Many people who survive a stroke or heart attack experience substantial tissue damage when the blood supply is restored. Much of this damage can be caused by the mitochondria inside the cells releasing a protein called cytochrome c that can cause cells to die in a process called apoptosis. The cytochrome c is released as the outer membrane of the mitochondria becomes permeable and pores called permeability transition pores open up in the inner membrane. Now Lin et al. explore if additional molecules released from the mitochondria might also initiate important cellular responses during the reoxygenation of oxygen-deprived tissue.

Lin and co-workers recently showed that the mitochondria of some cells can release a small enzyme cofactor, coenzyme A, which then promotes a cellular response called massive endocytosis. This process can cause up to 70% of the cell surface membrane to be absorbed into the interior of the cell in the form of membrane vesicles. Most forms of endocytosis involve a much smaller fraction of the cell membrane and employ a set of well-known endocytic proteins that are not involved in massive endocytosis. Now, Lin et al. investigate the role of massive endocytosis in cardiac muscle.

Electrical and optical measurements reveal that massive endocytosis occurs as cardiac cells that have been deprived of oxygen are reoxygenated. Lin et al. also find that an enzyme called DHHC5 must be present to allow endocytosis to take place during reoxygenation. DHHC5 is an enzyme that catalyzes a process called acylation – the transfer of acyl groups to proteins at the cell surface. Moreover, the deletion of DHHC5 has a beneficial impact on the performance of cardiac muscle after oxygen deprivation, which implies that molecules that inhibit protein acylation might protect the heart from damage during reoxygenation. Together, these results establish new pathological and physiological roles for the acylation, which is one of the most common biochemical modifications made to membrane proteins after they are synthesized.

Acute ischemic events are now a leading cause of death in the entire world (World Health Organization, 2013). Ironically, much of the damage from ischemic events can occur during reoxygenation of tissue when mitochondria begin to generate reactive oxygen species (ROS) at increased rates (Jennings, 2013). One of the hallmarks of reperfusion injury is a pronounced swelling of mitochondria, and the extensive opening of mitochondrial permeability transition pores (PTPs) that underlies this swelling (Haworth and Hunter, 1979) is a nearly decisive step on the path to cell demise (Halestrap et al., 2004). Associated with this progression, mitochondria release cytochrome c to the cytoplasm and thereby activate apoptosis programs that can definitively establish cell fate (Gottlieb, 2011).

Given that both outer and inner mitochondrial membranes become permeable during reperfusion injury, mitochondria with certainty release a large number of metabolites and small proteins. Therefore, the question arises whether, besides cytochrome c, other molecules released from mitochondria might serve signaling functions. Our analysis of Ca-activated endocytosis in BHK fibroblasts (Hilgemann et al., 2013) suggests that mitochondrial release of coenzyme A (CoA) may be important. This hypothesis arises from two facts. First, mitochondria accumulate CoA to high concentrations with respect to the cytoplasm (Leonardi et al., 2005). Second, many key cytoplasmic enzymes are modulated by binding long-chain acyl CoA (Faergeman and Knudsen, 1997), a CoA metabolite that will be generated immediately if CoA is released (Idell-Wenger et al., 1978). These include glycogen synthase (Wititsuwannakul and Kim, 1977), glucose-6-phosphate dehydrogenase (Kawaguchi and Bloch, 1974), and perhaps AMP kinase (Faergeman and Knudsen, 1997), as well as KATP potassium channels (Shumilina et al., 2006). Our study in BHK cells (Hilgemann et al., 2013) suggests that release of CoA from mitochondria may promote the palmitoylation of surface membrane proteins, subsequent to generation of acyl CoA. Further, our study suggests that extensive palmitoylation promotes internalization of membrane as a form of ‘lipid raft’-dependent endocytosis (Doherty and McMahon, 2009).

