*3.4. The Impact of Parkin Overexpression and Sepsis on the Expression of Genes and Proteins Regulating Mitochondrial Biology*

Since Parkin plays a key role in mitochondrial quality control [23–26,40], and because sepsis is well known to impair mitochondrial function, we investigated whether Parkin overexpression could attenuate the impact of sepsis on skeletal muscle mitochondria. To this end, we first quantified the expression levels of the key transcriptional regulators of mitochondrial biology. As shown in Figure 4A,B, sepsis resulted in an increase in *Nrf1, Nrf2,* and *Sirt1* mRNA expression levels. In contrast, sepsis resulted in a decrease in the expression of *Pgc1-*α*, Tfam and Sirt3*, as shown in Figure 4A,B. In the skeletal muscles of both Sham-operated and CLP mice, Parkin overexpression resulted in

a significant increase in *Nrf2* mRNA expression, depicted in Figure 4A. Parkin overexpression also led to an increased expression of *Sirt1* in the muscles of Sham-operated mice and an increase in *Tfam* expression in the muscles of CLP mice, as shown in Figure 4B.

**Figure 4.** The impact of Parkin overexpression and sepsis in skeletal muscle on genes regulating mitochondrial biogenesis and on mitochondrial protein contents. (**A**,**B**) qPCR analysis of genes involved in mitochondrial biology. (**C**) Representative immunoblots performed with primary antibodies against representative subunits of the OXPHOS complexes and VDAC. Ponceau stains were used as loading controls. (**D**,**E**) Quantification of the contents of (**D**) representative subunits of the OXPHOS complexes and (**E**) VDAC. 1 = Sham-AAV-GFP; 2 = Sham-AAV-Parkin; 3 = CLP-AAV-GFP; 4 = CLP-AAV-Parkin. Data are presented as mean ± SEM. (*n* = 6–9/group, \* = statistically significant; ns = not statistically significant).

We next assessed the impact of sepsis and Parkin overexpression on the content of proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system. As shown in Figure 4D, sepsis significantly decreased the content of the representative subunits of Complex I and Complex IV. This finding is consistent with previous reports, which documented decreased mitochondrial contents in septic skeletal muscles [10,12–14]. Similarly, sepsis lowered VDAC protein content by 58% in the GFP expressing skeletal muscles, as shown in Figure 4E. Importantly, no impact of sepsis was observed on Complex I, Complex IV and VDAC contents in the Parkin overexpressing muscles, as shown in Figure 4D,E. Taken together, these data strongly suggest that Parkin overexpression prevented the inhibitory effect of sepsis on muscle mitochondrial content.

#### *3.5. E*ff*ects of Parkin Overexpression and Sepsis on Mitochondrial Morphology and Dynamics*

To analyze the impact of sepsis and Parkin overexpression on skeletal muscle mitochondrial morphology, we used TEM to evaluate the morphology of intermyofibrillar (IMF) mitochondria of the GAS of sham and CLP mice. Representative TEM images obtained from the GAS of sham and CLP mice are shown in Figure 5A–D and Supplemental Figure S3. In the CLP group, IMF mitochondria of GFP expressing muscles were larger, less circular and more complex (i.e., higher values of aspect ratio and form factor) than IMF mitochondria of GFP expressing muscles of the sham group, shown in Figure 5E–J. In the sham group, Parkin overexpressing muscles had larger, less circular and more complex IMF mitochondria compared to GFP expressing muscles, as shown in Figure 5E–J. In the CLP group, Parkin overexpressing muscles had smaller, more circular and simpler IMF mitochondria compared to GFP expressing counterparts, shown in Figure 5 E–J. Taken together, these results indicate that sepsis results in enlarged and more complex mitochondria, an impact that is abolished by Parkin overexpression.

**Figure 5.** The impact of sepsis and Parkin overexpression on mitochondrial morphology in skeletal muscle. (**A**–**D**) Representative longitudinal TEM images from all groups that were used to assess mitochondrial morphology. Scale bar: 2μm. (**E**–**J**) Median values with 95% confidence interval (left) and relative frequencies (right) of multiple mitochondrial shape descriptors (Sham-AAV-GFP: *n* = 1246; Sham-AAV-Parkin: *n* = 728; CLP-AAV-GFP: *n* = 1149; CLP-AAV-Parkin: *n* = 1206). Groups not sharing a letter are significantly different (differences were tested using a Kruskal–Wallis test followed by a Dunn's multiple comparisons test; *p* < 0.05).

