Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis.

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.

Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis.

Superparamagnetic iron oxide (SPIO) particles have been used successfully as an intracellular contrast agent for nuclear MRI cell tracking in vivo. We present a method of detecting intracellular SPIO colloid uptake in live cells using cell magnetophoresis, with potential applications in measuring intracellular MRI contrast uptake. The method was evaluated by measuring shifts in mean and distribution of the cell magnetophoretic mobility, and the concomitant changes in population frequency of the magnetically positive cells when compared to the unmanipulated negative control. Seven different transfection agent (TA) -SPIO complexes based on dendrimer, lipid, and polyethylenimine compounds were used as test standards, in combination with 3 different cell types: mesenchymal stem cells, cardiac fibroblasts, and cultured KG-1a hematopoietic stem cells. Transfectol (TRA) -SPIO incubation resulted in the highest frequency of magnetically positive cells (>90%), and Fugene 6 (FUG) -SPIO incubation the lowest, below that when using SPIO alone. A highly regular process of cell magnetophoresis was amenable to intracellular iron mass calculations. The results were consistent in all the cell types studied and with other reports. The cell magnetophoresis depends on the presence of high-spin iron species and is therefore expected to be directly related to the cell MRI contrast level.

SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction.

Stem cell transplantation at the time of acute myocardial infarction (AMI) improves cardiac function. Whether the improved cardiac function results from regeneration of cardiac myocytes, modulation of remodeling, or preservation of injured tissue through paracrine mechanisms is actively debated. Because no specific stem cell population has been shown to be optimal, we investigated whether the benefit of stem cell transplantation could be attributed to a trophic effect on injured myocardium. Mesenchymal stem cells secrete SDF-1 and the interaction of SDF-1 with its receptor, CXCR4, increases survival of progenitor cells. Therefore, we compared the effects of MSC and MSC engineered to overexpress SDF-1 on cardiac function after AMI. Tail vein infusion of syngeneic MSC and MSC:SDF-1 1 day after AMI in the Lewis rat led to improved cardiac function by echocardiography by 70.7% and 238.8%, respectively, compared with saline controls 5 wk later. The beneficial effects of MSC and MSC:SDF-1 transplantation were mediated primarily through preservation, not regeneration of cardiac myocytes within the infarct zone. The direct effect of SDF-1 on cardiac myocytes was due to the observation that, between 24 and 48 h after AMI, SDF-1-expressing MSC increased cardiac myocyte survival, vascular density (18.2+/-4.0 vs. 7.6+/-2.3 vessels/mm2, P<0.01; SDF-1:MSC vs. MSC), and cardiac myosin-positive area (MSC: 49.5%; mSC:SDF-1: 162.1%) within the infarct zone. There was no evidence of cardiac regeneration by the infused MSC or endogenous cardiac stem cells based on lack of evidence for cardiac myocytes being derived from replicating cells. These results indicate that stem cell transplantation may have significant beneficial effects on injured organ function independent of tissue regeneration and identify SDF-1:CXCR4 binding as a novel target for myocardial preservation.

Speckle-tracking echocardiography correctly identifies segmental left ventricular dysfunction induced by scarring in a rat model of myocardial infarction.

Speckle-tracking echocardiography (STE) uses a two-dimensional echocardiographic image to estimate two orthogonal strain components. The aim of this study was to assess sensitivity of circumferential (S(circ)) and radial (S(rad)) strains to infarct-induced left ventricular (LV) remodeling and scarring of the LV in a rat. To assess the relationship among S(circ), S(rad), and scar size, two-dimensional echocardiographic LV short-axis images (12 MHz transducer, Vivid 7 echo machine) were collected in 34 Lewis rats 4 to 10 wk after ligation of the left anterior descending artery. Percent segmental fibrosis was assessed from histological LV cross sections stained by Masson trichrome. Ten normal rats served as echocardiographic controls. S(circ) and S(rad) were assessed by STE. Histological data showed consistent scarring of anterior and lateral segments with variable extension to posterior and inferior segments. Both S(circ) and S(rad) significantly decreased after myocardial infarction (P<0.0001 for both). As anticipated, S(circ) and S(rad) were lowest in the infarcted segments. Multiple linear regression showed that segmental S(circ) were similarly dependent on segmental fibrosis and end-systolic diameter (P<0.0001 for both), whereas segmental S(rad) measurements were more dependent on end-systolic diameter (P<0.0001) than on percent fibrosis (P<0.002). STE correctly identifies segmental LV dysfunction induced by scarring that follows myocardial infarction in rats.

Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor.

MSCs have received attention for their therapeutic potential in a number of disease states, including bone formation, diabetes, stem cell engraftment after marrow transplantation, graft-versus-host disease, and heart failure. Despite this diverse interest, the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors, we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts, which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs, cardiac function improved in MCP-3-expressing hearts (88.7%, p < .001) but not in control hearts (8.6%, p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather, MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration.

Stem cell therapy enhances electrical viability in myocardial infarction.

Clinical studies suggest increased arrhythmia risk associated with cell therapy for myocardial infarction (MI); however, the underlying mechanisms are poorly understood. We hypothesize that the degree of electrical viability in the infarct and border zone associated with skeletal myoblast (SKMB) or mesenchymal stem cell (MSC) therapy will determine arrhythmia vulnerability in the whole heart. Within 24 h of LAD ligation in rats, 2 million intramyocardially injected SKMB (n=6), intravenously infused MSC (n=7), or saline (n=7) was administered. One month after MI, cardiac function was determined and novel optical mapping techniques were used to assess electrical viability and arrhythmia inducibility. Shortening fraction was greater in rats receiving SKMB (17.8%+/-5.3%, p=0.05) or MSC (17.6%+/-3.0%, p<0.01) compared to MI alone (10.1%+/-2.2%). Arrhythmia inducibility score was significantly greater in SKMB (2.8+/-0.2) compared to MI (1.4+/-0.5, p=0.05). Inducibility score for MSC (0.6+/-0.4) was significantly lower than SKMB (p=0.01) and tended to be lower than MI. Optical mapping revealed that MSC therapy preserved electrical viability and impulse propagation in the border zone, but SKMB did not. In addition, injected SKMBs were localized to discrete cell clusters where connexin expression was absent. In contrast, infused MSCs engrafted in a more homogeneous pattern and expressed connexin proteins. Even though both MSC and SKMB therapy improved cardiac function following MI in rat, SKMB therapy significantly increased arrhythmia inducibility while MSC therapy tended to lower inducibility. In addition, only MSC therapy was associated with enhanced electrical viability, diffuse engraftment, and connexin expression, which may explain the differences in arrhythmia inducibility.

SDF-1 recruits cardiac stem cell-like cells that depolarize in vivo.

Prolongation or reestablishment of stem cell homing through the expression of SDF-1 in the myocardium has been shown to lead to homing of endothelial progenitor cells to the infarct zone with a subsequent increase in vascular density and cardiac function. While the increase in vascular density is important, there could clearly be other mechanisms involved. In a recent study we demonstrated that the infusion of mesenchymal stem cells (MSC) and MSC that were engineered to overexpress SDF-1 led to significant decreases in cardiac myocyte apoptosis and increases in vascular density and cardiac function compared to control. In that study there was no evidence of cardiac regeneration from either endogenous stem cells or the infused mesenchymal stem cells. In this study we performed further detailed immunohistochemistry on these tissues and demonstrate that the overexpression of SDF-1 in the newly infracted myocardium led to recruitment of small cardiac myosin-expressing cells that had proliferated within 2 weeks of acute MI. These cells did not differentiate into mature cardiac myocytes, at least by 5 weeks after acute MI. However, based on optical mapping studies, these cells appear capable of depolarizing. We observed greater optical action potential amplitude in the infarct border in those animals that received SDF-1 overexpressing MSC than observed in noninfarcted animals and those that received control MSC. Further immunohistochemistry revealed that these proliferated cardiac myosin-positive cells did not express connexin 43, but did express connexin 45. In summary, our study suggests that the prolongation of SDF-1 expression at the time of acute MI leads to the recruitment of endogenous cardiac myosin stem cells that may represent cardiac stem cells. These cells are capable of depolarizing and thus may contribute to increased contractile function even in the absence of maturation into a mature cardiac myocyte.

Stem cells in cardiovascular disease: methods and protocols.

Stem cells are cells capable of proliferation, self-renewal, and differentiation into various organ-specific cell types. Stem cells are subclassified based on their species of origin (mice, rat, human), developmental stage of the species (embryonic, fetal, or adult), tissue of origin (hematopoietic, mesenchymal, skeletal, neural), and potential to differentiate into one or more specific types of mature cells (totipotent, pluripotent, multipotent). Embryonic stem (ES) cells are totipotent, primitive cells derived from the embryo that have the potential to become all specialized cell types. Conversely, adult stem cells are undifferentiated cells found in differentiated tissue that retain the potential to renew themselves and differentiate to yield organ-specific tissues. Stem cells are attractive candidates for novel therapeutics for patients with different heart diseases, including congestive heart failure, most commonly caused by myocardial infarction. The remarkable proliferative and differentiation capacity of stem cells promises an almost unlimited supply of specific cell types including viable functioning cardiomyocytes to replace the scarred myocardium following transplantation.

