Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host

Abstract

Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function.

Figures

Fig. 1.

Differences in dye coupling between transplanted myoblasts and host cells. (a) eGFP-expressing myotubes form bundles when transplanted in infarcted myocardium. A 2D projection of a stack of 20 images (each separated by 2 μm) obtained with two-photon microscopy. (b) Immunostaining of the heavy chain of the fast (Left) and the slow (Right) myosin isoforms. (c) A myotube expressing eGFP recorded intracellularly with an Alexa Fluor 568-containing micropipette (Left, FITC filters to reveal eGFP; Right, rhodamine filters to reveal Alexa Fluor). Thirty minutes after the impalement (Right), no dye coupling was observed between the myotube and neighboring myocytes. (d) A myocyte recorded intracellularly with an Alexa Fluor-containing micropipette (rhodamine filters). Labeling of the myocyte 30 s (Left) and 8 min (Right) after penetration. Dye coupling and labeling of neighboring myocytes occurred within 1–2 min. Scale bars = 20 μm.

Fig. 2.

Emerging intrinsic membrane properties of eGFP-expressing myotubes. (a) Action potential characteristics of 5 types of muscle cell. (a1) Tibialis skeletal muscle cell; (a2) ventricular myocyte; (a3) eGFP-expressing myotube grafted for 28 days in the tibialis muscle; (a4) eGFP-expressing myotube grafted for 28 days in infarcted myocardium; (a5) eGFP-expressing myotube maintained in culture for 28 days. Whether grafted or in culture, eGFP-expressing myotubes fired brief action potentials (a3, a4, a5) in comparison with myocytes (a2). Note that the action potential of the myotubes grafted in infarcted myocardium (a4) is followed by a fast afterhyperpolarization. The action potentials were evoked either with extracellular stimulations (arrow heads point to the stimulation artifact) or with intracellular depolarizing current injections. (b) Firing properties of eGFP-expressing myotubes recorded in infarcted myocardium (Left and Center) or in culture (Right). (Left) Subthreshold depolarizing current pulses evoked a slow voltage-dependent depolarizing hump (arrow, middle trace) on top of which an action potential was triggered on higher stimulation (top trace). (Center) In another cell, a burst of action potentials was emitted on the slow depolarizing hump and followed by a slow afterhyperpolarization. (Right) Myotubes in culture do not fire bursts of action potentials.

Fig. 3.

Absence of electromechanical coupling between eGFP-expressing myotubes and myocytes. (Inset) Schematic representation of the experimental protocol used to detect contractions: movements of the recorded cell (red box) [i.e., an eGFP-expressing myotube (a and b) or a myocyte (c)] and of the neighboring myocardium (black box) were detected as local fluctuations in transilluminated light intensity. (a) Each action potential evoked in the myotube was accompanied by a local contraction (continuous red trace) that did not spread to the myocardium (continuous black trace). (b) The same myotube was electrically silent during spontaneous contractions of the entire explant. Light fluctuations detected in the red trace reflect only passive movements. (c) In opposition to the myotube, an intracellularly recorded myocyte fired with each contraction of the explant. The arrowheads point to the extracellular stimulations used to entrain the explant spontaneous contractions. The same calibration bars apply for a–c.