Calcium Signaling and Heart Failure: Defective Inter-Domain Interaction within the Ryanodine Receptor

Masafumi Yano
Yamaguchi University School of Medicine, Yamaguchi, Japan

Work by Yano and colleagues showed that the specific domain interaction with the ryanodine receptor (RyR) critically regulates the gating property of the RyR, and that its defectiveness may be involved in the common pathogenic mechanism of heart failure. The restoration of the defective domain interaction may provide a new clue for the development of a therapeutic strategy for heart failure.

Background

The RyR displays a micro-molecular complex, which includes 4 identical subunits and a variety of excessive proteins, such as phosphatase, PKA, and FK binding protein (FKBP). FKBP is known to stabilize channel gating with 1 RyR to 4 FKBP. In normal conditions, the channel is stabilized, but in heart failure, as this group reported previously, 3 of the 4 FKBPs are dissociated from the RyR and a comfirmation change of the RyR occurs and abnormal calcium leak is induced (Figure 1). 

The aberrant calcium leak is at the level of a single channel.  In heart failure, channel gating is hypersensitized to calcium: at a lower concentration of calcium, the channel is more activated. This can be seen in malignant hyperthermia, known as single point mutation disease of the skeletal type of RyR.

Many mutation sites have been reported for malignant hyperthermia or central core disease. Interestingly, these mutation sites cluster in 3 regions: N-terminal domain, central domain, and channel forming domain. Corresponding with these 3 regions, several mutations have been reported recently in patients with arrhythmogenic right ventricular dysplasia (ARVD) or polymorphic ventricular tachycardia (pVT). Because single point mutation at any region critically changes the channel gating, these regions are considered to be very important for the regression of channel gating.

Interestingly, the FKBP binding site is very closely located to the central region. Other mutations at the N-terminal domain or central domain induce hypersensitization of the channel. Ikemoto and colleagues proved their hypothesis, using skeletal type RyR, that there is an interaction between the N-terminal and the central domain as a regulatory switch for channel gating.

Study design and results

In the present study, Yano and colleagues assessed whether the abnormality in the inter-domain interaction induces the defective channel gating of RyR in heart failure. Under normal conditions, the N-terminal domain and the central domain interact, and then the channel is stabilized. The mode of the regulatory domain is called zipping.

They hypothesized that a mutation at either the N-terminal or the central domain interferes with the domain interaction and results in unzipping, which may de-stabilize the channel.

The domain peptide approach was used to prove their hypothesis. The domain peptide named DPc10 was used, which is a copy of the specific region of the central domain, has about 40 amino acid residues, and contains the reported mutation site. When DPc10 is introduced it is supposed to bind in the terminal region, and then in competition with native amino acid residue, unzipping may occur.

They confirmed that DPc10 binds to the RyR, as assessed by site-directed fluorescent labeling using the fluorescence confirmation probe SAED. Recently, in other work, they confirmed the fluorescence is located in the internal region. So, DPc10 binds to the N-terminal region.

To quantitate the mode of the domain interaction, they performed a chemical quencher experiment. In the unzipping state, a large chemical quencher, such as QSY-BSA, can easily access the gap between the regulatory domains and then efficiently quench the fluorescence. But, in the zipping state, the large chemical quencher cannot access the gap of the regulatory domain and the fluorescence cannot be quenched. In the presence of DPc10, the quenching extent was increased, indicating the shift of the mode from zipping to unzipping. FK506 also induced a shift of the mode from zipping to unzipping.

DPc10 dose-dependently induced calcium leak from the sarcoplasmic reticulum (SR). ATP was added, causing calcium uptake, and then after its completion, the calcium ATPase blocker thapsigargin was added to visualize the subsequent calcium leak. In the absence of DPc10, there was little calcium leak, but the leak appeared with an increase in the dose of DPc10.

DPc10 decreased the RyR-bound FKBP12.6, although DPc10 had no effect on the level of the phosphorylation of the RyR. Thus, this demonstrated that FKBP can be dissociated from the RyR, even without a change in the level of phosphorylation of the RyR.

Based on these findings, it is likely that FKBP dissociation induces domain unzipping and also that domain unzipping induces FKBP dissociation, as a regulating mechanism for channel gating.

