Activation of Ubiquitous Calpains Increases Cardiac Injury in Donation After Circulatory Death Donor Hearts by Degrading Mitofilin

1. Introduction

Heart transplantation is the definitive treatment for patients with end-stage heart failure. However, due to the limited availability of donor hearts from donation after brain death (DBD), hearts from donation after circulatory death (DCD) donors are also used for clinical transplantation [1,2]. Despite their potential, DCD hearts incur ischemia-reperfusion injury, which cause mitochondrial damage and potentially compromise their suitability for transplantation [3,4]. Ischemia-reperfusion injury impairs the electron transport chain and increases mitochondrial permeability transition pore (MPTP) opening in the DCD hearts [5,6]. While interventions before DCD heart procurement are not permitted, treatment during early reperfusion is a potential strategy to reduce cardiac injury. Reversible blockade of the electron transport chain with amobarbital during early reperfusion has been shown to decrease cardiac injury in DCD hearts [7,8]. Similarly, cyclosporine A treatment at the onset of reperfusion reduces cardiac injury by inhibiting MPTP opening [6,9]. These findings suggest that timely interventions during early reperfusion can effectively mitigate cardiac injury in DCD hearts, improving their potential for successful transplantation [10].

Calpain-1 (CPN1) and calpain-2 (CPN2) are two ubiquitous Ca²⁺-dependent cysteine proteases found in both the cytosol and mitochondria [11,12,13,14,15,16]. Activation of cytosolic CPN1 and CPN2 (cCPN1/2) increases cardiac injury during ischemia-reperfusion by degrading structural proteins, including spectrin [14,17,18,19]. Similarly, activation of mitochondria-localized CPN1 and CPN2 (mCPN1/2) impairs mitochondrial function during ischemia-reperfusion [19,20,21]. Specifically, mCPN1/2 activation contributes to decreased oxidative-phosphorylation and increases susceptibility to mitochondrial permeability transition pore (MPTP) opening in isolated rat hearts following ex vivo ischemia-reperfusion [14,20]. During the DCD process, hearts first undergo in vivo global ischemia before being procured for either ex vivo reperfusion. Thus, ischemia-reperfusion injury is inherent to DCD hearts [3,22,23]. However, it remains to be studied whether the activation of cCPN1/2 and mCPN1/2 contributes to ischemia-reperfusion injury in DCD hearts. In the current study, we investigated:(1) Whether the DCD process leads to the activation of cCPN1/2 and mCPN1/2. (2) Whether administering a CPN1/2 inhibitor at reperfusion would reduce cardiac injury in DCD hearts.

MPTP opening plays a central role in exacerbating cardiac injury in DCD hearts [6,9]. Recent studies indicate that alterations in mitofilin sensitize MPTP opening in hearts following ischemia-reperfusion [24,25,26]. Mitofilin, a key component of the mitochondrial inner membrane organizing system, is essential for maintaining mitochondrial structure and function [27]. It plays a critical role in preserving cristae architecture, which is crucial for sustaining oxidative phosphorylation and ATP production [27,28]. Additionally, mitofilin interacts with other components of the inner mitochondrial membrane organizing system to regulate mitochondrial dynamics, including fusion and fission [24,25,26]. A recent study demonstrated that mitofilin regulates MPTP opening by interacting with cyclophilin D through its C-terminal sequence. Cleaved mitofilin sensitizes MPTP opening due to its dissociation from cyclophilin D [25]. Furthermore, mitofilin content is decreased in rat hearts following ischemia-reperfusion [26]. However, it remains unclear whether the DCD process leads to alterations in mitofilin. Although MPTP opening is increased in DCD heart mitochondria [5], it is unknown whether this increase is due to mitofilin degradation. We hypothesize that mitofilin content is reduced in DCD heart mitochondria. Additionally, we will investigate whether this reduction in mitofilin content in DCD hearts is dependent on CPN1/2 activation.

