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Transgenic overexpression of the SUR2A-55 splice variant in mouse heart reduces infract size and promotes protective mitochondrial function

ATP-sensitive potassium channels found in both the sarcolemma (sarcKATP) and mitochondria (mitoKATP) of cardiomyocytes are important mediators of cardioprotection during ischemic heart disease. Sulfonylurea receptor isoforms (SUR2), encoded by Abcc9, an ATP-binding cassette family member, form regulatory subunits of the sarcKATP channel and are also thought to regulate mitoKATP channel activity. A short-form splice variant of SUR2 (SUR2A-55) was previously shown to target mitochondria and display diaxoxide and ATP insensitive KATP activity when co-expressed with the inward rectifier channels Kir6.2 and Kir6.1. We hypothesized that mice with cardiac specific overexpression of SUR2A-55 would mediate cardioprotection from ischemia by altering mitoKATP properties. Mice overexpressing SUR2A-55 (TGSUR2A-55) in cardiomyocytes were generated and showed no significant difference in echocardiographic measured chamber dimension, percent fractional shortening, heart to body weight ratio, or gross histologic features compared to normal mice at 11–14 weeks of age. TGSUR2A-55 had improved hemodynamic functional recovery and smaller infarct size after ischemia reperfusion injury compared to WT mice in an isolated hanging heart model. The mitochondrial membrane potential of TGSUR2A-55 mice was less sensitive to ATP, diazoxide, and Ca2+ loading. These data suggest that the SUR2A-55 splice variant favorably affects mitochondrial function leading to cardioprotection. These data support a role for the regulation of mitoKATP activity by SUR2A-55.


Introduction
Myocardial ATP-sensitive potassium channels (K ATP ) were first described more than 30 years ago [1] and are major contributors to pre-conditioning and cardioprotection [2,3,4]. They were first identified in the sarcolemma (sarcK ATP ) but soon after K ATP channels were also reported in the inner membrane of mitochondria (mi-toK ATP ) [5]. These channels are closed under normal physiologic conditions but open in response to acute stress leading to changes in cytoplasmic and mitochondrial function that promote cardioprotection [6,7,8].
The molecular composition of sarcK ATP has been identified as a hetero-octomer of four Kir6.x (Kir6.1 or Kir 6.2) pore forming subunits and four sulfonylurea receptor (SUR1 or SUR2) regulatory subunits from the ABC binding cassette family of membrane proteins [9]. In addition, both splice variants of the SUR2 gene (SUR2A and SUR2B) form functional sarcK ATP [10]. Different tissues display different subunit expression, giving rise to functionally distinct sarcK ATP [11,12,13]. The cardiac ventricular sarcK ATP is composed of SUR2A and Kir6.2 [14]. While there is a large body of literature studying the constituents of ventricular sarcK ATP , the molecular makeup of mitoK ATP in the heart has been more elusive. The most recent candidate for the pore forming subunit of mitoK ATP is the renal outer medullary potassium channel (ROMK or Kir1.1) which was first supported using a proteomic analysis of the mitochondrial inner membrane [15,16]. In addition, evidence exists that SUR2 gene products exist in mitochondria and play a role in mitoK ATP channel activity [17,18,19].
We described a 55 kDa splice variant (SUR2A-55) formed by intra-exonic splicing that is located predominantly in mitochondria [20] which other labs have also identified [21]. K ATP activity was recorded when SUR2A-55 was targeted to the sarcolemma by deleting a mitochondrial targeting sequence and when co-expressed with Kir6.2 and Kir6.1 in heterologous cells [20,22]. We also found that mice engineered with targeted disruption of exon 14e18 of SUR2 (SUR2 À/À ) lacked fulllength SUR2 but unexpectedly exhibited cardioprotection from ischemia reperfusion (IR) injury despite the absence of pinacidil, diazoxide, or glybenclamide sensitive sarcK ATP currents [23,24,25]. The gene product that encodes the SUR2A-55 splice variant was not perturbed by the deletion used to generate SUR2 À/À mice, and correspondingly mitochondria from these mice had favorable mitochondrial bioenergetics characterized by slight mitochondrial membrane depolarization at rest, insensitivity to ATP and diazoxide, increased reactive oxygen species (ROS), and tolerance to Ca 2þ loading [26]. Furthermore, K ATP currents in heterologous systems produced by co-expression of SUR2A-55 and Kir were less sensitive to block by ATP, more resistant to intracellular Ca 2þ , and less sensitive to K ATP drugs [22] compared to full-length SUR2A. Recently, a second SUR2 knockout mouse was created by removing exon 5, an exon found in both SUR2 and SUR2A-55. This mutation produced early lethality and failure to transition from fetal to adult myocardial metabolism [27]. These mice, unlike the prior SUR2 À/À mice, lacked both full-length SUR2 and the SUR2A-55 splice variant. From these mouse studies, we hypothesized that the SUR2A-55 splice variant plays an important role in mitochondrial function and cardioprotection.
To further understand the physiologic implication of SUR2A-55 we present here an investigation of novel mice overexpressing the SUR2A-55 splice variant in cardiac tissue, which we refer to as TG SUR2A-55 .

