Daniel A. Brenner

Cell Therapy Attenuates Deleterious Ventricular Remodeling and Improves Cardiac Performance After Myocardial Infarction

BackgroundMyocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. Methods and ResultsExperimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 106 myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of ≈30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. ConclusionsImplanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Human Amyloidogenic Light Chains Directly Impair Cardiomyocyte Function Through an Increase in Cellular Oxidant Stress

Primary amyloidosis is a systemic disorder characterized by the clonal production and tissue deposition of immunoglobulin light chain (LC) proteins. Congestive heart failure remains the greatest cause of death in primary amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Amyloid cardiomyopathy is largely unresponsive to current heart failure therapies, and is associated with a median survival of less than 6 months and a 5-year survival of less than 10%. The mechanisms underlying this disorder, however, remain unknown. In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy (cardiac-LC), specifically alter cellular redox state in isolated cardiomyocytes, marked by an increase in intracellular reactive oxygen species and upregulation of the redox-sensitive protein, heme oxygenase-1. In contrast, vehicle or control LC proteins isolated from patients without cardiac involvement did not alter cardiomyocyte redox status. Oxidant stress imposed by cardiac-LC proteins further resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intra-cellular calcium handling. Cardiomyocyte dysfunction induced by cardiac-LC proteins was independent of neurohormonal stimulants, vascular factors, or extracellular fibril deposition, and was prevented through treatment with a superoxide dismutase/catalase mimetic. This study suggests that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.

Amyloidogenic light chains induce cardiomyocyte contractile dysfunction and apoptosis via a non-canonical p38α MAPK pathway

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of <10%. Contributing to this poor prognosis is the fact that these patients generally do not tolerate standard heart failure therapies. The molecular mechanisms underlying this deadly form of heart disease remain unclear. Although interstitial amyloid fibril deposition of Ig light chain proteins is a major cause of cardiac dysfunction in AL cardiac amyloidosis, we have previously shown that amyloid precursor proteins directly impair cardiac function at the cellular and isolated organ levels, independent of fibril formation. In this study, we report that amyloidogenic light chain (AL-LC) proteins provoke oxidative stress, cellular dysfunction, and apoptosis in isolated adult cardiomyocytes through activation of p38 mitogen-activated protein kinase (MAPK). AL-LC–induced p38 activation was found to be independent of the upstream MAPK kinase, MKK3/6, and instead depends upon transforming growth factor-β-activated protein kinase-1 binding protein-1 (TAB1)-mediated p38α MAPK autophosphorylation. Treatment of cardiomyocytes with SB203580, a selective p38 MAPK inhibitor, significantly attenuated AL-LC–induced oxidative stress, cellular dysfunction, and apoptosis. Our data provide a unique mechanistic insight into the pathogenesis of AL-LC cardiac toxicity and suggest that TAB1-mediated p38α MAPK autophosphorylation may serve as an important event leading to cardiac dysfunction and subsequent heart failure.

Glucose-6-Phosphate Dehydrogenase Modulates Cytosolic Redox Status and Contractile Phenotype in Adult Cardiomyocytes

&NA; —Reactive oxygen species (ROS)‐mediated cell injury contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. Protection against ROS requires maintenance of endogenous thiol pools, most importantly, reduced glutathione (GSH), by NADPH. In cardiomyocytes, GSH resides in two separate cellular compartments: the mitochondria and cytosol. Although mitochondrial GSH is maintained largely by transhydrogenase and isocitrate dehydrogenase, the mechanisms responsible for sustaining cytosolic GSH remain unclear. Glucose‐6‐phosphate dehydrogenase (G6PD) functions as the first and rate‐limiting enzyme in the pentose phosphate pathway, responsible for the generation of NADPH in a reaction coupled to the de novo production of cellular ribose. We hypothesized that G6PD is required to maintain cytosolic GSH levels and protect against ROS injury in cardiomyocytes. We found that in adult cardiomyocytes, G6PD activity is rapidly increased in response to cellular oxidative stress, with translocation of G6PD to the cell membrane. Furthermore, inhibition of G6PD depletes cytosolic GSH levels and subsequently results in cardiomyocyte contractile dysfunction through dysregulation of calcium homeostasis. Cardiomyocyte dysfunction was reversed through treatment with either a thiol‐repleting agent (L‐2‐oxothiazolidine‐4‐carboxylic acid) or antioxidant treatment (Eukarion‐134), but not with exogenous ribose. Finally, in a murine model of G6PD deficiency, we demonstrate the development of in vivo adverse structural remodeling and impaired contractile function over time. We, therefore, conclude that G6PD is a critical cytosolic antioxidant enzyme, essential for maintenance of cytosolic redox status in adult cardiomyocytes. Deficiency of G6PD may contribute to cardiac dysfunction through increased susceptibility to free radical injury and impairment of intracellular calcium transport. The full text of this article is available online at http://www.circresaha.org. (Circ Res. 2003;93:e9‐e16.)

