Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin–protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Background: Calcium (Ca ²⁺ ) is a key regulator of energy metabolism. Impaired Ca ²⁺ homeostasis damages mitochondria, causing cardiomyocyte death, pathological hypertrophy, and heart failure. This study investigates the regulation and the role of the mitochondrial Ca ²⁺ uniporter (MCU) in chronic stress-induced pathological cardiac remodeling. Methods: MCU knockout or transgenic mice were infused with isoproterenol (ISO; 10 mg/kg per day, 4 weeks). Cardiac hypertrophy and remodeling were evaluated by echocardiography and histology. Primary cultured rodent adult cardiomyocytes were treated with ISO (1 nmol/L, 48 hours). Intracellular Ca ²⁺ handling and cell death pathways were monitored. Adenovirus-mediated gene manipulations were used in vitro. Results: Chronic administration of the β-adrenergic receptor agonist ISO increased the levels of the MCU and the MCU complex in cardiac mitochondria, raising mitochondrial Ca ²⁺ concentrations, in vivo and in vitro. ISO also upregulated MCU without affecting its regulatory proteins in adult cardiomyocytes. It is interesting that ISO-induced cardiac hypertrophy, fibrosis, contractile dysfunction, and cardiomyocyte death were exacerbated in global MCU knockout mice. Cardiomyocytes from knockout mice or overexpressing a dominant negative MCU exhibited defective intracellular Ca ²⁺ handling and activation of multiple cell death pathways. Conversely, cardiac-specific overexpression of MCU maintained intracellular Ca ²⁺ homeostasis and contractility, suppressed cell death, and prevented ISO-induced heart hypertrophy. ISO upregulated MCU expression through activation of Ca ²⁺ /calmodulin kinase II δB (CaMKIIδB) and promotion of its nuclear translocation via calcineurin-mediated dephosphorylation at serine 332. Nuclear CaMKIIδB phosphorylated CREB (cAMP-response element binding protein), which bound the Mcu promoter to enhance Mcu gene transcription. Conclusions: The β-adrenergic receptor/CaMKIIδB/CREB pathway upregulates Mcu gene expression in the heart. MCU upregulation is a compensatory mechanism that counteracts stress-induced pathological cardiac remodeling by preserving Ca ²⁺ homeostasis and cardiomyocyte viability.

Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging.

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K_d of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca²⁺]_mito) responded to the changes of cytosolic calcium ([Ca²⁺]_cyto) induced by histamine or thapasigargin. Moreover, external Ca²⁺ (100 μmol/L) directly induced an increase of [Ca²⁺]_mito in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca²⁺]_mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca²⁺ repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC^WT) or mutant HRC (HRC^S96A) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC^WT- and HRC^S96A-infected cells. The HRC^S96A mutant exacerbated the inhibitory effects of HRC^WT on the amplitude of Ca²⁺ transients, prolongation of Ca²⁺ decay time, and caffeine-induced sarcoplasmic reticulum Ca²⁺ release. Consistent with these findings, HRC^S96A reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC^WT. Furthermore, the frequency of spontaneous Ca²⁺ sparks, which was reduced by HRC^WT, was increased by mutant HRC^S96A under resting conditions although there were no spontaneous Ca²⁺ waves under stress conditions. However, expression of the HRC^S96A genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca²⁺ transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca²⁺ waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

Moderate exercise decreases the risk for atrial fibrillation (AF), an effect which is probably mediated via exercise-stimulated release of exerkines. β-Aminoisobutyric acid (BAIBA), a novel exerkine, has been reported to provide protective benefits against many cardiovascular diseases, yet its role in AF remains elusive. Herein, using a mouse model of obesity-related AF through high-fat diet (HFD) feeding, we found that 12-week drinking administration of BAIBA (170 mg/kg/day) decreased AF susceptibility in obese mice. Atrial remodeling assessment showed that BAIBA attenuated obesity-induced atrial hypertrophy and interstitial fibrosis, thereby ablating the substrate for AF. Of note, to our knowledge, this is the first report of the direct association of BAIBA and hypertrophy. BAIBA has been reported to be a key regulator of glucose and lipid metabolism, and we found that BAIBA alleviated insulin resistance in obese mice. Transcriptional analysis of metabolism-related genes showed that BAIBA increased the transcription of fatty acids metabolism-related genes in the atria of lean mice but not in that of obese mice. Mechanistic investigation showed that BAIBA stimulated AMP-activated protein kinase (AMPK) signaling in the atria of obese mice and palmitic acid (PA)-treated neonatal rat cardiomyocytes (NRCM), whereas inhibition of AMPK via Compound C attenuated BAIBA-conferred cardioprotection against hypertrophy and insulin resistance in PA-treated NRCM. Collectively, BAIBA attenuates AF susceptibility in obese mice via activated AMPK signaling and resultant improvement of insulin sensitivity, thereby providing perspectives on the potential therapeutic role of BAIBA in AF treatment.