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Deciphering the functions of Sox6 and myosin-encoded microRNAs in heart failure and hypertrophy

Final Report Summary - ROLE OF CARDIAC SOX6 (Deciphering the functions of Sox6 and myosin-encoded microRNAs in heart failure and hypertrophy)

1. Objective
Sox6, a member of the SoxD family of transcription factors, has recently been established as a mediator of the actions of myomiRs 208 and 499, small regulatory molecules, in skeletal muscle (van Rooij, 2009). Sox6 suppresses the expression of slow skeletal muscle genes and its deletion in mice promotes skeletal muscle fiber switching (Quiat, 2011). Some publications suggest that Sox6 and Sox5, another member of the SoxD transcription factor family, might be important players in the heart. However, there remains a need for a systematic investigation of the role of Sox6 and Sox5 and their potential target molecules in the adult mammalian heart.

2. Results
Using mutant mouse models, the role of Sox5 and Sox6 was investigated in the heart and potential downstream targets, such as Leucine-rich repeat containing 2 (Lrrc2), solute carrier 41a3 (Slc41a3) and biglycan (Bgn) were identified and functionally investigated. Results obtained from studying Sox5/6-deficient mice in the context of myomiRs 208 and 499 have been described in detail in the periodic report and will be summarized briefly only. The focus of this report is on the introduction of putative Sox5/6 target genes and the investigation of their functional relevance in mice.

Sox5, Sox6
Utilizing cardiomyocyte-specific Sox5,6 double knockout mice (DKO), we found that Sox5 and Sox6 are critical for maintaining cardiac contraction and relaxation velocity as well as blood pressure in-vivo (Fig. 1A-F). Upon cardiac stress by transverse aortic banding for 3 weeks, knockout mice but not control littermates developed cardiac failure evident from pronounced decrease in heart function (Fig. 1G,H), and the development of secondary signs of heart failure such as lung edema (Fig. 1I) and the appearance of so-called heart failure cells in the lungs (Fig. 1J). Cardiomyocyte-specific deletion of Sox5,6 did not influence cardiac morphology, histology or electrophysiology in-vivo. However, cardiomyocytes isolated from the heart showed altered action potential wave forms (Fig. 1K-P). Gene expression analyses revealed differentially expressed genes encoding for electrolyte channel subunits or calcium handling proteins (Fig. 1Q-R). Moreover, we found several muscle-specific or muscle-enriched, relatively understudied genes significantly regulated upon deletion of Sox5 and Sox6 in the heart / skeletal muscle. Amongst those were leucine-rich repeat containing 2 (Lrrc2), solute carrier 41a3 (Slc41a3), and biglycan (Bgn).

Leucine-rich repeat containing 2 (Lrrc2)
Domains within proteins with repeats of the amino acid leucine are very common in the animal kingdom (Ng, 2011). Their functions are diverse but the leucine-rich domain per se seems to foster protein-protein interactions (Kobe, 2011). Relatively little is known about Lrrc2, another member of the group of leucine-rich repeat containing proteins (Liu, 2010, Kiss, 2001, Kiss 2002). In a microarray performed in the outgoing host’s laboratory on skeletal muscle from Sox6 conditional knockout mice, Lrrc2 was one of the most significantly down-regulated genes (Quiat, 2011). Likewise, in a cardiac-specific overexpression model of one of the myomiRs, miR-499, Lrrc2 was significantly and robustly upregulated, whereas it was down regulated in miR-499 knockout animals. During this project, Lrrc2 was identified as a novel muscle-specific protein (Fig. 2A). Lrrc2 expression in the heart was restricted to cardiomyocytes and not in other cells of the heart (Fig. 2B). Expression of Lrrc2 in development was negligible in utero but induced after birth in the heart and tongue (Fig. 2C,D). Intracellular localization studies were conducted using plasmids, which contained the coding sequence of Lrrc2 with N- or C-terminal HA and Flag tags. Plasmids were electroporated into the tibialis anterior muscle and the muscle dissected, sectioned and stained thereafter revealing a striated staining pattern (Fig. 2E). Lrrc2 expression in skeletal muscle changes under certain conditions. In miR-499 transgenics, a mouse model with skeletal muscle-specific overexpression of the myomiR 499, Lrrc2 is significantly upregulated. Overexpression of miR-499 leads to a fiber type switch from fast to slow myofibers. During starvation in wild-type mice, Lrrc2 is also upregulated in skeletal muscle (Fig. 2F-H). This suggests a role for Lrrc2 in skeletal muscle fiber type specification and muscle atrophy.

