Micronutrient deficiencies in heart failure: Mitochondrial dysfunction as a common pathophysiological mechanism?

Heart failure is a devastating clinical syndrome, but current therapies are unable to abolish the disease burden. New strategies to treat or prevent heart failure are urgently needed. Over the past decades, a clear relationship has been established between poor cardiac performance and metabolic perturbations, including deficits in substrate uptake and utilization, reduction in mitochondrial oxidative phosphorylation and excessive reactive oxygen species production. Together, these perturbations result in progressive depletion of cardiac adenosine triphosphate (ATP) and cardiac energy deprivation. Increasing the delivery of energy substrates (e.g., fatty acids, glucose, ketones) to the mitochondria will be worthless if the mitochondria are unable to turn these energy substrates into fuel. Micronutrients (including coenzyme Q10, zinc, copper, selenium and iron) are required to efficiently convert macronutrients to ATP. However, up to 50% of patients with heart failure are deficient in one or more micronutrients in cross-sectional studies. Micronutrient deficiency has a high impact on mitochondrial energy production and should be considered an additional factor in the heart failure equation, moving our view of the failing myocardium away from an "an engine out of fuel" to "a defective engine on a path to self-destruction." This summary of evidence suggests that supplementation with micronutrients-preferably as a package rather than singly-might be a potential therapeutic strategy in the treatment of heart failure patients.

The erythropoietin receptor expressed in skeletal muscle is essential for mitochondrial biogenesis and physiological exercise.

Erythropoietin (EPO) is a haematopoietic hormone that regulates erythropoiesis, but the EPO-receptor (EpoR) is also expressed in non-haematopoietic tissues. Stimulation of the EpoR in cardiac and skeletal muscle provides protection from various forms of pathological stress, but its relevance for normal muscle physiology remains unclear. We aimed to determine the contribution of the tissue-specific EpoR to exercise-induced remodelling of cardiac and skeletal muscle. Baseline phenotyping was performed on left ventricle and m. gastrocnemius of mice that only express the EpoR in haematopoietic tissues (EpoR-tKO). Subsequently, mice were caged in the presence or absence of a running wheel for 4 weeks and exercise performance, cardiac function and histological and molecular markers for physiological adaptation were assessed. While gross morphology of both muscles was normal in EpoR-tKO mice, mitochondrial content in skeletal muscle was decreased by 50%, associated with similar reductions in mitochondrial biogenesis, while mitophagy was unaltered. When subjected to exercise, EpoR-tKO mice ran slower and covered less distance than wild-type (WT) mice (5.5 ± 0.6 vs. 8.0 ± 0.4 km/day, p < 0.01). The impaired exercise performance was paralleled by reductions in myocyte growth and angiogenesis in both muscle types. Our findings indicate that the endogenous EPO-EpoR system controls mitochondrial biogenesis in skeletal muscle. The reductions in mitochondrial content were associated with reduced exercise capacity in response to voluntary exercise, supporting a critical role for the extra-haematopoietic EpoR in exercise performance.

Selenoprotein DIO2 Is a Regulator of Mitochondrial Function, Morphology and UPRmt in Human Cardiomyocytes.

Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.

ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca2+ Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ.

ATPase inhibitory factor-1 (IF1) preserves cellular ATP under conditions of respiratory collapse, yet the function of IF1 under normal respiring conditions is unresolved. We tested the hypothesis that IF1 promotes mitochondrial dysfunction and pathological cardiomyocyte hypertrophy in the context of heart failure (HF). Methods and results: Cardiac expression of IF1 was increased in mice and in humans with HF, downstream of neurohumoral signaling pathways and in patterns that resembled the fetal-like gene program. Adenoviral expression of wild-type IF1 in primary cardiomyocytes resulted in pathological hypertrophy and metabolic remodeling as evidenced by enhanced mitochondrial oxidative stress, reduced mitochondrial respiratory capacity, and the augmentation of extramitochondrial glycolysis. Similar perturbations were observed with an IF1 mutant incapable of binding to ATP synthase (E55A mutation), an indication that these effects occurred independent of binding to ATP synthase. Instead, IF1 promoted mitochondrial fragmentation and compromised mitochondrial Ca2+ handling, which resulted in sarcoplasmic reticulum Ca2+ overloading. The effects of IF1 on Ca2+ handling were associated with the cytosolic activation of calcium-calmodulin kinase II (CaMKII) and inhibition of CaMKII or co-expression of catalytically dead CaMKIIδC was sufficient to prevent IF1 induced pathological hypertrophy. Conclusions: IF1 represents a novel member of the fetal-like gene program that contributes to mitochondrial dysfunction and pathological cardiac remodeling in HF. Furthermore, we present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial Ca2+ handling and mitochondrial-to-nuclear crosstalk involving CaMKII.

