Maike Krenz

Cell Biology of Ischemia/Reperfusion Injury

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of debilitating disease and death. Tissue injury and/or death occur as a result of the initial ischemic insult, which is determined primarily by the magnitude and duration of the interruption in the blood supply, and then subsequent damage induced by reperfusion. During prolonged ischemia, ATP levels and intracellular pH decrease as a result of anaerobic metabolism and lactate accumulation. As a consequence, ATPase-dependent ion transport mechanisms become dysfunctional, contributing to increased intracellular and mitochondrial calcium levels (calcium overload), cell swelling and rupture, and cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Although oxygen levels are restored upon reperfusion, a surge in the generation of reactive oxygen species occurs and proinflammatory neutrophils infiltrate ischemic tissues to exacerbate ischemic injury. The pathologic events induced by I/R orchestrate the opening of the mitochondrial permeability transition pore, which appears to represent a common end-effector of the pathologic events initiated by I/R. The aim of this treatise is to provide a comprehensive review of the mechanisms underlying the development of I/R injury, from which it should be apparent that a combination of molecular and cellular approaches targeting multiple pathologic processes to limit the extent of I/R injury must be adopted to enhance resistance to cell death and increase regenerative capacity in order to effect long-lasting repair of ischemic tissues.

Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice

Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.

Moderate Ethanol Ingestion and Cardiovascular Protection: From Epidemiologic Associations to Cellular Mechanisms

While ethanol intake at high levels (3-4 or more drinks), either in acute (occasional binge drinking) or chronic (daily) settings, increases the risk for myocardial infarction and stroke, an inverse relationship between regular consumption of alcoholic beverages at light to moderate levels (1-2 drinks per day) and cardiovascular risk has been consistently noted in a large number of epidemiologic studies. Although initially attributed to polyphenolic antioxidants in red wine, subsequent work has established that the ethanol component contributes to the beneficial effects associated with moderate intake of alcoholic beverages regardless of type (red versus white wine, beer, spirits). Concerns have been raised with regard to interpretation of epidemiologic evidence for this association including heterogeneity of the reference groups examined in many studies, different lifestyles of moderate drinkers versus abstainers, and favorable risk profiles in moderate drinkers. However, better controlled epidemiologic studies and especially work conducted in animal models and cell culture systems have substantiated this association and clearly established a cause and effect relationship between alcohol consumption and reductions in tissue injury induced by ischemia/reperfusion (I/R), respectively. The aims of this review are to summarize the epidemiologic evidence supporting the effectiveness of ethanol ingestion in reducing the likelihood of adverse cardiovascular events such as myocardial infarction and ischemic stroke, even in patients with co-existing risk factors, to discuss the ideal quantities, drinking patterns, and types of alcoholic beverages that confer protective effects in the cardiovascular system, and to review the findings of recent experimental studies directed at uncovering the mechanisms that underlie the cardiovascular protective effects of antecedent ethanol ingestion. Mechanistic interrogation of the signaling pathways invoked by antecedent ethanol ingestion may point the way towards development of new therapeutic approaches that mimic the powerful protective effects of socially relevant alcohol intake to limit I/R injury, but minimize the negative psychosocial impact and pathologic outcomes that also accompany consumption of ethanol.

Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder characterized by a wide spectrum of defects, which most frequently include proportionate short stature, craniofacial anomalies, and congenital heart disease (CHD). NS is the most common nonchromosomal cause of CHD, and 80%-90% of NS patients have cardiac involvement. Mutations within the protein tyrosine phosphatase Src homology region 2, phosphatase 2 (SHP2) are responsible for approximately 50% of the cases of NS with cardiac involvement. To understand the developmental stage- and cell type-specific consequences of the NS SHP2 gain-of-function mutation, Q79R, we generated transgenic mice in which the mutated protein was expressed during gestation or following birth in cardiomyocytes. Q79R SHP2 embryonic hearts showed altered cardiomyocyte cell cycling, ventricular noncompaction, and ventricular septal defects, while, in the postnatal cardiomyocyte, Q79R SHP2 expression was completely benign. Fetal expression of Q79R led to the specific activation of the ERK1/2 pathway, and breeding of the Q79R transgenics into ERK1/2-null backgrounds confirmed the pathways necessity and sufficiency in mediating mutant SHP2s effects. Our data establish the developmental stage-specific effects of Q79R cardiac expression in NS; show that ablation of subsequent ERK1/2 activation prevents the development of cardiac abnormalities; and suggest that ERK1/2 modulation could have important implications for developing therapeutic strategies in CHD.

