Abstract

   To rescue ischemic myocardium from progressing to myocardial
   infarction, timely identification of the infarct size and reperfusion
   is crucial. However, fast and accurate identification, as well as the
   targeted protection of injured cardiomyocytes following
   ischemia/reperfusion (I/R) injury, remain significantly challenging.
   Here, a near infrared heptamethine dye IR‐780 is shown that has the
   potential to quickly monitor the area at risk following I/R injury by
   selectively entering the cardiomyocytes of the at‐risk heart tissues.
   Preconditioning with IR‐780 or timely IR‐780 administration before
   reperfusion significantly protects the heart from ischemia and
   oxidative stress‐induced cell death, myocardial remodeling, and heart
   failure in both rat and pig models. Furthermore, IR‐780 can directly
   bind to F0F1‐ATP synthase of cardiomyocytes, rapidly decrease the
   mitochondrial membrane potential, and subsequently slow down the
   mitochondrial energy metabolism, which induces the mitochondria into a
   “quiescent state” and results in mitochondrial permeability transition
   pore inhibition by preventing mitochondrial calcium overload.
   Collectively, the findings show the feasibility of IR‐780‐based imaging
   and protection strategy for I/R injury in a preclinical context and
   indicate that moderate mitochondrial function depression is a mode of
   action that can be targeted in the development of cardioprotective
   reagents.

   Keywords: imaging, ischemia, mitochondria, protection, reperfusion
     __________________________________________________________________

   This work identifies a near infrared heptamethine dye, IR‐780,
   exhibiting stress‐induced selective enrichment in the at‐risk heart
   tissues following ischemia/reperfusion (I/R) injury and further
   protecting heart from I/R and oxidative stress induced cell death by
   targeting the mitochondrial F0F1‐ATP synthase of the cardiomyocytes,
   which promises IR‐780 as a potential ischemia‐selective agent for
   imaging and protection of myocardial I/R injury.

   graphic file with name ADVS-11-2307880-g001.jpg

1. Introduction

   Obstructive coronary artery disease‐induced myocardial infarction is
   one of the leading causes of morbidity and death worldwide.^[ [52]^1 ^]
   At present, the only method to rescue ischemic myocardium from
   myocardial infarction progression is the timely identification of the
   infarct size and reperfusion. However, in addition to salvage ischemic
   myocardium from infarction, reperfusion itself could also induce a
   specific additional component of irreversible injury.^[ [53]^2 ^] Thus,
   adjunct cardioprotection and reperfusion are urgently needed.
   Previously, several cardioprotective strategies including ischemic
   conditioning, ischemic conditioning‐derived pharmacological
   interventions, hypothermia, and vagal stimulation were developed but
   shown disappointingly poor translation into a clinical benefit in
   patients with acute myocardial infarction.^[ [54]^3 ^] The reason of
   the unsatisfactory cardioprotective strategies may be because of a poor
   understanding of the underlining pathologic mechanism and subsequently
   failing to accurately develop targeting drugs. Therefore, investigating
   the exact regulation signaling pathway following ischemia/reperfusion
   (I/R) and developing effective and preferably targeted cardioprotective
   strategy that can be administered before or during reperfusion remain
   major unmet medical needs.^[ [55]^3b ^]

   The mitochondria, the key organelle for most of the cell's energy
   production, are also the center of cell death regulation.^[ [56]^4 ^]
   According to previous studies of ischemic conditioning, the
   mitochondria have been shown as one of the most significant ending
   targets of all cardioprotective signaling pathways.^[ [57]^5 ^] Thus,
   developing mitochondria‐targeting cardioprotective strategies holds
   great potential. Previous evidence has indicated that irreversible
   opening of the mitochondrial permeability transition pore (mPTP) is the
   key molecular event during I/R to induce necrosis,^[ [58]^4 ^] which
   has been shown to be the main mode of cell death following I/R.^[
   [59]^6 ^] The mechanism studies of ischemic conditioning have shown
   that the protective effects of mitochondrial K [ATP] ^[ [60]^7 ^] and
   connexin 43,^[ [61]^8 ^] which are key targets of ischemic
   conditioning, are related to mPTP inhibition by decreasing
   mitochondrial calcium (Ca^2+) overload. Moreover, pharmaceutical mPTP
   opening inhibition before reperfusion could effectively decrease the
   infarcted area.^[ [62]^9 ^] Unfortunately, previous mPTP‐related
   cardioprotective drugs including cyclosporine A (CsA) have shown no
   significant cardioprotective effects in preclinical studies or clinical
   trials.^[ [63]^10 ^] The reason may be because of not fully
   understanding the molecular nature of the mPTP,^[ [64]^11 ^] thereby
   leading to the failure of a structure‐based drug design. Recent studies
   have shown that complex V of the mitochondrial oxidative
   phosphorylation (OXPHOS) electron transport chain (also named F0F1‐ATP
   synthase) is the key component of mPTP,^[ [65]^12 ^] suggesting that
   the mitochondrial OXPHOS electron transport chain is a potential
   cardioprotective target.^[ [66]^13 ^] It has shown cardioprotective
   effects by selectively binding with the S‐nitrosation of Cys39 on the
   ND3 subunit, thereby inhibiting complex I activation during
   reperfusion.^[ [67]^14 ^] Pharmaceutical inhibition of complex II
   activity during I/R has shown to be an effective cardioprotective
   strategy by succinate accumulation regulation during the ischemic stage
   and its oxidation during the subsequent reperfusion.^[ [68]^15 ^]
   However, whether complex V would be a potential cardioprotective target
   during I/R injury remains unclear owing to the lack of a direct complex
   V‐targeted agent or strategy.

   Here, we characterized IR‐780, a near infrared (NIR) heptamethine dye,
   which could be quickly and selectively uptaken by the injured
   cardiomyocytes of the area at risk. NIR imaging revealed that IR‐780
   would be a smart and practical probe for the early identification of
   infarcted areas. Furthermore, preconditioning with IR‐780 or timely
   IR‐780 administration before reperfusion significantly protects the
   heart from ischemia and oxidative stress‐induced cell death, myocardial
   remodeling, and heart failure in both rat and pig models. The
   protective effects of IR‐780 rely on binding to complex V of the
   mitochondrial OXPHOS electron transport chain, thereby rapidly
   decreasing the mitochondrial membrane potential and subsequently
   slowing down the mitochondrial energy metabolism, which induces the
   mitochondria into a “quiescent state.” Additionally, by inhibiting
   mitochondrial Ca^2+ overload, IR‐780 partially inhibited mPTP opening.
   These results show that IR‐780 is a potential ischemia‐selective agent
   for the imaging and protection of myocardial I/R injury.

