Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

Du, Yingzhen; Li, Jialei; Dai, Zhe; Chen, Yuxin; Zhao, Yao; Liu, **aoman; **a, Tian; Zhu, **jun; Wang, Yi**

doi:10.1007/s00018-024-05253-9

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

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Published: 10 June 2024

Volume 81, article number 254, (2024)
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Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

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Yingzhen Du¹,
Jialei Li²,
Zhe Dai²,
Yuxin Chen²,
Yao Zhao²,
**aoman Liu²,
Tian **a^3,4,
**jun Zhu¹ &
…
Yi** Wang ORCID: orcid.org/0009-0009-5507-3673¹

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Abstract

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2^TG) mice, PHB2^S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2^S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2^S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

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Introduction

As a severe complication of sepsis, septic cardiomyopathy (SC) manifests as reversible myocardial depression in patients with septic shock. Clinical features of SC include left ventricular dilation, decreased myocardial contractile function, and increased end-diastolic ventricular volume. Inflammatory injury, aberrant immune response, and cytokine storm have been proposed as the molecular bases of decreased myocardial function during sepsis-induced cardiomyopathy [1, 2]. However, findings from a randomized study in patients with virus-negative inflammatory cardiomyopathy suggest that immunosuppressive strategies may not provide additional clinical benefits for patients with SC [3, Full size image

In line with these findings, LPS-mediated PHB2 downregulation was nullified upon incubation of HL-1 cardiomyocytes with the proteasome inhibitor MG132 (Fig. 6F-G), suggesting involvement of the ubiquitin-proteasome system. On the contrary, and confirming proteasome involvement, PKM2 overexpression failed to sustain PHB2 expression in LPS-treated cardiomyocytes pre-incubated with betulinic acid (BA), a potent proteasome activator (Fig. 6H-I). Accordingly, protein stability analysis using pulse-chase assays showed that MG132 exposure counteracted LPS-stimulated PHB2 degradation (Fig. 6J), whereas PKM2 overexpression failed to extend the half-life of PHB2 in BA-treated cardiomyocytes (Fig. 6K). These results suggested that PKM2 overexpression in cardiomyocytes attenuates proteasome-mediated PHB2 degradation.

To elucidate the molecular basis underlying PKM2-mediated PHB2 stability, we evaluated the presence of constitutive interactions between PKM2 and PHB2 in cultured HL-1 cells. First, the active regions of PKM2 and PHB2 were screened by molecular docking simulations. We detected the existence of H-bond or hydrophobic interactions between PKM2 and PHB2 (Fig. 6L and M), with a minimum binding energy of -12.3 kcal·mol^− 1. Co-IP assays further confirmed an endogenous interaction between PKM2 and PHB2 under physiological condition or LPS stress (Fig. 6N and O). To define the structural moieties required for PKM2/PHB2 binding, we first mapped the regions of PKM2 that are required for protein-protein binding. Since PKM2 has four domains, we generated four different domain-deletion PKM2 mutants (Fig. 6P) and transfected them into HL-1 cardiomyocytes. Co-IP assays showed that PKM2ΔA, PKM2ΔC, and PKM2ΔN, but not PKM2ΔB, were able to pull-down PHB2 (Fig. 6Q).

PKM2 induces PHB2 phosphorylation

After we revealed an interaction between PKM2 and PHB2, we asked whether conformational changes occur in PHB2 for its stabilization after PKM2 binding. To address this question, we analyzed the regions of PHB2 that are required for its interaction with PKM2. As PHB2 has four major domains, we generated four different domain deletion PHB2 mutants (Fig. 7A) and transfected them into HL-1 cardiomyocytes. Co-IP assays showed that PHB2ΔN, PHB2ΔCC, and PHB2ΔC, but not PHB2ΔPHB, were able to pull-down PKM2 (Fig. 7B). This suggests that the PHB domain of PHB2 determines its binding to PKM2.

PKM2 has two conformations, a tetrameric one with high protein kinase (PK) activity that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP), and a dimeric one, which has lower PK activity and also exerts non-glycolytic enzymatic functions. Interestingly, co-IP results showed that PHB2 mainly interacted with dimeric, rather than tetrameric, PKM2 (Fig. 7C and F). Thus, LPS seems to interrupt the cooperation between PHB2 and dimeric PKM2. These results hinted that the kinase function of PKM2 is involved in the regulation of the PHB domain of PHB2. PKM2 has been reported to directly induce post-transcriptional phosphorylation of several proteins, such as ERK2^T202 [37], β-catenin^Y333 [38], histone H3^T11 [39], myosin light chain 2^Y118 [40], and protein kinase B substrate l (AKT1S1)^S202 [41]. On the other hand, PHB2 possesses three phosphorylation sites (S91, S176, and S243) which have been reported to enhance the anti-oxidative and anti-apoptotic properties of PHB2 through an undefined mechanism [42,43,44]. Since the PHB domain of PHB2 encompasses amino acids 68–185, we asked whether PKM2/PHB2 interaction results in PHB2 phosphorylation within this sequence fragment. Western blotting showed abundant expression of p-PHB2^S91 and little p-PHB2^S176 in heart tissues under baseline conditions. Neither LPS nor PKM2 overexpression had an impact on p-PHB2^S176 expression (Fig. 7G and I). In contrast, after exposure to LPS the expression of p-PHB2^S91 was downregulated in WT mice but remained at near normal levels in PKM2^Tg mice, compared with PBS treated mice (Fig. 7G and I). An in vitro kinase assay further showed that PKM2 induced PHB2 phosphorylation at S91, and this alteration was significantly inhibited by compound 3k, a PKM2 inhibitor (Fig. 7J and K).

