Elderly with Varying Extents of Cardiac Disease Show Interindividual Fluctuating Myocardial TRPC6-Immunoreactivity

Abstract

Both particular myocardial locations in the human heart and the canonical transient receptor potential 6 (TRPC6) cation channel have been linked with cardiac pathophysiologies. Thus, the present study mapped TRPC6-protein distribution in select anatomic locations associated with cardiac disease in the context of an orienting pathological assessment. Specimens were obtained from 5 body donors (4 formalin fixation, 1 nitrite pickling salt-ethanol-polyethylene glycol (NEP) fixation; median age 81 years; 2 females) and procured for basic histological stains and TRPC6-immunohistochemistry. The latter was analyzed descriptively regarding distribution and intensity of positive signals. The percentage of positively labelled myocardium was also determined (optical threshold method). Exclusively exploratory statistical analyses were performed. TRPC6-protein was distributed widespread and homogenously within each analyzed sample. TRPC6-immunoreactive myocardial area was comparable regarding the different anatomic regions and sex. A significantly larger area of TRPC6-immunoreactive myocardium was found in the NEP-fixed donor compared to the formalin fixed donors. Two donors with more severe heart disease showed smaller areas of myocardial TRPC6-immunoreactivity overall compared to the other 3 donors. In summary, in the elderly, TRPC6-protein is widely and homogenously distributed, and severe cardiac disease might be associated with less TRPC6-immunoreactive myocardial area. The tissue fixation method represents a potential confounder.

Keywords: TRPC6; heart; heart disease; histology; immunohistochemistry; morphology.

Figure 1

Representative findings in basic histological stains. Per row, one body donor and per column, one sampling site is displayed. Every field has a small heading indicating the body donor (D1—Donor 1, D2—Donor 2, D3—Donor 3, D4—Donor 4, D5—Donor 5), the sampling site (IVS—Interventricular septum, RAA—Right atrial appendage, SM—Septomarginal trabecula, PAP—Anterior papillary muscle, PV—Transition zone of left lower pulmonary vein to the left atrium), the magnification (100–100× magnification, 200–200× magnification), and the basic stain displayed in the picture (AB—Alcian blue, MG—Masson-Goldner-trichrome, HE—Hematoxylin-eosin).

Figure 2

Representative findings in TRPC6-immunohistochemistry. Per row, one body donor and per column, one sampling site is displayed. Every field has a small heading indicating the body donor (D1—Donor 1, D2—Donor 2, D3—Donor 3, D4—Donor 4, D5—Donor 5) and the sampling site (IVS—Interventricular septum, RAA—Right atrial appendage, SM—Septomarginal trabecula, PAP—Anterior papillary muscle, PV—Transition zone of left lower pulmonary vein to the left atrium). All 100× magnification.

Figure 3

Parasympathetic ganglion in the subepicardial fatty tissue of the left atrium. Every field has a small heading indicating the body donor (D5—Donor 5), the sampling site (PV—Transition zone of left lower pulmonary vein to the left atrium), the magnification (100–100× magnification, 200–200× magnification), and the staining (HE—Hematoxylin-eosin-stain, TRPC6–immunohistochemical labelling of TRPC6). A structure resembling a vegetative ganglion is seen in the center of each field. The two lower images show the repeatedly positive signals (red “*”) found within this structure. Additionally, the surrounding subepicardial fatty tissue presents with scattered positive signals (red ”>”) in areas not affected by wash out of the fat during tissue processing.

Figure 4

Boxplots showing the distribution of TRPC6-protein positively labelled myocardial area per specimen and donor. Per row, one body donor and per column, one sampling site is displayed. Every field has a small heading indicating the body donor (D1—Donor 1, D2—Donor 2, D3—Donor 3, D4—Donor 4, D5—Donor 5) and the sampling site (IVS—Interventricular septum, RAA—Right atrial appendage, SM—Septomarginal trabecula, PAP—Anterior papillary muscle, PV—Transition zone of left lower pulmonary vein to the left atrium) displayed by the respective box plot (thick line in the box—median; left border of the box—25th percentile; right border of the box—75th percentile; horizontal length of the box—interquartile range; left whisker—25th percentile minus 1.5× interquartile range; right whisker—75th percentile plus 1.5× interquartile range; dots outside the range of both whiskers and the box display outliers).

Figure 5

Scatter plots in the comparisons of TRPC6-positively labelled myocardial areas. Both (A) and (B) display summarizing scatter plots according to the subheading. (C–H) visualize the different comparative analyses, with the p-value of the respective test shown in the subheading of each field and the comparison shown as the label of the x-axis. Legends of colors and shapes are displayed on the right side of each diagram. Abbreviations: D1—Donor 1, D2—Donor 2, D3—Donor 3, D4—Donor 4, D5—Donor 5, IVS—Interventricular septum, RAA—Right atrial appendage, SM—Septomarginal trabecula, PAP—Anterior papillary muscle, PV—Transition zone of left lower pulmonary vein to the left atrium, NEP—Nitrite pickling salt-ethanol-polyethylene glycol fixation.