Next, the effect of DPc10 on the in vivo myocyte function was assessed. After delivering DPc10 into the cell, calciumtransient and cell shortening were measured using Fura 2. In normal myocytes, in response to forskolin, an adenyl cyclase activator, cell shortening and the calcium transient were increased. In the presence of Dpc10, cell shortening and the peak of the calcium transient were decreased, the duration of the calcium transient was prolonged, and the response to forskolin was very poor. However, when incubating the myocyte with the calcium channel stabilizer JTV519, cell shortening and calcium transient were restored and the response to forskolin was restored toward normal. Previously they reported that JTV519 restores the confirmation state of the RyR and prevents abnormal calcium leak through the RyR.

Because this is similar to what is seen in the failing myocyte, they measured cell shortening in the failing myocyte taken from the pacing-induced canine heart failure. In failing myocytes, cell shortening and calcium transient were deteriorated, and the response to forskolin was very poor. But, after incubating the failing myocytes in JTV519 for 12 hours, cell shortening and calcium transient were restored, and the response to forskolin was restored towards normal.

The regulatory domains in the failing myocytes were then evaluated. Interestingly, as in the case in which they introduced DPc10 in the normal myocyte, in the failing myocyte unzipping already occurred. The RyR is hyper-phosphorylated in association with a decreased amount of RyR-bound FKBP, and spontaneous calcium leak occurs. But in the presence of the channel stabilizer JTV519, the mode of the domain interaction shifted from the unzipping state to the zipping state. Spontaneous calcium leak disappeared.

Conclusion

Excessive beta-receptor stimulation induces PKA-mediated hyper-phosphorylation of the RyR, which induces the dissociation of FKBP. This may shift the mode of the domain interaction from zipping to unzipping. This domain zipping may lead to calcium leak and intracellular diastolic calcium overload, systolic and diastolic dysfunction, arrhythmia, and finally the development of heart failure. The mutations seen in AVRD or pVT directly induce the shift of the domain interaction from zipping to unzipping.

A beta blocker can prevent the abnormal flow at the beta-receptor level. JTV519 may directly affect the RyR and shift the mode of domain interaction from zipping to unzipping. JTV519 may inhibit domain unzipping.

Telomerase, Cyclins, and Cardiac Apoptosis

Michael D. Schneider
Baylor College of Medicine, Houston, TX

This lecture focused on the cytoprotective roles of telomerase and what has been learned about how telomere dysfunction kills cardiac myocytes.

The extrinsic apoptotic pathway involves death domain receptors and the intrinsic apoptotic pathway or mitochondrial-dependent pathway is triggered by familiar cardiovascular (CV) stressors such as oxidative stress and IGF receptors working AKT. Based on increasing evidence for the prevalence of apoptosis in acute myocardial damage in chronic heart failure states, investigators have sought to manipulate this cascade in a manner favorable for survival of cardiac myocytes. For example, by transgenic overexpression of Bcl2 family proteins or pharmacological manipulations with caspase inhibitors. However, particularly for long-term heart failure, the adverse effects of interfering with death surveillance pathways in bone marrow and other systems may be problematic.

Schneider and colleagues have chosen to focus primarily in the initiating signals for cardiac myocyte apoptosis, and have learned some new and interesting insights into how conventional CV stresses are transduced into apoptotic signals. Their work with 2 unpublished pathways is reviewed here: 1) telomere dysfunction through the activation of a proximal MAP kinase pathway, whose key players are HGK (MAP4K4) and TAK1 (MAP3K7. These are upstream activators of the JNKp38 pathways and lead to cell death via loss of mitochondrial potential. 2) the involvement of PAL-2 directed cyclin-dependent kinases, in particular, cyclin-dependent kinase 9 (T/cdka), that leads to mitochondrial dysfunction through the suppression of a key transcriptional coactivator for the expression of mitochondrial genes.

Telomere dysfunction

Telomeres are the DNA protein structure existing at the specialized ends of chromosomes and cap the linear ends of linear genomes. Telomerase reverse transcriptase (TERT) is an RNA-dependent DNA polymerase, using a specific RNA template. TERT maintains the telomeric repeat, preventing telomere erosion and “uncapping.” In adults, TERT is associated primarily with germ cells, tumor cells, and stem cells.