Materials and methods
2.1. Construction of a cardiomyocyte-specific SUR2A-55 overexpressing mouse (TG SUR2A-55 ) Transgenic mice expressing SUR2A-55 were generated in the University of Wisconsin Transgenic Animal facility. The 1.5 kb cDNA for SUR2A-55 bracketed by Xho I sites was cut with and ligated into the Sal I cloning site downstream of the 5.5 kb amyosin heavy chain (a-MHC) cardiac-specific promoter and upstream from the 0.6 kb human growth hormone poly A [28]. The a-MHC-SUR2A-55 construct was linearized with Sac II and gel purified and injected into the male pronucleus of FVB single-cell mouse embryos and implanted into psuedopregnant females. Offspring were screened by Southern blotting of genomic DNA using a 0.7 kb probe corresponding to the first 0.7 kb of the SUR2A-55 cDNA. Chimeric mice were bred with FVB mice and colonies were maintained by breeding hemizygous transgenic mice with WT FVB mice in accordance with the standards set by the Animal Care and Use Committees at the University of Wisconsin. All animal experiments were performed in compliance with relevant laws and institutional guidelines and approved by the local animal ethics committee of the University of Wisconsin. Male mice were selected for study owing to evidence of sex differences in myocardial protection [29,30]. Sex differences are likely to be important and will be the subject of additional study.

RNA analysis
Total RNA was extracted from mouse ventricle using TRIzol reagent (Invitrogen ThermoFisher, Waltham, MA) according to the manufacturer's instructions. Firststrand cDNA was synthesized from 3 mg of total RNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen ThermoFisher, Waltham, MA). The quantitative real-time PCR (qPCR) reaction was performed using a 1:2 dilution of cDNA in a total of 20 mL with 10 mL Gene Expression Master Mix (Integrated DNA Technologies, Coralville, IA) and a final concentration of 500 nM forward primer, 500 nM reverse primer, and 250 nM probe. Each cDNA sample analyzed for gene expression by qPCR using the BioRad CFX 96 Real-Time PCR Detection System. Amplification data were analyzed using the delta-delta Cq method with the relative standard curve method to obtain reaction efficiencies of 90e105%. The conditions of qPCR for all primer/probe sets were as follows: an initial denaturation at 95 C for 3 min followed by 40 cycles at 95 C for 15 s and 52 C for 1 min. Oligonucleotide primers were designed using published sequence data from GenBank with the aid of PrimeQuest Design Tool (Integrated DNA Technologies, Coralville, IA). The primer and hydrolysis probe sequences used are shown in Table 1. The mRNA expression was normalized to the levels of Actb and Gapdh mRNA as the reference genes. tibodies at 4 C overnight on a rocker. Immunoblots were performed with SUR2 specific antibodies that were generated as previously described [31] (1:500 dilution). Membranes were washed five times PBST for 5 min each and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibodies for 1 h at room temperature on a rocker. Blots were washed with PBST five times and developed with the ECL system (Pierce, Rockford, IL) according to the manufacturer's protocols.

Histopathology
Twelve week old TG SUR2A-55 and WT mice hearts were sectioned and stained with hematoxylin and eosin to assess myofiber architecture and Masson's trichrome to assess interstitial fibrosis.

Langendorff ischemia reperfusion experiments
Mice were anesthetized with inhaled 3% isoflurane and then euthanized by cervical dislocation. Hearts were rapidly excised and placed in chilled heparinized modified

Isolation of mitochondria
Mitochondria from murine ventricular cardiac tissue were isolated by homogenization and differential centrifugation as previously described [26,32]. Briefly, hearts were quickly excised after thoracotomy and placed in ice-cold isolation buffer (50 mM sucrose, 200 mM mannitol, 5 mM KH 2 PO 4 , 1 mM EGTA, and 5 mM 3-(N-morpholino)propanesulfonic acid). Hearts were manually minced, digested for 10 minutes with 0.1 mg/ml of trypsin and mitochondria were isolated with differential centrifugation. The final mitochondrial pellet was resuspended in ice cold isolation buffer with 0.1% bovine serum albumin. Protein concentrations were determined and 0.5 mg of mitochondria were used to perform each mitochondrial experimental run. Total isolation time was <2 hours and mitochondria were used within 1e2 hours of isolation.

