**1. An overview of Mitochondrial Bioenergetics and Function in the Healthy Heart**

Over recent years, mitochondrial dysfunction was recognized as the hallmark of manifold heart diseases (HDs) in their onset and development [1,2]. Despite the advancement of specific prevention guidelines and relevant therapeutic strategies, HD remains the main cause of death in Western countries [3]. Mitochondria occupy 30% of the total volume of cardiomyocytes and are localized in three different areas among cardiac fibers [4]; these structures are highly dynamic during all phases of heart development and exhibit continuous changes in terms of their bioenergetics and biology during cardiomyocyte differentiation [5]. This mitochondrial abundance is justified by the high-energy supply required by the heart that is provided by mitochondria, defined as "the powerhouse of the cells". In fact, via oxidative phosphorylation (OXPHOS), mitochondria generate 95% of the adenosine triphosphate (ATP) needed to maintain cardiac activities [6]. In cardiac tissue, the principal source of energy is generated by fatty acid oxidation (FAO), which occurs in the mitochondrial matrix. The products obtained by beta-oxidation enter the Krebs cycle and are ultimately utilized by the electron transport chain (ETC), which takes place along the inner mitochondrial membrane (IMM), and this phenomenon creates a proton electrochemical gradient and consequently generates the mitochondrial transmembrane potential (∆Ψm). Under physiological conditions, the ∆Ψ<sup>m</sup> is −180 mV across the IMM and thus acts as a driving force for cations, such as calcium (Ca 2+ ), which is considered the most important second messenger in cells. Mitochondrial Ca 2+ plays a crucial role in controlling cell physiopathology and cell fate [7]. In the heart, its importance is highlighted by the fact that it is fundamental for the contractile function of cardiomyocytes [8]; indeed, excitation and contraction are paired to cardiomyocyte depolarization and Ca 2+ fluxes.

**Citation:** Morciano, G.; Vitto, V.A.M.; Bouhamida, E.; Giorgi, C.; Pinton, P. Mitochondrial Bioenergetics and Dynamism in the Failing Heart. *Life* **2021**, *11*, 436. https://doi.org/ 10.3390/life11050436

Academic Editors: Giorgio Lenaz and Salvatore Nesci

Received: 24 March 2021 Accepted: 7 May 2021 Published: 12 May 2021

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Concomitant with an action potential, the sarcolemma depolarizes, and Ca2+ enters a dedicated area across the sarcolemma through L-type voltage-operated calcium channels (VOCCs). Here, the Ca2+ concentration increases from very low levels (nanomolar ranges) to approximately 10 µM to give rise to the so-called Ca2+ sparklet. These Ca2+ sparklets induce ryanodine receptor 2 (RyR2) opening and Ca2+ release from the main store inside cells, the sarcoplasmic reticulum (SR), which might generate higher Ca2+ sparks into the cytosol and thus allow cell shortening and blood pumping in the heart.

Mitochondrial Ca2+ also leads to the generation of ATP via the activation of dehydrogenases [9] and increasing the activity of the ATP synthase [10] when muscles are stimulated. ATP cycling, as well as Ca2+, constitutes a key point for the control of both muscle contraction and relaxation. Indeed, ATP is used by the sarcoendoplasmic reticulum Ca2+-ATPase cardiac isoform 2a (SERCA2a) to remove Ca2+ from the cytosol in the diastolic phase to ensure muscle relaxation and the correct Ca2+ reuptake into the SR for the next muscle contraction. Thousands of these events occur with each action potential. Although other systems of Ca2+ removal exist in the cells, such as Plasma membrane Ca2+ ATPase (PMCA) and Na+/Ca2+ exchanger (NCX), SERCA2a handles about 75% of cytosolic Ca2+ .

Ca2+ is also responsible for activating cell death pathways. Indeed, it is widely reported that persistent accumulation of Ca2+ into the cytosol and mitochondrial Ca2+ overload trigger the opening of the mitochondrial permeability transition pore complex (PTPC), a biological event that, if sustained, leads to cell death [11,12].

