Because the heart is a mitochondrion-rich tissue, we used the α-MHC-Cre transgenic mouse line (1
) to disrupt the Mfn-2
locus selectively in cardiac myocytes of Mfn-2loxP
). Cre-mediated excision of Mfn-2 exon 6 is associated with a predictable loss of Mfn-2 protein (Fig. A), which in the case of α-MHC-Cre × Mfn-2F/F
mice reaches ~90% efficiency (Fig. , right panel). We refer to these mice as F/F;cre and utilize their cre-negative F/F;− littermates or cre-only (+/+;cre) mice as age-matched controls. The protein levels of Mfn-1 did not appear to change significantly in these heart samples. Furthermore, the genetic recombination is found to be specific to the heart and is not detectable in any other tissue of F/F;cre mice, nor does it appear to occur in the absence of the cre
transgene (results not shown).
FIG. 1. Cardiac myocyte-specific deletion of Mfn-2. (A) Hearts from mice with the indicated Mfn-2 genotypes (F/F, homozygous for Mfn-2loxP; +/F, heterozygous for Mfn-2loxP) with or without the α-MHC transgene (+ or −) were analyzed (more ...)
Histological examination of F/F;cre hearts detected cardiac enlargement that was not accompanied by overt ventricular dilatation (Fig. ). Microscopic analysis revealed the presence of myocyte hypertrophy in F/F;cre hearts without significant increases in the collagen content (Fig. ). We analyzed the cardiac function of adult F/F;cre and F/F;− or +/+;cre mice using noninvasive echocardiography or cardiac catheterization (Tables and ). There were no significant differences in chamber dimensions, systolic function, or hemodynamic performance between the two groups, except for the detection of increased left ventricle (LV) mass in the F/F;cre group (Table ). To examine the heart function under conditions of β-adrenergic stress, we acutely infused isoproterenol (5 ng/kg/min) and monitored the hemodynamic response using LV catheterization. As shown in Fig. A and B and Table , this approach revealed small but statistically significant differences between F/F;cre and F/F;− or +/+;cre hearts in terms of systolic function (i.e., reductions in the end-systolic pressure and the maximum rate of LV pressure rise [dP/dtmax, where P is pressure and t is time] in the F/F;cre group [Table ]). Examination of contractility in isolated cardiac myocytes identified a small reduction in fractional shortening in the F/F;cre myocytes, while the intracellular Ca2+ ([Ca2+]i) transients appear to be similar between F/F;cre and F/F;− myocytes (Fig. ). Furthermore, the mRNA levels of various genes associated with stress, metabolism, and mitochondrial biogenesis or function were normal or showed small changes in expression in F/F;cre hearts, with the exception of the atrial natriuretic peptide (ANP) mRNA, which was upregulated by 2.9-fold (Table ). Taken together, these data suggest that the lack of Mfn-2 from the heart is associated with modest myocyte hypertrophy accompanied by mild deterioration of left ventricular function.
FIG. 2. Analysis of cardiac contractility in the presence or absence of Mfn-2. Pressure-volume loop recordings taken before (black line) or after (blue line) acute isoproterenol infusion in hearts with normal levels of Mfn-2 (F/F;−) (A) or in hearts where (more ...)
