Introduction
Mitochondria (MITO) are organelles with a well-known role in cellular metabolism and energy production, but they also play a critical role in cell growth and differentiation, cell signaling, and cell death or apoptosis [
1,
2]. Abnormalities in MITO morphology/function have been observed in a variety of metabolic and cardiac diseases, including heart failure (HF). Once thought of as inert, mitochondria are now known to be highly dynamic, constantly undergoing biogenesis, fission and fusion in response to changes in energy demands [
2,
3]. Fission and fusion are thought to be essential for normal mitochondrial function. A number of proteins and lipids have been shown to be important mediators of these dynamic processes [
3], particularly peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcription factor that drives MITO biogenesis, and cardiolipin (CL), a negatively charged phospholipid that is unique to MITO [
2]. The failing heart shows to dysregulation in both fission and fusion regulating proteins, and downregulation of MITO fusion proteins enhances apoptosis; therefore, a possible contributor to ongoing cardiomyocyte loss [
3] and potential mediator of progressive worsening of the HF state [
4,
5]. Thus, agents that can normalize MITO fission and fusion may have important therapeutic potential in the treatment of HF.
Elamipretide (ELAM) (also referred to as SS-31, MTP-131, or Bendavia) is a water-soluble tetrapeptide with structural motifs of natural and synthetic amino acids [
6]. The peptide enters the cell, crosses the MITO outer membrane, and localizes to the inner MITO membrane where it integrates with CL. In addition to modulating fission and fusion, CL plays an important role in the regulation of cristae formation, MITO DNA (mtDNA) stability and segregation, and the function and organization of the respiratory complexes into supercomplexes for oxidative phosphorylation [
7‐
11] ELAM has been shown to enhance adenosine triphosphate (ATP) synthesis in multiple organs, including the heart, kidney, neurons, and skeletal muscle [
12‐
21]. In dogs with coronary microembolization–induced HF, 3 months monotherapy with ELAM improved left ventricular (LV) systolic function and prevented progressive LV dilation. These improvements were associated with reduced reactive oxygen species (ROS) and overall improvement of MITO function that included improved MITO respiration, normalized membrane potential, and complex-I and -IV activities, as well as normalized rate of ATP synthesis [
22]. The objective of the current study was to evaluate determinants of MITO dynamics, including biogenesis, fission/fusion, and CL synthesis and remodeling in HF, and the effect of long-term therapy with ELAM on these processes.
Discussion
Results of this study indicate that in dogs and humans with HF, the LV myocardium manifests impaired MITO dynamics evidenced by impaired MITO biogenesis, dysregulation of the MITO fission and fusion machinery, and downregulation of mitofilin, a key protein necessary for the formation of tubular cristae and cristae junctions. In patients with HF, these abnormalities are present regardless of the etiology of HF, namely ICM or DCM. Importantly, results of this study showed that these defects can be reversed/normalized by long-term therapy with ELAM, a first-in-class cell-permeable tetrapeptide that selectively targets mitochondria. From a mechanism of action viewpoint, ELAM is known to integrate with and bind to CL, a constituent of the MITO inner membrane and site of the ETC.
In dysfunctional mitochondria typical of diseases such as HF, stability and integrity of CL are essential given the central role of CL in the regulation of MITO cristae formation, MITO fission and fusion, mtDNA stability and segregation, and in the function and organization of the respiratory complexes into supercomplexes for oxidative phosphorylation [
7‐
11]. Preserving the integrity of CL at sites of curvature along the MITO inner membrane serves to preserve constituent complex proteins of the ETC that are essential for efficient electron transfer along the ETC that led to normalization of energy production by the MITO and prevention of excess generation of ROS also by the mitochondria [
15,
20]. Efficient oxidative phosphorylation is also dependent on reducing ROS production by mitochondria. Excess formation of ROS is the primary culprit in the disruption of CL leading to MITO dysfunction. The (18:2)
4 acyl chain configuration of CL with the 18-carbon fatty alkyl chains with 2 unsaturated bonds on each are primary targets for ROS. Studies in dogs with chronic HF showed that long-term therapy with daily SC injections of ELAM can improve LV systolic function, a desirable outcome that was accompanied by normalization of plasma biomarkers and by improved MITO function evidenced by normalization of MITO respiration, membrane potential, complex-I and -IV activities, ROS formation, and maximum rate of ATP synthesis and ATP/adenosine diphosphate ratio [
22]. The current study builds on these findings, showing that ELAM in the setting of HF also normalizes MITO dynamics as evidenced by normalization of the following: (1) MITO biogenesis, (2) MITO fission and fusion, (3) MITO inner membrane integrity, and (4) CL synthesis and remodeling.
In the present study, dogs with HF demonstrated abnormalities of CL dynamics, specifically, decreased levels of total CL and (18:2)
4CL, decreased CLS-1, and abnormalities in the CL remodeling enzymes TAZ-1 (decreased) and ALCAT-1 (increased). CL is found in high concentrations at contact sites between the inner and outer membranes of mitochondria, where fusion and fission occur [
2]. CL plays a key role in MITO fusion via an interaction with OPA-1 and facilitates fission via recruitment and activation of Drp-1 [
2]. Previous studies have shown that CL is decreased in diseases associated with MITO dysfunction and that the CL remodeling enzymes are either upregulated (ALCAT-1) or downregulated (TAZ-1) [
2,
8]. The current study found that ELAM normalizes CL and (18:2)
4CL, as well as the regulatory enzymes in dogs with HF compared with control animals with HF.