2. Results

2.1. IR‐780 Identifies the Area at Risk of Infarction

   IR‐780 is a heptamethine dye with NIR fluorescent properties.^[ [69]^16
   ^] Thus, we first investigated the feasibility of its in vivo imaging.
   Following left coronary artery ligation of the pig heart, IR‐780 was
   intraperitoneally injected. Ten minutes later, the heart was harvested
   for NIR imaging. The ischemic area showed significantly low NIR signals
   (Figure [70]S1A–C, Supporting Information), which indicated successful
   ligation and IR‐780 as a workable in vivo imaging dye. Next, we
   detected the dynamic changes of IR‐780 in the injured heart tissues.
   Interestingly, the NIR signals in the area at risk was significantly
   increased as injury aggravated in both rat ischemia and I/R models (as
   early as 2 h following reperfusion or 4 h following ischemia, when the
   infarct tissues could be detected using triphenyl tetrazolium chloride
   [TTC] staining) (Figure [71]1A,B and Figure [72]S2A,B, Supporting
   Information). The comparison rate of the area at risk by Evans blue and
   IR‐780 and the infarct size by TTC staining and IR‐780 are
   96.79% ± 1.21 and 45.35% ± 4.34, respectively. Moreover, at 48 h
   following I/R, IR‐780 remained selectively retained in the infarcted
   area in both rat and pig I/R models (Figure [73]1C,E,F; Video [74]S1,
   Supporting Information), when obvious morphological infarct injury
   emerged.^[ [75]^17 ^] Laser confocal images of the IR‐780‐labeled
   infarct tissues showed that IR‐780 signals were selectively accumulated
   in the cardiomyocytes of the area at risk and the infarcted area,
   whereas they were significantly low in the cardiomyocytes of the normal
   area (Figure [76]1D,G; Figure [77]S2C, Supporting Information).
   Further, we tested the dynamic accumulation of IR‐780 in the injured
   heart tissues when IR‐780 was injected immediately following ischemia
   (Figure [78]S3A, Supporting Information). Thirty minutes later, low
   IR‐780 signals were detected in the ischemia area (Figure [79]S3B,
   Supporting Information), indicating IR‐780 infiltration even during
   ischemia. Following reperfusion, IR‐780 quickly accumulated in the
   injured heart tissues as early as 10 min (Figure [80]S3C,D, Supporting
   Information). Moreover, the NIR signal could be detected even at 5 days
   following IR‐780 injection (Figure [81]S4A,B, Supporting Information),
   which suggested the in vivo imaging stability of IR‐780. In addition,
   it showed much higher fluorescence intensity in the injured heart
   tissues than the main organs including liver, lung, spleen, kidney,
   muscle and intestine 2 h post the reperfusion, and the contrast value
   of fluorescence between the area at risk of heart and the main organs
   ranged from 1.07–6.15 (Figure [82]S4C,D, Supporting Information). These
   results demonstrated that IR‐780 is a potential NIR fluorescent small
   molecule to quickly identify the myocardial area at risk by targeting
   the injured cardiomyocytes.

Figure 1.

   Figure 1
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   IR‐780 identifies the area at risk of infarction following I/R. A)
   Schematic of the IR‐780 based imaging protocols in rat I/R model. B)
   Left: representative image of the NIR imaging 2 h following reperfusion
   in rat I/R model. Right: the corresponding bright‐field photograph of
   the same tissue following TTC staining. Scale bar, 5 mm. C) Left:
   representative image of the NIR imaging of the whole heart 48 h
   following reperfusion in rat I/R model. Right: the corresponding NIR
   imaging of the transverse sections. Scale bar, 5 mm. D) representative
   confocal images of the injured heart tissues 48 h following reperfusion
   in rat I/R model. AAR: area at risk, IFA: infarcted area. Scale bar,
   25 µm. E) Schematic of the IR‐780 based imaging protocols in pig I/R
   model. F) up: representative image of the in vivo NIR imaging and
   bright‐field photograph of the injured heart 48 h following reperfusion
   in pig I/R model; down: the corresponding NIR imaging and bright‐field
   photograph of the corresponding heart transverse sections. G)
   representative confocal images of the injured heart tissues 48 h
   following reperfusion in pig I/R model. Lg: ligation, AAR: area at
   risk, IFA: infarcted area, CtnT: Cardiac Troponin T. Scale bar, 25 µm.

2.2. IR‐780 Binds to Plasma Albumin Proteins and Releases in the Acidic
Microenvironment

   We subsequently investigated how IR‐780 accumulates in the injured
   heart tissues. A previous study has reported that some heptamethine
   indocyanine dyes exhibit high affinity to albumin proteins in the
   plasma, thereby resulting in enhanced fluorescence and permeability,
   and retention effects.^[ [84]^18 ^] First, we developed human serum
   albumin proteins‐IR‐780 (HSA‐IR‐780) nanoparticles, and the injection
   of these nanoparticles following reperfusion enhanced the NIR signals
   in the injured heart tissues following I/R injury (Figure [85]2A).
   Second, the absorbance spectra of IR‐780 dissolved in
   phosphate‐buffered saline (PBS) with HSA were significantly increased
   (Figure [86]2B). Moreover, the albumin‐bond site of IR‐780 was further
   determined using specific inhibitors (warfarin, ibuprofen, digoxin, and
   quinidine). Of them, ibuprofen (an albumin binding site II inhibitor)
   significantly decreased the ‐fluorescence intensity of the IR‐780
   solutions with HSA (Figure [87]2C,D). We further incubated the H9C2
   cells with IR‐780 solutions with or without HSA or ibuprofen; we
   observed a decreased uptake of IR‐780 with HSA, which was recovered by
   ibuprofen (Figure [88]S5A,B, Supporting Information). During ischemia,
   a metabolic switch to increase lactate production causes a decrease in
   tissue pH.^[ [89]^19 ^] With the decrease in pH levels from 7.19 to
   3.38, the characteristic spectra of HSA binding IR‐780 complexes
   shifted to that of free IR‐780 beginning at low pH levels (≈6.23)
   (Figure [90]2E), suggesting that heart ischemic acidic microenvironment
   benefits from its specific release. The abovementioned data suggest
   that IR‐780 binds to plasma albumin proteins and is released into the
   acidic microenvironment associated with ischemia.

Figure 2.

   Figure 2
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   The uptake mechanism of IR‐780 by cardiomyocytes following I/R. A)
   Representative NIR imaging and confocal images of the heart tissues 2 h
   following reperfusion in rats treated with IR‐780 or HSA‐IR‐780. Scale
   bar: 75 µm. B) The absorbance of the IR‐780 dispersed in PBS and HSA.
   C,D) The albumin‐bond site of IR‐780 was determined with specific
   inhibitors (warfarin, ibuprofen, digoxin, and quinidine, used as
   competitive inhibitors of albumin‐binding sites I, II, III, and
   α1‐glycolipoprotein, respectively) (n = 3 per group). E) The absorbance
   changes of IR‐780 dispersed in HSA within different pH conditions. F,G)
   Representative images and quantitative data of the NIR fluorescent
   signals in H9C2 cells exposed to IR‐780 pre‐treatment with ice
   condition, sulfobromophthalein (BSP), amiloride (an actin inhibitor),
   chlorpromazine (a clathrin inhibitor) and MβCD (a caveolae inhibitor)
   (n = 3 per group). Scale bar: 75 µm. H) Schematic of the in vivo
   protocols to verify the roles of OATPs on the IR‐780 uptake. I)
   Representative NIR photographs of the whole heart (left panel),
   transverse sections (middle panel) and micro‐photographs of the heart
   tissue received IR‐780 injection with or without pre‐BSP local
   administration in rat I/R models. Scale bar: 75 µm. J,K) Representative
   images and quantitative flow cytometric data of the NIR fluorescent
   signals in H9C2 cells exposed to IR‐780 within different pH conditions
   (n = 3 per group). Scale bar: 50 µm. L) Quantitative flow cytometric
   data of the mitochondrial membrane potential of the H9C2 cells exposed
   to hypoxia and I/R (n = 3 per group). M,N) Representative images and
   quantitative flow cytometric data of the NIR fluorescent signals in
   H9C2 cells exposed to IR‐780 pre‐treatment with FCCP, Rotenone,
   Oligomycin A (n = 3 per group). Scale bar: 50 µm. All the p values are
   present in the graphs.

