Joanne S. Ingwall

Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts

Transplantation of adult bone marrow–derived mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of 5 × 106 cells overexpressing Akt into the ischemic rat myocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition and cardiac myocyte hypertrophy, regenerated 80–90% of lost myocardial volume, and completely normalized systolic and diastolic cardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1 restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacZ. Thus, mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance.

Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells

Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells

Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement

We previously reported that intramyocardial injection of bone marrow‐derived mesenchymal stem cells overexpressing Akt (Akt‐MSCs) inhibits ventricular remodeling and restores cardiac function measured 2 wk after myocardial infarction. Here, we report that the functional improvement occurs in < 72 h. This early remarkable effect cannot be readily attributed to myocardial regeneration from the donor cells. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be important mechanisms of tissue repair and functional improvement after injection of the Akt‐MSCs. Indeed, in the current study we demonstrate that conditioned medium from hypoxic Akt‐MSCs markedly inhibits hypoxia‐induced apoptosis and triggers vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When injected into infarcted hearts, the Akt‐MSC conditioned medium significantly limits infarct size and improves ventricular function relative to controls. Sup‐port to the paracrine hypothesis is provided by data showing that several genes, coding for factors (VEGF, FGF‐2, HGF, IGF‐I, and TB4) that are potential mediators of the effects exerted by the Akt‐MSC conditioned medium, are significantly up‐regulated in the Akt‐MSCs, particularly in response to hypoxia. Taken together, our data support Akt‐MSC‐mediated para‐crine mechanisms of myocardial protection and functional improvement.‐Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L., Morello, F., Mu, H., Melo, L. G., Pratt, R. E., Ingwall, J. S., Dzau, V. J. Evidence supporting paracrine hypothesis for Akt‐modified mes‐enchymal stem cell‐mediated cardiac protection and functional improvement. FASEB J. 20, 661–669 (2006)

Myocardial Phosphocreatine-to-ATP Ratio Is a Predictor of Mortality in Patients With Dilated Cardiomyopathy

BACKGROUND In patients with heart failure due to dilated cardiomyopathy, cardiac energy metabolism is impaired, as indicated by a reduction of the myocardial phosphocreatine-to-ATP ratio, measured noninvasively by 31P-MR spectroscopy. The purpose of this study was to test whether the phosphocreatine-to-ATP ratio also offers prognostic information in terms of mortality prediction as well as how this index compares with well-known mortality predictors such as left ventricular ejection fraction (LVEF) or New York Heart Association (NYHA) class. METHODS AND RESULTS Thirty-nine patients with dilated cardiomyopathy were followed up for 928+/-85 days (2.5 years). At study entry, LVEF and NYHA class were determined, and the cardiac phosphocreatine-to-ATP ratio was measured by localized 31P-MR spectroscopy of the anterior myocardium. During the study period, total mortality was 26%. Patients were divided into two groups, one with a normal phosphocreatine-to-ATP ratio (>1.60; mean+/-SE, 1.98+/-0.07; n=19; healthy volunteers: 1.94+/-0.11, n=30) and one with a reduced phosphocreatine-to-ATP ratio (<1.60; 1.30+/-0.05; n=20). At re-evaluation (mean, 2.5 years), 8 of 20 patients with reduced phosphocreatine-to-ATP ratios had died, all of cardiovascular causes (total and cardiovascular mortality, 40%). Of the 19 patients with normal phosphocreatine-to-ATP ratios, 2 had died (total mortality, 11%), one of cardiovascular causes (cardiovascular mortality, 5%). Kaplan-Meier analysis showed significantly reduced total (P=.036) and cardiovascular (P=.016) mortality for patients with normal versus patients with low phosphocreatine-to-ATP ratios. A Cox model for multivariate analysis showed that the phosphocreatine-to-ATP ratio and NYHA class offered significant independent prognostic information on cardiovascular mortality. CONCLUSIONS The myocardial phosphocreatine-to-ATP ratio, measured noninvasively with 31P-MR spectroscopy, is a predictor of both total and cardiovascular mortality in patients with dilated cardiomyopathy.

