Objectives To quantify acute myocardial retention of cardiac-derived stem cells (CDCs) and evaluate different delivery methods using Positron Emission Tomography (PET). Background Success of stem cell transplantation for cardiac regeneration is partially limited by low retention/engraftment of the delivered cells. A clinically applicable method for accurate quantification of cell retention would enable optimization of cell delivery. Methods CDCs derived from syngeneic, male Wistar Kyoto (WK) rats were labeled with 18FDG and injected intramyocardially into the ischemic region of female WK rats following permanent left coronary artery ligation. The effects of fibrin glue, bradycardia (adenosine) and cardiac arrest were examined. 18FDG PET was performed for quantification of cell retention. Quantitative PCR for the male-specific SRY gene was performed to validate the PET results. Results Myocardial retention of cells suspended in PBS 1 hr after delivery was 17.6±11.5% by PCR and 17.8±7.3% by PET. When CDCs were injected immediately following induction of cardiac arrest, retention was increased to 75.6±18.6%. Adenosine slowed the ventricular rate and doubled CDC retention (35.4±5.3%). A similar increase in CDC retention was observed following epicardial application of fibrin glue at the injection site (37.5±8.2%). PCR revealed a significant increase in 3 week cell engraftment in the fibrin glue animals (22.1±18.6% vs 5.3±3.1%, for fibrin glue and PBS respectively). Conclusions In vivo PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with fibrin glue or lowering ventricular rate by adenosine may be clinically translatable methods for improving stem cell engraftment in a beating heart.

Background Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of chronic heart failure (HF) patients. Hallmarks of HF are depletion and disorganization of the transverse tubular system (t-system) in cardiomyocytes. Here, we investigated remodeling of the t-system in human end-stage HF and its role in cardiac recovery. Methods Left ventricular biopsies were obtained from 5 donors (CTRL) and 26 chronic HF patients undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-system structure, density, and distance of ryanodine receptor (RyR) clusters to the sarcolemma, including the t-system. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turn-down of the LVAD. Results The majority of HF myocytes showed remarkable t-system remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-system components was decreased in HF vs CTRL (0.37±0.01 vs 0.46±0.02, p<0.01), and the volume/length ratio was increased in HF (0.36±0.01μm2 vs 0.25±0.02μm2, p<0.0001). T-system density was reduced in HF, leading to increased RyR-sarcolemma distances (0.96±0.05μm vs 0.64±0.1μm, p<0.01). Low RyR-sarcolemma distances at time of LVAD implantation predicted high post-LVAD left-ventricular ejection fractions (EF, p<0.01) and EF increase during unloading (p<0.01). EF in patients with pre-LVAD RyR-sarcolemma distances larger than 1μm did not improve following mechanical unloading. Additionally, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-system density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9ms vs 10.3±1.7ms, p<0.05) and more asynchronous (18.1ms±1.5ms vs 8.9±2.2ms, p<0.01) in HF cells with low t-system density versus cells with high t-system density. Conclusions The t-system in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-system impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-system at time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery following mechanical unloading.

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Background Quantification of acute myocardial retention and lung bio-distribution of cardiosphere-derived cells (CDCs) following transplantation is important to improve engraftment. Methods and results We studied acute(1 hour) cardiac/lung retention in 4 groups (n = 25) of rats (normal—NL, acute ischemia-reperfusion—AI-RM, acute permanent ligation—PL, and chronic infarct by ischemia-reperfusion—CI-R) using intra-myocardial delivery, 1 group using intracoronary delivery (acute ischemia-reperfusion, AI-RC, n = 5) and 1 group using intravenous delivery (acute ischemia-reperfusion, AI-RV, n = 5) of CDCs by PET. Cardiac retention was similar in the NL, AI-RM, CI-R, and A-IRC groups (13.6% ± 2.3% vs 12.0% ± 3.9% vs 9.9 ± 2.8 vs 15.4% ± 5.5%; P = NS), but higher in PL animals (22.9% ± 5.2%; P < .05). Low cardiac retention was associated with significantly higher lung activity in NL and AI-RM groups (43.3% ± 5.6% and 39.9% ± 9.3%), compared to PL (28.5% ± 5.9%), CI-R (20.2% ± 9.3%), and A-IRC (19.9% ± 5.6%) animals (P < .05 vs AI-RM and NL). Lung activity was highest following intravenous CDC delivery (55.1% ± 9.3%, P < .001) and was associated with very low cardiac retention (0.8% ± 1.06%). Two-photon microscopy indicated that CDCs escaped to the lungs via the coronary veins following intra-myocardial injection. Conclusions Acute cardiac retention and lung bio-distribution vary with the myocardial substrate and injection route. Intra-myocardially injected CDCs escape into the lungs via coronary veins, an effect that is more pronounced in perfused myocardium.

Background Sepsis is the overwhelming host response to infection leading to shock and multiple organ dysfunction. Cardiovascular complications greatly increase sepsis‐associated mortality. Although murine models are routinely used for preclinical studies, the benefit of using genetically engineered mice in sepsis is countered by discrepancies between human and mouse sepsis pathophysiology. Therefore, recent guidelines have called for standardization of preclinical methods to document organ dysfunction. We investigated the course of cardiac dysfunction and myocardial load in different mouse models of sepsis to identify the optimal measurements for early systolic and diastolic dysfunction. Methods and Results We performed speckle‐tracking echocardiography and assessed blood pressure, plasma inflammatory cytokines, lactate, B‐type natriuretic peptide, and survival in mouse models of endotoxemia or polymicrobial infection (cecal ligation and puncture, [ CLP ]) of moderate and high severity. We observed that myocardial strain and cardiac output were consistently impaired early in both sepsis models. Suppression of cardiac output was associated with systolic dysfunction in endotoxemia or combined systolic dysfunction and reduced preload in the CLP model. We found that cardiac output at 2 hours post‐ CLP is a negative prognostic indicator with high sensitivity and specificity that predicts mortality at 48 hours. Using a known antibiotic (ertapenem) treatment, we confirmed that this approach can document recovery. Conclusions We propose a non‐invasive approach for assessment of cardiac function in sepsis and myocardial strain and strain rate as preferable measures for monitoring cardiovascular function in sepsis mouse models. We further show that the magnitude of cardiac output suppression 2 hours post‐ CLP can be used to predict mortality.