Lack of MG53 in human heart precludes utility as a biomarker of myocardial injury or endogenous cardioprotective factor.

AIMS: Mitsugumin-53 (MG53/TRIM72) is an E3-ubiquitin ligase that rapidly accumulates at sites of membrane injury and plays an important role in membrane repair of skeletal and cardiac muscle. MG53 has been implicated in cardiac ischaemia-reperfusion injury, and serum MG53 provides a biomarker of skeletal muscle injury in the mdx mouse model of Duchenne muscular dystrophy. We evaluated the clinical utility of MG53 as a biomarker of myocardial injury. METHODS AND RESULTS: We performed Langendorff ischaemia-reperfusion injury on wild-type and dysferlin-null murine hearts, using dysferlin deficiency to effectively model more severe outcomes from cardiac ischaemia-reperfusion injury. MG53 released into the coronary effluent correlated strongly and significantly (r = 0.79-0.85, P < 0.0001) with functional impairment after ischaemic injury. We initiated a clinical trial in paediatric patients undergoing corrective heart surgery, the first study of MG53 release with myocardial injury in humans. Unexpectedly, we reveal although MG53 is robustly expressed in rat and mouse hearts, MG53 is scant to absent in human, ovine, or porcine hearts. Absence of MG53 in 11 human heart specimens was confirmed using three separate antibodies to MG53, each subject to epitope mapping and confirmed immunospecificity using MG53-deficient muscle cells. CONCLUSION: MG53 is an effective biomarker of myocardial injury and dysfunction in murine hearts. However, MG53 is not expressed in human heart and therefore does not hold utility as a clinical biomarker of myocardial injury. Although cardioprotective roles for endogenous myocardial MG53 cannot be extrapolated from rodents to humans, potential therapeutic application of recombinant MG53 for myocardial membrane injury prevails.

A novel class of anticancer compounds targets the actin cytoskeleton in tumor cells.

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Somatic mutations in NKX2­5, GATA4, and HAND1 are not a common cause of tetralogy of Fallot or hypoplastic left heart.

The majority of congenital heart disease (CHD) occurs as a sporadic finding, with a minority of cases associated with a known genetic abnormality. Combinations of genetic and environmental factors are implicated, with the recent and intriguing hypothesis that an apparently high rate of somatic mutations might explain some sporadic CHD. We used samples of right ventricular myocardium from patients undergoing surgical repair of tetralogy of Fallot (TOF) and hypoplastic left heart (HLH) to examine the incidence of somatic mutation in cardiac tissue. TOF is a common form of cyanotic CHD, occurring in 3.3 per 10,000 live births. HLH is a rare defect in which the left side of the heart is severely under-developed. Both are severe malformations whose genetic etiology is largely unknown. We carried out direct sequence analysis of the NKX2­5 and GATA4 genes from fresh frozen cardiac tissues and matched blood samples of nine TOF patients. Analysis of NKX2­5, GATA4, and HAND1 was performed from cardiac tissue of 24 HLH patients and three matched blood samples. No somatic or germline mutations were identified in the TOF or HLH patients. Although limited by sample size, our study suggests that somatic mutations in NKX2­5 and GATA4 are not a common cause of isolated TOF or HLH.

Investigation of association between PFO complicated by cryptogenic stroke and a common variant of the cardiac transcription factor GATA4.

Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke.

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy.

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-ß1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

GATA4 mutations in 357 unrelated patients with congenital heart malformation.

Congenital heart disease (CHD) represents one of the most common birth defects, but the genetic causes remain largely unknown. Mutations in GATA4, encoding a zinc finger transcription factor with a pivotal role in heart development, have been associated with CHD in several familial cases and a small subset of sporadic patients. To estimate the pathogenetic role of GATA4 in CHD, we screened for mutations in 357 unrelated patients with different congenital heart malformations. In addition to nine synonymous changes, we identified two known (A411V and D425N) and two novel putative mutations (G69D and P163R) in five patients with atrial or ventricular septal defects that were not seen in control subjects. The four mutations did not show altered GATA4 transcriptional activity in synergy with the transcription factors NKX2-5 and TBX20. Our data expand the spectrum of mutations associated with cardiac septal defects but do not support GATA4 mutations as a common cause of CHD.