2.3. Uptake of IR‐780 by Cardiomyocytes is Dependent on Organic Anion
Transporting Polypeptides (OATPs) and Mitochondrial Membrane Potential

   Next, we investigated the uptake of IR‐780 by cardiomyocytes in the
   at‐risk heart tissues. In vitro, a significantly decreased IR‐780
   uptake was observed with ice incubation, indicating that the cellular
   uptake of IR‐780 is an energy‐dependent process (Figure [92]2F,G).
   Furthermore, sulfobromophthalein (BSP) (an OATP inhibitor) remarkably
   affected the cellular uptake of IR‐780, suggesting that IR‐780 uptake
   was actively mediated by OATP transporters. However, pretreatments with
   various endocytotic inhibitors including amiloride (actin inhibitor),
   chlorpromazine (clathrin inhibitor), and methyl‐β‐cyclodextrin (MβCD,
   caveolae inhibitor) had no effects on IR‐780 uptake (Figure [93]2F,G),
   suggesting that IR‐780 uptake was not mediated by endocytotic
   mechanism. Furthermore, the local injection of BSP into the left
   ventricular front wall 5 min before rat I/R significantly decreased
   IR‐780 signals detected by heart tissue NIR imaging and laser confocal
   imaging (Figure [94]2H,I). These data indicate that OATPs mainly
   mediated IR‐780 uptake by cardiomyocytes. A previous study has reported
   that OATP activity is closely related to pH levels.^[ [95]^20 ^] We
   subsequently detected IR‐780 uptake under four pH conditions from 5.2
   to 8.9. IR‐780 uptake was significantly increased in a pH level of 6.2,
   whereas it was decreased in pH levels of 5.2 and 8.9 (Figure [96]2J,K),
   indicating that the OATP activity was increased in a weakly acidic
   microenvironment associated with ischemia. Further, we verified that
   IR‐780 uptake was significantly increased when exposed to hypoxic
   conditions, whereas it was decreased when exposed to hypoxia and high
   pH levels (8.9) (Figure [97]S5C,D, Supporting Information). Moreover,
   during ischemia, the mitochondrial function was inhibited and exhibited
   high mitochondrial membrane potential (Figure [98]2L). The H9C2 cells
   treated with carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone
   (FCCP, an uncoupler of OXPHOS) and rotenone (a complex I inhibitor)
   exhibited significantly decreased IR‐780 uptake, whereas it was
   increased when treated with oligomycin A (a mitochondrial F0/F1‐ATPase
   inhibitor) (Figure [99]2M,N). These data indicate that IR‐780 uptake by
   cardiomyocytes is also dependent on the mitochondrial membrane
   potential.

2.4. IR‐780 Protects the Heart from I/R‐Induced Myocardial Death and Heart
Failure

   IR‐780 has been shown to target ischemic cardiomyocytes; subsequently,
   we further asked whether IR‐780 could affect the cell fate of these
   cardiomyocytes following I/R. Surprisingly, both IR‐780 administered
   72 h before ischemia and immediately following ischemia (Figure
   [100]3A) markedly decreased the I/R‐induced myocardial infarct size
   from 50.4% to 13.7% and 15.4% of the area at risk, respectively;
   however, the groups had comparable areas at risk (Figure [101]3B–D).
   Notably, IR‐780‐preconditioned and treatment hearts were resistant to
   I/R‐induced myocardial necrosis, as evidenced by the sharp decrease in
   Evans blue dye (EBD) penetration and lactate dehydrogenase (LDH)
   release (Figure [102]3E–G). Additionally, the results of TUNEL staining
   showed the protective effects of IR‐780 preconditioning and treatment
   on I/R‐induced myocardial apoptosis (Figure [103]3H;Figure [104]S6A,
   Supporting Information). Consistent with those of previous studies,^[
   [105]^6 ^] our results also indicated a relatively lower proportion of
   apoptosis than that of necrosis following I/R. Moreover, transmission
   electron microscope images showed that IR‐780 preconditioning and
   treatment significantly reduced mitochondrial rupture and myocardial
   fiber breakage following I/R (Figure [106]3I), whereas, in rats without
   I/R, no significant morphologic changes were observed following IR‐780
   preconditioning or treatment (Figure [107]S6B, Supporting Information).
   In an ischemic injury rat model (4 h ischemia without reperfusion),
   IR‐780 preconditioning and treatment also significantly decreased the
   ischemia‐induced myocardial infarct size from 45.15% to 6.7% and 15.4%,
   respectively (Figure [108]S7A–C, Supporting Information). Moreover, the
   decreased mitochondrial rupture or swelling following ischemia was
   shown in IR‐780‐preconditioned and treatment hearts (Figure [109]S7D,
   Supporting Information). These results suggest that besides imaging,
   IR‐780 is also an effective cardioprotective agent for decreasing the
   I/R‐induced myocardial cell death.

Figure 3.

   Figure 3
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   IR‐780 protects the heart from the I/R induced cell death and heart
   failure. A) Schematic of the IR‐780 based cardio‐protective protocols.
   B–D) Representative photographs and quantitative data for infarct size,
   area at risk (AAR) in rats of control (n = 5), IR‐780 treatment (n = 6)
   and IR‐780 precondition (n = 7) groups subjected to I/R; scale bar,
   2 mm. E, F) Representative photomicrographs and quantitative data from
   images of myocardial EBD uptake in rats of control (n = 5), IR‐780
   treatment(n = 6) and IR‐780 precondition (n = 8) groups subjected to
   I/R injury. Scale bar, 1 mm. G) Serum LDH concentrations in rats of
   control (n = 8), IR‐780 treatment (n = 7) and IR‐780 precondition (n =
   7) groups with I/R injury. H) Quantitative data of TUNEL staining
   images in rats of control, IR‐780 treatment and IR‐780 precondition
   groups with I/R injury (n = 6 per group). I) Representative
   Transmission Electron Microscope (TEM) photomicrographs of the heart
   tissues in rats of control, IR‐780 treatment and IR‐780 precondition
   groups with I/R injury. Scale bar, 0.5 µm; arrows, rupture
   mitochondria. J,K) Representative photographs and quantitative data of
   the fibrosis area in rats of control (n = 9), IR‐780 treatment (n = 9)
   and IR‐780 precondition (n = 12) groups with 30 min cardiac ischemia
   and 4 weeks reperfusion. Scale bar, 2 mm. L–N) Averaged data ejection
   fraction (EF, L), fractional shortening (FS, M) and systolic left
   ventricle internal diameter (LVID d, N) assessed by echocardiography in
   rats of sham(n = 10), control (n = 10), IR‐780 treatment (n = 9) and
   IR‐780 precondition (n = 10) groups with sham or 30 min cardiac
   ischemia and 4 weeks reperfusion. O,P) Representative photographs and
   quantitative data of the fibrosis area in male pigs (4‐month‐old
   weighing 28–32 kg) of control (n = 3), IR‐780 treatment (n = 3) and
   IR‐780 precondition (n = 4) groups with 90 min cardiac ischemia and 4
   weeks reperfusion. Scale bar, 2 mm. Q–S), Averaged data ejection
   fraction (EF, Q), fractional shortening (FS, R) and systolic left
   ventricle internal diameter (LVID, S) assessed by echocardiography in
   rats of sham(n = 2), control (n = 3), IR‐780 treatment (n = 3) and
   IR‐780 precondition (n = 4) groups with sham or 90 min cardiac ischemia
   and 4 weeks reperfusion. All the p values are present in the graphs.