Hemodynamic analysis before and after isoproterenol infusiona
Transcriptional regulation of genes of interest
Morphological analysis using electron microscopy of the LV wall revealed mostly round or rectangular mitochondria with diameters ranging from 0.5 to 2 μm in F/F;− hearts (Fig. A). In F/F;cre samples, however, some regions had mitochondria with normal morphology (Fig. ) while other areas contained enlarged mitochondria with diameters sometimes up to 3 or 4 μm and, more rarely, up to 5 or 6 μm that tended to form into clusters (Fig. ). In some cases, the enlarged mitochondria in the F/F;cre tissue displayed further abnormalities in their internal structure, such as loss of cristae and formation of inner membrane vesicles (Fig. ). However, as shown in Fig. , the gross mitochondrial cross-sectional area remains unchanged in the F/F;cre hearts, suggesting the maintenance of overall mitochondrial mass. The copy numbers of the mitochondrial gene for NADH dehydrogenase subunit 1 were not found to differ significantly between the two groups (results not shown), further suggesting normal mitochondrial biogenesis in F/F;cre myocytes. The myofibrillar compartment appears largely intact in F/F;cre sections (Fig. , myofibrils). Nevertheless, the cross-sectional area per individual mitochondrion is found to be, on average, significantly increased in the knockout group (Fig. ), in agreement with the presence of enlarged mitochondria. Furthermore, the distribution of the maximum and minimum mitochondrial diameters (Fig. , respectively) was found to be significantly altered in the Mfn-2 knockout group as it shifted to the right, indicative of mitochondria with increased diameters (4 to 5 μm in the major axis and 2 to 3 μm in the minor axis). This analysis also revealed that the number of detectable mitochondria per equal area analyzed was reduced in the knockout group (2,042 versus 1,186). Mfn-2 has been recently implicated in the bridging of the outer mitochondrial membrane with the endoplasmic reticulum (26
). Using electron microscopy on heart samples with or without Mfn-2, we examined the organization of the “Ca2+
release domains” that include the T-tubule, the junctional sarcoplasmic reticulum (jSR) and the outer mitochondrial membrane (13
). As shown in Fig. , the distance between the center of the T-tubule and the outer mitochondrial membrane does not appear to change significantly in the F/F;cre group, indicating that the gross distance between the jSR and the outer mitochondrial membrane is not likely to be affected by the absence of Mfn-2. Collectively, the electron microscopic analysis identified the propensity in Mfn-2-deficient mitochondria to become fewer and enlarged without changing their overall mass, indicating a defect in mitochondrial patterning and distribution rather than in biogenesis.
FIG. 3. Electron microscopy analysis of F/F;− and F/F;cre hearts. (A) The typical organization of mitochondria along the myofibrils is detected in F/F;− hearts. (B) Region of an F/F;cre heart containing mostly normal mitochondria. (C) Different (more ...)
To further examine mitochondrial morphology, we performed confocal microscopy on intact myocytes isolated from adult hearts. Mitochondria visualized with the membrane potential-sensitive dye TMRE have a rectangular shape and display the typical striated appearance in myocytes expressing normal levels of Mfn-2 (Fig. A). In contrast, the mitochondria from isolated F/F;cre myocytes are more heterogeneous in shape, often spherical, enlarged, and less precisely organized within the myocyte (Fig. ). Three-dimensional representation of mitochondria in the two groups further illustrates the presence of enlarged mitochondria within a given area of the myocyte (Fig. ). We examined, in addition to these morphological defects, the levels of the mitochondrial membrane potential (ΔΨm) in the absence of Mfn-2 using three different dyes that sequester in polarized mitochondria (Fig. ). As shown in the left panel of Fig. , the ratio of the JC-1 aggregate fluorescence to the JC-1 monomer fluorescence in F/F;cre myocytes is decreased, indicating a lower ΔΨm in mitochondria lacking Mfn-2. Consistently, fluorescence intensity due to accumulation of MitoTracker red or TMRM into mitochondria was found to be lower in myocytes without Mfn-2, again suggesting a partial decrease in ΔΨm (Fig. , middle and right panels). Taken together, results of the confocal microscopy analysis of isolated myocytes indicate that in the absence of Mfn-2, mitochondria lose their strict structural organization within the myocyte and display an increase in size which coincides with a partial reduction in membrane potential.
FIG. 4. Confocal analysis of adult cardiac myocytes indicates the presence of enlarged/spherical mitochondria. (A) F/F;− myocytes contain mitochondria with a rectangular shape that are highly ordered. (B) F/F;cre myocytes contain mitochondria with a heterogeneous (more ...)