Dysfunction induced by ischemia versus edema: does edema matter?

OBJECTIVES: Recovery from pediatric cardiac surgery is affected by ischemia-reperfusion injury, cardiac edema, and in some cases a low cardiac output syndrome. Although association has been made between the development of edema and dysfunction, modeling is confounded by intercurrent injurious stimuli that also cause cardiac edema and dysfunction. We tested whether a true causal relationship exists between edema and cardiac dysfunction. METHODS: We induced either ischemia or edema alone in isolated cardiomyocytes and whole Langendorff-perfused hearts. Function was measured as shortening dynamics and developed pressure, respectively. RESULTS: Ischemic injury impaired function in both cardiomyocytes and whole hearts. Isolated cells showed significant reduction in peak shortening and departure and relaxation velocities. Whole hearts displayed severely reduced developed pressures. Hyposmotic solution forced cardiomyocytes to swell to 7% greater than their normal size. No significant effect on shortening was seen. Similarly, Langendorff-perfused hearts were induced to take on 3% more water than control-perfused hearts and 9% more water than nonperfused hearts. This additional water was associated with mild dysfunction. CONCLUSIONS: We demonstrate the capacity of the heart to tolerate edema greater than that seen in clinical settings without residual effect. Ischemia results in ongoing contractile dysfunction of both isolated cardiomyocytes and whole hearts. We conclude that dysfunction resulting from edema in ex vivo cardiac models is mild and suggest review of the importance given to edema-mediated dysfunction after cardiac surgery.

Myocardial membrane injury in pediatric cardiac surgery: An animal model.

OBJECTIVE: Reduced myocardial performance invariably follows pediatric cardiac surgery and is manifested by a low cardiac output state in its severest form. The role of myocardial membrane proteins in this setting is unknown. Dystrophin and dysferlin are involved in membrane integrity, whereas aquaporins selectively transport water. These proteins were examined in a model of pediatric cardiac surgery, together with a trial of poloxamer 188, which may reduce membrane injury. METHODS: Eight lambs were randomized to saline with or without poloxamer 188. Lambs underwent 2 hours of cardiopulmonary bypass and aortic crossclamping. After a further 9 hours of monitoring, the hearts were assessed for water content, capillary leak, and protein expression. RESULTS: Dystrophin expression was unaffected by ischemia/reperfusion, but dysferlin expression was reduced. Aquaporin 1 protein increased after ischemia/reperfusion. Poloxamer 188 administration was associated with supranormal levels of dystrophin, preservation of dysferlin expression, and normalization of aquaporin 1 expression. Poloxamer 188 was associated with less capillary leak, maintained colloid osmotic pressure, and less hemodilution. Poloxamer 188 was associated with an improved hemodynamic profile (higher blood pressure, higher venous saturation, and lower lactate), although the heart rate tended to be higher. CONCLUSIONS: Changes in protein expression within the myocardial membrane were found in a clinically relevant model of pediatric cardiac surgery. Indicators of reduced performance, such as lower blood pressure and lower oxygen delivery, were lessened in association with the administration of the membrane protecting poloxamer 188. Poloxamer 188 was also associated with potentially beneficial changes in membrane protein expression, reduced capillary leakage, and less hemodilution.

Myocardial ischemia is more important than the effects of cardiopulmonary bypass on myocardial water handling and postoperative dysfunction: a pediatric animal model.

