Jochen Müller-Ehmsen

Rebuilding a Damaged Heart Long-Term Survival of Transplanted Neonatal Rat Cardiomyocytes After Myocardial Infarction and Effect on Cardiac Function

Background—The long-term effects of cardiac cell transplantation on cardiac function are unknown. Therefore, we tested the survival and functional impact of rat neonatal cardiac myocytes up to 6 months after transplantation into infarcted hearts. Methods and Results—Cardiomyocytes from male neonatal Fischer 344 rats (1 to 2 days, 3 to 5×106) or medium was injected into the infarcts of adult syngeneic female animals 1 week after left coronary artery ligation. Six months later, implanted cardiomyocytes were still present by quantitative TaqMan polymerase chain reaction and histology. In all treated hearts, discrete lumps of cells were present within the infarct scar, which was not observed in media-injected hearts typified by a transmural infarct scar. Infarct thickness was greater in treated animals versus control animals (909±97 versus 619±43 &mgr;m, P <0.02), whereas infarct size and left ventricular volumes were similar. By biplane angiography, left ventricular ejection fractions at 6 months were greater (0.36±0.03 versus 0.25±0.02, P <0.01) and significantly less infarct zone dyskinesis was seen (0.30±0.08 versus 0.55±0.07, P =0.035, lateral projection) in treated animals versus control animals. Conclusions—Grafted neonatal cardiomyocytes were present in infarcts 6 months after transplantation; they thickened the wall of the left ventricle and were associated with enhanced ejection fraction and reduced paradoxical systolic bulging of the infarct. Therefore, neonatal cardiac cell transplants exhibit long-term survival in a myocardial infarct model and contribute to long-term improved cardiac function. These results suggest that a damaged heart can be rebuilt.

Reduced Sodium Pump α1, α3, and β1-Isoform Protein Levels and Na+,K+-ATPase Activity but Unchanged Na+-Ca2+ Exchanger Protein Levels in Human Heart Failure

Background−Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+,K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. Methods and Results−Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic α1-, α2-, α3-, and glycoprotein β1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain–binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase α1- (0.62±0...

Effect of inotropic stimulation on the negative force-frequency relationship in the failing human heart.

BackgroundIn severe human heart failure, an increase in frequency of stimulation is accompanied by a reduced force of contraction in vivo and in vitro. The present study was aimed to investigate whether inotropic stimulation influences the inverse force-frequency relationship in failing human myocardium. Methods and ResultsThe effects of the cAMP-independent positive inotropic agents ouabain (0.01μmol/L) and BDF 9148 (0.1 μmol/L) as well as the 3-adrenoceptor agonist isoprenaline (0.01 μmol/L and 0.1 μmol/L) on the force-frequency relationship in electrically driven papillary muscle strips from nonfailing (brain death, n=5) and terminally failing (NYHA class IV, heart transplants, dilated cardiomyopathy, n=22) human myocardium were studied. For comparison, we examined the effect of elevation of the extracellular Ca2 concentration (3.2 mmol/L and 6.2 mmol/L). In nonfailing myocardium, force of contraction, peak rate of tension rise, and peak rate of tension decay increased, whereas time to peak tension and time to half relaxation decreased following an increase of stimulation frequency. In NYHA class IV, force of contraction gradually declined followed by changes of other parameters of isometric contraction. Moderate stimulation of contractility by isoprenaline (0.01μmol/L) partly reversed the negative force-frequency relationship in NYHA class IV and preserved the positive force-frequency relationship in nonfailing myocardium. The addition of ouabain and BDF 9148 together restored completely the force-frequency relationship in NYHA class IV. In contrast, high concentrations of isoprenaline (0.1 μmol/L) and an elevation of the extracellular Ca2 concentration enhanced the decline in force of contraction in the presence of higher stimulation frequencies. ConclusionsIt is concluded that functionally important changes occur in the intracellular Ca2 handling, leading to the negative force-frequency relationship in terminally failing human myocardium. Interestingly, the negative force-frequency relationship can be restored by agents producing positive inotropic effects by elevation of the intracellular Na+ concentration. These findings suggest that hitherto unknown changes in the intracellular ionic homeostasis occur in the failing human heart. Even though increasing [Ca2“J1 in failing heart cells may be detrimental, increasing [Na+], may be beneficial through a mechanism independent of an increase in [Ca2+J1.

The Na, K-ATPase in the failing human heart

The Na, K-ATPase consists of alpha- and beta-subunits and actively transports Na out and K into the myocyte. It is the receptor for cardiac glycosides exerting its positive inotropic effect by inhibiting enzyme activity, decreasing the driving force for the Na/Ca-exchange and increasing cellular content and release of Ca during depolarization. The specific binding capacity for cardiac glycosides is utilized as a tool for Na, K-ATPase quantification with high accuracy and precision. In treatment of patients with heart failure cardiac glycosides improve symptoms and reduce the need for hospitalization without affecting mortality. In endomyocardial biopsies from patients with compromised cardiac function total Na, K-ATPase concentration is decreased by approximately 40% and a correlation between decrease in heart function and decrease in Na, K-ATPase concentration exists. At the subunit level, the alpha1-, alpha3- and beta1-proteins are reduced in human heart failure. During digitalization approximately 30% of remaining Na, K-pumps are occupied by digoxin. Thus, a total of not less than half the Na, K-pumps may be out of function in the myocardium of digitalised heart failure patients. It is still a matter of debate whether a digitalis-like factor exists. There is a pressing need for the identification of its precise chemical structure, properties and quantitative relation to the Na, K-ATPase. It is recommended that cardiac glycosides are prescribed to heart failure patients who are still having heart failure symptoms after institution of mortality reducing therapy. Cardiac glycoside treatment is still the only safe inotropic drug for oral use that improves hemodynamics in patients with compromised cardiac function.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.