An important consequence of mitochondrial function is reactive oxygen species (ROS) production [13]. Indeed, ROS are physiologically generated by the ETC via the reduction of oxygen to superoxide and by the Krebs cycle in the mitochondrial matrix. Although a balance exists between ROS production and scavenging systems and that ROS regulate some physiological processes, a significant dysregulation may occur in or as a consequence of diseases; indeed, ROS bursts can damage proteins, lipids, and mitochondrial DNA (mtDNA), which trigger inflammation and ultimately cell death as well as PTPC opening [14].

Heart failure (HF) is a severe condition for which there is currently no cure, and only medications are administered to maintain a patient's life as normally as possible. Approximately 30 million people worldwide are suffering from HF, and these patients need new therapies that address the disease rather than only providing symptom relief. The clinical scenario is represented by two main conditions: either a reduction of left ventricular (LV) ejection fraction, named as heart failure with reduced ejection fraction (HFrEF) [15], which results in an enlarged LV that cannot contract as it should, or a preserved LV ejection fraction (HFpEF), where the LV preserves its ability to contract but, being less than the necessary volume of blood which enters in the left chamber during an improper diastole, it does not meet the body's requirement. Mitochondria, intracellular Ca2+ and ATP cycling not by chance are associated with significant alterations during HF, and understanding these changes might have a potential impact on the search for new pharmacological treatments. Two of the main intracellular alterations in HF are the elevated cytosolic Ca2+ levels in diastole and its decrease in availability in the SR during systole. This leads to an inefficient cycle of excitation contraction coupling (ECC). Several studies in human and animal models have associated these defects in Ca2+ cycling to SERCA2a dysfunction [16,17] in terms of reduced expression and impaired regulation [18].

HF develops through stages of cardiac adaptation in terms of macroscopic (e.g., hypertrophy) and microscopic (e.g., metabolism) changes. During cardiac hypertrophy, a metabolic shift from FAO to glycolysis occurs [19]. This finding is supported by a seminal paper that, using the carbon isotope labelling techniques [20], showed the importance of the glycolytic pathway in producing ATP in hypertrophic hearts to compensate for the significant decrease in OXPHOS [21]. Despite enhanced glycolysis, various studies showed either decreased or normal glucose oxidation, leading to an uncoupling between the uptake of glucose and its oxidation [19]. If the energy for low work load becomes inefficient, which means deeply impaired contractility, the advanced stage of HF begins which continues to be characterized by repression of FAO and high glycolysis. Here, the use of ketone

bodies and branched-chain amino acids (BCAAs) as alternative metabolic substrates are prevailing. These can be further converted to acetyl-CoA to enter the tricarboxylic acid cycle and ETC to produce ATP.

This review aims to describe mitochondrial dynamics and bioenergetics in the heart with references to HF.

#### **2. Mitochondria-Related Metabolic Abnormalities in the Failing Heart**

The mammalian heart is considered a metabolic omnivore with the capacity to oxidize fatty acids, ketone bodies, carbohydrates (glucose and lactate), and BCAAs to meet its high energy demand after birth [22]. Cardiac metabolism maintains a dynamic state of equilibrium for efficient energy transfer and is a highly concerted plethora of chemical reactions leading to the conversion of ATP both to sustain cell function and allow contraction, growth, repair, and regeneration. The metabolic alterations in the failing myocardium have been explored; from many points of view [23], HF is considered a return to fetal stages due to the shift from FAO-based metabolism (which mainly manifests as a 35% decrease in the ATP concentration and changes in substrate utilization) to glycolysis (the main active pathway during the fetal period) accompanied by progressive degeneration of the myocardium [24,25] (Figure 1). Accordingly, cardiac metabolism in HF was recognized as a field of active research for well over a century [26] and will be discussed in the first part of this review.

**Figure 1.** Metabolic and mitochondrial ultrastructural changes in HF. The figure summarizes the first part of the review which reports all metabolic changes and main mitochondrial ultrastructural abnormalities accompanying HF development in humans.
