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. 2007 Apr 15;580(Pt. 2):543-60.
doi: 10.1113/jphysiol.2006.123729. Epub 2006 Dec 21.

Propagation of the cardiac impulse in the diabetic rat heart: reduced conduction reserve

A Nygren  1 , M L Olson, K Y Chen, T Emmett, G Kargacin, Y Shimoni
Affiliations

Propagation of the cardiac impulse in the diabetic rat heart: reduced conduction reserve

A Nygren et al. J Physiol. .

Abstract

Diabetes mellitus is a growing epidemic with severe cardiovascular complications. Although much is known about mechanical and electrical cardiac dysfunction in diabetes, few studies have investigated propagation of the electrical signal in the diabetic heart and the associated changes in intercellular gap junctions. This study was designed to investigate these issues, using hearts from control and diabetic rats. Diabetic conditions were induced by streptozotocin (STZ), given i.v. 7-14 days before experiments. Optical mapping with the voltage-sensitive dye di-4-ANEPPS, using hearts perfused on a Langendorff apparatus, showed little change in baseline conduction velocity in diabetic hearts, reflecting the large reserve of function. However, both the gap junction uncoupler heptanol (0.5-1 mM) and elevated potassium (9 mM, to reduce cell excitability) produced a significantly greater slowing of impulse propagation in diabetic hearts than in controls. The maximal action potential upstroke velocity (an index of the sodium current) and resting potential was similar in single ventricular myocytes from control and diabetic rats, suggesting similar electrical excitability. Immunoblotting of connexin 43 (Cx43), a major gap junction component, showed no change in total expression. However, immunofluorescence labelling of Cx43 showed a significant redistribution, apparent as enhanced Cx43 lateralization. This was quantified and found to be significantly larger than in control myocytes. Labelling of two other gap junction proteins, N-cadherin and beta-catenin, showed a (partial) loss of co-localization with Cx43, indicating that enhancement of lateralized Cx43 is associated with non-functional gap junctions. In conclusion, conduction reserve is smaller in the diabetic heart, priming it for impaired conduction upon further challenges. This can desynchronize contraction and contribute to arrhythmogenesis.