To gain better insights into the mechanisms underlying the impact of sepsis and Parkin overexpression on mitochondrial morphology, we next assessed the expression and content of major genes and proteins regulating mitochondrial dynamics. In the Sham-operated mice, Parkin overexpression had no impact on the mRNA expression and protein levels of *Mfn2*, *Opa1* and *Drp1*, as shown in Figure 6A–G. As shown in Figure 6A, sepsis in GFP expressing muscles resulted in a decrease in the mRNA levels of pro-fusion *Mfn2* and *Opa1* and pro-fission *Drp1*. In Parkin overexpressing muscles, CLP resulted in a significant decrease in the mRNA levels of Mfn2 and Drp1, while Opa1 expression remained unaffected, shown in Figure 6A. At the protein level, no impact of sepsis or Parkin overexpression could be evidenced on MFN2 and OPA1 protein content. Interestingly, DRP1 protein levels were lower in the GFP and Parkin expressing muscles of CLP mice, relative to the sham group, as shown in Figure 6D,E. Similarly, DRP1 phosphorylation on Ser616, an activation site which triggers DRP1 translocation from the cytoplasm to mitochondria to promote mitochondrial fission [41], was also decreased in the GFP and Parkin expressing muscles of the CLP group, relative to the sham group, as shown in Figure 6D–G. These results indicate that sepsis seems to result in an inhibition of mitochondrial fission and that this effect was not influenced by Parkin overexpression.

**Figure 6.** The impact of sepsis and Parkin overexpression on mitochondrial dynamics in skeletal muscle. (**A**) qPCR analysis of mitochondrial dynamic-related gene expression in the GAS muscles of Sham and CLP animals injected with either AAV-GFP or AAV-Parkin. (**B**) Representative immunoblots of OPA1, GADPH and MFN2. Ponceau stains or GAPDH immunoblots were used as loading controls. (**C**) Quantification of OPA1, GADPH and MFN2 content. (**D**) Representative immunoblots performed with primary antibodies against pDRP1(ser616) and total DRP1. Ponceau stains or GAPDH immunoblots were used as loading controls. (**E**) Quantification of DRP1 content. (**F**) Quantification of the contents of pDRP1(ser 616) content. (**G**) Quantification of the pDRP1(ser 616) to total DRP1 ratio. 1 = Sham-AAV-GFP; 2 = Sham-AAV-Parkin; 3 = CLP-AAV-GFP; 4 = CLP-AAV-Parkin. Data are presented as mean ± SEM. (*n* = 6–9/group; \* = statistically significant; ns = not statistically significant).

#### **4. Discussion**

The accumulation of dysfunctional and injured mitochondria in skeletal muscles is believed to play a key role in the development of muscle weakness during sepsis [8,9]. In the current study, we investigated whether overexpressing Parkin, a key component of the PINK1-Parkin mitophagy pathway, could attenuate the negative impact of sepsis on skeletal muscles and their mitochondria. The current study indicates that Parkin overexpression prevented sepsis-induced accumulation of enlarged and complex mitochondria in the limb muscles of mice. Parkin overexpression also attenuated the sepsis-induced decrease in the content of complexes I and IV of the mitochondrial electron transfer system and prevented the development of limb muscle atrophy in septic mice. These results expand recent studies demonstrating that Parkin exerts protective effects on skeletal muscle health. Indeed, our group has recently reported that *Park2-*/*-* mice have decreased limb muscle contractility, depressed muscle mitochondrial respiration, increased mitochondrial uncoupling and enhanced susceptibility to the opening of mitochondrial permeability transition pore compared to wild-type (WT) mice [42]. *Park2-*/*-* mice also exhibit the impaired recovery of cardiac contractility and depressed cardiac mitochondrial functions in sepsis [29]. More recently, Peker et al. have reported that Parkin knockdown in C2C12 cells results in myotubular atrophy and that *Park2-*/*-* mice have decreased muscle mitochondrial respiration and increased levels of reactive oxygen species and fiber atrophy [43]. Parkin overexpression in the muscles of *Drosophila melanogaster* increased mitochondrial content, decreased proteotoxicity and extended lifespan [44]. Our finding that Parkin overexpression in the Sham group increased limb muscle fiber diameters is in accordance with our recent study documenting that Parkin overexpression for several months in young mice causes muscle hypertrophy, while in old mice, Parkin overexpression attenuates ageing-related loss of muscle mass and strength, increases mitochondrial content and enzymatic activities and protects from ageing-related oxidative stress, fibrosis and apoptosis [32]. Taken together, our current findings and published studies highlight the protective role that Parkin plays in skeletal muscle health.