Mitochondrial membrane potential and tolerance to Ca 2D
Mitochondrial membrane potential (Dψ m ) was monitored spectrophotometrically as previously described [26]. Briefly, with the use of rhodamine 123 (5 nM) and excitation l ex of 503-nm and emission l em of 527-nm, 0.5 mg of mitochondria were added to a cuvette containing malate (5 mM Comparison between 2 groups were made using the two-tailed Student's t-test. Ischemia-reperfusion experiments of Langendorff-perfused hearts were subjected to two-way ANOVA for comparison between groups.

Overexpression of SUR2A-55 in TG SUR2A-55 mice
Hearts from 10-14 week old mice were used for mRNA and protein analysis. Hearts from TG SUR2A-55 mice showed similar expression of full-length SUR2A mRNA but significantly increased SUR2A-55 mRNA compared to littermate WT mice (Fig. 1B). Qualitatively, the protein expression of SUR2A in the transgenic mice was not changed but there was increased protein expression of SUR2A-55 in TG SUR2A-55 mice compared to WT mice (Fig. 1C). While previous reports have detected SUR2A-55 in WT mouse hearts [20] and cardiomyoctyes [21], in our current FVB mouse line the level of SUR2A-55 was below the level of detection of western blotting and qPCR.

Cardiac structure and function in TG SUR2A-55 mice
Wall thickness and chamber dimensions were not significantly different between WT and TG SUR2A-55 mice during baseline echocardiography (Fig. 2). Heart rates   show any significant difference (Fig. 2). There were no gross histologic differences seen between WT and TG SUR2A-55 mice with hematoxylin and eosin staining (Fig. 2). Trichrome staining did not show any gross differences in cardiac fibrosis.

TG SUR2A-55 mice are protected against ischemia-reperfusion injury
Isolated hearts from TG SUR2A-55 and WT mice were subjected to 45 minutes of no flow ischemia and then 60 minutes of reperfusion. Baseline hemodynamic characteristics are shown in Table 2 and subsequent hemodynamic variables are presented as % recovery to baseline values (Fig. AeC). TG SUR2A-55 mice had improved left ventricular developed pressure (LVDP) compared to WT mice during reperfusion (P < 0.0001 by two-way ANOVA, Fig. 3A). TG SUR2A-55 mice also had improved þdp/dt and Àdp/dt compared to WT mice during reperfusion (P < 0.0001 by two-way AN-OVA, Fig. 3B and C). Representative heart sections stained with 1% TTC from TG SUR2A-55 and WT mice after IR injury (Fig. 4A) showed significantly reduced myocardial infarction in TG SUR2A-55 compared to WT mice (Fig. 4B).

The Dψ m of TG SUR2A-55 mice are less sensitive to ATP and diazoxide
The mitochondrial membrane potential (Dψ m ) was measured at low (10 mM) and high (500 mM) ATP concentrations in isolated mitochondria from TG SUR2A-55 and WT mice with the substrates malate and pyruvate. Representative traces show that the sensitivity of Dψ m to ATP was diminished in TG SUR2A-55 mitochondria compared to WT mitochondria ( Fig. 5A and B). There was a significant 14% difference in Dψ m for WT mitochondria measured at low ATP vs. high ATP concentrations (90.1 AE 6.7 afu vs. 104.7 AE 1.6 afu, P < 0.05), while there was only a 4% difference  [26]. However our current study did not find such a difference. In the presence of ATP, diazoxide decreased the Dψ m in WT mitochondria more than the Dψ m of TG SUR2A-55 mitochondria (Fig. 6A). Cumulative experiments showed that WT mitochondria had a significantly greater response to diazoxide compared to TG SUR2A-55 mice (18% AE 0.02 vs. 8% AE 0.02, P < 0.05, Fig. 6B).

The
Dψ m of TG SUR2A-55 mitochondria are more tolerant to Ca 2D To test Dψ m resistance to Ca 2þ , TG SUR2A-55 and WT mitochondria were challenged with 20 mM pulses of Ca 2þ while Dψ m was measured. Representative plots (Fig. 7A) show that WT mitochondria start to depolarize sooner and less Ca 2þ is needed to depolarize mitochondria from WT compared to TG SUR2A-55 hearts. (P < 0.05, Fig. 7B).

