TBC1D15-Drp1 interaction-mediated mitochondrial homeostasis confers cardioprotection against myocardial ischemia/reperfusion injury

Highlights

• Level of TBC1D15, a RAB7 GTPase-activating protein, is downregulated in human and rodent ischemic myocardium;

• Gain/loss-of-function, human sample and single cell RNAseq denote a mitochondrial and cardiac regulatory role for TBC1D15;

• TBC1D15 preserves mitochondrial integrity by combining with Drp1 to foster asymmetrical mitochondrial fission and clearance.

• Selective segregation of damaged mitochondria is mediated by Drp1 in coordination with Fis1/TBC1D15/Rab7

Abstract

Objective

Mitochondria are essential for myocardial ischemia/reperfusion (I/R) injury. TBC domain family member 15 (TBC1D15) participates in the regulation of mitochondrial homeostasis although its role remains elusive in I/R injury.

Methods and materials

This study examined the role of TBC1D15 in mitochondrial homeostasis under myocardial I/R injury using inducible cardiac-specific TBC1D15 knockin (TBC1D15^CKI) and knockout (TBC1D15^CKO) mice.

Results

TBC1D15 mRNA/protein levels were downregulated in human ischemic cardiomyopathy samples, mouse I/R hearts and neonatal mouse cardiomyocytes with H/R injury, consistent with scRNA sequencing finding from patients with coronary heart disease. Cardiac-specific knockin of TBC1D15 attenuated whereas cardiac-specific knockout of TBC1D15 overtly aggravated I/R-induced cardiomyocyte apoptosis and cardiac dysfunction. TBC1D15^CKI mice exhibited reduced mitochondrial damage and mitochondrial fragmentation following myocardial I/R injury, while TBC1D15^CKO mice displayed opposite results. TBC1D15 preserved mitochondrial function evidenced by safeguarding MMP and oxygen consumption capacity, antagonizing ROS accumulation and cytochrome C release, which were nullified by TBC1D15 knockdown. Time-lapse confocal microscopy revealed that TBC1D15 activated asymmetrical mitochondrial fission through promoting mitochondria-lysosome contacts untethering in NMCMs under H/R injury, whereas overexpression of TBC1D15 mutants (R400K and ∆231–240) failed to regulate asymmetrical fission and knockdown of TBC1D15 slowed down asymmetrical fission. Moreover, TBC1D15-offered benefits were mitigated by knockdown of Fis1 and Drp1. Mechanistically, TBC1D15 recruited Drp1 to mitochondria-lysosome contact sites via direct interaction with Drp1 through its C terminus (574–624) domain. Interfering with interaction between TBC1D15 and Drp1 abrogated asymmetrical mitochondrial fission and mitochondrial function. Cardiac phenotypes of TBC1D15^CKO mice upon I/R injury were rescued by adenovirus-mediated overexpression of wild-type but not mutants (R400K, ∆231–240 and ∆574–624) TBC1D15.

Conclusions

TBC1D15 ameliorated I/R injury through a novel modality to preserve mitochondrial homeostasis where mitochondria-lysosome contacts (through the TBC1D15/Fis1/RAB7 cascade) regulate asymmetrical mitochondrial fission (TBC1D15/Drp1 interaction), suggesting promises of targeting TBC1D15 in the management of myocardial I/R injury.

Introduction

Ischemic heart disease is one of the leading causes of disability and mortality worldwide [1], [2], [3]. Although clinical interventions such as angioplasty or thrombolytic agents have helped to re-establish coronary flow, their clinical efficacy and outcome are hampered by the presence of reperfusion injury [3]. Alleviation of ischemia/reperfusion (I/R) injury is pertinent to patient survival and hospitalization rate [4], [5]. Previous studies have revealed multiple mechanisms for myocardial I/R injury including mitochondrial injury, apoptosis, oxidative stress, endoplasmic reticulum (ER) stress, inflammation and microvascular obstruction [2], [3], [6]. Although the etiology of I/R injury seems complex, mitochondria are considered the predominant determinant for cardiomyocyte fate following I/R injury [3], [7]. In addition to sustaining the high energy demand of myocardium, mitochondria also trigger cell death as the main driving force for reactive oxygen species (ROS) production [3], [8], [9]. In this context, rescue of functional mitochondria and selective clearance of dysfunctional mitochondria in cardiomyocytes during I/R injury are critical to maintain mitochondrial and cellular homeostasis [3].