To evaluate whether PKM2-mediated PHB2 phosphorylation at S91 is necessary for PHB2 stabilization, phosphorylation-defective (PHB2^S91A) and phosphorylation-mimetic (PHB2^S91D) PHB2 mutants were transfected into HL-1 cells. After transfection with PHB2^S91D, LPS-mediated PHB2 degradation was delayed in control cells (Fig. 7L and M). In turn, in cells transfected with a PHB2^S91A mutant, PKM2 overexpression failed to maintain PHB2 stability (Fig. 7L and M). Taken together, our results showed that PKM2 binds to PHB2 and phosphorylates it at S91, leading to increased PHB2 stability.

PHB2 dephosphorylation abolishes PKM2-mediated mitochondrial protection

To evaluate whether PHB2 phosphorylation underlies PKM2-related MQC activation during SC, further assays were conducted in HL-1 cells transfected with PHB2^S91A and PHB2^S91D mutants. Immunofluorescent assessment of mitochondrial morphology showed that LPS-induced mitochondrial fragmentation was attenuated by overexpression of either PHB2^S91D or PKM2 (Fig. 8A and C). However, in HL-1 cells transfected with PHB2^S91A, PKM2 overexpression failed to maintain mitochondrial length and reduce the number of cardiomyocytes with plenty of fragmented mitochondria (Fig. 8A and C). Further, western blot analysis showed that LPS-mediated disruption of mitochondrial fission/fusion and mitophagy could be normalized by overexpression of PHB2^S91D or PKM2 (Fig. 8D and J). In contrast, in HL-1 cells transfected with PHB2^S91A, PKM2 overexpression failed to sustain mitochondrial fission/fusion and mitophagy (Fig. 8D and J). Similarly, mt-Keima assays confirmed that either PHB2^S91D or PKM2 overexpression was able to sustain mitophagy in the presence of LPS, while PKM2-induced mitophagy was nullified by PHB2^S91A transfection (Fig. 8K and L). Furthermore, mitochondrial biogenesis was stimulated by PHB2^S91D or PKM2 overexpression in LPS-treated cells, while the effect of PKM2 overexpression was abrogated by PHB2^S91A transfection (Fig. 8M and N). Lastly, transcriptions of mtUPR-related genes were slightly increased by LPS and partially restored toward baseline levels upon overexpression of either PHB2^S91D or PKM2 (Fig. 8O-P). However, in cells transfected with the PHB2^S91A mutant, the inhibitory effect of PKM2 on mtUPR was inhibited (Fig. 8O and P). These data indicate that PKM2-mediated mitochondrial protection occurs through PKM2-dependent PHB2 phosphorylation.

PKM2-mediated cardiomyocyte protection against septic insult requires PHB2 phosphorylation

To illustrate the necessary role of PHB2 phosphorylation in supporting PKM2-induced cardioprotection, HL-1 cardiomyocyte viability and function were determined after transfection with PHB2^S91D or PHB2^S91A mutants. LPS exposure promoted the release of cardiac injury markers, e.g. TnI, CK-MB, and LDH, into the culture medium in control cells, and this trend was decreased by either PKM2 overexpression or introduction of the phospho-mimetic PHB2^S91D mutant (Fig. 9A and C). In line with the findings described above, PKM2 overexpression failed to prevent the upregulation of cardiac damage markers in HL-1 cells co-transfected with the phospho-defective PHB2^S91A mutant after LPS challenge (Fig. 9A and C). In turn, both cell viability and survival assessed by CCK-8 assay (Fig. 9D) and TUNEL staining (Fig. 9E and F), respectively, were improved by either PKM2 overexpression or PHB2^S91D mutant transfection. In contrast, PKM2 overexpression-mediated cardiomyocyte survival was decreased in cells co-expressing PHB2^S91A (Fig. 9D and F). Moreover, LPS-stimulated transcription of pro-inflammatory factors was significantly suppressed by either PKM2 or PHB2^S91D overexpression, while the anti-inflammatory action of PKM2 was impaired upon transfection of PHB2^S91A (Fig. 9G-H). Finally, either PKM2 or PHB2^S91D, but not PHB2^S91D transfection, largely preserved myosin filament expression and organization in cardiomyocytes exposed to LPS (Fig. 9I-J). The above evidence thus confirms that PKM2-mediated cardioprotection during septic conditions requires PHB2 phosphorylation.

