Marcel G. J. Nederhoff

Toll-Like Receptor 4 Mediates Maladaptive Left Ventricular Remodeling and Impairs Cardiac Function After Myocardial Infarction

Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction. Therapeutic options to prevent LV remodeling are limited, which necessitates the exploration of alternative therapeutic targets. Toll-like receptors (TLRs) serve as pattern recognition receptors within the innate immune system. Activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling following myocardial infarction. To establish the role of TLR4 in postinfarct LV remodeling, myocardial infarction was induced in wild-type BALB/c mice and TLR4-defective C3H-Tlr4LPS−d mice. Without affecting infarct size, TLR4 defectiveness reduced the extent of LV remodeling (end-diastolic volume: 103.7±6.8 &mgr;L versus 128.5±5.7 &mgr;L; P<0.01) and preserved systolic function (ejection fraction: 28.2±3.1% versus 16.6±1.3%; P<0.01), as assessed by MRI. In the noninfarcted area, interstitial fibrosis, and myocardial hypertrophy were reduced in C3H-Tlr4LPS−d mice. In the infarcted area, however, collagen density was increased, which was accompanied by fewer macrophages, reduced inflammation regulating cytokine expression levels (interleukin [IL]-1&agr;, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, tumor necrosis factor-&agr;, interferon-&ggr;, granulocyte/macrophage colony-stimulating factor), and reduced matrix metalloproteinase-2 (4684±515 versus 7573±611; P=0.002) and matrix metalloproteinase-9 activity (76.0±14.3 versus 168.0±36.2; P=0.027). These data provide direct evidence for a causal role of TLR4 in postinfarct maladaptive LV remodeling, probably via inflammatory cytokine production and matrix degradation. TLR4 may therefore constitute a novel target in the treatment of ischemic heart failure.

Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction

We have developed a mouse severe combined immunodeficient (SCID) model of myocardial infarction based on permanent coronary artery occlusion that allows long-term functional analysis of engrafted human embryonic stem cell-derived cardiomyocytes, genetically marked with green fluorescent protein (GFP), in the mouse heart. We describe methods for delivery of dissociated cardiomyocytes to the left ventricle that minimize scar formation and visualization and validation of the identity of the engrafted cells using the GFP emission spectrum, and histological techniques compatible with GFP epifluorescence, for monitoring phenotypic changes in the grafts in vivo. In addition, we describe how magnetic resonance imaging can be adapted for use in mice to monitor cardiac function non-invasively and repeatedly. The model can be adapted to include multiple control or other cell populations. The procedure for a cohort of six mice can be completed in a maximum of 13 weeks, depending on follow-up, with 30 h of hands-on time.

Assessment of Myocardial Viability by Intracellular 23Na Magnetic Resonance Imaging

Background—Because of rapid changes in myocardial intracellular Na+ (Na+i) during ischemia and reperfusion (R), 23Na magnetic resonance imaging (MRI) appears to be an ideal diagnostic modality for early detection of myocardial ischemia and viability. So far, cardiac 23Na MRI data are limited and mostly concerned with imaging of total Na+. For proper interpretation, imaging of both Na+i and extracellular Na+ is essential. In this study, we tested whether Na+i imaging can be used to assess viability after low-flow (LF) ischemia. Methods and Results—Isolated rat hearts were subjected to LF (1%, 2%, or 3% of control coronary flow) and R. A shift reagent was used to separate Na+i and extracellular Na+ resonances. Acquisition-weighted 23Na chemical shift imaging (CSI) was alternated with 23Na MR spectroscopy. Already during control perfusion, Na+i could be clearly seen on the images. Na+i image intensity increased with increasing severity of ischemia. During R, Na+i image intensity remained highest in 1% LF hearts. Not only did we find very good correlations between Na+i image intensity at end-R and end-diastolic pressure (R=0.85, P<0.001) and recovery of the rate-pressure product (R=−0.88, P<0.001) at end-R, but most interestingly, also Na+i image intensity at end-LF was well correlated with end-diastolic pressure (R=0.78, P<0.01) and with recovery of the rate-pressure product (R=−0.81, P<0.01) at end-R. Furthermore, Na+i image intensity at end-LF was well correlated with creatine kinase release during R (R=0.79, P<0.05) as well as with infarct size (R=0.77, P<0.05). Conclusions—These data indicate that 23Na CSI is a promising tool for the assessment of myocardial viability.