Beyond the function of PTPs at the threshold of cell death, it is now established that transient mitochondrial PTP openings play important physiological functions by causing transient mitochondrial depolarizations that release matrix Ca to the cytoplasm accompanied by the generation of superoxide flashes (Brenner and Moulin, 2012; Zhang et al., 2013; Zhou and O’Rourke, 2012). In this light, a possibility emerges that cytoplasmic free CoA could physiologically play important second messenger functions in dependence on its release from mitochondria. On the one hand, mitochondrial depolarization will favor reverse CoA transport to the cytoplasm (Tahiliani, 1991), and on the other hand transient PTP openings may allow enough CoA to escape from the matrix space, down its more than 50-to-1 gradient, to significantly increase the low micromolar concentration of free cytoplasmic CoA (Idell-Wenger et al., 1978).

We describe here experiments that address these possibilities in cardiac myocytes and in intact cardiac tissue. First, we show that the function and abundance of cardiac Na/K pumps and Na/Ca exchangers is affected by the expression of the plasmalemma-directed acyl transferase, DHHC5, then we show that massive endocytic (MEND) responses in isolated myocytes depend on acyl CoA and DHHC5 activity, and that MEND responses occur when PTP openings occur during the reoxygenation of anoxic cardiac muscle. Finally, we show that two cardiac membrane proteins are indeed increasingly palmitoylated during reoxygentation, that internalized PLM (phospholemman) is more heavily palmitoylated than PLM on the cell surface, and that DHHC5 activity significantly impacts cardiac contractile recovery after anoxia.

In developing a tactic to characterize the MEND pathway in cardiac myocytes, we chose to focus on Na/K pumps. First, Na/K pumps are high density transmembrane protein complexes in the cardiac sarcolemma whose activity strongly modulates cardiac function (Hilgemann, 2004). As such, Na/K pumps likely represent a large fraction of membrane particles that have been visualized in freeze fracture studies and that cluster and decrease in number during anoxia/reoxygenation episodes (Frank et al., 1980). Second, it was described almost 40 years ago that the number of Na/K pumps, quantified as ouabain binding sites, decreases markedly during ischemia/reperfusion events (Beller et al., 1976). Recently, it has been shown that Na/K pumps are indeed internalized via endocytosis in a cardiac myocyte culture during ‘simulated’ ischemia/reperfusion (Belliard et al., 2012), and internalization still occurs during metabolic stress when a di-leucine motif of Na/K pump alpha subunits has been mutated to prevent pump involvement in conventional, clathrin-dependent trafficking (Pierre et al., 2010). Third, extensive internalization of Na/K pumps also occurs in response to metabolic stress in several other cell types (Mandel et al., 1994; Bortner et al., 2001; Brown et al., 2001; Mann et al., 2001). In alveolar epithelia the reversible internalization of Na/K pumps in hypoxia is initiated by ROS generation and the activation of protein kinase Cs (PKCs) (Dada et al., 2003), details that are reminiscent of our findings that PKCs and ROS promote MEND in BHK cells (Hilgemann et al., 2013). Fourth, the FXYD subunits of Na/K pumps can be dually palmitoylated and depalmitoylated at the surface membrane, and we have shown that overexpression of the major FXYD protein of cardiac muscle, phospholemman (PLM), promotes the occurrence of MEND in a PKC-dependent fashion in a TReX-293 cell line (Hilgemann et al., 2013).

As a second tactic, we developed a new electrical method to monitor surface membrane area as electrical cell capacitance (C_m) on-line in intact tissue over periods of hours. In results presented, we employ this non-invasive C_m (NIC) recording technique with superfused right ventricular strips (see ‘Materials and methods’). Briefly, NIC recording exploits the fact that myocyte sarcolemma constitutes the great majority of cellular surface membrane in the heart (Banerjee et al., 2007). Although non-myocytes can be as numerous as myocytes, all non-myocytes are much smaller than myocytes. Similar to skeletal muscle, therefore (Cole, 1976), rapid extracellular voltage oscillations (15 kHz/1 mV) can be used to monitor the composite sarcolemmal C_m. The use of this on-line method allowed us to rapidly test for the occurrence of MEND in intact cardiac tissue. As described subsequently, we have verified all findings from NIC recording with independent methods, specifically via an optical method in intact hearts to follow fluid phase uptake of fluorescent probes and via patch clamp of isolated myocytes. Additional results that validate NIC recording are provided in ‘Material and methods’.