Increased Myocardial Dysfunction After Ischemia-Reperfusion in Mice Lacking Glucose-6-Phosphate Dehydrogenase

Background—Free radical injury contributes to cardiac dysfunction during ischemia-reperfusion. Detoxification of free radicals requires maintenance of reduced glutathione (GSH) by NADPH. The principal mechanism responsible for generating NADPH and maintaining GSH during periods of myocardial ischemia-reperfusion remains unknown. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, generates NADPH in a reaction linked to the de novo production of ribose. We therefore hypothesized that G6PD is essential for maintaining GSH levels and protecting the heart during ischemia-reperfusion injury. Methods and Results—Susceptibility to myocardial ischemia-reperfusion injury was determined in Langendorff-perfused hearts isolated from wild-type mice (WT) and mice lacking G6PD (G6PDdef) (20% of WT myocardial G6PD activity). During global zero-flow ischemia, cardiac function was similar between WT and G6PDdef hearts. On reperfusion, however, cardiac relaxation and contractile performance were greatly impaired in G6PDdef myocardium, as demonstrated by elevated end-diastolic pressures and decreased percent recovery of developed pressure relative to WT hearts. Contractile dysfunction in G6PDdef hearts was associated with depletion of total glutathione stores and impaired generation of GSH from its oxidized form. Increased ischemia-reperfusion injury in G6PDdef hearts was reversed by treatment with the antioxidant MnTMPyP but unaffected by supplementation of ribose stores. Conclusions—These results demonstrate that G6PD is an essential myocardial antioxidant enzyme, required for maintaining cellular glutathione levels and protecting against oxidative stress-induced cardiac dysfunction during ischemia-reperfusion.

Exercise training attenuates age-associated diastolic dysfunction in rats.

Background—In contrast to systolic function, which is relatively well preserved with advancing age, diastolic function declines steadily after age 30. Our goal was to determine whether changes in diastolic function that occur with aging could be reversed with exercise training. Methods and Results—Adult (6-month-old) and old (24-month-old) Fischer 344/BNF1 rats were studied after either 12 weeks of treadmill training or normal sedentary cage life. Three aspects of diastolic function were studied: (1) left ventricular (LV) filling in vivo via Doppler echocardiograph, (2) LV passive compliance, and (3) the degree of ischemia-induced LV stiffening. Maximal exercise capacity was lower in the old rats (18±1 minutes to exhaustion on a standard treadmill) than in the adult rats (25±1 minutes). Training increased exercise capacity by 43% in the old rats and 46% in the adults (to 26±1 and 37±1 minutes, respectively). Echocardiographic indices of LV relaxation were significantly lower in the old rats, but with training, they increased back to the levels seen in the adults. LV stiffness measured in the isolated, perfused hearts was not affected by age or training. Also in the isolated hearts, the LV stiffened more rapidly during low-flow ischemia in the old hearts than in the adults, but training eliminated this age-associated difference in the response to ischemia. Conclusions—Our findings indicate that in rats, some age-associated changes in diastolic function are reversible and thus may not be intrinsic to aging but instead secondary to other processes, such as deconditioning.

Exercise Intolerance in Rats With Hypertensive Heart Disease Is Associated With Impaired Diastolic Relaxation