   As cardiomyocyte cell death plays significant roles in the pathological
   process of adverse cardiac remodeling and heart failure.^[ [111]^4 ,
   [112]^21 ^] we subsequently evaluated the potential effects of IR‐780
   on these sequelae of I/R injury. In a rat chronic I/R experimental
   setting (30 min ischemia followed by 4‐week reperfusion), IR‐780
   preconditioning and treatment effectively blocked I/R‐induced cardiac
   fibrosis (Figure [113]3J,K). Furthermore, the I/R‐induced cardiac
   contractile dysfunction (as indicated by decreases in fractional
   shortening and ejection fraction) and left ventricular dilation (as
   assessed by systolic left ventricular internal diameter) were
   significantly abolished by IR‐780 preconditioning and treatment
   (Figure [114]3L–N;Figure [115]S8A, Supporting Information). Notably, we
   evaluated the protective effects of IR‐780 in a pig chronic I/R model
   (90‐min ischemia followed by 4‐week reperfusion). Interestingly, IR‐780
   preconditioning nearly completely abolished the I/R‐induced cardiac
   fibrosis area from 24.1% to 2.1%, and two of the four pigs in the
   IR‐780 precondition group showed no fibrosis (Figure [116]2O,P).
   Furthermore, the I/R‐induced cardiac fibrosis area was significantly
   decreased by IR‐780 treatment from 24.1% to 10.1% (Figure [117]2O,P).
   Moreover, IR‐780 preconditioning completely restored the cardiac
   contractile dysfunction and left ventricular dilation; IR‐780 treatment
   improved the cardiac contractile dysfunction (Figure [118]2Q–S;Figure
   [119]S8B, Supporting Information). In addition, results of the blood
   routine, liver function, renal function indicated no significant
   changes of the main organ functions 1 month following IR‐780
   administration in both pig and rat I/R models (Figures [120]S9 and
   [121]S10, Supporting Information). Therefore, the in vivo data of both
   acute and chronic models of I/R or ischemia injury indicate that IR‐780
   is a potential candidate for protecting I/R or ischemia‐induced
   myocardial cell death and the sequelae of adverse cardiac remodeling
   and heart failure.

2.5. IR‐780 Targets the Mitochondria and Depresses the Mitochondrial Membrane
Potential

   Subsequently, we investigated how the protective effects of IR‐780
   (Figure [122]S11A, Supporting Information) work in cardiomyocytes. We
   first evaluated the IC50 of IR‐780, which was ≈2.5 µm (Figure [123]4A).
   Thus, we selected a relatively low concentration of 0.1 µm for the
   following in vitro experiments. Additionally, IR‐780 preconditioning
   and treatment protects the H9C2 cells from I/R‐induced necrosis and
   H[2]O[2]‐induced apoptosis, as evidenced by the significant reduction
   of LDH release and PI/Annexin V positive proportion, respectively
   (Figure [124]4B–D). As IR‐780 is an NIR fluorescent small molecule, we
   further investigated the subcellular localization of IR‐780 by
   detecting its fluorescent signals. Results indicated that IR‐780
   superiorly accumulated in the mitochondria as shown by co‐staining with
   MitoTracker green (Figure [125]4E;Figure [126]S11B, Supporting
   Information). A previous study has indicated that the mitochondria are
   one of the major sources of reactive oxygen species (ROS), which is one
   of the significant factors for inducing cell death during I/R.^[
   [127]^22 ^] We evaluated whether IR‐780 could inhibit ROS production.
   Reversely, IR‐780 increased the mitochondrial and total cellular ROS
   production (Figure [128]4F,G;Figure [129]S11C,E, Supporting
   Information). Twenty‐four hours following IR‐780 administration, the
   total cellular ROS significantly decreased, whereas the mitochondrial
   ROS remained higher than the control (Figure [130]4F,G). Data of the in
   vitro hypoxia or I/R models have also indicated no reduction of
   mitochondrial ROS in both IR‐780‐preconditioned and treatment groups
   (Figure [131]4H). Notably, IR‐780 administration sharply decreased the
   mitochondrial membrane potential even at a very low concentration of
   0.1 µm (Figure [132]4I;Figure [133]S11D,F, Supporting Information). The
   mitochondrial membrane potential could recover to the normal level at
   ≈6 h following a low‐dose IR‐780 administration (Figure [134]S11D,
   Supporting Information). A previous study reported that mitochondrial
   membrane potential reduction could induce the subsequent mitochondrial
   ROS burst.^[ [135]^22 ^] Conversely, excess mitochondrial ROS
   accumulation also decreased the mitochondrial membrane potential.^[
   [136]^23 ^] Our results of the mitochondrial ROS and mitochondrial
   membrane potential showed that the mitochondrial membrane potential
   decrease was accompanied with mitochondrial ROS increase, whereas the
   mitochondrial membrane potential recovered more rapidly than the
   mitochondrial ROS (Figure [137]S11C,D, Supporting Information),
   suggesting that the mitochondrial membrane potential reduction causes
   the mitochondrial ROS increase. A previous study has shown that the
   decrease in the mitochondrial membrane potential could induce
   mitochondrial swelling.^[ [138]^24 ^] IR‐780 induced mitochondrial
   swelling at 20 min and was sustained until ≈24 h (Figure [139]4J).
   These results suggest that the protective effects of IR‐780 are
   mediated by mitochondrial membrane potential regulation.

Figure 4.

   Figure 4
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   IR‐780 targets the mitochondria of the cardiomyocytes and affects the
   mitochondrial function. A) Cell viability assays for H9C2 cells exposed
   to increasing doses of IR‐780 over the course of 24 h (n = 6 pre
   group). B) LDH concentrations of cell culture medium in cells of
   control, IR‐780 treatment and IR‐780 precondition groups with 2 h
   ischemia and 1.5 h reperfusion (n = 6 per group). C,D) Representative
   images and quantitative data of the apoptosis in cells of sham, IR‐780,
   control, IR‐780 treatment and IR‐780 precondition groups with or
   without 400 µm H[2]O[2] for 2 h (n = 3 per group). E) Representative
   confocal images of the H9C2 cells treated with IR‐780 (0.1 µm) and
   co‐staining with Mito‐tracker green. Scale bar, 25 µm. F,G) Total cell
   (F) and mitochondrial (G) ROS production of H9C2 cells 20 min and 24 h
   exposed to IR‐780 (0.1 µm), (n = 3 per time point). H) mitochondrial
   ROS production of H9C2 cells in control, IR‐780 treatment, IR‐780
   precondition groups exposed to sham or ischemia (2 h) or ischemia (2 h)
   and reperfusion (5 min), n = 3 per group. I) Mitochondrial membrane
   potential test (TMRM staining) of H9C2 cells exposed to IR‐780 (0.1 or
   0.5 µm) for 20 min, n = 3 per group. J) Representative TEM
   photomicrographs of H9C2 cells exposed to IR‐780 (0.1 µm) over the
   course of 48 h. Scale bar, 0.5 µm. All the p values are present in the
   graphs.

2.6. IR‐780 Disrupts the Cell Metabolism of the Cardiomyocytes

   As the mitochondrial membrane potential is the basement of cell energy
   metabolism, we next performed a non‐targeted metabolomics analyses on
   cardiomyocytes at 20 min, 6, 24, and 72 h following IR‐780
   administration. The results of the metabolic profiles were clearly
   segregated in the different groups based on PLS‐DA analysis (Figure
   [141]5A). Pathway analysis identified central carbon metabolism in
   cancer, ABC transporters, and purine metabolism as the three greatest
   enrichment among the altered metabolites (Figure [142]5B). Importantly,
   among the identified metabolites involved in the three pathways,
   metabolites involved in tricarboxylic acid cycle and subsequent OXPHOS
   including pyruvate, acetyl coenzyme A, succinate, ATP, and ADP were
   significantly decreased following IR‐780 administration, whereas the
   metabolites in glutamine, uridine, and purine metabolisms were markedly
   increased (Figure [143]5C–E). Further analysis of the OXPHOS,
   glycolysis, and tricarboxylic acid cycle pathways indicated significant
   inhibition following IR‐780 administration (Figure [144]S11H–J,
   Supporting Information). Notably, these changes recovered ≈24 h
   following IR‐780 administration (Figure [145]5C–E; Figure [146]S11H–J,
   Supporting Information). Subsequently, we performed oxygen consumption
   rate (OCR) measurements and noted a dose‐dependent IR‐780 inhibition
   that was equally as potent as that of well‐characterized OXPHOS
   inhibitors (Figure [147]5F). However, prolonged (72 h) OCR measurements
   showed that cardiomyocytes fully recovered from this depletion (Figure
   [148]S11G, Supporting Information). These data indicate that low‐dose
   IR‐780 induced the mitochondria into a “quiescent state” and
   reprogramed the glucose metabolism into glutamine, uridine, and purine
   metabolisms, suggesting that the compensative metabolism pathways play
   positive roles in the protection of cardiomyocytes from I/R injury by
   potentially fueling the stressed cardiomyocytes, as recently
   reported.^[ [149]^25 ^]

Figure 5.

