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Published on: June 9, 2019
Quantitative proteomic analysis reveals high interference on protein expression of H9c2 cells activated with glucose
Erika Meneses-Romero1, Lorena Hernández-Orihuela1, Victoria Pando-Robles2
1Laboratorio Universitario de Proteómica (LUP), Instituto de Biotecnología-UNAM, Av. Universidad, 2001, Col Chamilpa, C.P. 62210 Cuernavaca, Morelos, Mexico.
Abstract:
In recent decades, the incidence of death and morbidity due to diabetes has increased worldwide, causing a high social and economic impact. Diabetes is a major cause of blindness, kidney failure, heart attack, stroke and lower limb amputation. However, the molecular mechanisms that make the heart and kidneys the main targets of diabetes are not completely understood. To better understand the complex biochemical mechanism of diabetic cardiomyopathy, we investigated the effects of hyperglycemia with concomitant digoxin and ouabain stimulation in H9c2 cells. Total extracted proteins were analyzed by label-free LC-MS/MS, quantified by Scaffold software and validated by parallel reaction monitoring (PRM) methodology. Here, we show that the eukaryotic initiation factors (Eifs) and elongation factors (Eefs) Eif3f, Eef2 and Eif4a1 are overexpressed following cardiotonic steroid (CTS) stimulation. Similarly, the expression of four 14-3-3 proteins that play a key role in cardiac ventricular compaction was altered after CTS stimulation. In total, the expression of nine protein groups was altered in response to the stimulation of H9c2 cells. Here, the biological consequences of these changes are discussed in depth. SIGNIFICANCE: Hyperglycemia is the main physiological condition that provokes tissue and vascular injuries in heart of diabetic patients. However, the changings at large scale in the expression of proteins of cardiomyocytes generated by this condition was not yet studied. Here we report for the first time the altered biosynthesis of nine groups of proteins of H9c2 cells activated by high glucose concentrations and by cardiotonic steroids (CTS). Furthermore, the increased biosynthesis of Eifs, Eefs and 14-3-3 protein groups by CTS, which play a crucial role in cardiomyopathies are original data reported in this work. These findings not only enhance our knowledge concerning to the effects of hyperglycemia and CTS on H9c2 cells but also indicate potential molecular targets to interfere in diabetes cardiomyopathy progression.
Insights
Diabetic cardiomyopathy involves molecular changes in heart cells. This study found that high glucose and cardiotonic steroids alter the expression of eukaryotic initiation factors (Eifs), elongation factors (Eefs), and 14-3-3 proteins in H9c2 cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Diabetes mellitus is a global health crisis, leading to significant morbidity and mortality, with the heart being a primary target organ.
- Diabetic cardiomyopathy, a serious complication, arises from complex molecular mechanisms not fully elucidated, necessitating further investigation into cellular responses.
- Understanding the impact of hyperglycemia on cardiac cells is crucial for developing effective therapeutic strategies against diabetes-related heart disease.
Purpose of the Study:
- To investigate the molecular mechanisms underlying diabetic cardiomyopathy by examining the effects of hyperglycemia and cardiotonic steroids (CTS) on H9c2 cardiac cells.
- To identify specific protein expression changes in response to high glucose and CTS stimulation in a cellular model of diabetic cardiomyopathy.
- To explore the role of eukaryotic initiation factors (Eifs), elongation factors (Eefs), and 14-3-3 proteins in the pathogenesis of diabetic heart conditions.
Main Methods:
- Utilized H9c2 cells to model the effects of hyperglycemia and cardiotonic steroid (CTS) stimulation.
- Employed label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive proteomic analysis.
- Quantified protein expression using Scaffold software and validated findings with parallel reaction monitoring (PRM).
Main Results:
- Identified overexpression of eukaryotic initiation factors (Eifs) and elongation factors (Eefs), specifically Eif3f, Eef2, and Eif4a1, upon CTS stimulation.
- Observed altered expression of four 14-3-3 proteins, known for their role in cardiac ventricular compaction, following CTS stimulation.
- Determined that a total of nine protein groups exhibited altered expression in H9c2 cells in response to hyperglycemia and CTS, indicating a broad molecular response.
Conclusions:
- Hyperglycemia and CTS significantly alter the expression of key protein groups involved in protein synthesis and cardiac function in H9c2 cells.
- The study provides novel insights into the molecular changes occurring in cardiomyocytes under diabetic conditions, highlighting Eifs, Eefs, and 14-3-3 proteins as potentially crucial players.
- These findings offer potential molecular targets for future therapeutic interventions aimed at mitigating the progression of diabetic cardiomyopathy.
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