To-date, multiple important mechanisms underlying the maintenance of mitochondrial homeostasis have been unveiled in mammalian cells [3]. Mitochondrial homeostasis is regulated by mitochondrial biogenesis, mitochondrial dynamics (fission, fusion and movement) and mitochondrial degradation. Mitochondrial autophagy (mitophagy) has long been considered the fundamental molecular machinery to maintain a healthy mitochondrial network, a process constituting segregation and subsequently elimination of damaged mitochondria [8], [10], [11]. It was demonstrated that mitochondrial fission driven by dynamin-related protein 1 (Drp1) facilitates the removal of dysfunctional portions by partitioning them to daughter mitochondria in an asymmetrical fashion to be selectively discarded by mitophagy [12], [13], [14]. Accordingly, cardiomyocyte-specific Drp1 knockout (Drp1-CKO) mice manifest buildup of damaged mitochondria and are more prone to I/R injury [15]. In contrast, previous work from Ong and colleagues described the utility of suppressing excessive mitochondrial fragmentation by dominant-negative Drp1 mutant or pharmacological Drp1 inhibitor in cardioprotection against I/R injury [16]. Therefore, the role of Drp1 in I/R injury remains to be further elucidated. Recent data also described a non-conventional mitophagy route driven by Drp1 where Rab9 protected the heart against ischemia by preserving healthy mitochondria [17]. Damaged mitochondria were also reported to be engulfed into multi-organellar lysosomal-like (LL) structures for degradation by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in I/R injury [18]. In addition, a recent study demonstrated that damaged mitochondria marked by Parkin-dependent ubiquitylation are sequestered into Rab5-positive early endosomes prior to the delivery to lysosomes for degradation [19]. Whether other machineries are present to participate in the elimination of dysfunctional mitochondria remains elusive.

Tre-2/Bub2/Cdc16 (TBC) domain family, member 15 (TBC1D15), a ubiquitously expressed protein, functions as a GTPase-activating protein (GAP) for Rab7 [20], [21]. TBC1D15 was originally found to regulate lysosomal morphology to confer protection against apoptosis in hematopoietic cell lines [22]. Emerging evidence has noted the involvement of TBC1D15 in the control of mitochondrial morphology as knockdown of TBC1D15 in HeLa cells provoked highly interconnected mitochondrial network structures [23]. In particular, TBC1D15 was found to foster untethering the GTP-bound Rab7-driven mitochondria–lysosome contact formation in mammalian cell lines [24], [25]. These findings from non-cardiac cells suggest that TBC1D15 may serve as a potential regulator of mitochondrial homeostasis. However, little is known with regards to the role of TBC1D15 in myocardial I/R injury. Previous study from our group also showed that TBC1D15 can improve autophagy flux via lysosomal regulation [26]. Although this observation has shed some lights towards a vital role of TBC1D15 in mitochondrial homeostasis [26], further study is still warranted to better elucidate the role of TBC1D15 in the governance of mitochondrial homeostasis in particular mitochondrial dynamics.

Herein, this study was designed to examine the role of TBC1D15 in I/R injury, particularly in the realm of mitochondrial quality control of cardiomyocytes. Our data suggested that TBC1D15 protected the heart against I/R injury through preserving mitochondrial homeostasis at the mitochondria–lysosome contact sites, denoting the role of TBC1D15 as a potential therapeutic target in I/R injury.