   Figure 5
   [150]Open in a new tab

   IR‐780 disrupts cell metabolism of the cardiomyocytes. A) The partial
   least squares‐discriminant analysis (PLS‐DA) score plot showing group
   distribution based on metabolite profiles of cell culture media samples
   of H9C2 cells exposed to IR‐780 (0.1 µm) over the course of 72 h, n = 6
   per time point. B) Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway enrichment analysis identified metabolites with −log10 (p
   value). The top 10 enriched pathways are shown. C–E) Heatmap of
   identified metabolites in the central carbon metabolism in cancer (C),
   ABC transporters (D) and Purine metabolism (E) pathways, n = 6 samples
   per group. F) Seahorse analyzer assays show that H9C2 cells undergo
   acute OCR inhibition. IR‐780 (introduced after 18 min) at four doses
   (0, 0.1, 0.5 and 1 µm; represented by four colored lines); FCCP
   (introduced after 48 min); and a mixture of rotenone and antimycin A
   (Rot and AA; introduced after 72 min). G‐K) Mitochondrial complex
   activities assay for isolated mitochondria exposure to different IR‐780
   concentrations, n = 3 per concentration. All the p values are present
   in the graphs.

2.7. IR‐780 Interacts with Respiratory Chain Complex V Proteins

   As OXPHOS is accomplished by respiratory chain complexes I–V, we
   further estimated the effects of IR‐780 on the activities of
   respiratory chain complexes. Complex V, also called mitochondrial F0F1
   ATP synthase, was mostly inhibited complex even at a very low
   concentration of 0.05 µm (≈20% decrease) (Figure [151]5G–K). We
   synthesized an active analog that was amenable to biotin moiety
   modification, which we named B‐IR‐780 (IC50 approximately equal to
   4.5 µm for H9C2 cells), to further investigate the targets of IR‐780
   (Figure [152]6A ;Figure [153]S12A, Supporting Information). B‐IR‐780
   conserves IR‐780 properties, including mitochondrial targeting, OCR
   inhibition, ROS induction, and reduction in mitochondrial membrane
   potential (Figure [154]6B–D; Figure [155]S12B,C, Supporting
   Information). Biotin‐mediated pull‐down experiments using
   B‐IR‐780‐pretreated H9C2 cells, followed by electrophoresis, resolved
   multiple Coomassie blue‐stained bands that were absent in biotin‐ or
   IR‐780‐incubated H9C2 cells, indicating IR‐780‐specific interactions
   (Figure [156]6E). We conducted mass spectrometry analysis of eluate
   from pull‐down samples from the mitochondria of B‐IR‐780‐treated H9C2
   cells and detected 33 mitochondrial proteins, of which eight were
   components of OXPHOS machinery ; components covering complexes III, IV,
   and V, and complex V was best represented (six proteins) (Figure
   [157]S12D, Supporting Information). Of them, the ATP synthesis subunits
   α, β, and the proton channel subunit, atp5mc2, were listed. Next, we
   used the whole cell lysis pull‐down assay to verify the interactions
   between the IR‐780 and complex V components. Results showed significant
   interactions between B‐IR‐780 and ATP synthesis subunits α, β, and
   atp5mc2 (Figure [158]6F), whereas it failed to pull down the other
   three components (data not shown). Moreover, the results of the PLA
   experiment supported the interactions between B‐IR‐780 with these three
   components of complex V (Figure [159]6G;Figure [160]S12E, Supporting
   Information). The abovementioned data indicate that complex V is the
   key target of IR‐780.

Figure 6.

   Figure 6
   [161]Open in a new tab

   B‐IR‐780 interacts with complex V proteins of OXPHOS. A) B‐IR‐780
   structure. B) Representative confocal images of the H9C2 cells treated
   with B‐IR‐780 (1 µm) and co‐staining with Mito‐tracker green. Scale
   bar, 25 µm. C) B‐IR‐780 induced OCR inhibition. B‐IR‐780 (0, 0.1, 5,
   10 µm) was added after 18 min, followed by FCCP (added after 48 min)
   and a mixture of rotenone and antimycin A (72 min). n = 3. D)
   Mitochondrial membrane potential test (TMRM staining) of H9C2 cells
   exposed to IR‐780 (5 or 10 µm) for 20 min. n = 3 per group. E) Sodium
   dodecyl sulfate gel protein Coomassie Blue stain from B‐IR‐780
   pull‐down assay using H9C2 cells treated with Biotin, B‐IR‐780, IR‐780,
   which demonstrates IR‐780‐specific interactions. N = 3. F) Biotin
   pull‐down followed by western blot with specific antibodies for
   atp5f1a, atp5f1b, and atp5mc2 validates complex V protein interaction
   with the IR‐780 moiety of B‐IR‐780. G) Proximity Ligation Assay for
   H9C2 cells exposed to biotin or B‐IR‐780 demonstrates the
   IR‐780‐specific interactions with complex V subunit atp5mc2. All the p
   values are present in the graphs.