Forced Expression of α-Myosin Heavy Chain in the Rabbit Ventricle Results in Cardioprotection Under Cardiomyopathic Conditions

Background—The biochemical differences between the 2 mammalian cardiac myosin heavy chains (MHCs), &agr;-MHC and &bgr;-MHC, are well described, but the physiological consequences of basal isoform expression and isoform shifts in response to altered cardiac load are not clearly understood. Mature human ventricle contains primarily the &bgr;-MHC isoform. However, the &agr;-MHC isoform can be detected in healthy human ventricle and appears to be significantly downregulated in failing hearts. The unique biochemical properties of the &agr;-MHC isoform might offer functional advantages in a failing heart that is expressing only the &bgr;-MHC isoform. This hypothesis cannot be tested in mice or rats because both species express &agr;-MHC as the predominant isoform. Methods and Results—To test the effects of persistent &agr;-MHC expression on the background of &bgr;-MHC, we made transgenic (TG) rabbits that expressed rabbit &agr;-MHC cDNA in the ventricle so that the endogenous myosin was partially replaced by the transgenically encoded species. Molecular, histological, and functional analyses showed no significant baseline effects in the TG rabbits compared with nontransgenic (NTG) littermates. To determine whether &agr;-MHC expression afforded any advantages to stressed myocardium, a cohort of TG and NTG rabbits was subjected to rapid ventricular pacing. Although both the TG and NTG rabbits developed dilated cardiomyopathy, the TG rabbits had a higher shortening fraction, less septal thinning, and more normal ±dP/dt than paced NTG rabbits. Conclusions—Transgenic expression of &agr;-MHC does not have any apparent detrimental effects under basal conditions and is cardioprotective in experimental tachycardia-induced cardiomyopathy.

Role of ERK1/2 signaling in congenital valve malformations in Noonan syndrome

Noonan syndrome (NS) is the most common nonchromosomal genetic disorder associated with cardiovascular malformations. The most prominent cardiac defects in NS are pulmonary valve stenosis and hypertrophic cardiomyopathy. Gain-of-function mutations in the protein tyrosine phosphatase Shp2 have been identified in 50% of NS families. We created a NS mouse model with selective overexpression of mutant Shp2 (Q79R-Shp2) in the developing endocardial cushions. In our model, Cre recombinase driven by the Tie2 promoter irreversibly activates transgenic Q79R-Shp2 expression in the endothelial-derived cell lineage. Q79R-Shp2 expression resulted in embryonic lethality by embryonic day 14.5. Importantly, mutant embryos showed significantly enlarged endocardial cushions in the atrioventricular canal and in the outflow tract. In contrast, overexpression of wild-type Shp2 protein at comparable levels did not enhance endocardial cushion growth or alter the morphology of the mature adult valves. Expression of Q79R-Shp2 was accompanied by increased ERK1/2 activation in a subset of cells within the cushion mesenchyme, suggesting that hyperactivation of this signaling pathway may play a pathogenic role. To test this hypothesis in vivo, Q79R-Shp2-expressing mice were crossed with mice carrying either a homozygous ERK1 or a heterozygous ERK2 deletion. Deletion of ERK1 completely rescued the endocardial cushion phenotype, whereas ERK2 protein reduction did not affect endocardial cushion size. Constitutive hyperactivation of ERK1/2 signaling alone with a transgenic approach resulted in a phenocopy of the valvular phenotype. The data demonstrate both necessity and sufficiency of increased ERK activation downstream of Shp2 in mediating abnormal valve development in a NS mouse model.

Noonan Syndrome Mutation Q79R in Shp2 Increases Proliferation of Valve Primordia Mesenchymal Cells via Extracellular Signal-Regulated Kinase 1/2 Signaling