Knockin mice expressing phospho-mimetic Phb2 ^S91D are less vulnerable to SC

To investigate whether PKM2-mediated PHB2 phosphorylation confers myocardial protection against SC in vivo, transgenic knockin mice carrying the Phb2^S91D variant were generated. Total-PHB2 was used as the loading control to analyze the changes in p-PHB2^S91. In comparison to WT mice, heart tissues from homozygous Phb2S91^D/D mice showed elevated levels of p-PHB2^S91 (Fig. 10A-B). Since PHB2 phosphorylation prevents PHB2 degradation, we further examined the alterations in total-PHB2 expression in WT, heterozygous Phb2S91^D/+, and homozygous Phb2S91^D/D mice. After normalizing to α-tubulin, the levels of total-PHB2 were significantly reduced in LPS-treated WT mice (Fig. 10C-D). However, these changes were partially prevented in Phb2S91^D/+ mice and fully restored in Phb2S91^D/D. Furthermore, the mtDNA/nDNA ratio was employed to assess potential alterations in mitochondrial content or mitochondrial copy numbers in transgenic knockin mice harboring the Phb2^S91D variant. As depicted in Fig. 10E, the mtDNA/nDNA ratio exhibited no significant changes among WT, heterozygous Phb2S91^D/+, and homozygous Phb2S91^D/D mice.

Heart function, assessed by echocardiography, was slightly improved in Phb2S91^D/+ mice (Fig. 10F and L), and largely normalized in Phb2S91^D/D mice upon LPS-induced SC. Similarly, LPS-stimulated TnI, CK-MB, and LDH were partially reduced in Phb2S91^D/+ mice and significantly inhibited in Phb2S91^D/D mice (Fig. 10M and O). Along with these changes, LPS-induced transcription of Mmp9, Mcp1, and Tnfα was downregulated in Phb2S91^D/+ mice, and further repressed in Phb2S91^D/D mice (Fig. 10P and R). In accordance with these alterations, myocardial accumulation of Gr-1 positive neutrophils was relived in Phb2S91^D/+ mice and completely prevented in Phb2S91^D/D mice (Fig. 10S and T). Lastly, LPS-induced cardiomyocyte death, as assessed by caspase-3 activity, was partly attenuated in Phb2S91^D/+ mice and remarkably inhibited in Phb2S91^D/D mice (Fig. 10U). Taken together, our results elucidated that PHB2 phosphorylation mediates myocardial protection against LPS-induced SC.

Discussion

The present study focused on the role of PKM2 in cardiomyocyte homeostasis and MQC regulation in the setting of SC. Our main findings showed that: (1) LPS reduced the levels of myocardial PKM2 mainly through suppressed expression of dimeric PKM2; (2) PKM2 overexpression in mice correlated with reduced myocardial inflammation and cardiomyocyte death after SC induction; (3) overexpression of PKM2 improved heart performance through repressing LPS-triggered mitochondrial damage; (4) PKM2-mediated mitochondrial protection in the presence of LPS correlates with normalized MQC, as evidenced by balanced mitochondrial fission/fusion events, activated mitophagy, increased mitochondrial biogenesis, and mitigated mtUPR; (5) upon LPS stress, PKM2 stimulated MQC through sustaining PHB2 expression; (6) PKM2 interacts directly with PHB2 and induces PHB2 phosphorylation at Ser91, resulting in increased PHB2 stability; (7) the protective actions of PKM2 on cardiomyocyte homeostasis and mitochondrial integrity are nullified upon expression of a phosphorylation-defective PHB2^S91A mutant construct. This study thus identified PHB2 dephosphorylation, leading to enhanced PHB2 protein degradation, as an initial signal determining MQC disruption during development of SC. Besides, we also showed that stabilized PKM2 expression is a prerequisite to sustain PHB2 phosphorylation after septic insult. These findings indicate that PKM2 downregulation, PKM2-PHB2 disassociation, PHB2 dephosphorylation, and MQC dysfunction are important determinators of sepsis-triggered myocardial injury.