A decrease in functional capacity is one of the most important clinical manifestations of hypertensive heart disease, but its cause is poorly understood. Our purpose was to evaluate potential causes of hypertension-induced exercise intolerance, focusing on identifying the type(s) of cardiac dysfunction associated with the first signs of exercise intolerance during the course of hypertensive heart disease. Exercise capacity was measured weekly in Dahl salt-sensitive rats as they developed hypertension as well as in Dahl salt-resistant control rats. Exercise capacity was unchanged from baseline during the first 8 weeks of hypertension, suggesting that hypertension itself did not cause exercise intolerance. After 9 to 12 weeks of hypertension, exercise capacity decreased in salt-sensitive rats but not in control rats. After 10 weeks of hypertension, indices of diastolic function (early truncation of the E wave), as assessed by echocardiography at rest, were decreased in the salt-sensitive rats. When exercise capacity had decreased by ≈25% in a rat, the heart was isolated, and left ventricular (LV) compliance and systolic function were measured. At that time point, LV hypertrophy was modest (an ≈20% increase in LV mass), and systolic function was normal or supernormal, indicating that exercise intolerance began during “compensated” LV hypertrophy. Passive LV compliance remained normal in salt-sensitive rats. Thus, in this model of hypertensive heart disease, exercise intolerance develops during the compensated stage of LV hypertrophy and appears to be due to changes in diastolic rather than systolic function. However, studies in which LV function is assessed during exercise are needed to conclusively define the roles of systolic and diastolic dysfunction in causing exercise intolerance.

Exercise Intolerance During Post-MI Heart Failure in Rats: Prevention with Supplemental Dietary Propionyl-L-Carnitine

Exercise capacity in patients with several types of cardiovascular disease can be improved with dietary carnitine, or carnitine derivatives. Mechanisms underlying this improvement remain largely unknown in part due to a lack of animal models of cardiac pathology in which carnitine derivatives improve exercise tolerance. Our goal was to evaluate the ability of propionyl-L-carnitine (PLC) to improve exercise tolerance in a rat model of exercise intolerance. Fischer 344 rats were followed after either a moderate size MI (n = 22) or sham MI surgery (n = 14). Starting 10 days post-surgery 10 of the MI and 7 of the sham rats received 100 mg/kg/day PLC in drinking water, which increased plasma and LV total l-carnitine concentrations 15–23% (p < 0.05). Rats were followed longitudinally until a statistically significant decrease in exercise capacity occurred in one of the groups, at which time all rats were sacrificed for study of the isolated perfused hearts. At 12-weeks post-MI exercise capacity had decreased 16 ± 7% (p < 0.05) in the MI group, but remained within 3% of baseline in the MI group that received PLC and the sham groups. Both MI groups exhibited the same degree of LV dilation, decrease in fractional shortening, and blunting of the response to isoproterenol. We conclude that supplemental dietary PLC attenuates the exercise intolerance that occurs secondary to post-MI heart failure in rats, but that this beneficial effect is not attributable to altered LV remodeling, an improved response to β-adrenergic stimulation, or increased skeletal muscle citrate synthase activity.

A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

Objective The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity. Methods Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34 / CD105 / CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b / CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR). Results A median of 4,212 (IQR: 2161 – 6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001). Conclusion This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

GDF-15 (Growth Differentiation Factor 15) Is Associated With Lack of Ventricular Recovery and Mortality After Transcatheter Aortic Valve Replacement

Background— Recent data suggest that circulating biomarkers may predict outcome in patients undergoing transcatheter aortic valve replacement (TAVR). We examined the association between inflammatory, myocardial, and renal biomarkers and their role in ventricular recovery and outcome after TAVR. Methods and Results— A total of 112 subjects undergoing TAVR were included in the prospective registry. Plasma levels of B-type natriuretic peptide, hs-TnI (high-sensitivity troponin I), CRP (C-reactive protein), GDF-15 (growth differentiation factor 15), GAL-3 (galectin-3), and Cys-C (cystatin-C) were assessed before TAVR and in 100 sex-matched healthy controls. Among echocardiographic parameters, we measured global longitudinal strain, indexed left ventricular mass, and indexed left atrial volume. The TAVR group included 59% male, with an average age of 84 years, and 1-year mortality of 18%. Among biomarkers, we found GDF-15 and CRP to be strongly associated with all-cause mortality (P<0.001). Inclusion of GDF-15 and CRP to the Society of Thoracic Surgeons score significantly improved C index (0.65–0.79; P<0.05) and provided a category-free net reclassification improvement of 106% at 2 years (P=0.01). Among survivors, functional recovery in global longitudinal strain (>15% improvement) and indexed left ventricular mass (>20% decrease) at 1 year occurred in 48% and 22%, respectively. On multivariate logistic regression, lower baseline GDF-15 was associated with improved global longitudinal strain at 1 year (hazard ratio=0.29; P<0.001). Furthermore, improvement in global longitudinal strain at 1 month correlated with lower overall mortality (hazard ratio=0.45; P=0.03). Conclusions— Elevated GDF-15 correlates with lack of reverse remodeling and increased mortality after TAVR and improves risk prediction of mortality when added to the Society of Thoracic Surgeons score.