TERT is markedly downregulated in myocardium after birth. Transgenic mice were made that expressed wild-type TERT or catalytically inactive TERT under the control of cardiac specific alpha-myosin heavy chain (HMC) promoter. In early hyperplasia, a larger than normal number of smaller than normal of cardiac myocytes were found. They also demonstrated that forced expression of telomerase was cytoprotective for the heart. TUNEL-positive cells post-infarction were reduced by 50%, with a slightly smaller decrease in infarct size, normalized for the area of risk. TERT also promotes myocyte survival after mechanical stress. In mice subjected to severe aortic banding, in the group with the greatest degree of constriction, TUNEL-positive cells, interstitial fibrosis, and systolic dysfunction are seen after just 1 week of load. Forced expression of telomerase prevents all 3 of those adverse aspects.

The biochemical consequences following mechanical load are telomere erosion within just 1 week and the transgene increases the basal telomere length and prevents its loss. This was accompanied by partial loss of 1 of those telomere repeating binding factors (TRF-2) and activation of known TRF-2 dependent responses, notably the phosphorylation and increased activity of the DNA damage checkpoint kinase, known as Chk2. All 3 of these responses were also seen in adult heart failure patients: telomere erosion, loss of TRF-2, and ATM-dependent phosphorylation of Chk2. Importantly, in age-matched patients with hypertrophic cardiomyopathy undergoing septal ablation surgery, and found neither apoptosis, telomere erosion, or the biochemical responses of the telomere were seen.

Studies for the mediators for the loss of TRF-2 and its ability to confer the apoptotic state led them to TGF-beta activated kinase 1, MAP3K7, and one of its upstream activators HGK (MAP4K4). TAK1 was originally described as an essential mediator of the BMP/TGF-beta cascade. Komuro and colleagues have shown that TAK1 acts in a divergent-convergent relationship with respect to the Smad transcription factors that are directly phosphorylated by these receptors. Further, they have shown an essential role for TAK1 in cardiac muscle specification. But, TAK1 also occupies a central role in signal transduction by other cascades, including cytokines and IRAKs. TAK1 also has a complex role as a negative regulator of the conical Wnt pathway. TAK1 activation by cytokines is thought to involve the upstream MAP kinase MAP4K4, which is a heart-enriched protein kinase for which little or no functional information was available.

Because existing antibodies to HGK were insufficient for their purposes, they used transgenic mice expressing epitope-tagged HGK in the heart. A number of CV stresses activate HGK in an immune complex assay, including transgenic expression of TNF-alpha and the G protein Gq, the essential signaling intermediate for angiotensin II, endothelin, and is an intermediate for mechanical signal transduction in the heart. Similar activation was seen by ischemia, reperfusion injury, and mechanical load in the epitope-tagged HGK mice.

Although HGK induced no discernible phenotype in cardiac muscle at baseline, it had a marked adverse synergy in relation to Gq. The Gq mice had mild concentric hypertrophy, and a very slight but significant increase apoptotic cells. The double transgenic mice had severely enlarged hearts with dilated cardiomyopathy (DCM), a marked increase in apoptosis measured by TUNEL staining and caspase activation, and severe early mortality.

They showed that a variety of stresses, including Gq, TNF-alpha, oxidative stress, and ceramide, result in downregulation of TRF-2, the essential telomere capping protein. The stress-induced downregulation of TRF-2 is occurring through the HGK-TAK1 module. The loss of TRF2 in ceramide-treated cells is partially rescued by dominant-negative HGK or dominant-negative TAK-1, with a larger rescue imposed by Bcl2, a conical apoptotic protein. So, stress pathways downregulate TRF2 via HGK-TAK1. Conversely, the loss of TRF2 function or expression activates HGK. This was shown using wild-type versus dominant-negative TRF2 or in antisense knockdown of the endogenous of TRF2.

To summarize the role of telomerase in cardiac protection, endogenous telomerase is downregulated in adult myocardium. Telomerase dysfunction is a consequence of cardiac stress cascades in tissue culture, in animal models, and in human heart failure.

