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

Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

<div><p>The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The <i>Mrps34</i> mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The <i>Mrps34</i> mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.</p></div

Nanoparticle-Mediated Dual Delivery of an Antioxidant and a Peptide against the L‑Type Ca²⁺ Channel Enables Simultaneous Reduction of Cardiac Ischemia-Reperfusion Injury

Increased reactive oxygen species (ROS) production and elevated intracellular Ca²⁺ following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca²⁺ channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca²⁺ channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca²⁺ channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca²⁺ channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluoresence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes

<i>Mrps34</i>^{<i>mut/mut</i>} mice have hypertrophic hearts and increased lipid accumulation in their livers.

<p>(A) Echocardiographic parameters of <i>Mrps34</i>^<i>wt/wt</i> (n = 5) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 5) mice. LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening; LVDPW, left ventricular posterior wall in diastole; LVSPW, left ventricular posterior wall in systole; IVDS, intraventricular septum in diastole; IVSS, intraventricular septum in systole. Values are means ± standard error. *p<0.05 compared with <i>Mrps34</i>^<i>wt/wt</i>. Liver sections cut at 8–12 μm thickness were stained with Haematoxylin and Eosin, oil red O and Haematoxylin or Gomori trichrome from young (B) and aged (C) <i>Mrps34</i>^<i>wt/wt</i> (n = 9) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 9) mice and visualized at 40X magnification. (D) Quantitative measurement of oil red staining using Image J. Data are means ± SEM of four different mice; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test. Serum ALT levels in young (E) and aged (F) <i>Mrps34</i>^<i>wt/wt</i> (n = 12) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 12) mice.</p

Decreased MRPS34 levels cause a reduction in mitochondrial protein synthesis and steady state levels of mitochondrial proteins.

<p>(A) Protein synthesis in heart and liver mitochondria isolated from <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} was measured by pulse incorporation of ³⁵S-labelled methionine and cysteine over time. Equal amounts of mitochondrial lysates (50 μg) were separated by SDS–PAGE, Coomassie stained (bottom panel) and visualized by autoradiography (top panel). Mitochondrial lysates (20 μg) from <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} livers and hearts of young (B) and aged (C) mice were resolved by SDS-PAGE and immunoblotted using antibodies to assess changes in mitochondrial protein abundance. Porin was used as a loading control. Data are means ± SEM of four separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

The <i>Mrps34</i> mutation causes reduced oxygen consumption and respiratory complex activities in heart and liver mitochondria.

<p>The activities of the five mitochondrial respiratory complexes were measured in mitochondria isolated from heart (A) and liver (B) of young <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice and aged mice (C and D), respectively. The respiratory complex activities were normalized relative to citrate synthase activity. Data are means ± SEM of three-four separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test. State 3 and 4 respiration was measured in mitochondria isolated from hearts (E) and livers (F) of aged <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice using an OROBOROS oxygen electrode. Data are means ± SEM of three-four separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

Decreased MRPS34 affects the stability of the 12S rRNA and specific mitochondrial mRNAs.

<p>(A) The abundance of mature mitochondrial transcripts in mitochondria isolated from young <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} livers and hearts was analyzed by northern blotting. (B) The abundance of mature mitochondrial transcripts in aged liver and heart was analyzed by northern blotting. 18S rRNA was used as a loading control. The data are representative of results obtained from at least 8 mice from each strain. Data are means ± SEM of three separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

A homozygous point mutation in the <i>Mrps34</i> gene causes a decrease in the MRPS34 protein.

<p>(A) Schematic showing the location of the mutation in the <i>Mrps34</i> gene, mRNA and protein. The 5′- and 3′-untranslated regions are shown as grey boxes and the predicted mitochondrial targeting sequence is shown as a black box (predicted using MitoProtII, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005089#pgen.1005089.ref040" target="_blank">40</a>]). Conservation of the protein sequence surrounding the mutation is highlighted with residues identical to those in the mouse sequence boxed (the mutated leucine is shown in red in the mouse sequence). Sequences used were obtained from GenBank at NCBI (human, <i>Homo sapiens</i>, NP_076425.1; chimp, <i>Pan troglodytes</i>, XP_001160122.1; monkey, <i>Macaca mulatta</i>, NP_001244544.1; mouse, <i>Mus musculus</i>, NP_081622.1; rat, <i>Rattus norvegicus</i>, NP_001099241.1; cow, <i>Bos taurus</i>, NP_001192540.1; dog, <i>Canis lupus familiaris</i>, XP_003639160.1; chicken, <i>Gallus gallus</i>, NP_001264526.1; frog, <i>Xenopus tropicalis</i>, NP_001004910.2; zebrafish, <i>Danio rerio</i>, NP_001007377.2) and the alignment was produced using ClustalW2 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005089#pgen.1005089.ref041" target="_blank">41</a>]. The secondary structure of mouse MRPS34 was predicted using PSI-PRED [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005089#pgen.1005089.ref042" target="_blank">42</a>] and the mutated region is shown. An alpha helical region is predicted to be lost when the L68P mutation is present. (B) The mutation was confirmed by Sanger sequencing of PCR amplicons from <i>Mrps34</i>^{<i>mut/mut</i>} and littermate matched <i>Mrps34</i>^<i>wt/wt</i> mice. (C) Mutation in the nuclear gene encoding the mitochondrial MRPS34 protein leads to its decreased abundance. MRPS34 and MRPL44 protein levels were investigated in mitochondria isolated from hearts and livers of <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice by immunoblotting. Porin was used as a loading control. The data are representative of results obtained from at least 8 mice from each strain. Data are means ± SEM of three separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

Reduced MRPS34 affects the abundance of mitochondrial respiratory complexes.

<p>BN-PAGE was performed on 75 μg of mitochondria isolated from liver and heart of young (A) and aged (B) <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice. Mitochondria lysates (75 μg) isolated from liver and heart of <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} young mice (C) and aged mice (D) were analyzed by BN-PAGE and the gels used for immunoblotting. Specific antibodies representing proteins of each of the mitochondrial complexes were used to compare abundance of protein in the wild type and mutant mice. Data are means ± SEM of five separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

MRPS34 is required for the small ribosomal subunit and the stability of the mitoribosome.

<p>(A) Mitochondrial ribosomal protein levels in mitochondria (50 μg) isolated from liver and heart of <i>Mrps34</i>^<i>wt/wt</i> compared to <i>Mrps34</i>^{<i>mut/mut</i>} mice was determined by immunoblotting. (B) Distribution of the MRPS34 protein in 10–30% sucrose gradients of heart (0.8 mg) and liver (1.2 mg) mitochondria from young and aged <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice. Mitochondrial protein lysates from heart (0.8 mg) and liver (1.2 mg) of young (C) and aged (D) <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} were fractionated on 10–30% sucrose gradients. The distribution of ribosomal proteins was analyzed by immunoblotting against antibodies that are markers of the small and large ribosomal subunits. The data are typical of results from at least three independent biological experiments.</p