Increasing evidence supports the beneficial effects offered by PKM2 in cardiovascular disorders. During cardiac development, loss of PKM2 in cardiomyocytes impairs cell cycling and reduces cardiomyocyte numbers and myocardium size [30]. By comparison, overexpression of PKM2 in heart tissues via cardiomyocyte-specific Pkm2 modified RNA protected the myocardium against acute or chronic myocardial infarction through enhancing heart function, inhibiting cell death, and repressing oxidative stress [30]. Mechanistically, PKM2-mediated cardioprotection seems to correlate with activation of anabolic pathways and β-catenin signaling, suggesting the dimeric PKM2, rather than tetrameric PKM2, is essential for maintaining heart performance [30]. Apart from its actions on cardiomyocytes, pharmacological activation of PKM2 in endothelial cells promotes proliferation and induces angiogenic differentiation [26]. Instead of mediating metabolic adaption, this beneficial effect has been reported to be associated with increased mitochondrial fusion/fission ratio, an effect accompanied by stabilized mitochondrial potential, decreased mitochondrial ROS generation, and angiogenesis-related gene expression [26]. Importantly, pulmonary hypertension [45], angiotensin II-triggered cardiac remodeling [46], and diabetic cardiomyopathy [47] were all shown to be relieved by PKM2 activation, either by genetic modification or pharmacological manipulation, through multiple mechanisms such as inhibition of TGF-β/Smad2/3 and Jak2/Stat3 pathways, oxidative stress, and NLRP3 inflammasome. These results highlight an unforeseen non-metabolic regulatory effect of PKM2 in cardioprotection. In accordance with the above results, our data also showed that SC is featured by a drop in cardiac levels of dimeric PKM2 and that re-introduction of PKM2 improves heart function through sustaining MQC. However, strategies aimed at modulating PKM2 expression require cautiousness, as the impact of its tetrameric and dimeric forms on heart performance would be different. Tetrameric PKM2 would increase glycolysis, a process less efficient than mitochondrial respiration in terms of ATP yield per glucose, despite demanding lower oxygen consumption. Of note, elevated PKM2-mediated glycolysis may result in lactic acidosis, a life-threatening medical condition during sepsis. Therefore, differential induction of dimeric PKM2 expression may be a safer approach to elicit cardioprotection.

A variety of regulatory mechanisms have been proposed to explain PHB2-mediated MQC control, including effects on total protein expression, post-transcriptional phosphorylation, and intracellular compartmentation. A reduction in total PHB2 expression in cardiac-specific PHB2 knockout mice impairs myocardial fatty acid oxidation (FAO) and induces heart failure through suppressing carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of cardiac mitochondrial FAO [48]. On the contrary, upregulation of PHB2 expression inhibits cardiomyocyte senescence and therefore delays heart aging through increased mitophagic flux [49]. In addition to altered PHB2 protein expression, phosphorylation of PHB2 at Ser91 is required for myocytic differentiation through MEF2-dependent transcription [44]. Meanwhile, experiments in leukemia cells showed that PHB2 phosphorylation at either S176 or S91 exerts anti-apoptotic actions by attenuating mitochondrial dysfunction [43]. In the nucleus, PHB2 regulates the expression of transcription factors involving in cell cycle progression [42], and increased nuclear PHB2 expression has been regarded as an early event for mitochondrial cristae disarrangement and disruption of inner mitochondrial membrane integrity [50]. Consistent with these findings, we found that experimental SC induces PHB2 downregulation and dephosphorylation in mouse cardiomyocytes. Importantly, our data further suggest that PHB2 dephosphorylation constitutes an endogenous mechanism inducing PHB2 degradation and downregulation. In fact, it is recognized that phosphorylation impacts protein degradation in a site-specific manner, influenced by surrounding amino acid composition, local structure, and phosphoprotein function [51]. In this regard, several proteins, including WRKY [52], p300 [53], c-Myb [54], and RhoA [55], may be degraded through post-transcriptional phosphorylation. Hence, our findings may serve to establish a novel cause-effect relationship between PHB2 phosphorylation and its total expression.

Limitation

Our present study has a few limitations that deserve mention. Firstly, although we observed that LPS had complex effect on PHB2 expression, additional experiments are required to figure out the mechanism by which LPS induces PHB2 degradation. Secondly, the evaluation of mitochondrial function primarily relied on in vivo experiments, with only a limited number of in vitro experiments analyzing mitochondrial respiration. Consequently, a more comprehensive assessment of mitochondrial function using additional in vitro techniques would strengthen our findings. Besides, the interactive effects between PKM2 and PHB2 were primarily examined in vitro. To better elucidate the underlying interplay mechanism of PKM2-mediated PHB2 phosphorylation, it is essential to conduct animal studies using genetically modified mice. Our present study has a few limitations that deserve mention. Firstly, although we observed that LPS had complex effect on PHB2 expression, additional experiments are required to figure out the mechanism by which LPS induces PHB2 degradation. Secondly, the evaluation of mitochondrial function primarily relied on in vivo experiments, with only a limited number of in vitro experiments analyzing mitochondrial respiration. Consequently, a more comprehensive assessment of mitochondrial function using additional in vitro techniques would strengthen our findings. Besides, the interactive effects between PKM2 and PHB2 were primarily examined in vitro. To better elucidate the underlying interplay mechanism of PKM2-mediated PHB2 phosphorylation, it is essential to conduct animal studies using genetically modified mice. Lastly, it appears that an additional regulatory mechanism is involved in the phosphorylation of PHB2 in Phb2^D/D knock-in mice during LPS exposure, as compared to LPS-treated WT mice and a phosphor-mimetic in vivo mutant seems to be attenuated by LPS due to undefined mechanism. We acknowledge these limitations and believe that addressing them in future studies will provide a more comprehensive understanding of the mechanisms involved.