Sarcolemmal significance of constitutive DHHC5 activity

Figure 1 demonstrates that expression of the DHHC5 acyl transferase strongly influences Na/K pump activity in cultured cardiac myocytes, namely human fibroblast-derived cardiac myocytes (iCell Cardiomyocytes, [Ma et al., 2011]). These myocytes are transfected easily to overexpress and knockdown regulatory proteins, they highly express cardiac-specific proteins, such as PLM and the Na/Ca exchanger, NCX1, and ion transport activities are equivalent to those of adult myocytes (Fine et al., 2013). Measuring maximal Na/K pump currents exactly as described in our study (Fine et al., 2013), the first two bar graphs of Figure 1 show that overexpression of DHHC5, together with green fluorescent GFP to identify transfected myocytes, decreases Na/K pump currents by 55%. This decrease becomes still somewhat larger with dual DHHC2/DHHC5 expression. Conversely, knockdown of DHHC5 by siRNA causes a 38% increase of Na/K pump currents, compared to myocytes transfected with scrambled siRNA (‘siRNA control’), and dual DHHC5/DHHC2 knockdown causes a 90% increase. Although these changes may arise from either a change of pump activity or a change of pump density in the sarcolemma, data presented next indicate that changes of transporter localization play a large role.

Figure 1

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We next describe myocytes and cardiac tissue from mice that are homozygous for a hypomorphic allele of DHHC5 (gene-trapped, GT) with cardiac DHHC5 expression decreased by >80% (Li et al., 2011). DHHC5-GT mice have significantly reduced growth rates. Between 4 and 5 weeks, the average weights of DHHC5-GT mice (14 ± 0.8 g) were 24% less than control litter mates (19 ± 0.9 g). Between 12 and 14 weeks, the average weights of DHHC5-GT mice (21 ± 0.2 g) were 13% less than control litter mates (24 ± 0.2 g). The dimensions of isolated myocytes from young DHHC5-GT animals were also significantly smaller than those from matched WT animals. Therefore, we employed animals 12 to 14 weeks of age for the studies to be described.

We used patch clamp with square wave voltage pulses to measure the myocyte surface area as cell electrical capacitance (C_m) (Lariccia et al., 2011), assuming that 1 pF constitutes 100 μm² of membrane. Cell area (i.e., C_m) /volume ratios of myocytes were then determined as described in Figure 2A. The volume of each myocyte was estimated from a micrograph of the myocyte still resting on the surface of the chamber employed. From the micrograph, the two-dimensional area of the myocyte was determined in square microns using Image-J software (http://rsbweb.nih.gov/ij/). The volume was then estimated by assuming an average 5-to-1 ratio of myocyte width to thickness (volume (μm³) = myocyte area²/myocyte length/5) and converted to pl. As shown in bar graphs in Figure 2B,C, the average surface areas of DHHC5-GT myocytes were modestly but significantly increased in comparison to WT myocytes from matched animals. The average volumes of DHHC5-GT myocytes were insignificantly decreased in myocytes from 12 to 14 week old animals. As shown in Figure 1D, the average myocyte area/volume ratio, calculated in pF/pl, was highly significantly increased by 24%. Figure 2E shows that comparable results were obtained via NIC recordings in right ventricles from DHHC5-GT and litter mate mice. The capacitive NIC signal (i.e., sarcolemma area in a hemisphere below the 0.6 mm recording electrode) was increased significantly by 21%, on average, in DHHC5-GT ventricles (n = 5) vs WT ventricles (n = 5).