Lrrc2 knockout mice
To study the function of Lrrc2, knockout animals were generated in the outgoing host’s laboratory. ES cells harboring a lacZ cassette inserted between two coding exons of Lrrc2, were ordered from KOMP and chimeric animals derived by blastocyst injections. Chimeric mice were bred with wildtype mice and 2 germline transmissions (out of 18 chimeric mice) were achieved. Heterozygous F1 animals were intercrossed to obtain animals used for experiments. Reporter knockout mice were viable,born according to expected Mendelian ratios and healthy until adulthood. The model was validated by Northern blot (Fig. 2I) and histology. Heart (Fig. 2J) and skeletal muscle tissue had β-galactosidase activity evident from blue staining upon reaction with X-gal. To explore the physiological function of Lrrc2, knockout mice were extensively phenotyped using different methodology: Echocardiography and cardiac catheterization, electrocardiograms, heart and muscle weights and histology, skeletal muscle fiber type determination. Deletion of Lrrc2 lead to increased cardiac contraction and relaxation velocity dp/dtmax (Fig. 2K) and dp/dtmin (Fig. 2L) during cardiac catheterization without any significant effects on cardiac pump function as assessed by echocardiography (Fig. 2M). Moreover, Lrrc2 reporter knockout mice had normal electrocardiograms. No differences were observed in skeletal muscle weights and histology between knockout and control mice at baseline and under starvation stress. In summary, Lrrc2 seemed to be dispensable for cardiac and skeletal muscle function, morphology and histology at baseline level.

Lrrc2 transgenic mice
Numerous transgenic mouse lines were generated by the researcher in the lab of the outgoing host, expressing the Lrrc2 coding sequence under the control of the alpha myosin heavy chain (αMHC), the beta myosin heavy chain (βMHC), the muscle creatine kinase (MCK) and the skeletal actin (SKA) promoter. Different promoters were chosen to achieve cardiomyocyte-specific expression of Lrrc2 during development (βMHC) or adulthood (αMHC), expression in heart and skeletal muscle (MCK) or skeletal-muscle only (SKA). All mouse lines were extensively validated for correct tissue- and time-specific expression of Lrrc2 mRNA and protein. In some cases Lrrc2 was tagged at the C-terminus with HA tag to allow detection by commercially available established antibodies.
Overall, overexpression of Lrrc2 in muscle tissue had only very subtle effects on muscle function and histology. For instance, mice with forced overexpression (15 fold) of Lrrc2 in the adult heart (αMHC-promoter) had decreased heart pump function as assessed by echocardiography (Fig. 2N). However, no changes in heart size (no hypertrophy) or histology were observed. Likewise, mice with forced overexpression of Lrrc2 in skeletal muscle had muscle weights comparable to control animals (Fig. 2O-Q) and reacted normally in starvation-induced muscle atrophy. Histologically, there was a mild increase in the percentage of slow fibers in soleus muscle of MCK- (Fig. 2R,S) and SKA-controlled overexpression of Lrrc2, which failed to reach significance due to biological variability.

Solute carrier 41a3 (Slc41a3)
The SLC41 family is a family of Magnesium transporters since at least two members, Slc41a1 and Slc41a2, have been shown to be involved in Magnesium uptake and transport (Sahni, 2013). My interest was focused on a poorly characterized member of this family, Slc41a3, which was expressed in skeletal muscle, the heart, testis and the adrenal gland as shown by Northern blot analysis (Fig. 3A), and significantly down-regulated upon deletion of Sox5 and Sox6 in the heart. Interestingly, expression of Slc41a3 in the heart was confined to cardiomyocytes (Fig. 3B) and underwent significant diurnal changes similar in extent to Bmal, one of the tissue clock genes (Fig. 3C). Its expression in the heart and in skeletal muscle was induced around weaning and significantly down regulated in skeletal muscle after denervation (Fig. 3D). Numerous open questions were addressed during this study of Slc41a3 including its putative action in Magnesium transport and its role in heart, skeletal muscle and adrenal gland function at baseline and during disease.