2. Material and Methods

2.1. Measurement of Cardiac Function and Infarct Size

Male Sprague Dawley rats (3–4 months old) were divided into three groups: CBD group: Rat hearts were procured without in vivo ischemia and then perfused on a Langendorff setup for 90 minutes using modified Krebs-Henseleit (K-H) buffer containing 115 mM NaCl, 4.0 mM KCl, 2.0 mM CaCl₂, 26 mM NaHCO₃, 1.1 mM MgSO₄, 0.9 mM KH₂PO₄, and 5.5 mM glucose. The K-H buffer was oxygenated with 95% O₂/5% CO₂ to maintain a pH of approximately 7.4, and its temperature was regulated at 37°C using a circulatory water bath. Hearts were perfused at a constant pressure of 72 mmHg. Vehicle-treated DCD group: Rat hearts underwent 25 minutes of in vivo global ischemia before being procured and perfused on a Langendorff setup for 90 minutes. MDL-treated DCD group: Rat hearts underwent 25 minutes of in vivo global ischemia before being procured and perfused on a Langendorff setup for 90 minutes with K-H buffer containing MDL (10 µM) (Figure 1A). In DCD hearts following 25 minutes of global ischemia, a latex balloon-tip catheter was inserted into the left ventricle to monitor left ventricular function after 10 minutes of reperfusion. Heart rate and left ventricular developed pressure (LVDP) were monitored and analyzed using LabChart software (ADInstruments Inc., Colorado Springs, CO). The rate-pressure product (RPP = HR (heart rate) × LVDP) was used to account for cardiac function variability associated with heart rate. Infarct size was measured in rat hearts following 90 minutes of reperfusion using triphenyl-tetrazolium chloride (TTC) staining [6,9].

2.2. Isolation of SSM and IFM

Both subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from CBD, untreated DCD, and MDL-treated DCD hearts [21]. In this study, rat hearts were perfused for only 60 minutes on a Langendorff setup, as our previous study showed that this duration of reperfusion causes significant mitochondrial dysfunction in DCD hearts. A single rat heart tissue sample was submerged in cold (4°C) buffer A [100 mM KCl, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mM EGTA, 5 mM MgSO₄·7H₂O, and 1 mM ATP, pH 7.4]. The heart was then blotted dry, weighed, and minced in a beaker on ice. The minced tissue was resuspended in buffer B (buffer A + 0.2% bovine serum albumin) and homogenized using a Polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 seconds at 10,000 rpm. The homogenate was centrifuged at 500 × g for 10 minutes to separate supernatant 1 and pellet 1. Supernatant 1 was further centrifuged at 3,000 × g for 10 minutes to generate supernatant 2 and pellet 2 (crude SSM). Supernatant 2 was then centrifuged at 100,000 × g for 30 minutes to obtain a particle-free cytosol. Pellet 1 from the Polytron homogenate was resuspended in buffer A, homogenized, and incubated with trypsin (5 mg/g wet weight) for 10 minutes at 4°C [15]. The homogenate was then centrifuged at 500 × g for 10 minutes to separate supernatant 3, which contained IFM, from the pellet (cell debris). Supernatant 3 was further centrifuged at 3,000 × g for 10 minutes to isolate crude IFM. The crude SSM and IFM were washed twice with buffer B and then resuspended in KME buffer (100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA). Mitochondrial protein content was measured using the Lowry method, with bovine serum albumin as a standard. Since SSM are more sensitive to ischemic damage in DCD hearts, we focused our study exclusively on SSM in the current investigation [3,5,7].

2.3. Assessing Calcium Retention Capacity (CRC) in the Isolated SSM

CRC was used to reflect the sensitivity of calcium-induced mitochondrial permeability transition pore (MPTP) opening in freshly isolated mitochondria [29]. SSM (400 μg/ml) were incubated in a buffer containing 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 5 mM succinate, and 20 mM Tris/HCl, pH 7.4. Sequential addition of a known amount of calcium (20 nmol/pulse) was used to induce MPTP opening in the incubated mitochondria [5]. CRC referred to the total amount of calcium additions required to trigger MPTP opening in the isolated mitochondria. The calcium retention capacity is greater in mitochondria oxidizing a complex II substrate compared to a complex I substrate [30]. Thus, succinate was used as the substrate for CRC measurement. Extra-mitochondrial Ca²⁺ concentration was recorded using 0.5 µM Calcium Green-5N, and fluorescence was monitored with excitation and emission wavelengths set at 500 and 530 nm, respectively [29].