Discussion
In this investigation we studied whether the cardiomyocyte specific overexpression of the SUR2A-55 splice variant affects the response to myocardial IR injury and alters mitoK ATP pharmacology. We found that at baseline there was no significant difference in echocardiographic parameters and gross histology between TG SUR2A-55 and littermate WT control mice. TG SUR2A-55 mice had improved infarct size and hemodynamic response when subjected to IR injury compared to WT mice. We further assessed isolated mitochondria from TG SUR2A-55 mice to see if they have a bioenergentic profile consistent with a protected phenotype [26,33]. The Dψ m in TG SUR2A-55 mitochondria were less responsive to ATP and diazoxide and exhibited an increased tolerance to Ca 2þ loading compared to the Dψ m in mitochondria from WT mice. K ATP channels composed of SUR2A-55 subunits have reduced sensitivity to ATP and diazoxide [22], which is predicted since alternative mRNA splicing removes putative ATP binding sites. These results show that overexpression of SUR2A-55 leads to preserved resting in vivo cardiac structure and function while promoting cardioprotection from myocardial stress associated with a favorable mitochondrial phenotype.

SUR2A-55 overexpression leads to a viable phenotype
While we were unable to identity significant amounts of SUR2A-55 in resting WT hearts, prior studies have shown the presence of SUR2A-55 in mouse heart and localization to the mitochondria [20,27]. In our study we found that baseline echocardiography-derived cardiac chamber size and function as well as heart to body weight ratio of TG SUR2A-55 mice compared to WT mice were not significantly different. We did, however, notice that in the Langendorff experiments, baseline þdp/dt and Àdp/dt values were significantly less in the TG SUR2A-55 compared to WT mice. The mechanism of the isolated heart baseline differences are unclear. It may be that TG SUR2A-55 mice are more susceptible to cardiac depression when ex vivo or that the mitochondrial changes observed translate to subtle changes in cardiac performance when measured ex vivo compared to in vivo. In addition, TG SUR2A-55 may be more sensitive to cardiac depression with the dose of anesthesia used for cervical dislocation. While the current overexpression model of SUR2A-55 leads to a viable mouse phenotype, recent data has suggested that deletion of all SUR2 products including the long-form and SUR2A-55 splice variant lead to lethality with a failure to transition from neonatal to mature adult metabolism [27]. Therefore, it is likely that the SUR2A-55 splice variant carries not only an important role in cardioprotection in adulthood but also in development during the neonatal period. Transgenic mice that conserve the SUR2A-55 variant but do not have the long-form SUR2 due to deletion of exons 14e18 are viable and live to adulthood [34]. While studying substrate utilization and cardiac metabolism were outside the scope of this investigation, future studies testing how SUR2A-55 affects myocardial metabolism may lead to greater understanding of how mitoK ATP affects both mitochondrial and cytoplasmic energy production.

Cardiomyocytes with increased expression of SUR2A-55 leads to cardioprotection
The location and significance of the SUR2-55 splice variant was originally presented by Ye et. al. [20] who described a SUR2-55 short-form based ATPsensitive potassium channel that was insensitive to diazoxide and targeted to the mitochondria. SUR2 À/À mice, which removed full length SUR2 but not SUR2A-55, displayed episodic vasospasm, sudden cardiac death, and enhanced insulin dependent glucose uptake [20,23,24]. The splice variant was formed through a nonconventional intraexonic splicing event within the 5 th and 30 th exons of the SUR2 mRNA. When subjected to IR injury, this SUR2 À/À mouse was found to have reduced infarction compared to controls [20,34]. In this current investigation we found that mice overexpressing the SUR2A-55 splice variant have improved tolerance to IR injury compared to controls. Unlike SUR2 À/À mice, the TG SUR2A-55 mice express the long form of SUR2 which is also found in mitochondria [20] and thus cardioprotection can occur with the expression of both splice variants. In addition, periodic vasospasm which is reported in the SUR2 À/À mouse, is not expected in the TG SUR2A-55 mouse and therefore does not explain the protected phenotype. We speculate from these studies that a switch from mitoK ATP formed predominantly with the long-form SUR2 splice variant to the short-form splice variant channel can cause a cardioprotective phenotype. Whether or not a switch to SUR2A-55 dominated mitoK ATP underlies cardioprotection in ischemic preconditioning requires further study. Therapies aimed at triggering a switch in SUR2 splice variant composition of mitoK ATP may be an option for cardioprotective drugs in the future.