A TRF2-MAP4K4 cycle amplifies apoptotic signals. Stress leading to activation of HGK, TAK1, and JNK lead independently to the loss of TRF2, and the circular action of these signaling molecules amplifies apoptotic signals. Exogenous telomerase prolongs cardiac myocyte cycling, and confers resistance to stress. One prediction from this model is that preventing the loss of TRF2 should be cytoprotective in myocardium. Recently they have confirmed this prediction. TRF2 was overexpressed under the control of the alpha-MHC promoter in doxirubicin-induced cardiomyopathy and showed the delay in mortality imposed by exogenous TRF2 and the significant rescue of cardiac myocytes from apoptosis. Conversely, they expressed the dominant-negative form of TRF2 in transgenic mice, which induces spontaneous cardiac myocyte apoptosis at a low level and spontaneous cardiomyopathy with onset at 8-9 months. Thus, they believe this is an essential pathway for cardiac myocyte protection and it is also a pathway that has therapeutic implications.

PAL2 directed cyclin-dependent kinases and mitochondrial dysfunction

The important role of atypical cyclin-dependent kinases (Cdk7, Cdk9) in cardiac myocyte hypertrophy was recently described by Sano and colleagues. The substrate for Cdk7 and Cdk9 is the large subunit of RNA polymerase II in a highly multimerized, serine-rich carboxy terminal domain (CTD). The unphosphorylated form of PAL2 is recruited to promoters, along with the basal transcription factors. Cyclin H and Cdk7 correspond to basal transcription factor TF2H and are important for PAL2 to engage splicing factors and execute pre-mRNA processing.

However, an additional phosphorylation is required for PAL2 to leave the promoter proximal region and enter the open reading frame, which occurs through Cdk9. This complex is also known as positive transcription elongation factor B (P-TEFb).

Both Cdk7 and Cdk9 are activated in vivo by hypertrophic signals, including Gq, calcineurin, and long-term mechanical load, in mice, as shown by Muroaki and colleagues. Cyclin T was expressed under the control of the alpha-MHC promoter and found a dose-dependent increase in Cdk9 activity, with concentric hypertrophy and myocycte enlargement at the higher levels. On the basis of these experiments, it might be inferred that activation of hypertrophic growth through Cdk9 might be potentially useful as a way to promote myocyte muscle restoration. However, that turned out not to be true.

The questions prompted by this work for RNA-polymerase II-directed cyclin-dependent kinases in heart failure are: Is hyperphosphorylation of RNA polymerase II germane to human heart failure, as extrapolated from mouse models?
Does hyperphosphorylation of RNAPII interact functionally with other hypertrophic pathways?
If synergistic or adverse effects exist, what are the mediators?

Phosphorylation of endogenous PAL2 on the preferred site by Cdk7, the preferred site by Cdk9, and immune complex kinase activity of both kinases are characteristic of human heart failure. In transgenic mice, the increase in endogenous Cdk9 activity can be seen conferred by the signaling protein Gq, with a somewhat greater increase induced by transgenic expression of cyclin T1. There is a synergistic effect on Cdk9 activity. The combined inheritance of Gq at doses that induce mild concentric hypertrophy plus cyclin T1 against mild concentric hypertrophy induces a florid DCM with extensive tissue fibrosis and apoptosis and early mortality. Microarray studies showed that even in the cyclin T1 mice that have little or no adverse phenotype, there was already a marked dysregulation of mitochondrial gene expression, including metabolic enzymes and mitochondrial transcription factors. One potential explanation for this generalized dysregulation of the mitochondrial program was the marked suppression for an essential coactivator of mitochondrial biogenesis, the PPAR gamma coactivator 1 (PGC-1).

Even in the cyclin T1 mice in the absence of overexpression of Gq, the mitochondrial had markedly diminished cristae and a decreased level of organization. Many of the mitochrondrial enzyme activities were depressed. By adenoviral delivery of cyclin T plus Cdk9 in tissue culture, they were able to test the functional consequences of this overexpression. They found that Cdk9 activity at physiological levels produced pronounced apoptosis and mitochondrial gene dysregulation. Repression of PGC-1 was a very early response to cyclin T plus Cdk9 in tissue culture. Maximal reduction of PGC-1 was achieved at 24 hours, whereas the other mitochondrial proteins continued to diminish over time. Cyclin T plus Cdk9 was also sufficient to suppress SERCA2 and certain other cardiac proteins. Importantly, forced expression of PGC1 by itself is sufficient to rescue cardiac myocytes from Cdk9-induced gene dysregulation. The suppression of Cox5b and the mitochondrial transcription factor Tfam and cardiac myocyte apoptosis were rescued by PGC-1 to baseline levels.

