Electrocardiography, echocardiography, myocyte shortening and histological data

Electrocardiography

Electrocardiography data obtained in diabetic and age-matched control rats using Bioamp (ADInstruments, Dunedin, New Zealand), 3 weeks after induction of diabetes.
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Echocardiography

Transthoracic echocardiography was performed in diabetic and age-matched control rats using VEVO 2100 (Visual Sonics, Toronto, ON, Canada) equipped with a 40-MHz transducer, 2 weeks after diabetes induction.
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Myocyte shortening
Cardiac
myocytes were placed in a perfusion chamber mounted on the stage of an inverted
microscope (Eclipse TS100; Nikon, Tokyo, Japan) equipped with an analog camera
(Myocam; IonOptix, Milton, MA, USA). Only rod-shaped myocytes with clear edges
and without spontaneous contractions were selected for analysis. Cells were
maintained at 37°C and field stimulated (MyoPacer; IonOptix) with 5 ms bipolar
pulses at 1 Hz frequency using platinum electrodes placed on the opposite sides
of the chamber. Contraction signals of load-free myocytes were recorded with
the help of a commercial software (IonWizard; IonOptix) and the sarcomere striation
pattern used to calculate changes in sarcomere spacing using a fast Fourier
transform algorithm (Sarclen Algorithm; IonOptix). The following parameters
were obtained: resting sarcomere length; amplitude of shortening (expressed
as % of resting sarcomere length); shortening and relaxation velocities and
time intervals to reach the peak of contraction (TPC); maximal shortening
velocity (TMS); maximal relaxation velocity (TMR) and 50% resting sarcomere
length (THALF). The parameters were calculated averaging at least 5 consecutive
contractions.
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Histology
For histological data, prepared sections were observed under microscopy (Leica Application suite). Magnification was set up at ×400. Micrographs were used to calculate the collagen content in the myocardial interstitium using the Image J software as the percentage of red-stained area in the section. An average of 15-20 images from each animal from each group was analyzed. To evaluate the perivascular fibrosis of intramyocardial arterioles of the ventricles, arterioles with a diameter between 100-200 μm were chosen. For each image of the arteriole, a region of interest was delimited around it to exclude areas of interstitial collagen which were not related to the arteriole. Perivascular fibrosis was determined by the ratio between the area occupied by collagen stained with picro-sirius within the region of interest and the area of ​​the lumen. The software Image J was used to calculate the area occupied by the collagen and to mark the regions of interest, and the software Zen (Zeiss, Germany) to calculate the area of ​​the lumen. The images were obtained with a 20x magnifying objective (Eclipse TE 300, Nikon, USA) with a digital camera (AxioCam, Zeiss, Germany).