Kinya Seo

In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

Responses of single-ventricular myocytes to dynamic axial stretching.

Mechano-electrical feedback (MEF) has mainly been studied in isolated single cardiomyocytes using the microelectrode and micropipette techniques, but information regarding its dynamic aspects at the cellular level is limited due to the technical difficulties associated with manipulating single cells and maintaining stable attachment of these devices. To overcome such difficulties, we have combined two experimental methods, namely a carbon fiber technique to hold single myocytes and a ratiometric fluorescence measurement technique to monitor Ca2+ transients or membrane potentials. Following an overview of the experimental technique for stretching myocytes, the results for single rat ventricular myocytes under axial stretching are presented. Ca2+ transients were influenced by the loading conditions and involvement of myofilaments was suspected in regulatory mechanism. Membrane potential measurements during dynamic axial stretching revealed that the action potential duration was prolonged when the stretch was applied during the late phase of twitch contraction, and that depolarization of the resting membrane potential depended on the phase, amplitude and speed of the applied stretch. The amplitude may also modulate the ion selectivity of stretch-activated channels. This combination of the carbon fiber technique with fluorescence measurement could represent a powerful tool for clarifying MEF at the cellular level.

Structural Heterogeneity in the Ventricular Wall Plays a Significant Role in the Initiation of Stretch-Induced Arrhythmias in Perfused Rabbit Right Ventricular Tissues and Whole Heart Preparations

Rationale: Mechanical stress is known to alter the electrophysiological properties of the myocardium and may trigger fatal arrhythmias when an abnormal load is applied to the heart. Objective: We tested the hypothesis that the structural heterogeneity of the ventricular wall modulates globally applied stretches to create heterogeneous strain distributions that lead to the initiation of arrhythmias. Methods and Results: We applied global stretches to arterially perfused rabbit right ventricular tissue preparations. The distribution of strain (determined by marker tracking) and the transmembrane potential (measured by optical mapping) were simultaneously recorded while accounting for motion artifacts. The 3D structure of the preparations was also examined using a laser displacement meter. To examine whether such observations can be translated to the physiological condition, we performed similar measurements in whole heart preparations while applying volume pulses to the right ventricle. At the tissue level, larger stretches (≥20%) caused synchronous excitation of the entire preparation, whereas medium stretches (10% and 15%) induced focal excitation. We found a significant correlation between the local strain and the local thickness, and the probability for focal excitation was highest for medium stretches. In the whole heart preparations, we observed that such focal excitations developed into reentrant arrhythmias. Conclusions: Global stretches of intermediate strength, rather than intense stretches, created heterogeneous strain (excitation) distributions in the ventricular wall, which can trigger fatal arrhythmias.

Hyperactive Adverse Mechanical Stress Responses in Dystrophic Heart Are Coupled to Transient Receptor Potential Canonical 6 and Blocked by cGMP–Protein Kinase G ModulationNovelty and Significance

Rationale: The heart is exquisitely sensitive to mechanical stimuli to adapt rapidly to physiological demands. In muscle lacking dystrophin, such as Duchenne muscular dystrophy, increased load during contraction triggers pathological responses thought to worsen the disease. The relevant mechanotransducers and therapies to target them remain unclear. Objectives: We tested the role of transient receptor potential canonical (TRPC) channels TRPC3 and TRPC6 and their modulation by protein kinase G (PKG) in controlling cardiac systolic mechanosensing and determined their pathophysiological relevance in an experimental model of Duchenne muscular dystrophy. Methods and Results: Contracting isolated papillary muscles and cardiomyocytes from controls and mice genetically lacking either TRPC3 or TRPC6 were subjected to auxotonic load to induce stress-stimulated contractility (SSC, gradual rise in force and intracellular Ca2+). Incubation with cGMP (PKG activator) markedly blunted SSC in controls and Trpc3−/−; whereas in Trpc6−/−, the resting SSC response was diminished and cGMP had no effect. In Duchenne muscular dystrophy myocytes (mdx/utrophin deficient), the SSC was excessive and arrhythmogenic. Gene deletion or selective drug blockade of TRPC6 or cGMP/PKG activation reversed this phenotype. Chronic phosphodiesterase 5A inhibition also normalized abnormal mechanosensing while blunting progressive chamber hypertrophy in Duchenne muscular dystrophy mice. Conclusions: PKG is a potent negative modulator of cardiac systolic mechanosignaling that requires TRPC6 as the target effector. In dystrophic hearts, excess SSC and arrhythmia are coupled to TRPC6 and are ameliorated by its targeted suppression or PKG activation. These results highlight novel therapeutic targets for this disease.

Thrombospondin-4 Is Required for Stretch-Mediated Contractility Augmentation in Cardiac MuscleNovelty and Significance

Rationale: One of the physiological mechanisms by which the heart adapts to a rise in blood pressure is by augmenting myocyte stretch-mediated intracellular calcium, with a subsequent increase in contractility. This slow force response was first described over a century ago and has long been considered compensatory, but its underlying mechanisms and link to chronic adaptations remain uncertain. Because levels of the matricellular protein thrombospondin-4 (TSP4) rapidly rise in hypertension and are elevated in cardiac stress overload and heart failure, we hypothesized that TSP4 is involved in this adaptive mechanism. Objective: To determine the mechano-transductive role that TSP4 plays in cardiac regulation to stress. Methods and results: In mice lacking TSP4 (Tsp4−/−), hearts failed to acutely augment contractility or activate stretch-response pathways (ERK1/2 and Akt) on exposure to acute pressure overload. Sustained pressure overload rapidly led to greater chamber dilation, reduced function, and increased heart mass. Unlike controls, Tsp4−/− cardiac trabeculae failed to enhance contractility and cellular calcium after a stretch. However, the contractility response was restored in Tsp4−/− muscle incubated with recombinant TSP4. Isolated Tsp4−/− myocytes responded normally to stretch, identifying a key role of matrix-myocyte interaction for TSP4 contractile modulation. Conclusion: These results identify TSP4 as myocyte-interstitial mechano-signaling molecule central to adaptive cardiac contractile responses to acute stress, which appears to play a crucial role in the transition to chronic cardiac dilatation and failure.