They currently postulate that hypertrophic signals that result in Cdk9 activation lead through a pathway that is not yet understood to marked suppression of PGC-1, a master regulator of mitochondrial biogenesis and function. The known partners for PGC-1 include PPAR gamma, nuclear respiratory factor, and MEF2. Downregulation has been seen of genes that are known targets for each of these dimers. This leads to marked suppression of genes required for mitochondrial metabolism and function, decreased resistance to apoptosis, and predisposition to heart failure in vivo.

To summarize, Cdk9 activation and hyper-phosphorylation of PAL2 are characteristic of mouse models, cultured cells, and human heart failure Benign levels of Cdk9 activity, which are well tolerated by themselves, cause florid heart failure when combined with other hypertrophic signals. Excess Cdk9 activity leads to global downregulation of genes for mitochondrial function, including its master regulator PGC-1. These levels repress PGC-1 acutely in culture, cause defective expression of genes for mitochondrial function in culture, and predispose to apoptosis. Restoring PGC-1 rescues cells from the adverse effects of Cdk9, and suggests that PGC-1 may be a useful therapeutic target in heart failure studies.

The Role of the Mitogen-Activated Protein Kinase Family in the Transition to Heart Failure

Kinya Otsu
Osaka University Graduate School of Medicine, Osaka, Japan

In their work with stress-activated MAP kinases, such as the ASK1 and p38 signaling pathways in the transition to heart failure, this group showed that mechanical stress, such as pressure overload or ischemic insult, activates the ASK1-JNK signaling pathway, leading to left ventricular (LV) remodeling and heart failure. P38 plays a protective role in the stress response. Thus, the balance of JNK and p38 may determine cell survival or cell death.

Background

Cardiac remodeling, including changes to the geometry, mass, volume, and function of the myocardium, is an adaptive response manifested as cardiac hypertrophy. The stimuli are continuous, excessive, and pathological, such as hypertension, infarction, and inflammation. The process can be maladaptive and the heart becomes dilated, resulting in dysfunction, necrosis, and apoptosis. Clinical symptoms of heart failure, such as congestion and dyspnea, then appear.  Figure 1 summarizes the working hypothesis of heart failure.

Extensive research has been conducted to elucidate the molecular mechanisms of cardiac remodeling. However, more remains to be elucidated. The trigger for cardiac remodeling is increased mechanical stress, by increased afterload or loss of contractile elements (Figure 2). Neurohormonal activation and cytokine release also are involved in this process.

Otsu and colleagues previously reported that apoptosis signal regulating kinase 1 (ASK1) is activated by angiotensin II, endothelin-I, TNF-alpha, and reactive oxygen species (ROS), which are known to be a mediator of cardiac remodeling. Thus, it can be hypothesized that ASK1 may play a role in cardiac remodeling. Their previous work showed that ASK1 is a ubiquitously expressed MAP3 kinase, an upstream kinase that activates MKK4/7-JNK and the MKK3/6-p38 signaling cascades. ASK1 plays a role in the mechanism of stress-induced apoptosis.

Study with ASK1 knockout mice and TAC

To elucidate the in vivo role of ASK1 in cardiac remodeling, this group used ASKI knockout mice (ASKKO). Two experimental models of cardiac remodeling were used: 1) a pressure overload-induced model, using thoracic transverse aortic constriction- (TAC) induced cardiac hypertrophy, with hypertrophy visible at 1 week and heart failure at 4 weeks; and 2) a myocardial infarction (MI) model, created by ligation of the left coronary artery. ASK1 activation was induced by TAC and MI, as shown by in vitro assay.

The baseline parameters of the ASKKO and wild-type (WT) mice showed no significant differences in body weight, heart weight, and hemodynamic parameters. The embryonic development and birth of the ASKKO was normal and they were indistinguishable in appearance from WT mice.

Surprisingly, although TAC induced an increase in heart weight, cross-sectional area in the tissue section, cell surface area in isolated cardiac myocyte, and heart weight/body weight ratio, there was no difference in these changes between the ASKKO and WT mice.