Conclusion

In summary, our study highlights a novel mechanism by which PKM2 downregulation impairs MQC in cardiomyocytes during SC. Specifically, our results show that PKM2-mediated phosphorylation of PHB2 at Ser91 is a key upstream event that serves to normalize mitochondrial homeostasis under septic challenge. Our findings may lay the foundation for research and development of new drugs aimed at preventing MQC dysfunction in SC by targeting the PKM2/PHB2 axis.

Materials and methods

Septic cardiopathy mouse model

C57BL/6J wild-type (WT) male mice (8–12 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). PKM2 transgenic (PKM2^Tg) mice were generated by Cyagen Biosciences (Santa Clara, CA, USA) according to a previous study [31] with a minor modification. In brief, mouse full-length PKM2 cDNA was amplified, ligated with a pGEMT-Easy vector, and transformed in NEB 5-α competent E. coli. Purified transgene constructs were microinjected into C57BL/6J egg pronuclei to generate PKM2^Tg mice. PHB2^S91D-knockin mice on a C57BL/6 background were also generated by Cyagen Biosciences. All mice were born and maintained under pathogen-free conditions. Male mice were used to induce SC. LPS (E. coli 0111: B4, #2630, Sigma-Aldrich, USA) was dissolved in phosphate buffered saline at 10 mg/kg and administered to mice intraperitoneally. Systolic blood pressure was determined by a noninvasive tail-cuff instrument (XBP 1000; Kent Scientific, Torrington, CT, USA). After 24 h, mice were sacrificed and serum was collected to analyze white blood cells (WBC), neutrophils, and monocyte numbers using an automatic blood analyzer (Sysmex XT-2000i; Sysmex Corp., Shanghai, China).

Echocardiography

Echocardiograms were obtained with a Vevo 2100 Ultrasound System (FUJIFILM VisualSonics Inc., Toronto, Ontario, Canada) equipped with a high-frequency (30 MHz) linear array transducer. Mice were lightly anesthetized with isoflurane (3% for induction and 1.0-1.5% for maintenance) mixed with 1 L/min O₂ via a facemask [56]. Hair was removed from the chest using a chemical hair remover, and the animals were placed in the left lateral decubitus position. During measurements, body temperature was carefully monitored with a rectal temperature probe and maintained close to 37 °C with the use of a heating pad. Ultrasound gel was applied to the chest and two-dimensional, M-mode, and spectral Doppler images were acquired from modified parasternal long-axis and short-axis views. All images were digitally stored for off-line analysis using Vevo LAB software (version 1.7.1, VisualSonics). Systolic and diastolic anatomic measurements were obtained from M-mode images at the mid-papillary level. LV mass was estimated by the area-length method. Analysis was performed by an investigator who was blind to treatment allocation and unaware of data from other modalities [57].

Histological analysis and immunohistochemistry

Cardiac mouse tissue was fixed, embedded in paraffin, cut into 4 μm-thick sections, and stained with hematoxylin and eosin (HE). For ICAM1 and VCAM1 immunohistochemistry, 5 μm-thick frozen heart sections were prepared on a cryostat, fixed with methanol/acetone, treated with 3% H₂O₂ to quench endogenous peroxidase activity, blocked with 2% bovine serum albumin (BSA), and incubated with anti-VCAM1 (1:500; #ab215380; Abcam, Cambridge, UK) and anti-ICAM1 (1:500; #ab171123; Abcam) antibodies overnight at 4 °C. After extensive washing, sections were incubated with suitable biotinylated secondary antibodies (ABC Kit, MX-200-4; Agilent Technologies, Santa Clara, CA, USA) [58]. Positive staining was visualized using DAB substrate (Solarbio, Bei**g, China).

Immunofluorescence

Heart sections were prepared according to a standard procedure and then incubated with blocking buffer containing 10% (w/v) BSA. Afterward, the sections were incubated overnight at 4 °C with primary antibodies and subsequently with a fluorophore-conjugated secondary antibody (Alexa Fluor® 568 goat anti-rabbit IgG (H + L); A11036; Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were then stained with DAPI and images captured with a fluorescence microscope (BX51, Olympus, Japan). The primary antibodies were listed in the Supplementary Table 1.