2.8. IR‐780 Inhibits the mPTP via Decreasing the Mitochondrial Calcium

   Next, we identified the missing link between the protective effects of
   IR‐780 and its interactions with complex V, which is well known to
   phosphorylate ADP and generate ATP by exploiting the proton gradient
   generated by complexes I, III, and IV.^[ [162]^26 ^] Furthermore,
   complex V is considered to be the one of the key components of the
   mPTP.^[ [163]^3 , [164]^12 , [165]^27 ^] The irreversible opening of
   the mPTP has been proven to be the leading cause of cardiomyocyte
   necrosis during I/R.^[ [166]^4 ^] We subsequently investigated whether
   IR‐780 protects cardiomyocytes from I/R injury via influencing the mPTP
   function. CsA, a well‐accepted mPTP inhibitor, significantly reversed
   the IR‐780‐induced mitochondrial membrane potential decrease (Figure
   [167]7A and Figure [168]S13A, Supporting Information). Interestingly,
   in another mPTP opening assessment using the calcein–cobalt quenching
   assay,^[ [169]^28 ^] IR‐780 increased the calcein fluorescence
   intensity, whereas ionomycin, a Ca^2+ ionophore and effective activator
   of mPTP, decreased it (Figure [170]7B,C;Figure [171]S14E,F, Supporting
   Information). The results in freshly isolated mitochondria from adult
   rat heart is consistent with that in cells (Figure [172]S14A,B,
   Supporting Information). However, Mito tracker, also a mitochondrial
   accumulated dye, has no effect on the mPTP activity (Figure
   [173]S14C,D, Supporting Information). Surprisingly, ionomycin did not
   decrease the mitochondrial membrane potential (Figure [174]7A;Figure
   [175]S13B, Supporting Information), and CsA could both inhibit mPTP
   opening and mitochondrial membrane potential decrease. Further, we
   continuously monitored the calcein fluorescence intensity of the cells
   with or without IR‐780 pretreatment followed by ionomycin
   administration. Data showed that the slope of the calcein fluorescence
   intensity reduction was significantly inhibited in the
   IR‐780‐pretreated cells (Figure [176]7D,E). These data indicate that
   the mPTP and the proton channel (known as ATP synthase c‐Subunit)
   targeted by IR‐780 are two independent channels, and both are regulated
   by CypD. IR‐780‐mediated proton channel opening could inhibit the mPTP
   opening. A previous study has reported that high Ca^2+ concentrations
   in the mitochondrial matrix is the major stimulus for mPTP opening
   during I/R.^[ [177]^4 ^] Thus, we evaluated whether IR‐780 could
   influence Ca^2+ concentrations in the mitochondrial matrix. Results of
   the adult rat cardiomyocytes staining with Rhod‐2 showed that IR‐780
   could reduce the fluorescence intensity of Rhod‐2 in cells with and
   without ionomycin (Figure [178]S14G,H, Supporting Information). We
   subsequently generated an H9C2 cell line expressing a mitochondrial
   localized, genetically encoded Ca^2+ indicator, 4mt‐GCaMP6, whose
   fluorescence intensity reflects the free Ca^2+ level in the
   mitochondria. Moreover, we developed a single‐cell optical signal
   detecting system, which could detect the real‐time optical signal
   dynamic at a single‐cell level (Figure [179]7F). Using this system, we
   monitored the real‐time mitochondrial Ca^2+ dynamics of H9C2 cells with
   or without IR‐780 pretreatment following ionomycin administration.
   Results showed that H9C2 cells with IR‐780 pretreatment significantly
   decreased the maximal mitochondrial Ca^2+ concentrations and delayed
   the time to reach the top following the ionomycin treatment
   (Figure [180]7G,H). Furthermore, using a traditional spinning disk
   confocal super resolution microscope in a real‐time model, we verified
   the results. Consistent with the results detected using the single‐cell
   system, data from the microscope also showed that IR‐780 significantly
   decreased the mitochondrial Ca^2+ concentrations and inhibited the
   mitochondrial Ca^2+ overload (Figure [181]S15A–D, Supporting
   Information). These results indicate that IR‐780 could inhibit mPTP
   opening by preventing mitochondrial Ca^2+ overload.

Figure 7.

   Figure 7
   [182]Open in a new tab

   IR‐780 inhibits the mPTP activity and decreases the mitochondrial
   calcium. A) Mitochondrial membrane potential test (TMRM staining) of
   H9C2 cells exposed to sham, IR‐780 (0.1 or 0.5 µm) with or without
   pre‐treatment of cyclosporin A (CsA, 10 µm), ionomycin (1or 2.5 µm).
   B,C) Representative confocal images (B) and quantitative flow cytometry
   data (C) of the mitochondrial Calcein in the H9C2 cells exposed to
   sham, IR‐780 (0.1 or 0.5 µm) with or without pre‐treatment of
   cyclosporin A (CsA, 10 µm), ionomycin (1 µm). Scale bar, 25 µm, n = 3
   per group. D,E) IR‐780 inhibits the mPTP activity. The mPTP activity
   assay (the decrease slope of the mitochondrial Calcein) (D) and
   representative traces of mitochondrial Calcein (E) during time
   monitored by the fluorescence of H9C2 cells exposed to ionomycin(1 µm)
   with or without pre‐IR‐780 (0.1 µm) (15 min before ionomycin
   administration), n = 3 per group. F) Schematic of the single cell‐based
   detecting of the fluorescence of 4mt‐GCamp6 in H9C2 cells. G,H) The
   maximal fluorescence of 4mt‐GCamp6 of each single cell (G) and the
   traces of mitochondrial Ca^2+ concentration during time monitored by
   the fluorescence of 4mt‐GCamp6 of each single cell (H) exposed to
   ionomycin(1 µm) with or without pre‐IR‐780 (0.1 µm) (15 min before
   ionomycin administration), n = 5 per group. All the p values are
   present in the graphs.

3. Conclusion

   The infarct size is a major determinant of the outcomes and prognosis
   of patients with acute myocardial infarction.^[ [183]^29 ^] It depends
   on the size of the ischemic area at risk of infarction. Thus, accurate
   identification of the area at risk and infarct size as early as
   possible is of significant benefit for the following therapy. In this
   study, we developed a novel methodology for assessing the area at risk
   of infarction at the early stage of the condition (as early as 10 min).
   The current methods for evaluating the area at risk following I/R
   injury, which mainly include positron emission tomography /computed
   tomography with radionuclides such as sodium [18F] fluoride,^[ [184]^30
   ^] radionuclides labeled molecules with injured related phospholipids
   binding activities,^[ [185]^31 ^] and T2‐weighted cardiac magnetic
   resonance imaging (T2‐CMR).^[ [186]^32 ^] Major limitations of the
   first two methodologies include adverse effects to hazardous ionizing
   radiation, intrinsically limited spatial resolutions,
   reconstruction‐dependent poor temporal resolution, high cost and lack
   of both exogenous and endogenous probes for molecular or functional
   imaging.^[ [187]^33 ^] The T2‐CMR is based on the edema at the injured
   sites. However, the experimental evidence points to substantial edema
   occurring in the infarcted region, with minimal or no edema occurring
   in the portion of the area at risk for a reversible injury.^[ [188]^34
   ^] NIR fluorescence imaging within the wavelength range of 700–900 nm
   (the “biological transparency NIR window”) has attracted immense
   attention for in vivo imaging in both fundamental scientific research
   and clinical practice. In contrast with the aforementioned tomographic
   imaging modalities, NIR fluorescence imaging does not suffer from the
   same drawbacks, and instead provides the benefits of real‐time
   wide‐field image acquisition and diffraction‐limited, high sensitivity,
   high spatial resolution in living organisms. ^[ [189]^16 , [190]^33 ,
   [191]^35 ^] However, there still lacks of favorable NIR probe for real
   time monitoring myocardial ischemia. In this study, we identified that
   IR‐780 as a stess‐indcued selective enrichment agent in the area at
   risk of myocardial infarction, which suggesting IR‐780 could be served
   as a myocardial injury detective probe. This is the first account of an
   ischemia‐selective cardioprotective fluorescent agent, utilizing the
   pathological differences between risk and non‐risk cardiomyocytes to
   drive uptake. However, to develop advanced NIR imaging devices, more
   effort is required.