31P NMR studies of creatine kinase flux in M-creatine kinase-deficient mouse heart

Hearts of wild-type and cytosolic muscle creatine kinase (M-CK)-knockout mice were perfused with Krebs-Henseleit buffer containing 10 mM glucose and 5 mM pyruvate and studied during pacing at 400 and 600 beats/min and during K+ arrest. Phosphocreatine (PCr) and ATP concentrations in M-CK-deficient hearts were not significantly different from those in wild-type hearts. With the use of31P NMR saturation transfer, the flux mediated predominantly by mitochondrial creatine kinase (Mi-CK) was clearly detected in M-CK-deficient hearts. Mi-CK flux was 4.8 ± 0.6 and 4.5 ± 0.6 mM/s during pacing at 400 and 600 beats/min, respectively, and was 3.5 ± 0.4 mM/s during cardiac arrest. In control hearts total CK flux was 7.8 ± 1.1 and 6.6 ± 1.3 mM/s during pacing at 400 and 600 beats/min, respectively, and decreased to 3.8 ± 0.5 mM/s during arrest. It is suggested that the relative contribution of Mi-CK to the total NMR-measured CK flux in the wild-type heart is higher than that of the homodimeric M-CK isoform (MM-CK).

Bio-energetic response of the heart to dopamine following brain death-related reduced myocardial workload: a phosphorus-31 magnetic resonance spectroscopy study in the cat.

OBJECTIVE Long-term exposure of the donor heart to high dosages of dopamine in the treatment of brain death-related hemodynamic deterioration has been shown to reduce myocardial phosphocreatine (PCr) and adenosine triphosphate (ATP) in myocardial biopsy specimens and may preclude heart donation for transplantation. Short-term exposure to the acute catecholamine release during the onset of brain death has shown an unchanged PCr/ATP ratio using in vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS). In this study 31P MRS was used to evaluate in vivo myocardial energy metabolism during long-term dopamine treatment. METHODS Twelve cats were studied in a 4.7 Tesla magnet for 360 minutes. At t = 0 minutes, brain death was induced (n = 6). At 210 minutes, when myocardial workload in the brain-death group was reduced significantly, dopamine was infused (n = 12) at 5 microg/kg/min and its dose was consecutively doubled every 30 minutes and was withheld during the last 30 minutes of the experiment. Phosphorus-31 magnetic resonance spectra were obtained from the left ventricular wall during 5-minute time frames, and PCr/ATP ratios were calculated. The hearts were histologically examined. RESULTS Although significant changes in myocardial workload were observed after the induction of brain death and during support and withdrawal of dopamine in both groups, the initial PCr/ATP ratio of 2.00+/-0.12 and the contents of PCr and ATP did not vary significantly. Histologically identified sub-endocardial hemorrhage was observed in 3 of 6 of the brain-dead animals and in 1 of 6 of the control animals. CONCLUSIONS High dosages of dopamine in the treatment of brain death-related reduced myocardial workload do not alter PCr/ATP ratios and the contents of PCr and ATP of the potential donor heart despite histologic damage.

Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR. (Mol Cell Biochem 174: 33–42, 1997)

THE EFFECT OF INHIBITION OF THE NA(+)/H+ EXCHANGER ON THE DEVELOPMENT OF HYPERTROPHY IN HYPERTROPHIC CARDIOMYOPATHY

Introduction In hypertrophic cardiomyopathy (HCM), the pathogenesis of asymmetrical left ventricular (LV) wall thickening is based upon a complex interplay of molecular pathways and is still largely unclarified. Since cariporide (an Na/H exchange blocker) has been proven effective in several pressure overload (animal) models by inhibiting the hypertrophic response, we hypothesized that cariporide might prevent or diminish the development of hypertrophy seen in HCM. Therefore, we treated transgenic mice (homozygous cMyBP-C null mice) with cariporide and subjected them to cardiomagnetic resonance imaging (CMR).