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. 2018 Feb 1;314(2):H311-H321.
doi: 10.1152/ajpheart.00515.2017. Epub 2017 Nov 3.

A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure

Jessica M Bradley  1 , Zhen Li  1 , Chelsea L Organ  1 , David J Polhemus  1 , Hiroyuki Otsuka  1 , Kazi N Islam  2 , Shashi Bhushan  1 , Olena M Gorodnya  3 , Mykhaylo V Ruchko  3 , Mark N Gillespie  3 , Glenn L Wilson  3   4 , David J Lefer  1
Affiliations

A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure

Jessica M Bradley et al. Am J Physiol Heart Circ Physiol. .

Abstract

Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10-12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6-12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.

Keywords: DNA repair; hypertrophic cardiomyopathy; mitochondria-targeted peptide; mitochondrial injury; myocardial infarction.

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Figures

Fig. 1.
Fig. 1.
Exscien1-III attenuates myocardial ischemic injury. A: schematic construction of Exscien1-III. Exscien1-III consists of a mitochondria targeting sequence (MTS), an endonuclease III protein transduction domain (i.e., DNA glycosylase), a hemagglutinin (HA) tag for immunological localization, a TAT sequence to facilitate cell uptake, and a polyhistidine tail (10-HIS). B: myocardial ischemia-reperfusion (MI/R) experimental protocol. Mice were subjected to 45 min of ischemia and 24 h of reperfusion. Exscien1-III, mExscien1-III, or vehicle (Veh) was administered via intracardiac injection at the time of reperfusion. C: assessment of the myocardial area at risk as a percentage of the left ventricle (AAR/LV) and myocardial infarct as a percentage of the area at risk (INF/AAR) in Exscien1-III-, mExscien1-III-, and Veh-treated animals. D: serum cardiac troponin I in Exscien1-III- and Veh-treated mice. Circles inside bars denote n values per study group.
Fig. 2.
Fig. 2.
Exscien1-III prevents left ventricular (LV) dilation and preserves LV function after myocardial ischemia (MI). A: experimental protocol for myocardial ischemia and extended reperfusion. Mice were subjected to 45 min of ischemia followed by 4 wk of reperfusion. Exscien1-III or vehicle (Veh) was administered via intracardiac injection at the time of reperfusion. Echocardiography was performed at baseline and at 1, 2, and 4 wk post-MI to assess LV end-diastolic dimension (LVEDD; B), LV end-systolic dimension (LVESD; C), interventricular septum thickness at diastole (IVDd; D), and LV ejection fraction (LVEF; E). *P < 0.05 vs. Veh; **P < 0.01 vs. Veh.
Fig. 3.
Fig. 3.
Exscien1-III prevents adverse cardiac remodeling and preserves cardiac function after transverse aortic constriction (TAC). A: experimental protocol for the TAC cardiac failure study. Mice were subjected to TAC surgery, and Exscien1-III or vehicle (Veh) treatment was administered daily via intraperitoneal injection starting 3 wk post-TAC and continued until the 12-wk end point. Echocardiography was performed at baseline and every 2 wk post-TAC to assess left ventricular (LV) end-diastolic dimension (LVEDD; B), LV end-systolic dimension (LVESD; C), LV posterior wall thichness at diastole (LVPWd; D), and LV ejection fraction (LVEF; E). *P < 0.05 vs. Veh; **P < 0.01 vs. Veh.
Fig. 4.
Fig. 4.
Treatment with Exscein1-III reduces transverse aortic constriction (TAC)-induced hypertrophy. Postmortem morphological assessment of ventricular weight to tibia length (in mg/mm; A), atria weight to tibia length (in mg/mm; B), and lung weight to tibia length (in mg/mm; C) from Exscien1-III- and vehicle (Veh)-treated mice. Circles inside bars denote n values per group. D: representative images of hematoxylin and eosin-stained cardiomyocytes from Exscien1-III- and Veh-treated mice.
Fig. 5.
Fig. 5.