A Mouse Model of Familial Hypertrophic Cardiomyopathy

A mouse model of familial hypertrophic cardiomyopathy (FHC) was generated by the introduction of an Arg403 → Gln mutation into the α cardiac myosin heavy chain (MHC) gene. Homozygous αMHC403/403 mice died 7 days after birth, and sedentary heterozygous αMHC403/+ mice survived for 1 year. Cardiac histopathology and dysfunction in the αMHC403/+ mice resembled human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fibrosis increased with age. Young male αMHC403/+ mice showed more evidence of disease than did their female counterparts. Preliminary results suggested that exercise capacity may have been compromised in the αMHC403/+ mice. This mouse model may help to define the natural history of FHC.

The Creatine Kinase System in Normal and Diseased Human Myocardium

We measured creatine kinase activity, isozyme composition, and total creatine content in biopsy samples of left ventricular myocardium from 34 adults in four groups: subjects with normal left ventricles, patients with left ventricular hypertrophy due to aortic stenosis, patients with coronary artery disease without left ventricular hypertrophy, and patients with coronary artery disease and left ventricular hypertrophy due to aortic stenosis. As compared with specimens of normal left ventricles, those from all patients with left ventricular hypertrophy had lower creatine kinase activity, higher MB creatine kinase isozyme content and activity, and lower creatine content. Specimens from the patients without left ventricular hypertrophy had normal creatine kinase activity, increased MB creatine kinase isozyme content and activity, and decreased total creatine content. The normal ventricles showed almost no MB isozyme content or activity. These data suggest that changes in the creatine kinase system occur in both pressure-overload hypertrophy and coronary artery disease. Patients with myocardial infarction who have mild or no preexisting fixed coronary artery disease or pressure-overload hypertrophy would not be expected to have elevation of serum MB creatine kinase.

Energy metabolism in heart failure and remodelling.

Myocytes of the failing heart undergo impressive metabolic remodelling. The time line for changes in the pathways for ATP synthesis in compensated hypertrophy is: flux through the creatine kinase (CK) reaction falls as both creatine concentration ([Cr]) and CK activity fall; increases in [ADP] and [AMP] lead to increases in glucose uptake and utilization; fatty acid oxidation either remains the same or decreases. In uncompensated hypertrophy and in other forms of heart failure, CK flux and fatty acid oxidation are both lower; any increases in glucose uptake and utilization are not sufficient to compensate for overall decreases in the capacity for ATP supply and [ATP] falls. Metabolic remodelling is under transcriptional and post-transcriptional control. The lower metabolic reserve of the failing heart contributes to impaired contractile reserve.

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Creatine Kinase System in Failing and Nonfailing Human Myocardium

BACKGROUND The creatine kinase (CK) reaction is important for rapid resynthesis of ATP when the heart increases its work. Studies defining the CK system in human failing and nonfailing myocardium are limited and in conflict. To resolve this conflict, we measured the activities of CK and its isoenzymes and the contents of creatine and CK-B in homogenates of human myocardium. METHODS AND RESULTS Myocardium was sampled from 23 subjects who underwent heart transplant, 36 subjects maintained in an intensive care unit before heart harvesting, 13 accident victims, and 2 patients undergoing heart surgery. Since the characteristics of myocardium of potential organ donors differed from those of myocardium of accident victims, data are presented for three groups: failing, donor, and control. CK activity was 7.7 +/- 1.9 and 6.0 +/- 1.4 IU/mg protein in left (LV) and right (RV) ventricles of failing, 9.4 +/- 2.5 and 10.7 +/- 2 IU/mg protein in LV and RV of donor, and 11.6 +/- 2.4 IU/mg protein in LV of control hearts. CK-MM and the mitochondrial isoenzyme activities were lower in failing and donor LV, and CK-MB activity and CK-B content were higher in failing and donor hearts. Creatine contents were 64 +/- 25 and 56 +/- 18.6 nmol/mg protein in LV and RV of failing, 96 +/- 30 and 110 +/- 24 nmol/mg protein in LV and RV of donor, and 131 +/- 28 nmol/mg protein in LV of control hearts. CONCLUSIONS In failing and nonfailing donor human myocardium, there is a combined decrease of CK activity and creatine that may impair the ability to deliver ATP to energy-consuming systems.

Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy.

An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.