysis of other master regulatory loci will reveal additional genes and improve our understanding of the transcriptional networks that direct the progression towards heart failure.

Background: Cardiac fibrosis is a common pathology in the diseased heart, which can cause a loss of elasticity and contractile dysfunction. Cardiac fibrosis is a complex process driven by many pathological triggers which involve numerous genes, pathways and cell types. Despite its importance, the genetic basis for the development of cardiac fibrosis has not been systematically explored. Methods and Results: We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) for high resolution GWAS and systems genetics study of quantitative traits in mice. Eight week old female mice from 80 unique inbred strains of the HMDP were given 30 ug/g/day of isoproterenol (ISO) for three weeks and cardiac fibrosis was assessed by Masson Trichrome staining which revealed a wide spectrum in the degree of fibrosis among the HMDP strains both before and after treatment. Using the Efficient Mixed Model Algorithm, we identified 13 significant or suggestive loci contributing to cardiac fibrosis, many containing numerous gene candidates. Within one of these loci, Abcc6, an orphan ABC transporter linked to the human disease pseudoxanthoma elasticum, was identified as a possible candidate for ISO-induced cardiac fibrosis. A splice-site mutation present in 19 strains of the HMDP was significantly linked to a higher degree of ISO-induced cardiac fibrosis(P=1E-4) but was not linked to increased fibrosis in untreated animals(P=0.25). Targeted genetic knockout of Abcc6 promoted ISO-induced cardiac fibrosis while reintroducing the wildtype Abcc6 allele to an genetic strain homozygous for the Abcc6 splice site mutation significantly alleviated ISO-induced cardiac fibrosis. Conclusion: A GWAS performed on levels of cardiac fibrosis observed in ISO treated animals using HMDP mice as model system uncovered significant genetic contributions to stress-induced cardiac fibrosis. Abcc6 is a novel gene contributing to ISO-induced cardiac fibrosis in the heart.