OBJECTIVES: Low cardiac output state is the principal cause of morbidity after surgical intervention for congenital heart disease. Myocardial ischemia-reperfusion injury, apoptosis, capillary leak syndrome, and myocardial edema are associated factors. We established a clinically relevant model to examine relationships between myocardial ischemia, edema, and cardiac dysfunction and to assess the role of the water transport proteins aquaporins. METHODS: Sixteen lambs were studied. Seven were control animals not undergoing cardiopulmonary bypass, and 9 underwent bypass. Six had 90 minutes of aortic crossclamping with blood cardioplegia and moderate hypothermia. The remaining 3 underwent cardiopulmonary bypass without aortic crossclamping. Hemodynamic and biochemical data were recorded, and myocardial edema, apoptotic markers, and aquaporin expression were determined after death. RESULTS: The group undergoing cardiopulmonary bypass with aortic crossclamping had a low cardiac output state, with early postoperative tachycardia, hypotension, increased serum lactate levels, and impaired tissue oxygen delivery (P < .05) compared with the group undergoing cardiopulmonary bypass without aortic crossclamping. The lambs undergoing cardiopulmonary bypass with aortic crossclamping had increased myocardial water (P < .05) compared with those not undergoing cardiopulmonary bypass and a 2-fold increase in aquaporin 1 mRNA expression (P < .05) compared with those not undergoing cardiopulmonary bypass and those undergoing cardiopulmonary bypass without aortic crossclamping. CONCLUSIONS: A temporal association between hemodynamic dysfunction, myocardial edema, and increased aquaporin 1 expression was demonstrated. Cardiopulmonary bypass without ischemia was associated with minimal edema, negligible myocardial dysfunction, and static aquaporin expression. Ischemic reperfusion injury is the main cause of myocardial edema and myocardial dysfunction, but a causal relationship between edema and dysfunction remains to be proved.

Changes in skeletal muscle expression of AQP1 and AQP4 in dystrophinopathy and dysferlinopathy patients.

Transmembrane water transport is mediated by aquaporins (AQPs), of which AQP1 and AQP4 are expressed in skeletal muscle. AQP4 expression is reduced in Duchenne muscular dystrophy (DMD) patients, and is reported to correlate with decreased alpha1-syntrophin and altered osmotic permeability. In this study, we assessed the relationship between AQP1, AQP4, dystrophin and alpha1-syntrophin in dystrophinopathy and dysferlinopathy patients. Muscle biopsies of patients with DMD (n = 8) and limb-girdle muscular dystrophy type 2B (LGMD2B; n = 5) were screened for AQP1 and AQP4 expression by real-time quantitative RT-PCR or Western blot and immunohistochemistry. AQP expression was further analyzed in primary myotubes derived from DMD and LGMD2B patients by cell culture and immunohistochemistry. AQP1 transcript and protein expression was significantly elevated in DMD biopsies, and was localized to the sarcolemma of muscle fibers and endothelia of muscle capillaries. AQP4 was significantly reduced despite normal dystrophin and alpha1-syntrophin in dysferlinopathy patients, while expression of AQP1 was variably upregulated. Expression of AQP1 and AQP4 was normal in patient-derived primary myotubes, suggesting that altered AQPs observed in biopsies are likely secondary to the dystrophic process. Our study shows that AQP4 downregulation can occur in muscular dystrophies with either normal or disrupted expression of dystrophin-associated proteins, and that this might be associated with upregulation of AQP1.

Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy.

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Myocardial water handling and the role of aquaporins.

Cardiac surgery is performed in approximately 770,000 adults and 30,000 children in the United States of America annually. In this review we outline the mechanistic links between post-operative myocardial stunning and the development of myocardial edema. These interrelated processes cause a decline in myocardial performance that account for significant morbidity and mortality after cardiac surgery. Factors leading to myocardial edema include hemodilution, ischemia and reperfusion as well as osmotic gradients arising from pathological change. Several members of the aquaporin family of water transport proteins have been described in the myocardium although their role in the pathogenesis and resolution of cardiac edema is not established. This review examines evidence for the involvement of aquaporins in myocardial water handling during normal and pathological conditions.

Cardiac aquaporin expression in humans, rats, and mice.

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation.

The transcriptional response to hypoxic insult controlled by FRA-2.