This group had previously reported that ASK1 plays a role in cardiac hypertrophy with an in vitro system. That data showed that angiotensin II, endothelin, and PE are induced in the cardiac hypertrophic response, but the dominant-negative form of ASK1 inhibited this response. In contrast, constitutively active ASK1 can induce the hypertrophic response, suggesting that ASK1 plays a role in cardiac hypertrophy. The precise mechanism to explain this discrepancy is not known, but one possibility is that the signaling pathways leading to hypertrophy are multifold and one signaling molecule is insufficient to prevent pressure-overload hypertrophy. 

Pathological analysis at 4 weeks after TAC showed that the heart dilation with fibrosis seen in the WT was completely prevented in the ASKKO mice. Functional analysis at 4 weeks after TAC showed that the WT mice hearts are dilated and percent fractional shortening was significantly decreased, but in the ASKKO mice the heart dilation and decrease in percent fractional shortening was prevented (Figure 3). This suggests that ASKI 1 may play a role in cardiac remodeling by pressure overload.

Study with ASKKO mice and MI

In this model, 4 weeks post-MI, the infarct area was extended to the free wall, apex, and some of the septum in the WT mice, and fibrosis replaced the ischemia injury in the remote area. However, in ASKKO the infarct was limited to the initial ischemic insult region and the remote area appeared to be normal (Figure 4).

Pathological analysis 4 weeks post- MI revealed that LVDd, LVDs, and percent fractional shortening were increased in WT mice, but the cardiac remodeling was significantly prevented in the ASKKO mice (Figure 5).

These data suggest that ASK1 plays an important role in cardiac remodeling in pressure overload and in MI. ASK1 is involved in stress-induced apoptosis. At 1 week and 4 weeks after TAC, a large increase in TUNEL-positive cells was seen, but this increase was significantly attenuated in the ASKKO mice. The TUNEL-positive cells are alpha-sarcomeric actin positive cells, suggesting that an apoptotic cell is a cardiac myocyte. These same findings were seen in the MI model. There was an increase in apoptosis at 1 week and 4 weeks in the border and remote areas, but this increase was significantly inhibited in the ASKKO mice in both areas.

To confirm the involvement of ASK1 in apoptosis, they isolated cardiac myocytes from the ASKKO mice. An increase in hydrogen peroxide concentration was associated with a decrease in the number of surviving cells. However, ASKKO mice showed greater resistance to hydrogen peroxide (Figure 6).

Constitutively active ASK1 can induce cardiac hypertrophy. After infection of rat neonatal cardiomyocytes with a higher titre of constitutively active ASK1, apoptosis was induced, indicating that the extent of ASK1 activation may determine the direction toward cardiac hypertrophy or apoptosis.

Cardiac-specific knockout mice experiments

After TAC or MI, p38 is activated in WT mice, but there was no significant difference in the activation level in the ASKKO mice. However, there was significant activation of JNK in WT mice after TAC and MI, but this activation was significantly attenuated in ASKKO mice (Figure 7). So, the ASK1-JNK signaling pathway may lead to LV remodeling and heart failure. Apoptosis may be involved in this process.

To elucidate the in vivo role of p38 in the stress response, they generated p38 floxed allele mice, with a functional exon containing the ATP binding site, and they generated mice overexpressing Cre recombinase under the control of the alpha myosin heavy chain (MHC) promoter. Cross-breeding these mice resulted in a cardiac-specific p38 KO mice, with a 90% reduction of p38 alpha, without any significant differences in the p38 isoform or MAP kinase.

No significant difference was found in the microscopic or macroscopic appearance in the p38KO mice heart. No significant difference was found in the function or structure of the heart. So, there was no requirement for p38 alpha during embryonic development. No significant difference was seen in any of the measured physiological parameters in p38KO mice.

At 1 week after TAC, there was no significant difference in the hypertrophic response between the WT and p38KO mice, such as LV weight, cross-sectional area, or biochemical markers. This suggests that p38 does not have an essential role in cardiac hypertrophy by pressure overload. However, the p38KO mice hearts became dilated with massive fibrosis, suggesting that p38 plays a protective role in the stress response.

In the p38KO mice, 1 week after TAC, an increase in LVd and LVds was found, and a decrease in percent fractional shortening. In the p38KO mice there was a large increase in the TUNEL-positive cells, which were alpha-sarcomeric actin-positive, indicating they are cardiomyocytes. An increase in the release of cytochrome C into the cytosol was detected, as well as an increase in the Bax/Bcl ratio, suggesting mitochondrial pathways are involved in apoptosis in p38KO hearts.