Our findings that sepsis elicits distinct changes in skeletal muscle mitochondria, such as decreased VDAC level (a marker of mitochondrial content [45–47]), the downregulation of three mitochondrial biogenesis genes (*Pgc1-*α, *Tfam* and *Sirt3*) and decreased complexes I and IV levels, are in agreement with published studies on septic humans and experimental animals [10,12–14,20]. We report for the first time that Parkin overexpression in skeletal muscle prevents the inhibitory effects of sepsis on the expression of *Tfam* and on the content of complexes I and IV, as well as VDAC. Based on these results, we speculate that Parkin overexpression might have improved mitochondrial functions in septic muscles. This speculation is supported by the observation that Parkin overexpression increases mitochondrial content and enzymatic activities in normal skeletal muscles [32,44]. We should emphasize that, in the current study, Parkin overexpression increased *Nrf2* expression in the skeletal muscles of septic animals. Considering the role that this transcription factor has in the regulation of the expression of several anti-oxidant enzymes [48], we anticipate that increased *Nrf2* levels in muscles overexpressing Parkin might have contributed to the protection of mitochondrial morphology and contents in septic animals.

Mitochondria form a dynamic network constantly undergoing fusion and fission events that tightly regulate the shape (i.e., morphology), size and number of mitochondria [41,49]. In the present study, we show that sepsis significantly alters mitochondrial morphology by increasing the proportion of enlarged and more complex IMF mitochondria. These results extend previous observations, showing that sepsis causes major alterations of the mitochondrial ultrastructure in skeletal muscle [13,19–21]. This increase in mitochondrial size and complexity in septic muscles might have been caused by decreased DRP1 contents and activation [41], which are expected to alter the fusion/fission balance towards increased mitochondrial fusion. Since mitochondrial fission is required for mitochondrial degradation through mitophagy [50], it is possible that decreased DRP1 content and activation may play a role in the accumulation of damaged and dysfunctional mitochondria in septic muscles by impairing muscle capacity to recycle dysfunctional mitochondria through the mitophagy pathway. It should also be noted that a decrease in DRP1 content per se might have also played a role in myofiber atrophy. Indeed, a recent study showed that muscle-specific DRP1 deletion results in severe muscle dysfunction, characterized by atrophy, weakness, fiber degeneration and mitochondrial dysfunction [51]. Importantly, we found that Parkin overexpression attenuated sepsis-induced changes in mitochondrial morphology and rendered muscle mitochondria to be smaller, more circular and simpler, relative to muscles expressing GFP. These findings are in agreement with previous reports, indicating that Parkin overexpression in skeletal muscles and neurons stimulates mitochondrial fragmentation [44,52]. We speculate that the decrease in mitochondrial size and complexity in septic muscles overexpressing Parkin might have facilitated the recycling of damaged/dysfunctional mitochondria.

A puzzling result of the present study is the increase in the proportion of enlarged and more complex mitochondria in the Parkin overexpressing muscles of sham-operated mice. The mechanisms behind the differences in the effects of Parkin on mitochondrial morphology in the sham and CLP groups remain unclear. We should point out, however, that although Parkin overexpression altered the mitochondrial morphology in skeletal muscles, none of the parameters related to mitochondrial dynamics that we investigated were affected by Parkin overexpression. Indeed, no significant impact of Parkin overexpression on the expression and protein content of MFN2, OPA1 and DRP1 was evident. Furthermore, Parkin overexpression had no effect on DRP1 phosphorylation on Ser616, suggesting that there was no change in DRP1 activity. Further studies are therefore required to identify the mechanisms underlying the differential impact of Parkin overexpression on skeletal muscle mitochondrial morphology in healthy and septic animals.