Electron microscopy

Transmission electron microscopy was performed according to a previous report [59]. In brief, samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 1 h, and then washed in 100 mM phosphate buffer without CaCl₂. All samples were post-fixed in 1% osmium tetroxide for 1 h and stained in 2% uranyl acetate in maleate buffer pH 5.2 for an additional hour. The samples were rinsed, dehydrated in an ethanol series, embedded in resin (EMbed 812, EMS, Hatfield, PA, USA), and baked overnight at 60 °C. Hardened blocks were cut using a Leica Ultra Cut UCT microtome. Ultrathin Sect. (60 nm) were collected on carbon-coated grids and contrast-stained using 2% uranyl acetate and lead citrate. Embedded samples were analyzed by a JEM-1400 Plus electron microscope at 80 KV (Jeol Ltd., Tokyo, Japan).

Primary cardiomyocyte culture and treatment

Mouse hearts were removed and perfused using a Langendorff system for ∼ 3 min with Ca²⁺-free bicarbonate-based buffer. Enzymatic digestion was initiated by adding collagenase type B/D to the perfusion solution. After approximately 7 min, the left ventricle was removed and discarded. Male mice cardiomyocytes from the left ventricular wall were isolated as reported previously by us [60] and plated at 1 ∼ 2 × 10⁴ cells/cm² in mouse laminin pre-coated culture dishes. After 1 h culturing in a 5% CO₂ incubator at 37 °C, cardiomyocytes were incubated with Fura 2-AM (Invitrogen) for 10 min and then subjected to field stimulation at 2 Hz with platinum electrodes. Contraction and relaxation measures were determined as previously described [59]. Additional experiments were performed using the cardiac mouse HL-1 cell line (American Type Culture Collection, Manassas, VA, USA). To induce inflammation-mediated damage, HL-1 cells were treated with 10 µg/ml LPS for 24 h [35].

RNA extraction, reverse transcription, and qRT-PCR

Total RNA extraction was performed on heart tissues and HL-1 cells using TRIzol® reagent (Ambion® by Life Technologies, Austin, TX, USA) [14]. Purified RNA samples were reverse transcribed for cDNA synthesis using the GeneAmp™ RNA PCR Core Kit (Applied Biosystems, Waltham, MA, USA). qRT-PCR amplification of target genes and GAPDH (internal control) from heart tissues was performed using the TaqMan® Gene Expression Assay (Applied Biosystems). Amplification of target genes and GAPDH from HL-1 cells was performed using SYBR™ Green qPCR Master Mix (Applied Biosystems). The primers used are listed in Supplementary Table 2. An StepOnePlus™ Fast Real-Time PCR System (Applied Biosystems) was used for all qRT-PCR assays. The Cytochrome c oxidase subunit I (CO1) gene of the mtDNA was amplified by qPCR to determine the mtDNA copy. The transcript level of mtDNA was reflected by NADH dehydrogenase subunit 1 (ND1). The CO1 gene, representing the cytochrome c oxidase subunit I in mtDNA, and the NDUFV1 gene in nDNA were amplified using qPCR. The amplification process began with an initial step at 94 °C for 10 min, followed by 40 cycles consisting of 94 °C for 10 s, 60 °C for 30 s, and 94 °C for 10 s. All reactions were performed in duplicate. Subsequently, the amplification curves were analyzed using SDS 1.9.1 software (Applied Biosystems). Based on a previous study [61], these curves were utilized to determine the relative mtDNA: nDNA ratio in each sample.

Protein extraction and western blotting

Whole-cell protein lysates were extracted from heart tissues or HL-1 cells using RIPA lysis buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) supplemented with Complete Protease and PhosSTOP phosphatase inhibitors (both from Roche Diagnostics, Basel, Switzerland) [62]. To prepare mitochondrial protein lysates, mitochondrial fractions from human HL-1 cells were isolated using the Mitochondria Isolation Kit for Mammalian Cells (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Equal volumes of protein lysates were separated by SDS-PAGE and transferred onto PVDF membranes (GE Healthcare, Little Chalfont, UK). The antibodies used for western blotting are listed in Supplementary Table 1.

Seahorse assay

The Seahorse XFe96 extracellular flux analyzer (Agilent) was used to assess respiration and mitochondrial function. Cells were seeded on Matrigel-coated assay plates 7 days before measurement at a density of 20,000 cells per XFe96 well. Cells were washed twice in Agilent Seahorse XFe DMEM Basal Medium (Agilent Technologies, cat. no. 103575-100) supplemented with 2 mM glutamine, 10 mM glucose and 1 mM sodium pyruvate 1 h before the assay and for the duration of the measurement. For the standard profiling (Mito Stress test), oligomycin was injected at 1.5 µM, FCCP at 0.5 µM and rotenone/antimycin A were added at 0.5 µM. The oxygen consumption rate (OCR) was normalized to the number of nuclei quantified by DAPI staining. OCR and ATP production rates were obtained using the Seahorse Wave controller Software 2.6.1 (Agilent) as our previously described [63].

