APVV-21-0443

Principal Investigator: Alexandra Zahradníková jr.

Duration: July 2022 – June 2026
Coordinating Organization: Institute of Experimental Endocrinology BRC SAS

Annotation:

Several mutations in mitochondrial genes of respiratory chain proteins are associated with heart failure (HF). The extent of their contribution to the development of HF at the level of cardiomyocytes (CM) is unknown. Recent findings indicate that various HF etiologies share similar remodeling of CM, especially of their membrane system and calcium signaling. The project is aimed to validate the in vitro model of HF development in a genetic disorder with reduced function of mitochondrial respiratory chain and to verify at the cellular level prevention of HF development by metabolic therapy with nutritional supplements. We will use the HL-1 CM cell line with allotopic expression of a mutant form of the MT-ND5 gene for subunit 5 of respiratory complex I (mG13513A), associated with human hypertrophic cardiomyopathy, to determine the impact of diseased mitochondria on cytoarchitecture and contractile function of cardiac myocytes. By methods of electron and confocal microscopy, cell electrophysiology, oxygraphy, and molecular biology, we will monitor expression and localization of dyadic proteins and changes in mitochondrial structure, localization, and oxidative capacity. Since genetic cardiomyopathy cannot yet be addressed by gene therapy, we will investigate the benefits of metabolic therapy for the compensation of invalidated mitochondrial function. We will compare in HL-1 CM the effect of the hereditary mitochondrial insufficiency with that of HF development imposed by overstimulation. We expect that nutritional supplementation of mitochondrial function will improve CM performance and suppress the development of HF. Results will be published in leading experimental cardiology and physiology journals and publicize them on the projec t website and in the media. Three PhD students and several M.S. students will participate in the project.

Keywords:

myocardium, heart failure, cellular energetics, cardiac myocyte, calcium signalling, cytoarchitecture, protein expression

Objectives:

The overall project objective is to identify common links in the development of the heart failure phenotype in two cellular models – a model of the hereditary disorder leading to a reduced function of the mitochondrial respiratory chain and a model of an acquired mitochondrial dysfunction induced by tachypacing, and to test the potential of nutritional supplementation for metabolic therapy of developing heart failure. We will focus on the impairment of mitochondrial oxidative function that leads to dilated or hypertrophic cardiomyopathy in genetic diseases such as MELAS, Leigh’s syndrome, MERRF, and MIDD. We will test the hypotheses that
(i) impairment of mitochondrial respiration by expression of non-functional respiratory complex 5 subunit I in mitochondria of cardiomyocytes will evoke HF phenotype by changes in the cytoarchitecture, contractility, and calcium signaling;
(ii) the ultrastructural remodeling results from impaired balance of mitochondrial metabolites, which slows down the degradation of HIF1α and thus decrease the expression of junctophilin, the key structural protein necessary for formation of dyads and their proper calcium releasing function; and
(iii) the development of HF phenotype resulting from mitochondrial dysfunction of cardiomyocytes can be partially compensated for by metabolic therapy. We will test these hypotheses by comparing the morphological and functional characteristics of the cardiomyocytes expressing the impaired mitochondrial gene MT-ND5 G13513A, cardiomyocytes with impaired mitochondrial oxidation induced by tachypacing, and cardiomyocytes developing the HF phenotype exposed to metabolic therapy. Metabolic therapy means supplementation of the incubation medium by specific mixtures of vitamins and coenzymes.

Specific aim 1: To validate HF phenotype development in cellular models of mitochondrial dysfunction in the cardiomyocyte cell line HL-1 by
(a) expressing mutated gene MT-ND5 G13513A in the mitochondria and
(b) tachypacing the HL-1 cardiomyocytes during cell culture.

Specific aim 2: To characterize the impact of mitochondrial dysfunction on the structure and function of cardiomyocytes. This aim will be achieved by examining
(a) the morphology, calcium signaling, and contractility of model cardiomyocytes, and
(b) the expression of key proteins of excitation-contraction coupling.

Specific aim 3: To examine the potential of metabolic therapy in the two models of heart failure development by supporting mitochondrial respiration. This aim will be achieved by examining the effect of metabolic therapy on the morphology, respiration, calcium signaling, and contractility of model cardiomyocytes.

Publications: