TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

Abstract 475: Beta-adrenoceptor Stimulation Uniquely Regulates Exosome Micro-rna Content Secreted From Cardiomyocytes in vitro and Those Found in vivo Circulating in Blood

Background and Purpose: The influence of β-adrenoceptor (βAR) signaling on the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether βARs can induce specific changes in the content of circulating blood exosomes in HF. In this study, we have evaluated whether βAR stimulation by isoproterenol (ISO) on neonatal rat ventricle myocytes (NRVMs) and in vivo in mice can alter the number, size and microRNA (miR) content of secreted exosomes. Methods and Results: ISO treatment of NRVMs did not change exosome number and size of compared than vehicle (PBS). After purifying total RNA from treated myocytes and secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. We found that ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) in exosomes and in the myocytes themselves, mir-292a and -1947 were up regulated and mir-7b, down regulated. Further, ISO was then used to treat C57 mice via osmotic pump chronically. The number and size of exosomes purified from circulating blood was not changed after 2 and 8 weeks. We evaluated expression of the 14 miRs down-regulated in myocytes as well miR-1 and -21 (important cardiac miRs) both in the blood and hearts of ISO-treated mice. MiR-1 did not change in both blood exosomes and heart tissue and miR-21 was up-regulated in heart tissue but not in blood both at 2 and 8 weeks after ISO treatment. In addition, miRNA-489 and -7b was down-regulated in hearts with miR-214 and -292a up regulated. In blood exosomes, we found only down-regulation of miR-489 and -7b but not miR-214 and 292a. Conclusions: We found that the βAR agonist ISO altered exosomal miR contents but not exosome size and number from myocytes. Importantly, this was found in myocytes in culture and in vivo blood and myocardium that were treated with ISO but several differences were found and changes in blood and myocytes were not homogeneous. However, two miRs, mir-7b and -miR-489, both changes similarly from their origin (NRVMs or mouse hearts) and also in circulating blood exosomes. Therefore, these miRs may represent biomarkers for sympathetic nervous system abnormalities in HF and their therapeutic potential should be evaluated.

Abstract 401: The γ2-AMPK Regulates Glucose and Glycogen Metabolism in the Heart by Modulating Cytosolic Hexokinase II Level

Mutations in the γ2-subunit of AMPK (encoded by PRKAG2 gene) cause glycogen storage cardiomyopathy. We generated a mouse model with cardiac specific deletion of γ2-AMPK (cKO) to define its function in the heart. The cKO mice were born normal and remained indistinguishable from control mice under baseline conditions but developed greater cardiac hypertrophy and dysfunction after transverse aortic constriction (TAC). The total AMPK activity was normal in cKO hearts and it increased after TAC to the same extent as that of the controls. However, the γ2-AMPK activity was abolished in the cKO and the overall AMPK activity was maintained by a compensatory increase in the γ1-AMPK activity. The lack of γ2-AMPK selectively reduced hexokinase II in the cytosol resulting in decreases in glycogen content and NADPH levels. Knock-down of γ2-AMPK subunit in neonatal rat ventricle cardiomyocytes resulted in increased oxidative stress and exacerbated response to phenylephrine and H 2 O 2 , both were rescued by HKII overexpression. Taken together, these data identify isoform-specific functions of γ2-AMPK in regulating glucose and glycogen metabolism that are critical for cardiac response to stress. The results also indicate that the specific activation of γ2-AMPK is responsible for the glycogen storage cardiomyopathy associated with PRKAG2 mutations.