METHODS: In this study, a dystrophin-deficient myoblast cell line established from the skeletal muscle of a dystrophic (mdx) mouse was used as a model. The dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation. The cells were subjected to total protein extraction prior to Western blotting assay technique. Protein sub-fractionation has been conducted to determine protein localization. The live-cell analysis of autophagy assay was done using a flow cytometer.
RESULTS: In our culture system, the dfd13 myoblasts did not achieve terminal differentiation. PTEN expression was profoundly increased in dfd13 myoblasts throughout the differentiation day subsequently indicates perturbation of PI3K/Akt/mTOR regulation. In addition, rictor-mTORC2 was also found inactivated in this event. This occurrence has caused FoxO3 misregulation leads to higher activation of autophagy-related genes in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, the ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features.
CONCLUSION: Perturbation of the PTEN-PI3K/Akt pathway triggers excessive autophagosome formation and subsequently reduced autophagic flux within dystrophin-deficient myoblasts where these findings are of importance to understand Duchenne Muscular Dystrophy (DMD) patients. We believe that some manipulation within its regulatory signaling reported in this study could help restore muscle homeostasis and attenuate disease progression. Video Abstract.
METHODS AND RESULTS: The Efficacy of a Standardized Diuretic Protocol in Acute Heart Failure (ENACT-HF) study is an international, multicentre, non-randomized, open-label, pragmatic study in AHF patients on chronic loop diuretic therapy, admitted to the hospital for intravenous loop diuretic therapy, aiming to enrol 500 patients. Inclusion criteria are as follows: at least one sign of volume overload (oedema, ascites, or pleural effusion), use ≥ 40 mg of furosemide or equivalent for >1 month, and a BNP > 250 ng/L or an N-terminal pro-B-type natriuretic peptide > 1000 pg/L. The study is designed in two sequential phases. During Phase 1, all centres will treat consecutive patients according to the local standard of care. In the Phase 2 of the study, all centres will implement a standardized diuretic protocol in the next cohort of consecutive patients. The protocol is based upon the recently published HFA algorithm on diuretic use and starts with intravenous administration of two times the oral home dose. It includes early assessment of diuretic response with a spot urinary sodium measurement after 2 h and urine output after 6 h. Diuretics will be tailored further based upon these measurements. The study is powered for its primary endpoint of natriuresis after 1 day and will be able to detect a 15% difference with 80% power. Secondary endpoints are natriuresis and diuresis after 2 days, change in congestion score, change in weight, in-hospital mortality, and length of hospitalization.
CONCLUSIONS: The ENACT-HF study will investigate whether a step-wise diuretic approach, based upon early assessment of urinary sodium and urine output as proposed by the HFA, is feasible and able to improve decongestion in AHF with volume overload.