Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium

Abstract

Objective The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. Methods Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. Results The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower stimulation frequencies (preconditioned engineered tissue constructs vs maximum embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). Conclusions We have generated skeletal myoblast–based transplantable grafts that electrically couple to myocardium.

Preconditioning of skeletal myoblast-based engineered tissue constructs enables functional coupling to myocardium in vivo

“…In agreement with our previous in vitro studies, 19 myocardial APs could not be evoked by graft stimulation in NP-ETC transplanted hearts (Figure 4, D ). The conduction velocity within the murine myocardium is proportional to the number of functional gap junctions, 26 mainly formed by connexin 43 proteins.…”

“…25 Immunohistochemical analyses showed robust expression of both markers irrespective of preconditioning (Figure 3, A – D ), suggesting potential for either graft type to provide functional mechanical action and interaction. In agreement with our previous in vitro coupling studies, 19 there is an apparent difference in the level of expression of both markers between transplanted P-ETCs and NP-ETCs. This emphasizes the inability of NP-ETCs to provide mechanical responses to stimulation in vitro, which was further confirmed by stimulating viable heart slices carrying either type of graft ex vivo (data not shown).…”

“…However, foci of expression in the graft-to-host interface were however predominantly found in P-ETC transplanted heart sections (Figure 3, E ), suggestive of a heightened potential for electrophysiologic coupling between host myocardium and transplanted P-ETCs. The former finding and the apparent presence of connexin 43 in the cytoplasma of grafted cells is corroborated by the quantitative data generated in our previous in vitro 19 and in vivo 21 studies.…”

“…19,31 This maximum capture frequency from stimulation within the graft (Figure 5, A and D ) was lower than in native heart tissue, but within the physiologic range of adult mice heart rates. 32 Stimulation above the capture frequency resulted in 2:1 conduction (Figure 5, B ).…”

“…18 In addition, we confirmed that mechanical preconditioning led to preservation of the electromechanical competence of SMs, as they coupled to cardiomyocytes in vitro. 19 …”