2.4. Western Blotting

Cytosol or mitochondrial proteins were separated using non-staining 12% or 4-15% Tris-glycine gels (Bio-Rad, Hercules, CA). The total protein content in the gel was digitally analyzed after UV activation using Image Lab 6.0 software (Bio-Rad, Hercules, CA). Proteins were then transferred from the gel to a PVDF membrane (Fisher Scientific, Hampton, NH) using semi-dry transfer (Bio-Rad) for 60 minutes at 20 V. The blots were incubated for 1 hour at room temperature in 5% (w/v) non-fat dry milk (Bio-Rad) and 1% BSA in TBST (Tris-buffered saline with Tween 20) buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20), followed by an overnight incubation at 4°C with primary antibodies (see information in Table 1). The membrane was washed four times (5 minutes each time) with TBST buffer. Then, the membrane was incubated at room temperature for 1 hour in the presence of a 1:10,000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG F(ab)₂ (GE Healthcare Life Sciences, Piscataway, NJ) secondary antibodies. The membrane was washed three times (10 minutes each time) with TBST buffer. Finally, the blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ). The protein images on the membranes were digitally recorded and analyzed using Image Lab 6.0 software (Bio-Rad, Hercules, CA) [31].

2.5. Statistical Analysis

Data are expressed as the mean ± standard error [23]. For all analyses, differences between groups (≥ 3 groups) were compared using one-way ANOVA when the data passed normality and equal distribution tests. When a significant F value was obtained, means were compared using the Student-Newman-Keuls test for multiple comparisons. Statistical significance was defined as a p-value of < 0.05.

3. Results

3.1. Inhibition of CPN1/2 Decreases Cardiac Injury in DCD Hearts

The heart rate (HR) and left ventricular developed pressure (LVDP) were decreased in DCD hearts during reperfusion compared to CBD hearts (Figure 1B,C). The rate pressure product (RPP, LVDPxHR) was also decreased in DCD hearts compared to CBD hearts (Figure 1D). These results suggest that cardiac function was impaired in DCD hearts following ischemia and reperfusion. HR, LVDP, and RPP in MDL-treated hearts were still decreased compared to CBD hearts (Figure 1B–D). Although MDL treatment did not improve HR in DCD hearts during reperfusion compared to untreated DCD hearts (Figure 1B), it did slightly improve LVDP and RPP in DCD hearts, especially during late reperfusion, compared to untreated DCD hearts (Figure 1C,D). Our previous study shows that infarct size is increased in DCD hearts compared to CBD hearts [6]. Here we found that MDL treatment led to a decreased infarct size in DCD hearts compared to untreated DCD hearts (Figure 2). These results indicate that inhibition of CPN1/2 reduces cardiac injury in DCD rat hearts following ischemia-reperfusion.

3.2. MDL Treatment Decreased MPTP Opening in DCD Hearts

The CRC was used to assess the sensitivity of MPTP opening in isolated SSM. Figure 3A shows the original tracing for the CRC measurement in the CBD, DCD, and DCD+MDL groups. The CRC in SSM isolated from untreated DCD hearts was decreased compared to the corresponding SSM from CBD hearts (Figure 3A,B), suggesting that the sensitivity of MPTP opening is increased in DCD heart mitochondria. MDL treatment led to an increased CRC in the SSM from treated DCD hearts compared to the SSM from untreated DCD hearts (Figure 3A,B), indicating that activation of CPN1/2 sensitizes MPTP opening in DCD heart mitochondria.

3.3. MDL Treatment Decreased the Activation of cCPN1/2 and mCPN1/2 in DCD Hearts

Spectrin is a substrate of cytosolic CPN1/2 (cCPN1/2) [18], and its activation leads to spectrin cleavage. An increase in cleaved spectrin at 150 kDa serves as a biomarker of cCPN1/2 activation [18,19]. There were no significant differences in spectrin content between the CBD, DCD, and DCD+MDL groups (Figure 4A,B), but the content of cleaved spectrin was significantly higher in DCD hearts compared to CBD hearts (Figure 4A,C), indicating that the DCD process activates cCPN1/2. MDL treatment reduced the content of cleaved spectrin in DCD hearts (Figure 4A,C), supporting its effectiveness in decreasing cCPN1/2 activation in DCD hearts. Total protein was used as a loading control.