Figure 2

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Next we show in Figure 2F,G that the average current density of Na/K pumps is significantly increased by 37% in DHHC5-GT myocytes, while a 17% increase of Na/Ca exchange (NCX1) current density was not significant. Finally, we tested directly whether pumps and exchangers are on average more retained in the sarcolemma vs internal membranes in DHHC5-deficient vs WT hearts. To do so, we employed a PEGylation assay described in ‘Materials and methods’ to determine the fractions of NCX1 and the regulatory Na/K pump subunit, PLM, resident in the cell surface. Both of these proteins are advantageous for amine PEGylation because they have extracellular N-termini (Geering, 2006; Ren and Philipson, 2013). Using a 5 kD NHS-PEGylation reagent to label the outer sarcolemma of intact, perfused hearts, Figure 2H shows typical Western blot density profiles from this assay for PLM and NCX1. Using a least squares Gaussian fit to determine relative protein densities, 34 ± 3% of PLM and 31 ± 2% of NCX1 bands were shifted 5 kD to higher molecular weights in hearts from WT animals. As shown in Figure 3B, the calculated surface fractions of both membrane proteins were significantly increased in DHHC5-GT hearts vs WT hearts, namely by 27 ± 5 and 23 ± 2%, respectively.

Figure 3

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DHHC5 deficiency inhibits MEND in four protocols in murine myocytes

In BHK cells, large MEND responses can be evoked by rapidly perfusing the cytoplasm of cells via the patch clamp pipette with solutions containing metabolites that promote mitochondria to open PTPs (‘KSP solution’, containing potassium [100 mM], succinate [5 mM], and inorganic phosphate [Pi, 1 mM]). As shown in Figure 3A, WT myocytes generate 33% MEND responses upon pipette perfusion of KSP solution with rough time constants of 2.5 min, and these responses are decreased significantly by 75% in DHHC5-GT myocytes. When the cytoplasmic solution employed in BHK cells contains myristoyl CoA (mCoA), application of H₂O₂ (hydrogen peroxide) (80 μM) for a few minutes primes cells to undergo MEND when the oxidative stress is removed. As shown in Figure 3B, transient H₂O₂ application (80 μM) also causes large MEND responses in cardiac myocytes (∼35%) when mCoA (15 μM) is included in the pipette. These responses were also strongly reduced in DHHC5-GT myocytes (p<0.01).

In BHK cells, activation of PKCs with diacylglycerol surrogates induces MEND responses that constitute 20–40% of the plasmalemma when mCoA is present in cytoplasmic solutions (Hilgemann et al., 2013). The equivalent experiments caused much slower responses in myocytes, as well as when phospholipase C-coupled GPCRs (G-protein coupled receptor) were activated (e.g., by adenosine or phenylephrine) in the presence of mCoA. Figure 3C illustrates MEND responses that occur when the alpha receptor agonist, phenylephrine (50 μM) (Puceat et al., 1994), is used in the presence of mCoA (20 μM). MEND did not occur unless both phenylephrine and mCoA were present (n = 10). While slower than responses to KSP solution and transient oxidative stress, sarcolemma area decreases on average by about 40% over 30 min in WTl myocytes, but only 14% in DHHC5-GT myocytes (p<0.05).

