The two early cardiac enzymes for injury TnT and CK-MB were measured; both enzymes remained within normal range after <i>in vivo</i> electroporation mediated gene transfer with 8 and 16 pulses, but the values were altered with the 32 pulses protocol.

<p>(a) TnT levels (b) CK-MB levels. The levels were below detection levels in the group in which invivo electroporation was not performed.</p

Left ventricular contractile function of heart following electroporation mediated gene transfer using 8, 16 and 32 pulses protocols (n = 5 each group).

<p>Indices of contractility and relaxation determined respectively by the maximum (dP/dt max) (a) and the minimum (dP/dt min) (b) and developed LVP (dLVP) (c) of the first derivative of ventricular pressure with respect to time. These parameters remained unchanged before (time 0) and after electroporation except for the 32 pulses group where the values are transiently altered. A complete normalization of the values occurred within 10–20 minutes. No abnormality was detected in the group in which invivo electroporation was not performed.</p

Graphical representation of transgene expression measured over time.

<p>After electroporation mediated gene transfer the animals were sacrificed at different time points.</p

The <i>in vivo</i> electroporation mediated gene transfer to the heart is a two step procedure.

<p>(a) First, the coronary sinus is occluded with a 6-0 prolene tourniquet and the plasmid is injected into the coronary vein. (b) The heart is then positioned between the plate electrodes and the electric field is applied.</p

<i>In vivo</i> electroporation mediated gene transfer to the beating heart.

<p>(a) BLI images at day 1, 3 and 5 after <i>in vivo</i> electroporation mediated gene transfer to the normal heart. Graphical representation of the quantification of the luciferase activity, with electroporated compared to non-electroporated hearts; (b) BLI measurements, the photon counts per heart field are expressed in relative light units (RLU) 16479±4338 RLU for electroporated heart vs. 3539±1555 RLU for non electroporated heart (p = 0.018) at day 1. (c) Luciferase assay, RLU per milligram (mg) of protein was measured using a luminometer in the same sample 6594±1806 RLU/mg for electroporated heart vs 1684±760 RLU/mg of total protein for non electroporated heart (p = 0.011) at day 1. (d) Control heart, plasmid was injected but not elecroporated. (e) Expression of EGFP was found on the cell surface.</p

For histological analysis the hearts were evaluated at 24 hrs post gene transfer.

<p>The following criteria were considered: vascular congestion, infiltration and polymorphonuclear infiltrates. No haemorrhage or infiltration was noticed with any pulsing protocol. (a) 8 pulses (b) 16 pulses (c) 32 pulses (Magnification = ×200).</p