Co-immunoprecipitation

Co-immunoprecipitation (co-IP) was performed as described previously [20]. Briefly, HL-1 cells were harvested and then solubilized with IP buffer (150 mM NaCl, 10% glycerol, 20 mM Tris–HCl pH 7.4, 2 mM EDTA, 0.5% Nonidet P-40, 0.5% Triton X-100, and complete protease inhibitor) for 1.5 h at 4 °C. The insoluble material was removed by centrifugation at 12,000 × g for 10 min, and the supernatant was collected. One-tenth of the supernatant was pipetted out and used as ‘Input’. The remaining supernatant fraction was subsequently incubated with anti-Flag M2 affinity gel (Sigma-Aldrich) at 4 °C overnight. The beads were washed six times with lysis buffer, and the proteins (IP products) were eluted by boiling the beads in SDS sample buffer. Finally, the samples were analyzed by western blotting [64].

In vitro kinase assay

In vitro kinase assays were conducted at 27 °C for 90 min in 20 mM HEPES pH 7.0, 1 mM TCEP, 10 mM MgCl₂, 0.1 mM Na₃VO₄, and 0.6 µM BSA, with 1.6 µM recombinant mouse PHB2 protein (#MBS1037998; MyBioSource, Inc., San Diego, CA, USA), 12.5 µM ATP, 2 nM recombinant mouse PKM2 protein (#ab95474, Abcam), and 20 nM ADP-heptose or 20 nM DF-006 A, according to our previous reports [65].

Analysis of myocardial inflammation and myocardial injury markers

Quantitative measurements of myocardial inflammation and myocardial injury biomarkers in cell culture medium or mouse serum were performed using mouse ELISA kits: Serum Creatine Kinase MB (CK-MB) (#E4607; BioVision, Waltham, MA, USA), Troponin I (TNI) (#MBS854284; MyBioSource, Inc.), and Lactate Dehydrogenase (LDH) (#MBS720560; MyBioSource, Inc.) [66]. Apoptosis induction was evaluated with a Caspase-3 Colorimetric Assay Kit (#K106; BioVision, Inc.) and a Caspase-9 Colorimetric Assay Kit (#K119; BioVision, Inc.), as per manufacturer’s instructions [67].

Mitochondrial membrane potential, mitochondrial ROS, and mitochondrial permeability transition pore (mPTP) detection

For mitochondrial membrane potential quantification, HL-1 cells were incubated in media containing 10 nM JC-1 (#E-CK-A301; Elabscience, Houston, TX, USA) at 37 °C for 10 min. Images were captured under a fluorescence microscope (BX51, Olympus, Japan). MitoSOX™ Red Mitochondrial Superoxide Indicator (#M36008; Thermo Fisher Scientific) was used to evaluate the production of mitochondrial ROS (mROS) in cells under LPS treatment. In brief, cells were incubated with 5 nM MitoSOX™ Red at 37 °C for 10 min and then observed using a fluorescence microscope (BX51, Olympus, Japan). mPTP detection was performed using an Image-IT™ LIVE Mitochondrial Transition Pore Assay Kit (#I35103, Thermo Fisher Scientific) as per manufacturer’s instructions [68].

Adenovirus vector construction

A PKM2 overexpression plasmid was cloned into pcDNA3.1 (Invitrogen) vector for adenovirus production. PHB2 sequences were translated from HL-1 cell cDNA. PHB2 has four domains, including an N-terminal mitochondrial targeting domain (N, 1–50 aa), a PHB domain (PHB, 68–185 aa), a coiled coil domain (CC, 190–264 aa), and a C-terminal region (C, 265–299 aa). Full length PHB2, PHB2 without N-terminal domain (PHB2ΔN), PHB2 without C-terminal domain (PHB2ΔC), PHB2 without CC domain (PHB2ΔCC), and PHB2 without PHB domain (PHB2ΔPHB) were generated by PCR and ligated into pHAGE-HA vectors for adenovirus production. PKM2 contains an N-terminal domain (N, 1–43 aa), an A domain (44–116 aa and 219–389 aa), a B domain (117–218 aa), and a C region (390–531 aa). Full length PKM2, PKM2 without N domain (PKM2ΔN), PKM2 without A domain (PKM2ΔA), PKM2 without C domain (PKM2ΔC), and PKM2 without N domain (PKM2ΔN) were generated by PCR and ligated into pHAGE-Myc vectors for adenovirus production. The constitutively active (PHB2^S91D) and inactive (PHB2^S91A) PHB2 mutant plasmids were obtained to construct lenti-PHB2 viruses as previously described by us [69]. The adenovirus was packaged in 293T cells using Opti-MEM reduced serum medium, along with helper plasmids and PEI reagent. adenovirus supernatant was collected 48 h later and used to infect HL-1 cells after addition of polybrene. Positive clones were then selected by puromycin [69].