In murine myocytes, Ca influx can activate MEND responses that occur much more rapidly than those just described (Lariccia et al., 2011). When activated by reverse Na/Ca exchange, MEND stops abruptly when Ca influx is terminated (Lariccia et al., 2011), suggesting that Ca is acting via a rather direct mechanism that is likely different from MEND responses just described. Nevertheless, Figure 4A demonstrates that Ca-activated MEND is highly significantly reduced in DHHC5-GT myocytes vs WT myocytes. Figure 4A illustrates Ca-activated MEND in a WT myocyte, the top trace being C_m (i.e., membrane area) and the bottom trace being membrane current. When Ca influx is activated, C_m begins to decline after a 3 to 4 s delay and then declines further by 28% with a rough time constant of 8 s. As documented in Figure 2G, exchange currents were on average larger in DHHC5-GT myocytes. However, as shown in Figure 4B, MEND responses were decreased from 27% on average in WT myocytes to only 3% in DHHC5-GT myocytes. Figure 4B shows further that MEND responses in WT myocytes were decreased by 70% when myocytes were preincubated with the PTP/cyclophillin D-specific cyclosporine, NIM811 (N-methyl-4-isoleucine cyclosporine) (3 μM) (Waldmeier et al., 2002), for 1 hr. However, inclusion of a high CoA concentration (4 mM) in the cytoplasmic solution to acutely inhibit DHHC acyl-transferease activity (Hilgemann et al., 2013) did not significantly inhibit Ca-activated MEND in WT myocytes. We conclude therefore that palmitoylation reactions do not occur during the protocol of Figure 4A, although this form of MEND nevertheless depends on the palmitoylation state of the sarcolemma.

Figure 4

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Figure 4C solidifies this interpretation using myocytes that overexpress Na/Ca exchangers (NCX1) by 10-fold. Both the time course of MEND and the decay of NCX1 current during Ca influx are accelerated nearly 10-fold (τ <1 s), and myocytes internalize more than 50% of their sarcolemma within 1–2 s. Using the myosin II inhibitor blebbistatin (Farman et al., 2008) (10 μM in all solutions) to inhibit contraction, myocytes did not contract or show obvious morphological changes during these profound responses. As shown in Figure 4D, neither pretreatment with NIM811 for 1 hr (3 μM) nor inclusion of a high CoA concentration (4 mM) in the cytoplasmic solution significantly affected these responses. Clearly, Ca can promote MEND in myocytes by a mechanism that is promoted by, but does not require, membrane protein palmitoylation.

MEND occurs during reoxygenation of anoxic cardiac muscle

Reperfusion of ischemic cardiac tissue is one of the best defined circumstances in which PTP openings occur profusely and correlate with a significant amount of reoxygenation injury after ischemic or anoxic episodes (Halestrap, 2009). Importantly, much of this damage can be prevented by chemical preconditioning of cardiac tissue with hormones, such as adenosine, that activate PKCε and likely inhibit PTP openings (Baines et al., 2003). Given evidence that significant endocytosis can occur in cardiac ischemia/reperfusion via unconventional mechanisms (Pierre et al., 2010), we tested next whether MEND indeed occurs during reoxygenation of anoxic cardiac tissue.

Figure 5A illustrates NIC recording in rapidly superfused right ventricular strips (0.5–0.7 mm thick; 36°C). Using 15 kHz sinusoidal voltage oscillations, the conductance signal component (I_G) decreases and the capacitive signal component (I_CAP) increases as the perturbing electrode (a 0.6 mm Ag/AgCl pellet, recessed 0.4 mm in a glass capillary) is brought close to and touches the muscle surface. The conductance signal (I_GX) then reflects conductance of the extracellular space, which constitutes about 30% of the heart volume. Capacitive signals reflect the relative amount of surface membrane in a hemisphere of tissue below the electrode. The capacitive signal showed little change over 30 min after oxygen-saturated solution was switched to oxygen-free, glucose-free solution with 5 mM deoxyglucose to promote nucleotide depletion. However, when oxygen-saturated, glucose-containing solution was reintroduced, NIC signals decreased over 25 min by 21% on average.

Figure 5

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Composite results from similar recordings, shown in Figure 5B, support the interpretation that MEND is occurring in intact cardiac muscle by mechanisms that are related to those described in BHK cells (Hilgemann et al., 2013). First, the cardiac MEND responses were strongly decreased when muscles were taken from hearts that had been FA (fatty acid)-depleted by 15 min arterial perfusion of FA-free albumin (60 μM) prior to the experiments. Second, a low concentration of staurosporine (STS; 0.1 μM), which promotes MEND responses in BHK cells (Hilgemann et al., 2013), increased sarcolemma loss to 35%. Third, cyclosporine A (CsA; 1 μM) strongly decreased sarcolemma loss. Fourth, the preconditioning hormone, adenosine (0.1 mM) (Ytrehus et al., 1994), decreased sarcolemma loss by 52%. And fifth, MEND was nearly ablated in muscles from DHHC5-GT animals.