Slc41a3 knockout mice
Global deletion of Slc41a3 in reporter knockout mice was validated using Northern blot analysis (Fig. 3E), qPCR and lacZ staining. In line with previous results, lacZ staining was most prominent in heart, skeletal muscle and adrenal gland (Fig. 3F). Reporter knockout mice were born close to expected Mendelian ratios. No alterations were observed upon deletion of Slc41a3 in cardiac function assessed by echocardiography (Fig. 3G), cardiac catheterization or EKGs. Serum Mg2+ levels were undistinguishable between knockout and control mice at baseline (Fig. 3H). The same was true for the development of body weight under normal chow and for body fat composition assessed by echoMRI (Fig. 3I,J). Muscle weights normalized to tibia length were increased by trend but this tendency didn’t reach statistical significance. Interestingly, adrenal glands of reporter knockout mice showed significantly decreased adrenalin, dopamine and serotonin levels per gland (Fig. 3K-M) and decreased protein content while size and weight of adrenal glands were normal. The biological relevance of this decrease is unclear since Slc41a3 reporter mice displayed normal response towards cardiac stress imposed by pressure overload. We furthermore challenged Slc41a3 reporter knockout mice metabolically by 1) monitoring cardiac pump function and electrophysiological properties while animals were on low-magnesium diet and 2) following body fat composition and glucose intolerance while on a high fat diet. 1) Low magnesium levels in the blood had similar effects on cardiac function or EKGs in reporter mice and control mice. 2) Blood glucose levels after glucose challenge were slightly higher in knockout mice fed with a high fat diet but failed to reach statistically significant levels (Fig. 3N,O). Denervation was performed to assess whether Slc41a3 is relevant for skeletal muscle atrophy but no significant differences were observed between reporter knockout and control animals. In summary, we did not find robust hints for an important role of Slc41a3 in heart and skeletal muscle. At this stage, we cannot rule out functional redundancy between family members.

Biglycan (Bgn)
Biglycan, together with decorin, lumican and other members of this class, are small leucine-rich repeat proteoglycans, which have major roles during development and disease (reviewed in Iozzo, 2010, Theocharis, 2010, Chen 2013). Through interaction with collagen, biglycan has been shown to promote collagen stability (Ameye, 2002). Mice with a targeted deletion of biglycan suffer from heart ruptures after experimentally induced myocardial infarction due to inappropriate stabilization of the fibrotic scar (Westermann, 2008). Biglycan was one of the few extracellular matrix genes significantly regulated in the hearts of mice lacking Sox5 and Sox6. In this study, we sought to investigate whether biglycan-deficiency in mice influences the development of fibrosis during cardiac pressure-overload and whether it facilitates the regression of myocardial fibrosis during cardiac unloading. We established a new model of reversible transverse aortic constriction in mice. We observed that cardiac fibrosis, induced by cardiac pressure overload, was irreversible during cardiac unloading (Fig. 4A-C). Cardiac function (Fig. 4D), hypertrophy (Fig. 4E) and hypertrophic gene expression pattern (Fig. 4F), however, were almost normalized to baseline levels by relieving pressure from the heart. We found mRNA levels of biglycan to follow the same pattern (Fig. 4G) but protein levels to remain stably high during cardiac unloading (Fig. 4H-J).

Biglycan-deficient mice and in-vitro results
We first subjected biglycan-deficient mice to long-term cardiac pressure overload and found that knockout mice developed less cardiac fibrosis (Fig. 4M,N) and hypertrophy (Fig. 4L) and displayed better cardiac function than their wildtype controls (Fig. 4K). However, genetic loss of biglycan did not enable a regression of fibrosis during cardiac unloading suggesting a role of biglycan for the establishment but not for the dissolution of fibrosis (Fig. 4O,P). Soluble biglycan was able to induce cardiomyocyte hypertrophy in cultivated neonatal rat cardiomyocytes (Fig. 4Q) and activated pro-hypertrophic and pro-fibrotic genes, which partially overlapped with a gene program induced by the pro-hypertrophic agents phenylephrine and isoproterenol. Amongst those genes were Abra and Nr4a1 (Fig. 4R,S). We conclude that besides its role as a structural extracellular matrix protein, biglycan has an important function as a signaling molecule during cardiac remodeling.

3. Outlook
During the study of the cardiac function of Sox5 and Sox6 we found several putative direct or indirect target genes in the heart, which we studied using different mutant mouse models. Biglycan, which might not be a direct target but is indirectly regulated upon deletion of Sox5 and Sox6 in the heart, is one of the most promising targets for therapeutic purposes since it seems to influence the development of cardiac fibrosis during cardiovascular disease with cardiac pressure overload such as with aortic valve stenosis or hypertension. Targeting biglycan early in disease might provide a way to avoid excess deposition of fibrosis during cardiac pathology. This approach may thus positively influence cardiac performance in patients with heart disease.


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