Exscien1-III is localized in cardiac mitochondria after daily intraperitoneal injections and reduces transverse aortic constriction (TAC)-induced mtDNA damage. A and B: protein expression of the hemagglutinin (HA) tag found on the Exscien1-III fusion peptide measured in total ventricular (A) and mitochondrial tissue fractions (B) isolated from mice treated with Exscien1-III or vehicle (Veh) at 12 wk post-TAC. C: protein expression of HA-tagged Exscien1-III measured in the total cell lysate, cytosol, nuclease, and mitochondria at 6 wk post-TAC. D: fraction of intact mtDNA in the heart measured in mice treated with Exscien1-III or Veh at 6 wk post-TAC or Sham. Circles inside bars denote n values per group. COX IV, cytochrome c oxidase subunit IV.
Fig. 6.
Fig. 6.
Exscien1-III attenuates myocardial fibrosis after transverse aortic constriction (TAC). A: representative images of cardiac sections at 12 wk post-TAC stained for picrosirus red (PSR) or Masson’s trichrome for fibrosis. Assessment of collagen volume fraction was measured via fluorescence quantification of PSR-stained slides. B: schematic construction of Exscien1-III. Exscien1-III consists of a mitochondria targeting sequence (MTS), an endonuclease III protein transduction domain, a hemagglutinin (HA) tag for immunological localization, a TAT sequence to facilitate cell uptake, and a polyhistidine tail (10-HIS), interstitial and perivascular collagen (C). Circles inside bars denote n values per group.
Fig. 7.
Fig. 7.
Exscien1-III increases superoxide dismutase (SOD)2. A: levels of total circulating 8-isoprostane in plasma from mice treated with Exscien1-III or vehicle (Veh) at 12 wk after transverse aortic constriction (TAC). B: mRNA levels of SOD2 in ventricular homogenates from mice treated with Exscien1-III or Veh at 12 wk post-TAC. C and D: protein expression of SOD2 was measured in total ventricular homogenates (C) and cardiac mitochondrial fraction (D). E: protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was measured in the cardiac mitochondrial fraction. Circles inside bars denote n values per group. COX IV, cytochrome c oxidase subunit IV.
Fig. 8.
Fig. 8.
Exscien1-III reduced mitochondrial apoptotic markers. A: mRNA levels of p53 in ventricular homogenates from mice treated with Exscien1-III or vehicle (Veh). B−D: protein expression of Bax (B), cleaved caspase-3 (C), and cleaved caspase-9 (D) in cardiac mitochondrial fractions from mice treated with Exscien1-III or Veh at 12 wk after transverse aortic constriction (TAC). Circles inside bars denote n values per group. COX IV, cytochrome c oxidase subunit IV.
Fig. 9.
Fig. 9.
A simplified version of Exscien1-III, which does not contain the hemagglutinin (HA) tag, also attenuates myocardial ischemia-reperfusion (MI/R) injury. Mice were subjected to 45 min of ischemia and 24 h of reperfusion. Exscien1-III or vehicle (Veh) was administered via intracardiac injection at the time of reperfusion. A: schematic construction of Exscien1-III without the HA tag. B: data for the myocardial area at risk (AAR) as a percentage of the left ventricle (AAR/LV) and infarct size as a percentage of the area at risk (INF/AAR) in Exscien1-III without HA tag- and Veh-treated animals.
Fig. 10.
Fig. 10.
8-Oxoguanine DNA glycosylase (OGG1) attenuates myocardial ischemic injury and reduces myocardial infarct size. Mice were subjected to 45 min of ischemia and 24 h of reperfusion. OGG1, mOGG1, or vehicle (Veh) was administered via intracardiac injection at the time of reperfusion. A: data for the myocardial area at risk as a percentage of the left ventricle (AAR/LV) and infarct size as a percentage of the area at risk (INF/AAR) in OGG1-, mOGG1-, and Veh-treated animals. B: serum cardiac troponin I in OGG1- and Veh-treated mice. Circles inside bars denote n values per group.
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