Pulse-chase analysis

Protein stability was determined through pulse-chase analysis as described by us previously [59]. In brief, HL-1 cells were first radiolabeled with [35 S]-Met (100 mCi) under complete medium for the indicated times at 37 °C in the absence or presence of LPS. After washing twice with PBS, cells were cultured under complete medium for the indicated duration. Then, cell samples were lysed and immunoprecipitated with protein G Plus beads coated with the indicated antibodies, followed by western blot analysis [70].

Cell viability

Cell viability was determined using a Cell Counting Kit-8 (CCK-8) (Do**do, Kumamoto, Japan) according to the manufacturer’s instructions [71]. HCC cells were seeded in 96-well plates (2 × 10³ cells per well) in a final volume of 100 µl. At the indicated time point, 10 µl of CCK-8 reagent was added into each well for 1 h at 37 °C, and absorbance was measured at 450 nm using a multimode plate reader (BioTek, USA).

Statistical analysis

Data were analyzed using GraphPad Prism 5 software and are shown as mean values and SEM. Differences between two groups were analyzed by unpaired t test if data were normally distributed. Otherwise, Mann-Whitney U test was used. One or two-way ANOVA was used for comparisons between more than two groups. Bonferroni post-hoc test was further performed to compare two specific groups. Two-tailed tests were performed in all statistical analyses, and p < 0.05 was considered significant.

Data availability

The data supporting the findings of this study are found within the article and the supplementary material. All relevant raw data will be made available from the corresponding author upon reasonable request.

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Funding

This study was supported by grants from Bei**g Natural Science Foundation (Nos. 7232150, 7242029, 7242028 and 7244449) NSFC (Nos. 82370083, 82200091, 82341089, 82270279 and 82200296).

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The Second Medical Center & National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital, Medical School of Chinese PLA, Bei**g, China
Yingzhen Du, **jun Zhu & Yi** Wang
School of Medicine, Southern Medical University, Guangzhou, Guangdong, China
Jialei Li, Zhe Dai, Yuxin Chen, Yao Zhao & **aoman Liu
Department of Clinical Laboratory Medicine, The First Medical Centre, Medical School of Chinese People’s Liberation Army, Bei**g, China
Tian **a
**anning Medical College, Hubei University of Science & Technology, **anning, China
Tian **a

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Yingzhen Du
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Jialei Li
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Yuxin Chen
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**aoman Liu
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Tian **a
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**jun Zhu
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Contributions

Yingzhen Du, Jialei Li, Zhe Dai, Yao Zhao, **jun Zhu conceived the original experiments. Jialei Li, Yuxin Chen, **aoman Liu and Tian **a contributed to the manuscript revision. Zhe Dai and Jialei Li carried out all the in vivo experiments and molecular investigation in vitro. **jun Zhu and Tian **a wrote the whole manuscript. **jun Zhu contributed to study concept and design, obtained funding, supervision of the study, literature search, writing of the manuscript and revised the final version of manuscript. All the authors read the article and approved the submission.

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Correspondence to **jun Zhu or Yi** Wang.

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Du, Y., Li, J., Dai, Z. et al. Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation. Cell. Mol. Life Sci. 81, 254 (2024). https://doi.org/10.1007/s00018-024-05253-9

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Received: 10 July 2023
Revised: 11 April 2024
Accepted: 20 April 2024
Published: 10 June 2024
DOI: https://doi.org/10.1007/s00018-024-05253-9

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Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation

Abstract

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Introduction

PKM2 induces PHB2 phosphorylation

PHB2 dephosphorylation abolishes PKM2-mediated mitochondrial protection

PKM2-mediated cardiomyocyte protection against septic insult requires PHB2 phosphorylation

Knockin mice expressing phospho-mimetic Phb2 S91D are less vulnerable to SC

Discussion

Limitation

Conclusion

Materials and methods

Septic cardiopathy mouse model

Echocardiography

Histological analysis and immunohistochemistry

Immunofluorescence

Electron microscopy

Primary cardiomyocyte culture and treatment

RNA extraction, reverse transcription, and qRT-PCR

Protein extraction and western blotting

Seahorse assay

Co-immunoprecipitation

In vitro kinase assay

Analysis of myocardial inflammation and myocardial injury markers

Mitochondrial membrane potential, mitochondrial ROS, and mitochondrial permeability transition pore (mPTP) detection

Adenovirus vector construction

Pulse-chase analysis

Cell viability

Statistical analysis

Data availability

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

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Ethics approval and consent to participate

Conflict of interest

Additional information

Publisher’s Note

Electronic supplementary material

Supplementary Material 1

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Knockin mice expressing phospho-mimetic Phb2 ^S91D are less vulnerable to SC