Activation of mitochondrial CPN1/2 (mCPN1/2) contributes to mitochondrial damage during ischemia-reperfusion . Apoptosis-inducing factor (AIF) is a known substrate of mCPN1, and mCPN1 activation cleaves AIF, producing truncated AIF (tAIF) [32,33,34,35]. Mitochondrial CPN2 (mCPN2) may also play a role in AIF cleavage [12]. Thus, a decrease in AIF content or an increase in tAIF content serves as an indicator of mCPN1/2 activation. Compared to CBD hearts, AIF content (62 kDa) was reduced in DCD hearts (Figure 4D,E). However, MDL treatment preserved AIF content in DCD hearts (Figure 4D,E). A reduction in AIF should lead to an increase in tAIF (57 kDa) levels. However, no differences in tAIF content were observed among the groups (Figure 4D,F). AIF is anchored to the inner mitochondrial membrane, whereas tAIF is detached from the membrane [12]. Increased MPTP opening can cause tAIF to be released into the cytosol, potentially explaining the absence of elevated tAIF levels in DCD heart mitochondria [12,36]. These results support the activation of both cCPN1/2 and mCPN1/2 in DCD hearts. MDL treatment during reperfusion can still decrease CPN1/2 activation in the DCD hearts.

3.4. MDL Treatment Decreased the Degradation of Mitofilin in DCD Hearts

Recent studies indicate that mitofilin regulates MPTP opening through its interaction with cyclophilin D [24,25,26]. We investigated whether mitofilin content was reduced in DCD hearts subjected to 25 minutes of ischemia followed by 60 minutes of reperfusion. MDL treatment improved CRC in DCD hearts (Figure 3A,B), prompting us to examine whether MDL treatment also preserved mitofilin content in DCD hearts. Mitofilin content in isolated mitochondria was assessed using immunoblotting. Compared to CBD hearts, mitofilin content was significantly reduced in DCD hearts (Figure 5A,B). MDL treatment preserved mitofilin levels in DCD hearts (Figure 5A,B). These results support that the DCD process leads to decreased mitofilin content in a CPN1/2-dependent manner.

Cyclophilin D is a key regulator of MPTP opening [37]. Therefore, the content of cyclophilin D was measured in CBD and DCD heart mitochondria. There were no significant differences in the content of cyclophilin D between the CBD, DCD, and DCD+MDL groups (Figure 5C,D), indicating that the increased MPTP opening in DCD heart mitochondria was not due to a decrease in cyclophilin D content.

4. Discussion

In this study, we found that both cCPN1/2 and mCPN1/2 are activated in the DCD hearts. Reperfusion following ischemia leads to increased MPTP opening and decreased mitofilin content in DCD heart mitochondria. Inhibition of CPN1/2 using MDL reduces cardiac injury in DCD hearts. Additionally, inhibition of CPN1/2 decreases MPTP opening and preserves mitofilin content. These results suggest that activation of CPN1/2 increases MPTP opening in DCD hearts by degrading mitofilin. Mitofilin is a critical target for preserving mitochondrial integrity during ischemia-reperfusion injury. Protecting its function could be a key strategy to minimize cardiac damage and improve outcomes in ischemia-reperfusion injury related heart conditions.

Ischemia-reperfusion leads to activation of cytosolic and mitochondrial calpains in isolated mouse and rat hearts [32,33,38]. In the present study, we found that the formation of cleaved spectrin was increased in the DCD hearts, supporting the idea that the DCD process leads to activation of cytosolic CPN1/2. Mitochondrial CPN1 is identified in the mitochondrial intermembrane space and matrix [11,12], and CPN2 also exists in the matrix of cardiac mitochondria [36]. Activation of mitochondrial CPN1 within the intermembrane space augments cardiac injury during reperfusion by inducing the translocation of truncated AIF from mitochondria to the cytosol and nucleus [11,12]. Activation of mitochondrial CPN2 may also be involved in AIF cleavage [12]. Therefore, a reduced AIF content or an increased truncated AIF content is used as an indicator of mitochondrial CPN1/2 activation, at least in the intermembrane space compartment. AIF content was decreased in DCD hearts compared to CBD hearts, indicating that mitochondrial CPN1/2 was activated in the DCD heart mitochondria. A decrease in AIF content should lead to an increase in truncated AIF content. However, truncated AIF is not increased in the DCD hearts compared to CBD. Although AIF is bound to the inner mitochondrial membrane, truncated AIF is detached from it [12]. Thus, truncated AIF can be released from mitochondria to the cytosol and nucleus when the permeability of the mitochondrial membrane is increased in pathological conditions, including ischemia-reperfusion [39]. MPTP opening is increased in the DCD heart mitochondria, leading to increased permeation of the outer membrane. Consequently, some truncated AIF content can be released into the cytosol in DCD heart mitochondria through increased MPTP opening. This may explain why the DCD process does not lead to an increased content of truncated AIF. MDL treatment decreased spectrin cleavage and maintained AIF content in DCD hearts, supporting that MDL treatment given during reperfusion can effectively prevent both cytosolic and mitochondrial CPN1/2 activation. Thus, inhibition of CPN1/2 is a promising strategy to decrease cardiac injury in DCD hearts.

