Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Elevated expression of activated Na+/H+ exchanger protein induces hypertrophy in isolated rat neonatal ventricular cardiomyocytes

The plasma membrane protein the Na(+)/H(+) exchanger isoform1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. Levels of NHE1 protein and activity are elevated in cardiac disease; however, the mechanism by which these factors contribute to the accompanying hypertrophy in the myocardium is still not clear. To investigate the mechanism of NHE1-induced hypertrophy in the myocardium we constructed two adenoviral vectors expressing either wild type NHE1 protein or a constitutively active NHE1 protein. Infection of neonatal rat ventricular cardiomyocytes (NRVM) resulted in elevated expression of both wild type NHE1 or constitutively active NHE1. Only expression of activated NHE1 protein resulted in an increase in cell size and in an increase in protein synthesis in isolated cardiomyocyte cells. The results demonstrate that expression of activated NHE1 promotes cardiac hypertrophy in isolated cardiac cells and that simple elevation of levels of wild type NHE1 protein does not have a significant hypertrophic effect in NRVM. The results suggest that regulation of NHE1 activity is a critical direct effector of the hypertrophic effect induced in the myocardium by the NHE1 protein.     

Sustained Na+/H+ Exchanger Activation Promotes Gliotransmitter Release from Reactive Hippocampal Astrocytes following Oxygen-Glucose Deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na⁺/H⁺ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H⁺ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na⁺ and Ca²⁺ overload. The latter was mediated by reversal of Na⁺/Ca²⁺ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na⁺ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H⁺ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na⁺ and Ca²⁺ homeostasis, which reduces Na⁺-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.     

Sustained intracellular acidosis activates the myocardial Na/H exchanger independent of amino acid Ser and p90

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia-reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser⁷⁰³, Ser⁷⁷⁰, and Ser⁷⁷¹. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser⁷⁰³ and p90^rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser⁷⁰³; however, phosphorylation by p90^rsk required Ser⁷⁰³. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90^rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser⁷⁰³ and p90^rsk are not required for activation by sustained intracellular acidosis and that p90^rsk phosphorylation of Ser⁷⁰³ is independent of this type of activation.     

Recovery from hypoxia-induced internalization of cardiac Na+/H + exchanger 1 requires an adequate intracellular store of antioxidants

The heart is highly active metabolically but relatively underperfused and, therefore, vulnerable to ischemia. In addition to acidosis, a key component of ischemia is hypoxia that can modulate gene expression and protein function as part of an adaptive or even maladaptive response. Here, using cardiac-derived HL-1 cells, we investigate the effect of various hypoxic stimuli on the expression and activity of Na⁺/H ⁺ exchanger 1 (NHE1), a principal regulator of intracellular pH. Acute (10 min) anoxia produced a reversible decrease in the sarcolemmal NHE1 activity attributable to NHE1 internalization. Treatment with either 1% O ₂ or dimethyloxaloylglycine (DMOG; 1 mM) for 48-hr stabilized hypoxia-inducible factor 1 and reduced the sarcolemmal NHE1 activity by internalization, but without a change in total NHE1 immunoreactivity or message levels of the coding gene ( SLC9A1) determined in whole-cell lysates. Unlike the effect of DMOG, which was rapidly reversed on washout, reoxygenation after a prolonged period of hypoxia did not reverse the effects on NHE1, unless media were also supplemented with a membrane-permeant derivative of glutathione (GSH). Without a prior hypoxic episode, GSH supplementation had no effect on the NHE1 activity. Thus, posthypoxic NHE1 reinsertion can only take place if cells have a sufficient reservoir of a reducing agent. We propose that oxidative stress under prolonged hypoxia depletes intracellular GSH to an extent that curtails NHE1 reinsertion once the hypoxic stimulus is withdrawn. This effect may be cardioprotective, as rapid postischaemic restoration of the NHE1 activity is known to trigger reperfusion injury by producing an intracellular Na ⁺-overload, which is proarrhythmogenic.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: Homology and conservation of splice sites

The Na⁺/H⁺ exchanger is a widely distributed integral membrane protein that is responsible for pH regulation in mammalian tissues. We have cloned and analyzed the NHE1 isoform of the mouse genomic Na⁺/H⁺exchanger. A clone from a mouse genomic library contained the NHE1 promoter region and the 5-untranslated region. It also contained the first 121 amino acids of the coding region of the Na⁺/H⁺ exchanger. A splice site occurred after amino acid 121, at the same region as in the human NHE1 gene. The deduced amino terminal coding sequence was 76 and 88% identical to the human and rat NHE1 sequences respectively. The 5-untranslated region was highly homologous to that of other species and two minicistrons contained in the human Na⁺/H⁺ exchanger were present in the mouse sequence. The results show that the deduced protein sequence of the mouse NHE1 gene has a high level of homology with other species and that the splice site of the first intron is conserved. These results suggest that the first large intron may play an important role in the NHE1 gene expression.     