FRA-2 is involved in cellular differentiation and is also upregulated in response to ischemic injury to the brain. To shed light on the function of this transcription factor, a novel microarray analysis was utilized to identify FRA-2-dependent gene expression increased in the hypoxic response. Genes were identified that were upregulated by exposure of neuronally differentiated PC12 cells to hypoxia. Using a dominant negative construct to block FRA-2, a second subset of genes that were FRA-2 dependent was found. Cross comparison then allowed isolation of a list of genes that were induced in response to hypoxia in a FRA-2-dependent manner. These data suggest that FRA-2 is involved in the transcriptional control of neuroprotective genes and in the switch from aerobic to anaerobic metabolism.

Temporary focal ischemia in the mouse: technical aspects and patterns of Fluoro-Jade evident neurodegeneration.

Animal models of cerebral infarction are crucial to understanding the mechanisms of neuronal survival following ischemic brain injury and to the development of therapeutic interventions for victims of all types of stroke. Rodents have been used extensively in such research. One rodent model of stroke utilizes either permanent or temporary occlusion of the middle cerebral artery (MCAO) to produce ischemia. Since the development of an endovascular method for this was published in 1989, MCAO has been applied commonly to the rat, and often paired with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining for stroke volume measurement. Meanwhile, advances in the ability to genetically alter mice have allowed exciting lines of research into ischemia. Because of technical demands and issues with survival, relatively few laboratories have investigated the MCAO method in the mouse. Our present work utilizes a mouse middle cerebral occlusion (MCAO) model of embolic stroke to study neuronal degeneration following temporary focal cerebral ischemia. C57Bl/6J mice were used to examine the exact effects of MCAO using Fluoro-Jade, a marker of neurodegeneration that allows observation of specific brain regions and cells destined to die. A time course of escalating neuronal degeneration from 10 min to 7 days following MCAO was established. Technical aspects of this popular method for transient focal ischemia as it applies to the mouse are discussed.

Temporal and regional expression of Fos-related proteins in response to ischemic injury.

The AP-1 transcription factor family has been widely studied in the response to ischemic brain injury. The data to date have demonstrated a complex involvement that depends on stimulus, subunit composition and brain region. One member in particular, the Fos-related antigen FRA-2, has demonstrated a potential for controlling neuroprotective gene expression. This study characterized the temporal and regional expression of a variety of proteins following ischemic injury induced by occlusion of the middle cerebral artery in rats. The results demonstrated upregulation of both c-Fos and FRA-2 in penumbral regions that preceded upregulation of the classic injury-associated proteins expressed by astrocytes and microglia and, in the case of FRA-2, appeared to correlate with neuronal survival. A further, previously undescribed, expression of FRA-2 in endothelial cells of the core ischemic region was also demonstrated.

Neurodegeneration in the rat hippocampus and striatum after middle cerebral artery occlusion.

Animal models of ischemia are in wide use to elucidate the molecular mechanisms of brain injury that result from cardiovascular disease in humans. We have used the fluorescent, anionic dye, Fluoro-Jade, to examine cellular degeneration that occurs in association with the middle cerebral artery occlusion (MCAO) model. MCAO results in cortical infarction as well as damage to the hippocampus leading to a delayed form of death of hippocampal neurons. We examined brain sections at 6 h, 12 h, 1, 4, 7, 14 and 21 days after injury. Fluoro-Jade labeling of the striatum was seen over a protracted time-course, with degeneration beginning by 6 h after injury. Neuronal degeneration in the hippocampus, in contrast, occurs between 12 h and 7 days after injury with neuronal death reaching a peak at 4 days. GFAP/Fluoro-Jade double labeling revealed that the Fluoro-Jade positive staining at late time-points in the striatum included astrocytic cells. Together, the results show Fluoro-Jade to be a useful marker of cellular degeneration following ischemic injury. Further, the use of this dye has enabled us to demonstrate previously undescribed events of cellular injury resulting from ischemia.