HF, regardless of etiology, is associated with increased sympathetic drive as evidenced by a sustained increase in PNE concentration. The increase in PNE can lead to downregulation of eNOS, decreased levels of cGMP, and finally to downregulation of PGC-1α, as illustrated in Fig.
1. Because PGC-1α is an important co-transcriptional regulator of MITO biogenesis, its downregulation can have a major adverse impact on MITO biogenesis and, therefore, lead to disruption of needed organelle turnover. Results from the present study indicate that long-term therapy with ELAM was associated with normalization of PNE concentration along with normalization of eNOS expression, cGMP levels, and expression of PGC-1α. Normalization of this signaling pathway affords a potential cardioprotective effect. In addition to its fundamental role in MITO biogenesis, PGC-1α is also an important regulator of lipid and glucose metabolism and data suggest that PGC-1α agonists can improve cardiac function, decrease fibrosis, and improve contractility and endothelial function in models of HF [
25].
As alluded to earlier, results of the present study also demonstrated that MITO fission-regulating proteins, namely Fis-1 and Drp-1, are markedly increased whereas fusion-regulating proteins, namely Mfn2, OPA-1, are markedly decreased in dogs with HF and human hearts with DCM or ICM etiology. These findings are consistent with those reported in other animal models [
1,
3,
26,
27]. Chen et al. [
3] found that OPA-1 is decreased in HF in human and rat idiopathic cardiomyopathy and that the reduction was associated with increased apoptosis. Another group found that Mfn2/Drp-1 ratio (i.e., fusion/fission ratio) is decreased during HF and that treatment with a MITO division inhibitor improved cardiac function by normalizing the ratio [
1]. The current study found that in dogs with HF, treatment with ELAM significantly increased Mfn2 and OPA-1 and significantly decreased Fis-1 and Drp-1 compared with HF-CON dogs. Phosphorylation of Mfn2 has also been shown to be an important mediator of mitophagy [
28]. Phosphorylation of Mfn2 mediates the cytosolic ubiquitin ligase Parkin recruitment to damaged mitochondria [
28]. Ablation of Mfn2 in mouse cardiomyocytes was shown to suppress mitophagy [
28]. In the present study, pMfn2 was significantly downregulated, a condition that likely suppressed mitophagy and gave rise, in part, to the accumulation of morphologically and functionally abnormal mitochondria.
Our results also indicate that the inner membrane protein mitofilin was significantly reduced in HF-CON dogs compared with normal dogs and in human DCM and ICM hearts compared with DNR human hearts. Mitofilin is a protein of the inner MITO membrane and is associated with a large multimeric protein complex of about 1200 kDa. Mitofilin has critical functions in MITO morphology and MITO fission and fusion, specifically in the formation of tubular cristae and cristae junctions. Mitofilin also regulates cytochrome C release during apoptosis. Downregulation of mitofilin in HeLa cells has been shown to lead to decreased cellular proliferation and increased apoptosis, along with ultrastructural evidence of disorganized MITO inner membrane; abnormalities that are also manifested in HF. Mitofilin is one of the most abundant MITO proteins and is highly expressed in heart muscle [
29]. Downregulation of mitofilin is invariably associated with MITO dysfunction. Mitofilin has been shown to be diminished in diabetic cardiomyopathy [
30]. Results from our study demonstrate that administration of ELAM is associated with a normalization of mitofilin abundance in dogs with HF compared with untreated dogs with HF.
The present study has some limitations to be considered. We previously showed that the dog model of chronic HF from which LV tissue was obtained for the present study manifest abnormalities of cardiomyocyte MITO ultrastructure characterized by small mitochondria with loss of electron dense matrix and disrupted inner membrane [
31]. We also showed that these ultrastructural abnormalities are associated with abnormal MITO function characterized by poor MITO respiration, reduced membrane potential and reduced maximum rate of ATP synthesis [
22,
32]. In the present study, we did not obtain and specifically prepare tissue to evaluate MITO ultrastructure by transmission electron microscopy (TEM). Therefore, we were unable to demonstrate that normalization of fission and fusion proteins, mitofilin, and cardiolipin synthesis and remodeling proteins after treatment with ELAM also resulted in normalization of MITO ultrastructure. Nonetheless, several studies by other investigators have shown MITO ultrastructure normalization in multiple organs following therapy with ELAM [
32‐
36].. In mice with HF produced by transverse aortic constriction (TAC), saline treated control animals showed increased number of damaged MITO with disrupted cristae compared to normal cristae in animals treated with SS-31 or ELAM [
33].. Treatment with ELAM was also shown to normalize MITO morphology in kidneys of aging mice, in rats with acute kidney injury due to ischemia reperfusion, and improved MITO ultrastructure and morphology in lymphoblasts and fibroblasts derived from patients with Friedreich ataxia [
34‐
37]
.
Taken together, the results of the current study provide further evidence of the role of MITO abnormalities in the progression of the HF state and demonstrate that long-term therapy with ELAM reverses these abnormalities. The results provide additional mechanistic framework for the improvements in LV function associated with ELAM therapy in dogs with coronary microembolization-induced HF and suggest that future investigations of ELAM in the treatment of HF in humans are warranted.