Mechanical and electrical effects of cell-based gene therapy for ischemic cardiomyopathy are independent.

Cell-based gene therapy to alter the myocardial tissue microenvironment has been shown to improve mechanical cardiac function, but little is known regarding its effects on arrhythmogenic risk. Clinical studies with skeletal myoblasts (SKMBs) have suggested a potential increase in arrhythmogenic risk. Therefore, we studied the functional mechanical and electrical effects of transient reestablishment of stem cell homing via transplantation of stromal-cell derived factor-1 (SDF-1)-expressing SKMBs. Eight weeks after anterior myocardial infarction, rats received in five divided doses into the periinfarct zone 1 million SKMBs transfected with AdSDF-1 (n=15) or AdGFP (n=8). Echocardiography was used to quantify changes in cardiac function, and optical mapping was used to determine the arrhythmogenic risk. Eight weeks after cell therapy, we observed a 54% (p=0.004) increase in shortening fraction in AdSDF-1:SKMB-treated rats, but only an 18.8% increase (p=not significant) with GFP:SKMB. SDF-1-treated hearts exhibited an increase in vascular density compared with control SKMBs (34.9+/-7.1 vs. 20.7+/-5.6 vessels/mm2; p<0.01). Optical mapping performed 8 weeks after cell therapy revealed that all animals that received SKMBs regardless of viral transfection had inducible ventricular tachycardia (VT) whereas only 50% of saline-treated animals had inducible VT (p<0.05). Transient reestablishment of stem cell homing via transplantation of modified SKMBs is sufficient to improve cardiac function. However, despite improved mechanical function, the risk of ventricular tachycardia increased. We propose that future studies on functional effects of cell-based gene therapies should address both mechanical and electrical consequences.

Scaling of diastolic intraventricular pressure gradients is related to filling time duration.

In early diastole, pressure is lower in the apex than in the base of the left ventricle (LV). This early intraventricular pressure difference (IVPD) facilitates LV filling. We assessed how LV diastolic IVPD and intraventricular pressure gradient (IVPG), defined as IVPD divided by length, scale to the heart size and other physiological variables. We studied 10 mice, 10 rats, 5 rabbits, 12 dogs, and 21 humans by echocardiography. Color Doppler M-mode data were postprocessed to reconstruct IVPD and IVPG. Normalized LV filling time was calculated by dividing filling time by RR interval. The relationship between IVPD, IVPG, normalized LV filling time, and LV end-diastolic volume (or mass) as fit to the general scaling equation Y = kM beta, where M is LV heart size parameter, Y is a dependent variable, k is a constant, and beta is the power of the scaling exponent. LV mass varied from 0.049 to 194 g, whereas end-diastolic volume varied from 0.011 to 149 ml. The beta values relating normalized LV filling time with LV mass and end-diastolic volume were 0.091 (SD 0.011) and 0.083 (SD 0.009), respectively (P < 0.0001 vs. 0 for both). The beta values relating IVPD with LV mass and end-diastolic volume were similarly significant at 0.271 (SD 0.039) and 0.243 (SD 0.0361), respectively (P < 0.0001 vs. 0 for both). Finally, beta values relating IVPG with LV mass and end-diastolic volume were -0.118 (SD 0.013) and -0.104 (SD 0.011), respectively (P < 0.0001 vs. 0 for both). As a result, there was an inverse relationship between IVPG and normalized LV filling time (r = -0.65, P < 0.001). We conclude that IVPD decrease, while IVPG increase with decreasing animal size. High IVPG in small mammals may be an adaptive mechanism to short filling times.

Engineered cell therapy for sustained local myocardial delivery of nonsecreted proteins.

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene- and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.

Electrophysiological consequence of skeletal myoblast transplantation in normal and infarcted canine myocardium.

