High resolution mapping of the cardiac transmural proteome using reverse phase protein microarrays

Abstract

The expression level of proteins governing the electrical excitability of and conduction within ventricular myocardium are known to vary as a function of distance through the heart wall. The expression patterns of a subset of these proteins are altered in disease. Precise measurement of such patterns is therefore essential to understanding structure-function relationships within the heart in health and disease. Here, we report a new experimental approach using reverse-phase protein microarrays to map the left ventricular transmural proteome. This approach can yield submillimeter spatial resolution, and when coupled with the method of array microenvironment normalization, reduces nonbiological components of variability to ∼10% of overall study variability. In addition, the experimental design provides sufficient statistical power to detect small, yet potentially biologically significant expression changes on the order of 1.1-fold. The usefulness of this technique is demonstrated by mapping the transmural expression of Serca2a in the left ventricle of 12 canine hearts, each in one of three states: normal, dyssynchronous heart failure, and dyssynchronous heart failure followed by cardiac resynchronization therapy. We confirm the existence of a 40% transmural gradient (epi>endo) of Serca2a, and demonstrate the ability of this technique to yield highly significant transmural expression differences within each individual heart.

Fig. 1.

Experimental overview. A, A depiction of the acquisition of an anterior transmural wedge from a left ventricle (z axis represents the transmural dimension), which is immediately snap frozen. B, An individual transmural wedge being sectioned at 1-mm resolution (dotted line represents the sectioning plane x-y), at −24°C in a cryostat. Sample tissue from each disparate 1-mm region is lysed to create a whole-cell lysate. All 1-mm whole-cell lysates are then robotically printed on a RPMA in unique spots, as demonstrated by the arrows. C, A sypro ruby total protein stain of a single HRSH array is shown (the left side of the array contains samples printed at 600 μg/ml, and the right side at 150 μg/ml). The array is magnified to display the AMN format of RPMA printing. D, The structure of replicates printed on the RPMAs for each 1 mm transmural depth of wedge tissue. In total, sixteen replicates are printed that consist of independent sectioning and lysing replicates (reps) as well as repeated spotting on the array.

Fig. 2.

Total protein normalization. A, The primary intensity plotted versus the sypro ruby intensity. The red line is the fit total protein dependence using the total protein controls, slope = 1.34. B, A plot of the TPN adjusted intensity versus the same sypro ruby intensity, total protein control slope = –0.0017.

Fig. 3.

Nested ANOVA displaying the four normal hearts assayed using this technique. All replicates from all samples are displayed, grouped according to their origin as seen on the bottom of the graph. Unprocessed Serca2a primary intensity is on top, TPN adjusted intensity in the middle, and the final TPN and AMN adjusted intensity is shown on the bottom.

Fig. 4.

LRMH Serca2a staining results (A) relative to a negative stain (B) (lateral wedge only). The 16-replicate mean and standard deviation are shown for each depth of each heart, grouped by N-normal (green), d-DHF (black), and C-CRT (red). Four shapes within each state are used to represent each unique heart (filled square, filled triangle, open triangle, and open circle). The within-state means of the four hearts at each depth are shown by a horizontal line. A, shows the result of staining a LRMH array with a SERCA2a antibody, B, is an identical array that was used as a negative control (stained with secondary antibody only).

Fig. 5.

HRSH RPMA results and correlation to LRMH RPMAs. A, Serca2a expression of a single DHF heart (lateral wedge shown as black open triangle in Fig. 3) at full 1-mm resolution. B, Negative stain transmural expression of an identical HRSH RPMA from the same printing run. C, Scatter plot of serca2a expression for samples that were on the LRMH array and the twelve HRSH arrays (R² = 0.85).