In the current study, we report data indicating that autophagy was induced in septic skeletal muscles, as evidenced by the increased expression of several autophagy-related genes, including *Lc3b*, *Gabarapl1*, and *Sqstm1*, and by the increase in the LC3B-II/LC3B-I ratio in the muscles of CLP mice. These findings are in agreement with previous reports, which documented increased muscle autophagy in various models of sepsis [20,30,53]. An interesting finding in our study is that Parkin overexpression had no effects on the expression of autophagy-related genes and the LC3B-II/LC3B-I ratios in the sham and CLP groups. Given the key role that Parkin plays in the recycling of dysfunctional mitochondria by autophagosomes [23,50], our results indicate that basal and activated autophagy levels in normal and septic muscles, respectively, were sufficient to deal with increased mitophagy in muscles overexpressing Parkin. We also observed that BNIP3 mRNA and protein levels increased significantly in septic skeletal muscles, and that this induction was not influenced by Parkin overexpression. The BNIP3 protein localizes to the mitochondria and promotes PINK1-Parkin-independent mitophagy by interacting with the LC3 protein, resulting in the recruitment of autophagosomes to damaged mitochondria [54]. The lack of changes in BNIP3 levels in response to Parkin overexpression suggests that BNIP3-mediated mitophagy functions in an independent fashion to that of the PINK1-Parkin pathway. It is worth mentioning that the present study suffers from several limitations. First, we did not directly assess whether Parkin overexpression actually translates into increased mitophagic flux. Although it was recently reported that Parkin overexpression is sufficient to trigger higher mitochondrial clearance in cardiomyocytes [55], further studies should investigate whether Parkin overexpression is sufficient to increase mitophagy in healthy and septic skeletal muscles. Another important limitation arises from the fact that muscle contractility was not assessed in the present study. Further studies are therefore required to define whether Parkin overexpression can attenuate sepsis-induced skeletal muscle weakness.

#### **5. Conclusions**

The present study provides evidence that Parkin overexpression attenuates sepsis-induced myofiber atrophy and prevents sepsis-induced changes in mitochondrial morphology and protein contents. These findings suggest that targeting mitophagy may represent a promising therapeutic strategy to attenuate sepsis-induced skeletal muscle wasting.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/6/1454/s1: Figure S1, Construction of the AAV1 designed to overexpress Parkin; Figure S2, The impact of sepsis and Parkin overexpression on the content and phosphorylation levels of proteins regulating protein synthesis; Figure S3, Additional TEM images; Table S1, List of antibodies; Table S2, List of qPCR primers.

**Author Contributions:** Designed and Conceived this Study, J.-P.L.-G., G.G. and S.N.A.H.; Collected, Analyzed and Interpreted the Data, Prepared all figures and tables and Wrote the first draft of the manuscript, J.-P.L.-G.; O.R., T.J.C., D.M. and F.E.B. were involved in data collection and analyses; G.G. and S.N.A.H. supervised the research, contributed to data analysis and interpretation and wrote the final version of the manuscript with J.-P.L.-G. Funding Acquisition: G.G. and S.N.A.H. All authors have read and approved the final version of the manuscript.

**Funding:** This work was funded by grants from the Natural Sciences and Engineering Council of Canada (NSERC, #RGPIN-2014-04668 awarded to Gilles Gouspillou) and from the Canadian Institute of Health Research (CIHR; MOP-93760 awarded to Sabah N. A. Hussain and MOV-409262 awarded to Sabah N. A. Hussain and Gilles Gouspillou). Gilles Gouspillou is also supported by a Chercheur Boursier Junior 1 salary award from the Fonds de Recherche du Québec en Santé (FRQS-35184). Tomer J Chaffer was supported by a Natural Sciences and Engineering Council of Canada Undergraduate Student Research Award (NSERC USRA). Jean-Philippe Leduc-Gaudet was supported by a CIHR Vanier Fellowship and currently holds a RI-MUHC Fellowship. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Acknowledgments:** We thank Jeannie Mui from the Facility for Electron Microscopy Research (FEMR, McGill University, Montreal, QC, Canada) for her support and expertise. We are grateful for the technical support provided by Laurent Huck and the staff of the Meakins-Christie Laboratories at the Research Institute of the McGill University Health Center. We would like to thank Basil Petrof (McGill University and RI-MUHC) for his thoughtful discussions related to this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
