The high energy demand of the heart muscle requires functional mitochondria. Consequently, genetic mutations in mitochondria-related genes, are often causative to hypertrophic cardiomyopathy (HCM). The underlying molecular mechanisms often remain unclear, but reveal important insights into the pathomechanism in heart failure. Our study focused on a familial case of non-classical mitochondrial HCM. We sought to identify the impact on metabolism and mitochondrial function and its role in the pathology of this familial cardiomyopathy.
Whole exome sequencing revealed a homozygous premature stop codon variant p.Y47* in the UQCR11 gene, which encodes the small 6 kDa subunit XI of respiratory chain complex III, suggesting a functional knockout (KO) of this subunit. To investigate the disease mechanism, we employed cellular models to study the pathophysiology related to this genetic defect. Using CRISPR/Cas9, we generated a KO of UQCR11 in HeLa and induced pluripotent stem cells (iPS), confirmed through Sanger sequencing and western blotting. The UQCR11-KO HeLa cells displayed normal growth under standard conditions but showed increased apoptosis when cultured in glucose-deprived media, compared to the healthy control. Subsequently, we performed Seahorse Mito Stress test and could observe similar oxygen consumption rates, but lower ATP production-coupled respiration under standard culture conditions. Glucose depletion exacerbated the phenotype in UQCR11-KO cells, leading to decreased basal and maximal respiration as well as reduced ATP production-coupled respiration compared to controls. To gain further insights, we analyzed metabolites from isolated mitochondria using NMR metabolomics, which revealed distinct clustering between healthy and UQCR11-KO cells. Significant alterations were observed in amino acid and metabolites of the one carbon metabolism, highlighting the mutation's impact on mitochondrial function. As the patient’s phenotype was predominantly observed in cardiac cells, we differentiated our generated iPS cells into iPS-derived cardiomyocytes (iPS-CMs) to specifically examine cardiac effects. Under standard culture conditions, an automated macro-based analysis showed decreased beating frequency and slower contraction and relaxation in the cells. Morphologically, immunofluorescent staining revealed hypertrophic cardiomyocytes and disrupted mitochondrial networks. Additionally, western blot analysis of iPS-CMs demonstrated altered expression of respiratory chain proteins.
In summary, we established an in vitro model of UQCR11-related hypertrophic cardiomyopathy to investigate the effects of this loss-of-function variant. Mitochondrial dysfunction induces a comprehensive remodeling of cellular metabolism. UQCR11-KO iPS-CMs showed reduced contractility, hypertrophic morphology, and altered expression of respiratory chain complexes, consistent with patient’s phenotype. Future work will focus on elucidating the mechanistic link between the metabolic alterations and cardiac dysfunction.