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Figures

Figure 7
Figure 7. Analysis of lateralization of Cx43
Panels a–b′, differential interference contrast (a) and immunofluorescence (b and b′) images of an isolated ventricular myocyte from an STZ-diabetic rat heart labelled with anti-Cx43. Panels c and c′: immunofluorescence images of an isolated ventricular myocyte from a non-diabetic (control) rat heart labelled with anti-Cx43. White rectangles in b′ and c′ surround the intercalated discs. To identify the intercalated discs in images, the images were first temporarily rescaled (using Photoshop software) so that the entire cell could be seen in an image. This made the disc regions more evident. The disc regions were then surrounded by boxes (panels b′ and c′) and the pixel intensities within the boxes were set to the background level for the analysis of lateralization (using locally written software) as described in the text. To rule out possible biasing in choosing the cellular regions associated with the intercalated discs in the images, the same set of images was analysed separately by two individuals. Results from the two separate analyses were not significantly different from one another. Scale bars in panels a and c represent 10 μm. Cells were labelled with anti-Cx43 primary antibody and secondary antibody conjugated to Alexa 488.
Figure 1
Figure 1
Fluorescence images showing the activation wavefronts during activation in response to pacing near the base of the left ventricle (top of image) in a control heart in normal Krebs solution (A); the same control heart in the presence of 0.5 mm heptanol (B); a diabetic heart in normal Krebs solution (C); and the same diabetic heart in the presence of 0.5 mm heptanol (D). All images show the state of activation 23.6 ms after the onset of the stimulus pulse. Note the considerably more pronounced slowing in response to heptanol in the diabetic heart (C and D) compared to the control heart (A and B). The colour scale shows fluorescence after background subtraction, a measure of membrane potential, with red corresponding to the most depolarized potentials. All data were signal averaged over approximately 20 activation cycles (see Supplemental material, Appendix A). The insets show optically recorded action potentials (APs) from a pixel near the apex of the left ventricle of the two hearts shown. Green lines show APs in normal Krebs solution, and red lines show APs in the presence of 0.5 mm heptanol. (Upper panel, APs in the control heart; lower panel, APs in the diabetic heart.) Note the considerably larger delay in response to heptanol in the diabetic heart compared to the control heart. Data were signal averaged over approximately 20 cycles.
Figure 2
Figure 2
Isochronal maps (1 ms isochrones) showing activation in response to pacing near the base of the left ventricle (top of image) in a control heart in normal Krebs solution (A); the same control heart in the presence of 0.5 mm heptanol (B); a diabetic heart in normal Krebs solution (C); and the same diabetic heart in the presence of 0.5 mm heptanol (D). Insets in B and D show histograms constructed from the times of activation for individual points on the preparation (the data shown in the colour maps). Green histograms represent activation in normal Krebs solution and red histograms activation in the presence of 0.5 mm heptanol. The widths of these histograms at 20% of peak levels were used as a measure of the total activation time of the preparation. Note the considerably more pronounced broadening of the histogram in response to heptanol in the diabetic heart (inset in D) compared to the control heart (inset in B). The colour scale in the isochronal maps shows the time of activation at each point on the preparation, with red corresponding to the points activated the earliest. All times are with reference to the onset of the stimulus pulse for example (a point with time of activation=5 ms is activated 5 ms after the onset of the stimulus pulse). Activation data were averaged over approximately 20 activation cycles.
Figure 3
Figure 3
Summary data showing the effect of increased [K+] (A) and increasing concentrations of heptanol on the activation time (width of histograms shown in Fig. 3) (B). Activation times for each preparation were normalized to the value obtained in normal Krebs solution to account for differences in heart size. *Statistical significance (t test with P < 0.05). Slowing in response to elevated [K+], as well as heptanol application, was significantly more pronounced in diabetic hearts than in control hearts under the same conditions. Data shown are based on n=6–7 for [K+] (A) and n=8–9 for heptanol concentrations of 0.5 mm or less (B). Data for heptanol concentrations greater than 0.5 mm are based on n=4–6 due to lower success rate (complete conduction failure in some preparations) at these higher concentrations.
Figure 4
Figure 4
A, sample action potentials recorded from single ventricular myocytes obtained from a control (left) and a diabetic (right) rat. Horizontal lines depict 0 mV level. B, summary data for maximal upstroke rate (left) and resting potentials (right) for cells from control (open columns) and diabetic (hatched columns) rats. No significant differences were measured in either parameter.
Figure 5
Figure 5. Effect of stimulation rate on onset of activation
A, action potentials (in a heart from a diabetic rat) obtained from one pixel at cycle lengths of 200–80 ms (top to bottom, 20 ms intervals). The first discernible delay occurs at 140 ms. B, summary data obtained from 3 hearts.
Figure 6
Figure 6
A, thin sections of the left ventricle from a non-diabetic (left panel) and STZ-diabetic (right panel) rat heart showing the structural changes associated with diabetes. Sections were labelled with anti-Cx43 (primary antibody) and secondary antibody conjugated to Cy3. Scale bar in right panel represents 20 μm; arrows are drawn parallel to the long axis of the cells in these sections. B, Western blot of ventricular cardiac myocyte homogenates from control and diabetic rats labelled with anti-Cx43. Molecular mass is shown to the left. C, analysis of the Western blot shown in B. Densities of the bands labelled with anti-Cx43 were normalized to the total protein transferred to the blot (determined from densitometric analysis of the blot after labelling of all proteins with amido black). Results are expressed relative to the mean normalized density of the Cx43 bands from the control samples. The relative density of the Cx43 bands from the control and diabetic samples were not significantly different from one another (P=0.11).
Figure 8
Figure 8. Summary of analysis of lateralization
The fraction of anti-Cx43 immunofluorescence associated with the intercalated discs and that associated with lateralized Cx43 was determined as described in the text. Results shown were compiled from two separate cell preparations from non-diabetic and two from diabetic rats. Forty individual control cell images and 41 STZ cell images were analysed. The fraction of lateralized Cx43 fluorescence was significantly higher in the STZ cells than that in the control cells (P < 0.0005).
Figure 9
Figure 9. Distribution of N-cadherin and connexin 43 in isolated left ventricular cardiac myocytes from STZ-diabetic rats
A, differential interference contrast (DIC) micrograph of an isolated myocyte (a) and immunofluorescence images showing the distribution of N-cadherin (b) and Cx43 (c) in the myocyte. Panel d is an overlay of the images in b (pseudo-coloured red) and c (pseudo-coloured green). Green arrows in c point to lateralized Cx43 labelling that is not co-localized with N-cadherin; red arrows point to lateralized Cx43 labelling that is co-localized with N-cadherin labelling. B, DIC micrograph (a′) and three image planes (b′–d′) of a second cell dual-labelled with anti-connexin 43 (green) and anti-N-cadherin (red). Cells were dual-labelled as described in Methods; scale bars in a and a′ represent 10 μm. Cells were dual-labelled with polyclonal anti-connexin 43 (secondary antibody was conjugated to Alexa fluor 488) and monoclonal anti-N-cadherin (secondary antibody was conjugated to Cy3).
Figure 10
Figure 10. Distribution of β-catenin and Cx43 in isolated left ventricular cardiac myocytes from STZ-diabetic rats
A, DIC micrograph of an isolated myocyte (a) and immunofluorescence images showing the distribution of β-catenin (b) and Cx43 (c) in the myocyte. Panel d is an overlay of the images in b (pseudo-coloured red) and c (pseudo-coloured green). Green arrows in c point to lateralized Cx43 labelling that is not co-localized with β-catenin; red arrows point to lateralized Cx43 labelling that is co-localized with β-catenin labelling. B, DIC micrograph (a′) and three image planes (b′–d′) of a second cell dual-labelled with anti-Cx43 (green) and anti-β-catenin (red). Cells were dual-labelled as described in Methods; scale bars in a and a′ represent 10 μm. Cells were dual-labelled with polyclonal anti-Cx43 (secondary antibody was conjugated to Alexa fluor 488) and monoclonal anti-β-catenin (secondary antibody was conjugated to Cy3).
Figure 11
Figure 11. Analysis of the collagen content of ventricular tissue from control and diabetic rats
Aa, immunofluorescence image of a thin section from a control rat ventricle labelled with anti-collagen III; Ab, similarly labelled image from a diabetic rat ventricle (scale bar in a, 100 μm). B, summary of analysis of images of anti-collagen III-labelled ventricular sections. Results are expressed as percentage of tissue area occupied by collagen III. Area occupied by collagen in the control sections was not significantly different from that occupied by collagen in the diabetic sections (P=0.9). Thirty control and 30 diabetic images were analysed (10 of each were collected with a ×40 objective and 20 with a ×10 objective).
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