As described in the accompanying article (Hilgemann et al., 2013), MEND appears to be ‘cargo-dependent’, being promoted by overexpression of the palmitoylated Na/K subunit, PLM (Tulloch et al., 2011), in T-Rex293 cells. Therefore, we examined whether the occurrence of MEND would be altered in cardiac muscle from PLM-deficient mice. As shown in the last bar graph of Figure 5B, reoxygenation-induced MEND was reduced by 58% in right ventricles from PLM knockout mice (Bell et al., 2008).

As an independent verification of the occurrence of MEND, we describe in Figure 6 the fluid-phase uptake of FITC-labeled dextran (4000 MW) by myocytes in arterially perfused hearts. Hearts were perfused retrograde with either FA free- or 2:1 myristate-loaded albumin (60 μM) during the same anoxia-reoxygenation protocol. During reoxygenation, hearts were perfused with FITC-dextran (0.5 mM) for 25 min followed by washout at room temperature for 10 min. Subsequently, epicardial myocytes were imaged several cell layers below the outer cardiac surface; the outer most myocytes were excluded because they are atypical, being enriched in cardiac stem cells (Schlueter and Brand, 2012). Hearts perfused with FA—free albumin reveal diffuse staining of a limited fraction of myocytes. This reflects nonspecific dextran uptake as a result of reperfusion stress. Hearts perfused with FA reveal, in addition to diffuse staining of a fraction of myocytes, bright punctate staining that is indicative of dextran uptake into large endosomes and vacuoles. Punctate staining becomes very profuse in hearts perfused with STS (0.1 μM) and is absent when CsA (3 μM) is perfused with albumin and FA. Statistical analysis, performed as described in Methods, is provided in Figure 6B, together with the additional result that dextran uptake was negligible in the same protocol in ventricles from DHHC5-GT mice.

Figure 6

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Palmitoylation is activated during reoxygenation-induced cardiac MEND

To test whether palmitoylation of membrane-associated proteins is indeed enhanced during reoxygenation of anoxic cardiac tissue, we employed a resin assisted capture assay for acylated proteins (acyl-RAC) (Forrester et al., 2010) to determine the palmitoylation status of proteins that might be important in MEND. In addition to PLM, we examined flotillin-2 because flotillins may be involved in the ordering of proteins into Lo domains (Langhorst et al., 2005). In this assay, free cysteines are initially blocked, followed by deacylation of palmitoylated residues with hydroxylamine (NH₂OH), cysteine-specific pull-down, and quantification of captured, deacylated proteins via Western blotting. The presence of a single acylation site is ‘positive’ and multiple vs single acylations are not distinguished. Proteins that are stably palmitoylated, such as caveolins (Parat and Fox, 2001), can be used as loading controls in the assay (Tulloch et al., 2011). Usually, we analyzed caveolin-3 (CAV3) content after stripping secondary antibodies employed in initial blots, but results were very similar when analyzed in relation to Western blot loading controls of initial lysates.

Figure 7A illustrates Western blots for a pair of hearts. One was subjected to the anoxia/reoxygenation protocol of Figure 5A, and the second heart was perfused with oxygen-containing perfusate for an equivalent time (1 hr at 1.5 ml/min; 37°C). For PLM blotting, we employed an N-terminal (extracellular) antibody (see ‘Materials and methods’) to avoid effects of cytoplasmic (C-terminal) phosphorylation or palmitoylation on antibody binding. As shown in Figure 7B, anoxia/reoxygenation caused a 71% increase of palmitoylated PLM (n = 5; p<0.01) and a 77% increase of palmitoylated flotillin-2 (n = 5; p<0.01) relative to caveolin-3. Consistent with the idea that DHHC5 constitutively affects palmitoylation of these proteins, Figure 7C shows that palmitoylated PLM and flotillin-2 were decreased by 27% and by 51%, respectively, in DHHC5-GT hearts that were perfused briefly to remove blood and immediately frozen.