4.3.
Overexpression of SUR2A-55 changes Dψ m response to pharmacology and has implication for the regulation of mitoK ATP Mitochondria play a paramount role in cell death. During myocardial ischemia reperfusion injury, Ca 2þ entry into the mitochondria ultimately leads to the mitochondrial transition pore (MPTP) opening and cell death [35,36]. In our investigation, we found that mitochondria from TG SUR2A-55 are more resistant to Ca 2þ loading. In addition, we observed that the Dψ m from TG SUR2A-55 mitochondria are less sensitive to ATP concentration and have less responsiveness to diazoxide. Interestingly, the percent difference in Dψ m at low and high ATP concentrations in this report is similar to prior studies on the SUR2 À/À mice [26] in which the SUR2A-55 isoform was preserved. While the electron transport chain and a number of channels in the mitochondria may be the reason for changes in Dψ m [37], the change in sensitivity to ATP and diazoxide, as well as prior studies on SUR2A-55 make mitoK ATP the most likely target responsible for our observed data. However, direct measurements of mitoK ATP currents were not assessed. These data support prior work that SUR2A-55 promotes a mitoK ATP channel that is insentive to diaxoxide and ATP. The lack of ATP or diazoxide to change Dψ m suggest a persistent potassium leak and constituently active mitoK ATP channel [22,26].The mitochondrial membrane depolarization that ensues even in times of increased ATP, such as reperfusion, can result in lower mitochondrial calcium entry and reduced MPTP opening. Prior reports have suggested this as a mechanisms for protection from mitoK ATP activation and our data would continue to support this theory [38,39,40].
Further study in this area is challenging given the elusive nature of the mitoK ATP channel. However, recent data suggested that ROMK2 may be the pore forming unit of mitoK ATP [15]. Molecular reconstitution studies and gene deletion studies show that ROMK interacts with an ABC binding cassette to manifest native channel properties [41]. Additional studies have shown that ROMK2 binds to an another SUR2 splice variant, SUR2B, to form sulfonylurea sensitive K þ channels that were not observed with ROMK1 or ROMK3. It is unknown if other splice variants of SUR can create native functional channels with ROMK. Further investigations studying interactions of ROMK2 and SUR2A splice variants may shed light on the molecular identity of mitoK ATP . Our research suggests considerable plasticity of the mitoK ATP channel with different subunits affecting the channels response to pharmacology. This may also explain the elusive nature of the mitoK ATP channel and conjecture surrounding its existence.
While our current investigations describe the response of SUR2A-55 overexpression in mice, certain limitations exist. For practical reasons of scope, we restricted study to overexpression of the A isoform of SUR2. Prior studies have shown that both SUR2A-55 and SUR2B-55 isoforms exist [20,21]. Therefore the SUR2B isoforms may also have physiologic significance and play a role in mitochondrial function. We concentrated on the SUR2A-55 isoform as our prior work showed qualitiatvely increased expression of SUR2A-55 over SUR2B-55 in mouse hearts [20]. In addition, due to the scope of our research, we restricted our study to male mice. While previous studies have shown targeting of the SUR2A-55 protein to mitochondria, we did not assess its location in TG SUR2A-55 and did not measure potassium flux in mitochondria. However, our mitochondrial biophysical studies suggest a direct effect for SUR2A-55 on mitochondria.We also used a global ischemia ex vivo model for IR injury which has different physiologic stress than a coronary ligation model and may not be as translational to human disease. In addition, while we report cardioprotection to IR injury and changes in mitonchondrial biophysical properties in TG SUR2A-55 mice, there may be other cellular mechanisms including changes in sarcK ATP activity that could be responsible for the cardioprotective response. Further studies on the role of SUR2A-55 in sarcK ATP activity are needed.

Conclusion
In conclusion, elucidating pathways in cardioprotection may lead to improved clinical treatment of heart disease. While great advances have been made in the treatment of ischemic heart disease, it remains the leading cause for the development of heart failure [42,43] and imposes a large burden on society [44]. In this research investigation, we showed that SUR2 splice variants offer an interesting area of further research to clarify mitochondrial function in ischemic heart disease. By altering the expression of SUR2 splice variants, different cardiac phenotypes arise. The overexpression of the SUR2A-55 splice variant leads to cardioprotection likely through a constitutively active mitoK ATP channel. Further research to identify the components of the mitoK ATP channel that enhance the cardioprotective phenotype should be considered using the TG SUR2A-55 mouse.

Declarations
Author contribution statement