Recent Advances in Modeling Mitochondrial Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Around one third of patients with mitochondrial disorders develop a kind of cardiomyopathy. In these cases, severity is quite variable ranging from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. ATP is primarily generated in the mitochondrial respiratory chain via oxidative phosphorylation by utilizing fatty acids and carbohydrates. Genes in both the nuclear and the mitochondrial DNA encode components of this metabolic route and, although mutations in these genes are extremely rare, the risk to develop cardiac symptoms is significantly higher in this patient cohort. Additionally, infants with cardiovascular compromise in mitochondrial deficiency display a worse late survival compared to patients without cardiac symptoms. At this point, the mechanisms behind cardiac disease progression related to mitochondrial gene mutations are poorly understood and current therapies are unable to substantially restore the cardiac performance and to reduce the disease burden. Therefore, new strategies are needed to uncover the pathophysiological mechanisms and to identify new therapeutic options for mitochondrial cardiomyopathies. Here, human induced pluripotent stem cell (iPSC) technology has emerged to provide a suitable patient-specific model system by recapitulating major characteristics of the disease in vitro, as well as to offer a powerful platform for pre-clinical drug development and for the testing of novel therapeutic options. In the present review, we summarize recent advances in iPSC-based disease modeling of mitochondrial cardiomyopathies and explore the patho-mechanistic insights as well as new therapeutic approaches that were uncovered with this experimental platform. Further, we discuss the challenges and limitations of this technology and provide an overview of the latest techniques to promote metabolic and functional maturation of iPSC-derived cardiomyocytes that might be necessary for modeling of mitochondrial disorders.

Gain-of-function mutation in ubiquitin-ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues.

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin-ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from two patients and three (non)familial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients' explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, that can be prevented by restoring desmin protein levels.

Sodium-glucose co-transporter 2 inhibition with empagliflozin improves cardiac function in non-diabetic rats with left ventricular dysfunction after myocardial infarction.

AIMS: Sodium-glucose co-transporter 2 (SGLT2) inhibition reduces heart failure hospitalizations in patients with diabetes, irrespective of glycaemic control. We examined the effect of SGLT2 inhibition with empagliflozin (EMPA) on cardiac function in non-diabetic rats with left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Non-diabetic male Sprague-Dawley rats underwent permanent coronary artery ligation to induce MI, or sham surgery. Rats received chow containing EMPA that resulted in an average daily intake of 30 mg/kg/day or control chow, starting before surgery (EMPA-early) or 2 weeks after surgery (EMPA-late). Cardiac function was assessed using echocardiography and histological and molecular markers of cardiac remodelling and metabolism were assessed in the left ventricle. Renal function was assessed in metabolic cages. EMPA increased urine production by two-fold without affecting creatinine clearance and serum electrolytes. EMPA did not influence MI size, but LV ejection fraction (LVEF) was significantly higher in the EMPA-early and EMPA-late treated MI groups compared to the MI group treated with vehicle (LVEF 54%, 52% and 43%, respectively, all P < 0.05). EMPA also attenuated cardiomyocyte hypertrophy, diminished interstitial fibrosis and reduced myocardial oxidative stress. EMPA treatment reduced mitochondrial DNA damage and stimulated mitochondrial biogenesis, which was associated with the normalization of myocardial uptake and oxidation of glucose and fatty acids. EMPA increased circulating ketone levels as well as myocardial expression of the ketone body transporter and two critical ketogenic enzymes, indicating that myocardial utilization of ketone bodies was increased. Together these metabolic changes were associated with an increase in cardiac ATP production. CONCLUSION: Empagliflozin favourably affects cardiac function and remodelling in non-diabetic rats with LV dysfunction after MI, associated with substantial improvements in cardiac metabolism and cardiac ATP production. Importantly, it did so without renal adverse effects. Our data suggest that EMPA might be of benefit in heart failure patients without diabetes.