Examining Efficacy of “TAT-less” Delivery of a Peptide against the L‑Type Calcium Channel in Cardiac Ischemia–Reperfusion Injury

Increased calcium influx through the L-type Ca²⁺ channel or overexpression of the alpha subunit of the channel induces cardiac hypertrophy. Cardiac hypertrophy results from increased oxidative stress and alterations in cell calcium levels following ischemia–reperfusion injury and is an independent risk factor for increased morbidity and mortality. We find that decreasing the movement of the auxiliary beta subunit with a peptide derived against the alpha-interacting domain (AID) of the channel attenuates ischemia–reperfusion injury. We compared the efficacy of delivering the AID peptide using a trans-activator of transcription (TAT) sequence with that of the peptide complexed to multifunctional polymeric nanoparticles. The AID-tethered nanoparticles perfused through the myocardium more diffusely and associated with cardiac myocytes more rapidly than the TAT-labeled peptide but had similar effects on intracellular calcium levels. The AID-complexed nanoparticles resulted in a similar reduction in release of creatine kinase and lactate dehydrogenase after ischemia–reperfusion to the TAT-labeled peptide. Since nanoparticle delivery also holds the potential for dual drug delivery, we conclude that AID-complexed nanoparticles may provide an effective platform for peptide delivery in cardiac ischemia–reperfusion injuries

Limits of agreement between liver fat fraction measurements.

<p>A) MRI HIST-MORPH fat fraction plotted against HIST-MORPH fat fraction for the 59 subjects. The solid line is the line of equivalence. B) The difference between the natural logarithm of HIST-MORPH and the natural logarithm of MRI HIST-MORPH plotted against the mean of the two logarithms for the 59 subjects. The solid lines indicate the 95% limits of agreement and the dashed line is the mean difference between the logarithms of the two methods.</p

Analysis of the area under the receiver operating characteristic curve using the histopathologist’s visual estimate of fat in the histological sections.

*<p>Cut-offs are defined according to the NASH CRN grading system. Abbreviations: AUC, Area under receiver operating characteristic curve; CI, confidence interval; MRI, magnetic resonance imaging.</p

Analysis of the area under the receiver operating characteristic curve using the morphometric image analysis estimate of fat in the histological sections.

<p>Abbreviations: AUC, Area under receiver operating characteristic curve; CI, confidence interval; MRI, magnetic resonance imaging.</p

Example of histology images and morphometric image analysis.

<p>A) Histologic image of a liver (Masson trichrome, ×20 objective), B) Binary image of same image after application of threshold, C) Mask showing fat vacuoles after application of size and structural criteria, D) Examples of binary histology images with measured fat percentage areas. These images have been thresholded as in 1B, but not masked (as in 1C), so as to keep the additional white spaces that are not represented in the areal fat estimate, but are visible in a histology image. Each square is 500 microns across.</p

Plot of the MRI derived α value versus the fractional area of fat vacuoles in the histological section (HIST-MORPH).

<p>The solid line is a fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059287#pone.0059287.e003" target="_blank">Equation 3</a> to the data (r² = 0.84).</p

Axial magnetic resonance images of a patient with a typical ROI (solid grey circle) used for fat analysis.

<p>A) 2.38 ms (opposed phase), B) 4.76 ms (in phase), C) 7.14 ms (opposed phase).</p

Axial slices of the longest echo time (TE = 18 ms) for subjects from the four different groups characterized.

<p>(A) Control, (B) SCD, (C) PNH and (D) NTDT.</p

Correlations between serum ferritin concentration and liver R2 (A-C), bone marrow R2 (D-F), spleen R2 (G-I) and heart R2* (J-L) in SCD, PNH and NTDT patients.

<p>Correlations between serum ferritin concentration and liver R2 (A-C), bone marrow R2 (D-F), spleen R2 (G-I) and heart R2* (J-L) in SCD, PNH and NTDT patients.</p

Comparison of tissue R2 ratios for four different groups of subjects: controls (black) and SCD (blue), PNH (purple) and NTDT (green) patients.

<p>(A) Spleen R2 / Liver R2, (B) Bone Marrow R2 / Liver R2 and (C) Bone Marrow R2 / Spleen R2. Mean values ± SD are represented by the horizontal bars. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 for Kruskal Wallis analysis with Dunn post test. The asterisk color indicates the group with which the difference was found. The whole SCD data set has been used for the analysis.</p