OBJECTIVE: The purpose of this study is to test our hypothesis that injection of skeletal myoblasts (SkMbs) into viable tissue may alter impulse conduction but that injections into nonviable tissue (scar) will have negligible impact. BACKGROUND: Myocardial infarction (MI) is a major public health problem. SkMb transplantation after MI has been shown to have some beneficial effect on hemodynamic function. Previous studies have indicated that SkMbs do not electrically couple with viable host myocardium in vivo. METHODS: We used optical mapping to measure impulse propagation and arrhythmia inducibility in the canine left ventricular wedge preparation with and without MI. MI was created by temporary ligation of a branch of the left anterior descending coronary artery (LAD) (7.0 +/- 3.8 ng/mL troponin 24 hours after MI). Labeled SkMbs (10(8) in 4 mL of serum-free basal solution) were injected from the epicardium (20-40 0.1 mL injections) into normal myocardium (n = 8) or the central zone of the MI (n = 6). RESULTS: During endocardial pacing in the absence of MI, transmural conduction velocity was similar with (35.75 +/- 3.4 cm/s) and without (37.42 +/- 3.6 cm/s) SkMb transplantation. However, pacing from the epicardium resulted in conduction slowing in regions that were DiI-positive and associated with the expression of skeletal myosin (fast) but not connexin-43. In all preparations with MI (n = 13), abnormal impulse propagation was seen regardless of SkMb transplantation. Arrhythmias (at least one extra beat after standard programmed stimulation) occurred most frequently in preparations with MI independent of SkMb transplantation. In preparations without MI (n = 8), SkMb transplantation did not significantly increase arrhythmia inducibility. CONCLUSION: We conclude that SkMbs transplanted into normal myocardium can cause abnormal impulse propagation. These data suggest that the location of SkMb transplantation may influence arrhythmia vulnerability associated with MI.

Optical mapping of late myocardial infarction in rats.

Late myocardial infarction (MI) is associated with ventricular arrhythmias and sudden cardiac death. The exact mechanistic relationship between abnormal cellular electrophysiology, conduction abnormalities, and arrhythmogenesis associated with late MI is not completely understood. We report a novel, rapid dye superfusion technique to enable whole heart, high-resolution optical mapping of late MI. Optical mapping of action potentials was performed in normal rats and rats with anterior MI 7 days after left anterior descending artery ligation. Hearts from normal rats exhibited normal action potentials and impulse conduction. With the use of programmed stimulation to assess arrhythmia inducibility, 29% of hearts with late MI had inducible sustained ventricular tachycardia, compared with 0% in normal rats. A causal relationship between the site of infarction, abnormal action potential conduction (i.e., block and slow conduction), and arrhythmogenesis was observed. Optical mapping techniques can be used to measure high-resolution action potentials in a whole heart model of late MI. This experimental model reproduces many of the electrophysiological characteristics (i.e., conduction slowing, block, and ventricular tachycardia) associated with MI in patients. Importantly, the results of this study can enhance our ability to understand the interplay between cellular heterogeneity, conduction abnormalities, and arrhythmogenesis associated with MI.

Platelet, not endothelial, P-selectin is required for neointimal formation after vascular injury.

BACKGROUND: P-selectin blockade significantly inhibits inflammation and neointimal formation after arterial injury; however, the independent roles of platelet and endothelial P-selectins in this process are unknown. In atherosclerosis, both platelet and endothelial cell P-selectins are important. This study was designed to determine whether P-selectin expression on platelet, endothelial, or both surfaces is critical to the inflammatory response and neointimal formation after arterial injury. METHODS AND RESULTS: Using wild-type (WT) and P-selectin-knockout (Psel(-/-)) mice, we performed bone marrow transplantation to generate chimeric mice that expressed either platelet P-selectin (Plt-Psel) or endothelial P-selectin (EC-Psel). Double injury of the carotid artery was performed in these mice as well as in WT and Psel(-/-) mice. Animals were euthanized 4 or 21 days after arterial injury. Morphometric data showed that there was more neointimal formation in the WT mouse group when compared with the Psel(-/-) mouse group (0.015+/-0.004 vs 0.004+/-0.004 mm2, P<0.001). Further comparison showed significantly less neointimal area in EC-Psel mice (0.006+/-0.004 mm2) compared with Plt-Psel mice (0.011+/-0.005 mm2, P=0.026) and WT mice (0.015+/-0.004 mm2, P=0.001). No significant differences were observed between WT and Plt-Psel mice or between Psel(-/-) and EC-Psel mice. Decreased neointimal formation was accompanied by a reduced inflammatory response, as evidenced by immunostaining of RANTES and MCP-1 4 days after injury. CONCLUSIONS: Platelet P-selectin expression, but not endothelial P-selectin, plays a crucial role in the development of neointimal formation after arterial injury, and therapeutic strategies targeting leukocyte-platelet interactions could be effective in inhibiting restenosis.