   Notably, our findings showed that IR‐780 could not only identify the
   area at risk of infarction but also more significantly protect
   cardiomyocytes from I/R‐induced cell death (Figure [192]8 ). The
   mitochondria, which is also the key regulator of both necrosis and
   apoptosis, are the primary target of IR‐780.^[ [193]^4 ^] Of note, the
   changes in mitochondrial membrane permeability were the most
   significant events for inducing apoptosis and necrosis. The
   permeabilization of the outer mitochondrial membrane allows the release
   of cytochrome c into the cytosol where it functions as a cofactor in
   apoptosome assembly. The Ca^2+‐dependent mPTP opening located in the
   inner mitochondrial membrane mediated mitochondrial necrosis. Notably,
   IR‐780 could significantly decrease cardiomyocyte necrosis and
   apoptosis. Consistent with the results of a previous study,^[ [194]^4 ,
   [195]^6 ^] our results indicated that compared with apoptosis, necrosis
   was the major mode of cell death during I/R. Thus, strategies to block
   the mPTP opening during I/R were sufficient to inhibit the I/R‐induced
   cell death. Previously, several drugs (reviewed in ref^[ [196]^11 ^])
   have been reported to inhibit the mPTP opening, which have shown
   protective roles in animal I/R models. However, the candidate drugs
   failed to induce a consistent cardioprotective effect in further
   clinical trials.^[ [197]^10 , [198]^36 ^] Thus, developing novel
   mPTP‐targeting drugs remains urgently needed. The results of this study
   showed that mitochondrial respiratory chain complex V (also named
   F0F1‐ATP synthase) was the primary target of IR‐780, which could induce
   a rapid mitochondrial membrane potential decrease. Recent studies have
   reported that respiratory chain complex V is also the key component of
   the permeability transition pore.^[ [199]^11 , [200]^37 ^]
   Surprisingly, IR‐780 decreased the mitochondrial membrane potential
   (evaluated using TMRM, which is also used to reflect mPTP activity^[
   [201]^38 ^]) and conversely increased the calcein fluorescence
   intensity (another experiment to evaluate mPTP activity^[ [202]^39 ^]).
   Moreover, our results showed that ionomycin, a Ca^2+ ionophore,
   significantly decreased calcein fluorescence intensity, whereas it had
   little effects on TMRM intensity. This suggests that the pore response
   to Ca^2+ and the pore targeted by IR‐780 are two different channels.
   Interestingly, these two channels were inhibited by CsA, suggesting
   that they abut in space. Further analysis of the IR‐780‐targeting
   components of complex V showed that the ATP synthase c‐subunit was in
   the list, suggesting that IR‐780 induced the c‐ring opening, dispersed
   the electron gradient, and more importantly inhibited the
   Ca^2+‐regulated mPTP opening. However, the exact interactions of these
   two pores require further investigation.

Figure 8.

   Figure 8
   [203]Open in a new tab

   Schematic illustration of IR‐780 based imaging and cardioprotection of
   myocardial I/R injury. IR‐780 based imaging: IR‐780 is administrated
   immediately after reperfusion, and IR‐780 directly binds to plasma
   albumin proteins, go into the area at risk through  enhanced
   permeability and retention effects and releases in the acidic
   microenvironment associated with ischemia. Then, accumulated in the
   ischemic cardiomyocytes of the infarct area as the injury aggravation,
   and the infarct size could be assessed by a mobile NIR image Scope
   system. IR‐780 based cardioprotection: IR‐780 is administrated 72 h
   before ischemia or immediately after ischemia. Then, IR‐780 is
   increased uptake by the ischemic cardiomyocytes through enhanced
   activity of OATPs within the acidic microenvironment associated with
   ischemia, accumulated in the mitochondria of the cardiomyocytes
   dependent on the mitochondrial membrane potential, and binds to the
   respiratory chain complex V components (including ATP synthesis subunit
   α, β, the proton channel subunit atp5mc2), which leads to rapidly
   decrease of the mitochondrial membrane potential, subsequently slowing
   down of the mitochondrial energy metabolism, preventing the
   mitochondrial Ca^2+ overload and inhibition of the mPTP. Thereby, this
   mitochondrial “quiescent state” reduces cell death from I/R injury.
   NIR: Near Infrared; OATPs: organic anion transporting polypeptides;
   IMS: intermembrane space; mPTP: mitochondrial permeability transition
   pore.

   In addition to mPTP opening inhibition, IR‐780 also markedly curbed the
   mitochondrial metabolism, including the TCA cycle, OXPHOS, and even
   glycolysis, which induced the mitochondria into a relative “quiescent
   state,” which additionally exhibited a slightly swollen morphology,
   decreased mitochondrial membrane potential, and ROS production. These
   properties were also observed in cells with mitoK[ATP] activation[.] ^[
   [204]^40 ^] The activated mitoK[ATP] channels induce a K^+ influx into
   the mitochondrion that caused mild mitochondrial swelling and ROS
   production.^[ [205]^41 ^] The mitoK[ATP] opening mediates the
   protective roles of ischemic conditioning and has shown great potential
   in I/R treatment.^[ [206]^3b ^] Additionally, this “quiescent state” of
   the mitochondria was shown in drug inducible uncoupling, wherein the
   low FCCP concentration (100 nm) could induce ROS‐dependent
   cardioprotection^[ [207]^42 ^] although without detectable
   depolarization.^[ [208]^43 ^] Thus, we supposed that the
   cardioprotection of IR‐780 may be partly mediated by the ROS‐activated
   intrinsic protection mechanism. However, how IR‐780 induces
   mitochondrial ROS production remains unclear.

   In summary, we have shown for the first time that an NIR heptamethine
   dye IR‐780 has the potential to rapidly monitor the area at risk
   following I/R injury by selectively accumulating in the cardiomyocytes
   of the at‐risk heart tissues. IR‐780 could bind to plasma albumin
   proteins and be released into the acidic microenvironment of ischemic
   tissues. The IR‐780 uptake by cardiomyocytes is dependent on OATPs and
   mitochondrial membrane potential. Moreover, preconditioning with IR‐780
   or timely IR‐780 administration before reperfusion significantly
   reduces ischemia and oxidative stress‐induced cell death, as well as
   avoids myocardial remodeling and heart failure in both rat and pig
   models. The protective effects of IR‐780 relies on its binding to the
   subunits α, β, and the proton channel subunit (atp5mc2) of the
   mitochondrial OXPHOS electron transport chain complex V, thereby
   rapidly decreasing the mitochondrial membrane potential and
   subsequently slowing down the mitochondrial energy metabolism, further
   inducing the mitochondria into a “quiescent state.” Moreover, IR‐780
   binding to complex V resulted in mPTP opening inhibition by preventing
   mitochondrial Ca^2+ overload. Our findings identify IR‐780 as a
   promising injured cardiomyocyte‐targeting agent for both rapid imaging
   and cardioprotection of I/R injury.

4. Experimental Section

Cell Culture

   Rat H9C2 cells were purchased from American Type Culture Collection and
   cultured at 37 °C and 5% CO[2] in Dulbecco's Modified Eagle Medium
   (DMEM) with low NaHCO[3] (1.5 g L^−1) containing 10% fetal bovine serum
   (Gibco) with 100‐U mL^−1 penicillin and 0.1 mg mL^−1 streptomycin
   (Beyotime). The cells of passages 3–8 were used for further
   experiments. The 4mt‐GCamp6 lentiviral vector was purchased from
   BrainVTA (Wuhan, China). Transfection of the 4mt‐GCamp6 in H9C2 cells
   was performed according to the manufacturer's instructions. Following
   7‐day culturing, successfully transfected 4mt‐GCamp6‐positive cells
   were sorted using a Beckman Moflo XDP flow cytometer with 488 nm
   excitation and 525 nm emission.

IR‐780 Uptake In Vitro

   H9C2 cells were seeded into six‐well plates at a density of
   5 × 10^5 cells and were used for further experiments when up to 90% of
   confluence was reached. The cells were treated with DMEM with amiloride
   (75 µg mL^−1, HY‐B0285, MCE), BSP (250 µm, S0252, Sigma),
   chlorpromazine (15 µg mL^−1, 31 679, Sigma), methyl‐β‐cyclodextrin
   (MβCD, 7.5 mm, 779 776, Sigma), FCCP (10 µm, C2920, Sigma), rotenone
   (10 µm, 45 656, Sigma), oligomycin A (10 µm, 495 455, Sigma), HSA
   (1 µm, A1887, Sigma), HSA (1 µm, A1887, Sigma) + ibuprofen (100 µm,
   I0415, TCI), or ice or hypoxic conditions (5% O[2]) or different pH
   conditions (5.2, 6.2, 7.4, and 8.9) for 30 min; subsequently, IR‐780
   (0.1 µm, 425 311, Sigma) was added into the medium for another 20 min.
   Next, the cells were washed thrice with PBS and were for further NIR
   imaging using the Leica NIR fluorescent microscope and flow cytometric
   analysis using a BD Accuri C6 Flow Cytometer.