The molecular pathways regulating valve development are only partially understood. Recent studies indicate that dysregulation of mitogen-activated protein kinase (MAPK) signaling might play a major role in the pathogenesis of congenital valvular malformations, and, in this study, we explored the role of extracellular signal–regulated kinase (ERK) 1/2 activation in valve primordia expressing the Noonan syndrome mutation Q79R-Shp2. Noonan syndrome is an autosomal dominant disease characterized by dysmorphic features and cardiac abnormalities, with frequent pulmonic stenosis. The Q79R mutation of PTPN11 previously identified in Noonan syndrome families results in a gain-of-function of the encoded protein tyrosine phosphatase Shp2. We compared the effects of wild-type Shp2 and Q79R-Shp2 on endocardial cushion development. Atrioventricular and outflow tract endocardial cushions were excised from chick embryos, infected with wild-type Shp2 or Q79R-Shp2 adenovirus and embedded in a gel matrix. Q79R-Shp2, but not wild-type-Shp2, expression resulted in increased outgrowth of cells into the gel. The dependence of the Q79R-Shp2 effect on ERK1/2 and p38 MAPK signaling was then determined. The MAPK/ERK kinase (MEK)-1 inhibitor U0126, but not the p38-MAPK pathway inhibitor SB203580, abolished the effect of Q79R-Shp2 on cushion outgrowth. Coinfection with Q79R-Shp2 and dominant negative MEK-1 prevented enhanced endocardial cushion outgrowth, whereas expression of constitutively active MEK-1 mimicked the effect of Q79R-Shp2. Furthermore, dissociated cushion cells displayed increased 5-bromodeoxyuridine incorporation when infected with Q79R-Shp2 but not with wild-type Shp2. This promitotic effect was eliminated by U0126. Our results demonstrate that ERK1/2 activation is both necessary and sufficient to mediate the hyperproliferative effect of a gain-of-function mutation of Shp2 on mesenchymal cells in valve primordia.

Heart failure with preserved ejection fraction: chronic low-intensity interval exercise training preserves myocardial O2 balance and diastolic function

We have previously reported chronic low-intensity interval exercise training attenuates fibrosis, impaired cardiac mitochondrial function, and coronary vascular dysfunction in miniature swine with left ventricular (LV) hypertrophy (Emter CA, Baines CP. Am J Physiol Heart Circ Physiol 299: H1348-H1356, 2010; Emter CA, et al. Am J Physiol Heart Circ Physiol 301: H1687-H1694, 2011). The purpose of this study was to test two hypotheses: 1) chronic low-intensity interval training preserves normal myocardial oxygen supply/demand balance; and 2) training-dependent attenuation of LV fibrotic remodeling improves diastolic function in aortic-banded sedentary, exercise-trained (HF-TR), and control sedentary male Yucatan miniature swine displaying symptoms of heart failure with preserved ejection fraction. Pressure-volume loops, coronary blood flow, and two-dimensional speckle tracking ultrasound were utilized in vivo under conditions of increasing peripheral mean arterial pressure and β-adrenergic stimulation 6 mo postsurgery to evaluate cardiac function. Normal diastolic function in HF-TR animals was characterized by prevention of increased time constant of isovolumic relaxation, normal LV untwisting rate, and enhanced apical circumferential and radial strain rate. Reduced fibrosis, normal matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-4 mRNA expression, and increased collagen III isoform mRNA levels (P < 0.05) accompanied improved diastolic function following chronic training. Exercise-dependent improvements in coronary blood flow for a given myocardial oxygen consumption (P < 0.05) and cardiac efficiency (stroke work to myocardial oxygen consumption, P < 0.05) were associated with preserved contractile reserve. LV hypertrophy in HF-TR animals was associated with increased activation of Akt and preservation of activated JNK/SAPK. In conclusion, chronic low-intensity interval exercise training attenuates diastolic impairment by promoting compliant extracellular matrix fibrotic components and preserving extracellular matrix regulatory mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype in a large animal model resembling heart failure with preserved ejection fraction.

The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.

Distribution and Structure-Function Relationship of Myosin Heavy Chain Isoforms in the Adult Mouse Heart

The two cardiac myosin heavy chain isoforms, α and β, exhibit distinct functional characteristics and therefore may be distributed regionally within the heart to match the functional demands of a specific region. In adult mouse hearts, which predominantly express α-myosin heavy chain, we observed high concentrations of β-myosin in distinct areas such as at the tip of papillary muscles and at the base close to the valvular annulus. In light of these distinct distribution patterns of the myosin isoforms, we subsequently explored the isoform-specific structure-function relationships of the myosins. The α- and β-isoforms are 93% identical in amino acid sequence, but it remains unclear which of the nonidentical residues determines isoform functionality. We hypothesized that residues situated within or close to the actin-binding interface of the myosin head influence actin binding and thereby modulate actin-activated ATPase activity. A chimeric myosin was created containing β-sequence from amino acid 417 to 682 within the α-backbone. In mice, ∼70% of the endogenous cardiac protein was replaced with the chimeric myosin. Myofibrils containing chimeric myosin exhibited ATPase activities that were depressed to the levels observed in hearts expressing ∼70% β-myosin. In vitro motility assays showed that the actin filament sliding velocity generated by chimeric myosin was similar to that of α-myosin, almost twice the velocities observed with β-myosin. These data indicate that this large domain sequence switch conferred β-like actin-activated ATPase activities to the chimeric myosin, suggesting that this region is responsible for the distinct hydrolytic properties of these myosin isoforms.