Biology subjects

Koutsifeli, P.

Publications and source records attributed to Koutsifeli, P..

5 recordsLinked to original sources

bioRxiv2024

Targeted glycophagy ATG8 therapy for diabetic heart disease

Diabetic heart disease is highly prevalent and is associated with the early development of impaired diastolic relaxation. The mechanisms of diabetic heart disease are poorly understood and it is a condition for which there are no targeted therapies. Recently, disrupted glycogen-autophagy (glycophagy) and glycogen accumulation have been identified in the diabetic heart. Glycophagy involves glycogen receptor binding and linking with an ATG8 protein to locate and degrade glycogen within an intracellular phago-lysosome. Here we show that glycogen receptor protein STBD1 (starch-binding-domain-protein-1) is mobilized early in the cardiac glycogen response to metabolic challenge in vivo, and that deficiency of a specific ATG8 family protein, Gabarapl1 ({gamma}-aminobutyric-acid-receptor-associated-protein-like-1) is associated with diastolic dysfunction in diabetes. Gabarapl1 gene delivery treatment remediated cardiomyocyte and cardiac diastolic dysfunction in type 2 diabetic mice and diastolic performance of diabetic human iPSC-derived cardiac organoids. We identify glycophagy dysregulation as a mechanism and potential treatment target for diabetic heart disease.

physiology↗

bioRxiv2024

Methods for detection of cardiac glycogen-autophagy

Glycogen-autophagy ( glycophagy) is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

molecular biology↗

bioRxiv2024

Mechanical loading reveals an intrinsic cardiomyocyte stiffness contribution to diastolic dysfunction in murine cardiometabolic disease

Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for diastolic dysfunction, and therapies to manage symptoms have limited efficacy. Understanding of the cardiomyocyte origins of diastolic dysfunction is an important priority to identify new therapeutics. The investigative goal was to experimentally define in vitro stiffness properties of isolated cardiomyocytes derived from rodent hearts exhibiting diastolic dysfunction in vivo in response to dietary induction of cardiometabolic disease. Male mice fed a high fat/sugar diet (HFSD vs control) exhibited diastolic dysfunction (echo E/e doppler ratio). Intact paced cardiomyocytes were functionally investigated in three conditions: non-loaded, loaded and stretched. Mean stiffness of HFSD cardiomyocytes was 70% higher than control. E/e for the origin hearts was elevated by 35%. A significant relationship was identified between in vitro cardiomyocyte stiffness and in vivo dysfunction severity. With conversion from non-loaded to loaded condition, the decrement in maximal sarcomere lengthening rate was more accentuated in HFSD cardiomyocytes (vs control). With stretch, the Ca2+ transient decay time course was prolonged. With increased pacing, cardiomyocyte stiffness was elevated, yet diastolic Ca2+ elevation was attenuated. Our findings show unequivocally that cardiomyocyte mechanical dysfunction cannot be detected by analysis of non-loaded shortening. Collectively, these findings demonstrate that a component of cardiac diastolic dysfunction in cardiometabolic disease is derived from cardiomyocyte stiffness. Differential responses to load, stretch and pacing suggest that a previously undescribed alteration in myofilament-Ca2+ interaction contributes to intrinsic cardiomyocyte stiffness in cardiometabolic disease. KEY POINTSO_LIUnderstanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. C_LIO_LIIn this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, [≥]25weeks). Performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro. C_LIO_LICalibrated cardiomyocyte stretches demonstrated that stiffness (stress/strain) was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e) in vivo. HFSD cardiomyocyte Ca2+ transient decay was prolonged in response to stretch. Stiffness was accentuated with pacing increase while the elevation in diastolic Ca2+ was attenuated. C_LIO_LIData show unequivocally that cardiomyocyte mechanical dysfunction cannot be detected by analysis of non-loaded shortening. C_LIO_LIThese findings suggest that stretch-dependent augmentation of the myofilament-Ca2+ response during diastole partially underlies elevated cardiomyocyte stiffness and diastolic dysfunction of hearts of animals with cardiometabolic disease. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/581448v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a2fd4corg.highwire.dtl.DTLVardef@1a398ceorg.highwire.dtl.DTLVardef@184ff46org.highwire.dtl.DTLVardef@936923_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure LegendC_FLOATNO Understanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. In this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, [≥]25weeks). Performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro. Calibrated cardiomyocyte stretches demonstrated that stiffness was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e) in vivo. These findings show that stiff hearts are characterized by stiff cardiomyocytes in metabolic disease. C_FIG

physiology↗

bioRxiv2022

Myocardial deformation imaging by 2D speckle tracking echocardiography for assessment of diastolic dysfunction in murine cardiopathology

Diastolic dysfunction is increasingly identified as a key, early onset subclinical condition characterizing cardiopathologies of rising prevalence, including diabetic heart disease and heart failure with preserved ejection fraction (HFpEF). Diastolic dysfunction characterization has important prognostic value in management of disease outcomes. Validated tools for in vivo monitoring of diastolic function in rodent models of diabetes are required for progress in pre-clinical cardiology studies. 2D speckle tracking echocardiography has emerged as a powerful tool for evaluating cardiac wall deformation throughout the cardiac cycle. The aim of this study was to examine the applicability of 2D speckle tracking echocardiography for comprehensive global and regional assessment of diastolic function in a pre-clinical murine model of cardio-metabolic disease. Type 2 diabetes (T2D) was induced in C57Bl/6 male mice using a high fat high sugar dietary intervention for 20 weeks. Significant impairment in left ventricle peak diastolic strain rate was evident in longitudinal, radial and circumferential planes in T2D mice. Peak diastolic velocity was similarly impaired in the longitudinal and radial planes. Regional analysis of longitudinal peak diastolic strain rate revealed that the anterior free left ventricular wall is particularly susceptible to T2D-induced diastolic dysfunction. These findings provide a significant advance on characterization of diastolic dysfunction in a pre-clinical mouse model of cardiopathology and offer a comprehensive suite of benchmark values for future pre-clinical cardiology studies.

physiology↗

bioRxiv2021

Protective role of the Atg8 1 homologue Gabarapl1 in regulating cardiomyocyte glycophagy in diabetic heart disease

Diabetic heart disease is highly prevalent and characterized by diastolic dysfunction. The mechanisms of diabetic heart disease are poorly understood and no targeted therapies are available. Here we show that the diabetic myocardium (type 1 and type 2) is characterized by marked glycogen elevation and ectopic cellular localization - a paradoxical metabolic pathology given suppressed cardiomyocyte glucose uptake in diabetes. We demonstrate involvement of a glycogen-selective autophagy pathway ( glycophagy) defect in mediating this pathology. Genetically manipulated deficiency of Gabarapl1, an Atg8 autophagy homologue, induces cardiac glycogen accumulation and diastolic dysfunction. Stbd1, the Gabarapl1 cognate autophagosome partner is identified as a unique component of the early glycoproteome response to hyperglycemia in cardiac, but not skeletal muscle. Cardiac-targeted in vivo Gabarapl1 gene delivery normalizes glycogen levels, diastolic function and cardiomyocyte mechanics. These findings reveal that cardiac glycophagy is a key metabolic homeostatic process perturbed in diabetes that can be remediated by Gabarapl1 intervention.

molecular biology↗