Animals

   In this study, 6–10‐week‐old male SD rats weighing ≈250–300 g and
   3–4‐month‐old male Bama miniature pigs weighing 28–32 kg were used from
   the Animal Center of Army Medical University (AMU). In vivo experiments
   were conducted in accordance with the Guidelines for the Care and Use
   of Laboratory Animals of the AMU, and all procedures were approved by
   the Animal Care and Use Committee of the AMU (approval number:
   AMUWEC2020690).

SD Rat I/R and Ischemia Model

   Rats were anesthetized with pentobarbital (40 mg kg^−1, i.p.) and
   ventilated on a Rayward rodent respirator via a tracheostomy.
   Parasternotomy was performed, and a reversible suture was placed around
   the left anterior descending (LAD) coronary artery. Myocardial I/R was
   induced by tightening the suture for 30 min and subsequently loosening
   it (2 h reperfusion in acute I/R injury or 4‐week reperfusion in
   chronic I/R experimental setting or without loosening in the ischemia
   model). The animals were allowed to recover from the surgery.

Bama Miniature Pig I/R Model

   The male Bama miniature pigs were sedated with a cocktail of 4 mg kg^−1
   zolazepam hydrochloride and 4 mg kg^−1 tiletamine hydrochloride
   injected intramuscularly. A mixture of 1%–2% isoflurane dissolved in
   40% air and 60% oxygen was continuously administered for inhalatory
   anesthesia via a Rayward respiratory anesthesia machine for large
   animals. Electrocardiogram, heart rate, and arterial pressure were
   constantly monitored. Thoracotomy was performed in the left fourth
   intercostal space; subsequently, the pericardial sac was opened to
   expose the heart. Myocardial I/R was induced by coronary occlusion. The
   LAD coronary artery was encircled by a suture thread; subsequently, the
   suture was tightened to occlude the vessel, which was confirmed by the
   presence of regional myocardial cyanosis (Figure 1A, Supporting
   Information). Moreover, vessel occlusion was confirmed by NIR imaging,
   wherein IR‐780 (0.667 mg kg^−1) was intravenously injected immediately
   following occlusion. Ten minutes later, the hearts were harvested and
   sectioned through four horizontal planes. The sections were
   subsequently photographed using an NIR imaging instrument, and NIR
   signals were absent in the regions supplied by the LAD coronary artery
   ( Figure 1A, Supporting Information). Myocardial I/R was induced by
   tightening the suture for 90 min and subsequently removed it to start
   the reperfusion phase.

Infarct Size Quantification

   Briefly, following the 2 h reperfusion, the LAD coronary artery was
   re‐occluded at the same position as the original infarction. To
   delineate the area at risk, the left ventricle was injected with 1 mL
   of 1% EBD. The heart was then excised and cut into four transverse
   slices. To differentiate infarction from viable tissues, the slices
   were subsequently incubated with fresh 1% TTC at 37 °C for 15 min.
   Next, the slices were photographed, and the regions of the left
   ventricle, which were negative for EBD staining and positive for TTC,
   were calculated.

IR‐780‐Based Imaging

   In the SD rat I/R or ischemia model, myocardial infarction was induced
   in the male SD rats as previously described; immediately following
   ischemia or reperfusion, they received intraperitoneal injection of
   IR‐780 (0.667 mg kg^−1) or HSA‐IR‐780 (containing the equivalent dose
   of IR‐780 [0.667 mg kg^−1]). The hearts were harvested at indicated
   timepoints following reperfusion or ischemia. NIR imaging was
   subsequently performed on the main organs including liver, lung,
   spleen, kidney, muscle, intestine, the whole heart and transverse
   slices using a mobile NIR image scope (developed by Professor Wang Ping
   from Huazhong University of Science and Technology) or an in vivo
   imaging system (VISQUE Smart‐LF, Vieworks). For the BSP blocking
   experiment, animals received direct intramyocardial injections of
   200‐uL BSP (0.25 mg kg^−1) or PBS in the LV anterior wall 5 min before
   I/R. To compare the area at risk by Evans blue and IR‐780 and infarct
   size by TTC and IR‐780, 8 heart transverse slices of 2 h post
   reperfusion were selected, and the following areas were analyzed by
   Image J software: the area at risk of Evans blue negative (A), infarct
   size of TTC (B) and the whole heart area (C) in the photographs, and
   the IR‐780 accumulated area (D) and the whole heart area (E) in the NIR
   images. The comparison (F) of the area at risk by Evans blue and IR‐780
   were calculated as follows: F = (D/E)/(A/C) × 100. The comparison (G)
   of infarct size by TTC and IR‐780 were calculated as follows:
   G = (B/E)/(A/C) × 100.

IR‐780‐Based Protection

   The in vivo IR‐780 precondition group consisted of rats or pigs that
   received IR‐780 (0.667 mg kg^−1 for rats and pigs) intraperitoneal
   injection once daily for 3 days before I/R or ischemia. In the chronic
   I/R models, rats or pigs were administered another IR‐780
   (0.667 mg kg^−1) once a week. The in vivo IR‐780 treatment group
   comprised rats or pigs that received IR‐780 (0.667 mg kg^−1 for rats
   and pigs) intraperitoneal injection immediately following ischemia. In
   the chronic I/R models, rats or pigs were administered another IR‐780
   (0.667 mg kg^−1) once a week. The control group included rats or pigs
   that received an equivalent volume of PBS intraperitoneal injection
   once daily for 3 days before I/R or ischemia. In the chronic I/R
   models, rats or pigs received another equivalent volume of PBS
   injection once a week. The whole blood and serum samples of the rats or
   pigs at 1 month following reperfusion in the chronic I/R models were
   seeded for blood routine, liver function, kidney function analysis.

   For the in vitro H9C2 cell preconditioning, the cells were treated with
   IR‐780 (0.1 µm in DMEM) at 37 °C for 20 min; subsequently, IR‐780 was
   washed out. After 24 h, the cells were used for subsequent experiments.
   For the in vitro H9C2 cell treatment, the cells were treated with
   IR‐780 (0.1 µm in DMEM) at 37 °C for 20 min; subsequently, IR‐780 was
   washed out for succeeding experiments. For the H9C2 cell control, the
   cells were treated with an equivalent volume of DMEM at 37 °C for
   20 min and subsequently changed to full medium (DMEM with 10% FBS) for
   subsequent experiments.

Statistical Analysis

   All statistical analyses were performed using Statistical Package for
   the Social Sciences (version 13, SPSS Inc., Chicago, IL, USA). Data
   were expressed as means ± SD. Data of two groups were compared using
   two‐sided unpaired Student's t‐test. Multiple group comparisons were
   performed using one‐way analysis of variance. Analysis of variances
   were followed by LSD post hoc tests for multiple comparisons when the
   normality and homogeneity of the variance assumptions were satisfied,
   while Tamhane with heterogeneous variance. A p‐value <0.05 was
   considered statistically significant. All experimental n numbers are
   provided in the figure legends.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   Z.C., X.T., T.J. contributed equally to this work. Z.C. contributed to
   conception and design, data analysis and interpretation, financial
   support, manuscript writing; X.T., T.J., Y.W. contributed to provision
   of study material, collection and assembly of data, data analysis and
   interpretation; L.D., C.S., J.W., X.Y., H.L. contributed to thoracotomy
   of the pigs; G.S., C.Z., L.Q. contributed to collection and assembly of
   data; L.L., X.C. contributed to chemical synthesis. C.S. contributed to
   conception and design, financial support, final approval of manuscript.

Supporting information

   Supporting Information
   [209]ADVS-11-2307880-s001.pdf^ (2.5MB, pdf)

   Supplemental Video 1
   [210]Download video file^ (3.1MB, mp4)

Acknowledgements