Decreased number of circulating progenitor cells in obesity: beneficial effects of weight reduction

AIMS Cardiovascular risk factors are associated with decreased levels of circulating progenitor cells (CPC). The aim of this study was to determine whether the number of CPC is an independent correlate of body mass index (BMI) and whether weight loss leads to an increase in CPC. METHODS AND RESULTS CD34 positive and KDR/CD34, CD133/CD34, and CD117/CD34 double positive cells were measured by fluorescence activated cell sorting (FACS) analysis in peripheral blood of 149 volunteers (52.5 +/- 12.0 years, BMI 21.5-52.7 kg/m(2), mean 31.6 +/- 5.1 kg/m(2)) participating in a weight reduction program offered by German pharmacies. In addition, carotid intima media thickness (IMT) and brachial artery flow-mediated dilatation were determined. After a diet and sports program for 6 months, 86 representing subjects were re-evaluated (mean weight loss 5.8 +/- 5.2 kg). There was an inverse correlation between BMI as well as waist circumference and CPC, especially CD34 positive, KDR/CD34 positive, CD133/CD34 positive, and CD117/CD34 positive cells. This decrease in CPC in obesity held true not only for the absolute cell numbers, but also for the relative fractions of KDR, CD133, and CD117 positive cells within the CD34 positive cells, indicating a specific down regulation of these progenitor cell types. Multiple regression analysis revealed that BMI was a more prominent predictor of CPC regulation than blood pressure, LDL cholesterol, triglycerides, fasting glucose, and smoking. IMT increased in dependence on BMI (P < 0.001) and was inversely correlated with the number of CD34 positive cell (P < 0.05). After diet, there was a significant increase of CD34 and CD117/CD34 positive cells, which correlated with the decrease in BMI. Also, weight loss was accompanied by a decrease in IMT (P = 0.015), which also correlated with the increase in CPC (P < 0.001). The increase in the number of CPC was independent from whether weight loss was achieved by increased physical exercise or by reduced calorie intake only. CONCLUSION Obesity is associated with decreased numbers of CPC and increased IMT. Diet and weight loss lead to an increase in CPC count, which might contribute to regression of IMT.

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs: Effects of β-Blockade and Angiotensin-Converting Enzyme Inhibition

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by 86Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5±0.5 to 86.0±27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3±1.7 to 129.8±51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1±1.2 nmol/L and at 30±1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7±0.8 to 166.9±91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the &bgr;1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

Electrophysiological Maturation and Integration of Murine Fetal Cardiomyocytes After Transplantation

In the present study, we investigated the electrophysiological maturation and integration of immature cardiomyocytes after transplantation; maturation and integration are essential to achieve the cardiac regeneration. Murine fetal cardiomyocytes (FCMs) (d12.5-d15.5) expressing enhanced green fluorescent protein under the control of the &agr;-actin promoter were injected into cryoinjured areas and adjacent myocardium of cryoinjured mouse ventricles. Viable short axis tissue slices (thickness, 150 &mgr;m) of the ventricles were prepared 5 to 6 days after transplantation. Glass microelectrodes were used for measurements of action potentials in transplanted FCMs and host cardiomyocytes within the slices. Stimulation at frequencies of up to 10 Hz was performed via a unipolar electrode placed in viable host tissue. Transplanted FCMs could be distinguished clearly from host tissue by their green fluorescence and their electrophysiological properties: maximal upstroke velocity (Vmax) was significantly lower and action potential duration at 50% repolarization (APD50) was significantly longer compared with values of adult cardiomyocytes. Transplanted FCMs surrounded by cryoinjured tissue showed spontaneous electrical and contractile activity, which was in no case synchronous with host tissue. Vmax and APD50 of these nonintegrated cells matched values of cultivated dissociated FCMs. In contrast, 82% of transplanted FCMs surrounded by viable host tissue were electrically integrated; ie, electrical and contractile activity was synchronous with host tissue and these cells had more mature action potential parameters (significantly higher Vmax and shorter APD50) compared with nonintegrated FCMs. In conclusion, electrophysiological maturation and integration of transplanted FCMs depend on an embedment in viable host myocardium. FCMs surrounded by cryoinjured tissue maintain physiological but immature AP properties.

Poor Cell Survival Limits the Beneficial Impact of Mesenchymal Stem Cell Transplantation on Acute Kidney Injury

Background: Although renal tubular epithelium has a great capacity for repair it has been suggested that the administration of mesenchymal stem cells may accelerate the recovery following severe ischemic injury. Methods: Here we analyzed the survival rate and organ distribution of transplanted mesenchymal stem cells as well as their contribution to kidney regeneration after ischemic renal injury using functional tests, histological examination as well as quantitative real-time PCR. Results: Intravenously injected stem cells were mainly trapped in lungs and liver. One hour after injection, less than 1% of the injected stem cells could be detected in the injured kidneys. These cells disappeared within the first few days and did not replace renal epithelial cells precluding substantial transdifferentiation. To clarify whether reinforced stem cell delivery might promote sustained survival or conversion to tubular epithelia, stem cells were directly injected into the injured kidneys. Although these grafted cells also did not show sustained survival or contribute to structural renal repair, stem cell injection was associated with a significant but transient initial decrease in serum creatinine. Conclusion: These data suggest that mesenchymal stem cells do not significantly contribute to epithelial renewal after ischemic injury, promoting the idea that the major impact of cell-based therapy for acute kidney injury may result from paracrine or endocrine effects unrelated to stem cell transdifferentiation.

Ouabain as a Mammalian Hormone

Abstract: Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the μ chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na+,K+‐ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.