Synthesis and Na+/H+ Exchanger‐1 Inhibitory Activity of Substituted (Quinolinecarbonyl)guanidine Derivatives

The Na⁺/H⁺ exchanger (NHE) is a protein expressed in many mammalian cell types. It is involved in intracellular pH (pH_i) homeostasis by exchanging extracellular Na⁺ for intracellular H⁺. To date, nine NHE isoforms (NHE1–NHE9) have been identified. NHE1 is the most predominant isoform expressed in mammalian cardiac muscle. A novel series of substituted (quinolinecarbonyl)guanidine derivatives were designed and synthesized as NHE inhibitors. Most compounds can inhibit NHE1‐mediated platelet swelling in a concentration‐dependent manner, among which compound 7f was the most active and more potent than cariporide. Furthermore, compound 7f has also been demonstrated to exhibit the in vivo cardioprotective effects against SD rat myocardial ischemic‐reperfusion injury superior to those of cariporide.     

Gender-specific effects of exercise on cardiac pathology in Na+/H+ exchanger overexpressing mice

The Na(+)/H(+) exchanger isoform 1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. It is known that NHE1 levels increase in cardiac disease and we have recently demonstrated that expression of an activated NHE1 protein promotes cardiac hypertrophy in the mouse myocardium. We examined the gender-specific effects of exercise in combination with elevated cardiac expression of NHE1 on the myocardium in mice. Control mice and transgenic mice expressing elevated levels of wild type NHE1 and activated NHE1 were examined. There were gender-specific differences in the effects of NHE1 with exercise. Exercised wild type male mice showed a tendency toward increased heart weight. This was not apparent in female mice expressing elevated NHE1 levels. In some transgenic female mice, there was a significant decrease in the size of the exercised hearts, which was different from what occurred with male mice. Body weight was maintained in exercised control and transgenic male mice; however, it decreased in female mice with exercise more so in transgenic female mice expressing elevated levels of NHE1. Female mice expressing activated NHE1 had elevated HW/BW ratios compared to males, and this was exaggerated by exercise. These results suggest that gender-specific activation of NHE1 may be critical and that NHE1 plays a more critical role in promoting some types of hypertrophy in females in comparison with males.     

The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation

The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO(2)(-)/NO(3)(-) and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO(2)(-)/NO(3)(-), but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO(2)(-)/NO(3)(-) and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.     

NHE and ICAM-1 expression in hypoxic/reoxygenated coronary microvascular endothelial cells

Although Na+/H+ exchange (NHE) has been implicated in myocardial reperfusion injury, participation of coronary microvascular endothelial cells (CMECs) in this pathogenesis has been poorly understood. NHE-induced intracellular Ca2+ concentration ([Ca2+]i) overload in CMECs may increase the synthesis of intercellular adhesion molecules (ICAM), which is potentially involved in myocardial reperfusion injury. The present study tested the hypothesis that NHE plays a crucial role in [Ca2+]i overload and ICAM-1 synthesis in CMECs. Primary cultures of CMECs isolated from adult rat hearts were subjected to acidic hypoxia for 30 min followed by reoxygenation. Two structurally distinct NHE inhibitors, cariporide and 5-(N-N-dimethyl)-amiloride (DMA), had no significant effect on the acidic hypoxia-induced decrease in intracellular pH (pH(i)) of CMECs but significantly retarded pH(i) recovery after reoxygenation. These NHE inhibitors abolished the hypoxia- and reoxygenation-induced increase in [Ca2+]i. Expression of ICAM-1 mRNA was markedly increased in the vehicle-treated CMECs 3 h after reoxygenation, and this was significantly inhibited by treatment with cariporide, DMA, or Ca2+-free buffer. In addition, enhanced ICAM-I protein expression on the cell surface of CMECs 8 h after reoxygenation was attenuated by treatment with cariporide, DMA, or Ca2+-free buffer. These results suggest that NHE plays a crucial role in the rise of [Ca2+]i and ICAM-1 expression during acidic hypoxia/reoxygenation in CMECs. We propose that inhibition of ICAM-1 expression in CMECs may represent a novel mechanism of action of NHE inhibitors against ischemia-reperfusion injury.     

Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.     

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.