Figure 7

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We next combined the acyl-RAC assay with the amine PEGylation assay, illustrated in Figure 2H, to determine the fraction of PLM at the cell surface and then additionally determine the fraction of PLM that is palmitoylated at the surface membrane vs internal membrane pools. Using the PEGylation protocol described in ‘Materials and methods’, Figure 7D shows the PEGylation and acyl-RAC results for four hearts, two of which were PEGylated and two of which were not. The blots shown include a loading control and the acyl-RAC result for each heart with loading amounts adjusted to generate similar densities on the acyl-RAC blots. Densities of the loading controls from PEGylated hearts (second lanes from the left and from the right) indicate that 40 and 46% of PLM was PEGylated, respectively, and is therefore sarcolemmal. In the acyl-RAC pull-downs from the same hearts, however, only 17 and 23% of PLM is PEGylated. Therewith, a key tenet of our working hypothesis is verified. Although palmitoylation can target proteins to the surface membrane (Greaves et al., 2009), enhanced palmitoylation may favor the removal of palmitoylated proteins from the surface membrane.

MEND impacts post-anoxia cardiac function

As described in Figure 5B, MEND is decreased in intact cardiac tissue by treatments that protect the heart from reperfusion damage, namely CsA and adenosine (Halestrap et al., 2004). Furthermore, we find that MEND is promoted by an agent that prevents ischemic preconditioning and promotes PTP openings, STS (Ytrehus et al., 1994). These results therefore suggest that the occurrence of MEND might negatively impact the recovery of the heart from anoxia-reoxygenation episodes. To test if this is indeed the case, we analyzed contractile function of right ventricular strips paced at 0.25 Hz and subjected to 30 min of anoxia in the absence of glucose, followed by reoxygenation and administration of glucose (15 mM). As shown in the examples in Figure 8A and in the composite data in Figure 8B, contraction became negligible within 8 min of anoxia without glucose. During reoxygenation for 25 min, contractile function of WT ventricles recovered only little, 12% on average. However, contractile function of right ventricles from DHHC5-GT animals recovered substantially, 60% on average, equivalent to some of the most effective ‘preconditioning’ protocols (Halestrap et al., 2004). Thus, as already suggested by others (Belliard et al., 2012), internalization of sarcolemma may contribute to the acute failure of cardiac function during ischemia/reperfusion episodes.

Figure 8

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This article lends support to a working hypothesis that mitochondrial PTP openings can initiate surface membrane remodeling by promoting palmitoylation-dependent endocytosis (Hilgemann et al., 2013). We have demonstrated here that this form of endocytosis occurs extensively during the reoxygenation of anoxic cardiac tissue and therefore is relevant to the cellular consequences of ischemia/reperfusion events. In addition, DHHC5-dependent palmitoylation constitutively regulates the protein content of the cardiac sarcolemma and affects how cardiac myocytes respond to cell signals, such as oxidative stress.

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Author details

1. Mei-Jung Lin

   Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   M-JL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Michael Fine

   Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   MF, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Jui-Yun Lu

   Department of Internal Medicine and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   J-YL, Provided DHHC5-GT mice and DHHC5 reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Sandra L Hofmann

   Department of Internal Medicine and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   SLH, Provided DHHC5-GT mice and DHHC5 reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Gary Frazier

   Department of Physics, University of Texas at Dallas, Richardson, United States

   Contribution

   GF, Conception and design, Designed and built the NIC-recording device employed

   Competing interests

   The authors declare that no competing interests exist.

6. Donald W Hilgemann

   Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   DWH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   donald.hilgemann@utsouthwestern.edu

   Competing interests

   The authors declare that no competing interests exist.

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