Role of Oxidative Stress in Post-Infarct Left Ventricular Remodeling and Failure

Hiroyuki Tsutsui
Hokkaido University Graduate School of Medicine, Sapporo, Japan

Studies in transgenic mice by this group showed that mitochondrial antioxidants, including glutathione peroxidase (GSHPx) and peroxiredoxin-3 (Prx-3), and mitochondrial transcription factor A (mtTFA) are novel therapeutic target genes in heart failure. MtTFA is important in the replication and maintenance of mitochondrial DNA.

Background

Myocardial infarction is the most important cause of heart failure. Post-infarct myocardial remodeling plays an important role in the pathophysiology of heart failure, and includes infarct expansion that occurs hours to days post-infarct and the subsequent global remodeling process that occurs days to months post-infarct. Activation of the renin-angiotensin system (RAS) in myocardial remodeling has led to the use of ACE inhibitors as the first-line drug for the treatment of the acute and chronic phases of heart failure. The 23% risk reduction in mortality with ACE inhibitors found in the SOLVD treatment trial is insufficient. Elucidation is necessary of novel contributing factors that must be downstream of neurohormones that are activated in the setting of heart failure and upstream of myocardial remodeling, which includes hypertrophy, fibrosis, and apoptosis (Figure 1).

Thus, oxidative stress in the pathophysiology of heart failure has been the focus of research by Tsutsui and colleagues. Previous work by this group showed that TNF-alpha and angiotensin II can generate reactive oxidant species (ROS) in cardiac myocytes. In the post-infarct mouse model they showed that 1) mitochondrial superoxide generation was increased in the failing heart, 2) mitochondrial DNA injury can be observed, and 3) the mitochondrial copy number was significantly decreased with a parallel decrease in mitochondrial DNA-derived transcript messages encoding the mitochondrial electron transport complexes, leading to a decrease in the activity of Complex I, III, and IV (Figure 2). However, Complex II and citrate synthase are encoded by nuclear DNA and were preserved at normal levels.

Based on these results, mitochrondrial ROS leads to mitochondrial DNA damage and electron transport gene dysfunction, which then leads to additional ROS generation—a vicious cycle between mitochondrial damage and mitochondrial dysfunction (Figure 3). Therefore, the modulation of mitochondrial oxidative stress can be a novel therapeutic strategy for heart failure.

Study with GSHPx in transgenic mice \

GSHPx is a key antioxidant enzyme, which scavenges hydrogen peroxide (H2O2) and prevents the formation of other more toxic radicals such as the hydroxy radical (OH). GSHPx possesses a higher affinity for H2O2 than catalase, also a scavenger for H2O2 and OH. GSHPx is present in high amounts within the heart, especially in the cytosolic and mitochondrial compartments. Therefore, GSHPx can exert greater protective effects against oxidative damage than SOD, catalase, or their combination.

Myocardial infarction was created in GSHPx transgenic mice (C57BL/6xCBA/J hybrid mice overexpressing GSHPx; TG+MI group, n=44) and in wild-type mice (WT+M group, n=46).  Myocardial TBARS, an index of myocardial oxidative stress, was increased in the WT+MI group, to about 70 nmol/g, but this increase was inhibited in the TG+MI group (40 nmol/g). Survival was improved in the TG+MI group compared to the WT+MI group (about 85% versus 60%, p<0.01). Infarct size measured by TTC staining showed no significant different between the groups for the risk area (about 40% in each) or the infarct area (about 80% in each).

Study with MtTFA in transgenic mice

In the transgenic mice, human mtTFA was highly expressed in the heart, assessed by Western blot analysis. Importantly, endogenous mouse mtTFA was not altered in transgenic mice. After MI was created in the mtTFA transgenic mice and WT mice, none of the transgenic mice died, despite comparable MI size to the WT mice.

Conclusion

Current work by this group includes the physiology, pathology, and molecular biology in these transgenic mice. The preliminary results are very promising, showing that mtTFA is a very novel and promising target in the treatment of heart failure. Based on the current results, mtTFA can be a novel target to maintain mitochondrial biogenesis and function and ultimately for the treatment of heart failure.