Opening of the MPTP increases cardiac injury during ischemia-reperfusion [40,41]. Our studies show that MPTP opening is also a key mechanism of cardiac injury in DCD hearts [6]. Recent studies have made significant advances in understanding the structure of the MPTP. The initial components of the MPTP include voltage dependent anion channels, the inorganic phosphate carrier, and the translocator protein. More recent studies suggest that ATP synthase is another key component of the MPTP [42,43]. However, the exact subunits of ATP synthase involved in MPTP formation remain uncertain. An increase in calcium levels within mitochondria leads to calcium binding to ATP synthase, resulting in a conformational change in ATP synthase that favors MPTP opening [42,43]. Cyclophilin D, a soluble protein in the mitochondrial matrix, is a key regulator of the MPTP. The binding of Cyclophilin D to MPTP components, including ATP synthase, markedly decreases the calcium threshold required to trigger MPTP opening [42,43]. Genetic elimination of Cyclophilin D inhibits MPTP opening [37], supporting the key role of Cyclophilin D in the regulation of MPTP opening. Pharmacologic inhibition of Cyclophilin D using cyclosporin A is also well-known to decrease MPTP opening. Treatment with cyclosporin A during early reperfusion leads to decreased cardiac injury in DCD hearts [6,9], supporting that inhibition of MPTP is a promising approach to reduce cardiac injury in DCD hearts.

Recent studies also indicate that mitofilin plays a crucial role in cardiac injury during ischemia-reperfusion by regulating MPTP opening through its interaction with Cyclophilin D via its C-terminal sequence [24,25]. Cleavage of mitofilin dissociates it from Cyclophilin D, which binds to MPTP components and promotes MPTP opening [24,25,26]. Reperfusion, especially following prolonged ischemia, leads to decreased mitofilin content and increased infarct size [26]. In this study, we found that mitofilin content was reduced in DCD hearts compared to CBD hearts following reperfusion. MDL treatment improved mitofilin content and decreased infarct size, supporting the notion that restoring mitofilin content is essential for reducing cardiac injury in DCD hearts.

Inhibition of CPN1/2 leads to decreased MPTP opening during ischemia-reperfusion [33,44]. The mechanisms are related to the protection of complex I, ATP synthase, and other components [20]. In this study, we show that inhibition of CPN1/2 helps preserve mitofilin. MDL treatment preserved mitofilin content, which in turn decreased Cyclophilin D binding to MPTP components, leading to reduced MPTP opening (Graphic abstract). Our results suggest that mitofilin is a substrate of CPN1/2, and activation of CPN1/2 sensitizes MPTP opening by degrading mitofilin in DCD hearts.

There are several limitations to our study. First, only male rats were used in this experiment. Our previous study showed that mitochondrial dysfunction and MPTP opening also occur in female rat DCD hearts [45]. Therefore, we anticipate that CPN1/2 will also be activated in female DCD hearts, and inhibition of CPN1/2 will reduce cardiac injury in these hearts as well. Although MDL is a well-known CPN1/2 inhibitor, it does have some off-target effects [46]. To clarify the relationship between CPN1/2 activation and mitofilin cleavage, future in vitro studies using purified CPN1/2 and mitofilin peptides are needed. Additionally, post-translational modifications of Cyclophilin D may influence MPTP opening [47,48]. While the content of Cyclophilin D did not change in DCD hearts, its post-translational modifications may be altered in these hearts, warranting further investigation.

5. Conclusion

Our study shows that CPN1/2 are activated in DCD hearts, leading to mitofilin degradation, which results in sensitizing the MPTP opening. Administering CPN1/2 inhibitor during early reperfusion is a promising strategy to decrease cardiac injury in the DCD hearts.