The effect of Mfn-2 on mitochondrial morphology was also examined in cultured neonatal rat cardiac myocytes treated with Mfn-2-specific siRNAs. This approach led to significant reductions in Mfn-2 mRNA and protein levels (results not shown). Mfn-2 downregulation was associated with fragmentation of the elongated and interconnected mitochondria into numerous smaller spherical mitochondria (Fig. , upper panels). However, this was not the case in adult myocytes, where Mfn-2 ablation led to increased mitochondrial size (Fig. , lower panels). These data suggest that the effects of Mfn-2 on mitochondrial morphology can be greatly affected by the cellular context.
To assess the effects of Mfn-2 ablation on mitochondrial function, we examined the activities of mitochondrial enzymes in whole-heart preparations or the respiratory activity of isolated interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) (Fig. ). Deletion of Mfn-2 did not affect the activities of citrate synthase (CS), isocitrate dehydrogenase (IDH), or medium-chain acyl-CoA dehydrogenase (MCAD) in whole tissue (Fig. ) and in IFM and SSM (results not shown), consistent with normal biogenesis of mitochondrial mass in the absence of Mfn-2. The sizes of isolated mitochondria were assessed using a flowmetric approach. As shown in Fig. , the mitochondrial volume does not change significantly upon deletion of Mfn-2 when comparisons are made between IFM. Nevertheless, the absence of Mfn-2 is associated with a significant increase in volume in SSM (Fig. ), in agreement with the notion that loss of Mfn-2 results in the formation of a subset of enlarged mitochondria that coexist with structurally normal mitochondria. Using a number of different substrates, the rates of ADP-driven (state III) oxygen consumption in isolated IFM and SSM were found to be similar, regardless of the presence/absence of Mfn-2 (Fig. ). Furthermore, levels of state IV respiration were found to be similar between the two groups, and the respiratory control ratio was unaffected (results not shown), indicating that Mfn-2 is dispensable for normal coupling of the respiratory chain.
FIG. 5. Functional evaluation of mitochondria in F/F;− and F/F;cre hearts. (A) Enzymatic activities in total myocardial extracts. CS, citrate synthase; IDH, isocitrate dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase (6 and 7 preparations per (more ...)
Despite the relatively normal metabolic function of Mfn-2-deficient mitochondria, there was a pronounced resistance to Ca2+
-induced mitochondrial permeability transition (MPT). As shown in Fig. A, the incremental infusion of Ca2+
in isolated SSM and IFM can lead to a marked increase in extramitochondrial Ca2+
. This is attributed to the formation of the high-conductance MPT pore (MPTP), which mediates an exponential release of the previously accumulated Ca2+
). This assay shows that the loss of Mfn-2 can significantly delay the MPTP, as judged by the comparison of the Ca2+
release curves in control and Mfn-2-deficient mitochondria, an effect that can be observed in both SSM and IFM (Fig. ). The cumulative Ca2+
load required for MPTP opening (mitochondrial Ca2+
tolerance) was also increased in Mfn-2 SSM and IFM (Fig. ). This comparison shows that Mfn-2-deficient mitochondria required approximately twice the Ca2+
load applied to wild-type mitochondria to induce MPTP opening.
FIG. 6. Ca2+ retention capacity is increased in Mfn-2-depleted mitochondria. (A) Subsarcolemmal mitochondria (SSM) (upper panel) and interfibrillar mitochondria (IFM) (lower panel) were isolated from F/F;− and F/F;cre hearts and assessed for their (more ...)
MPTP opening was also assessed by measuring the gradual swelling of isolated mitochondria in the presence of Ca2+. Mitochondria with or without Mfn-2 were exposed to 200 μM Ca2+, and swelling was monitored as a decrease in absorbance over time. As shown in Fig. A and B, the change in absorbance (relative to the baseline absorbance) is more pronounced in wild-type mitochondria than in mutant mitochondria, indicating that the absence of Mfn-2 is associated with an attenuated MPT response. In the presence of cyclosporine (CsA), mitochondria from both groups maintained their optical density throughout the assay (results not shown), suggesting that the decrease in absorbance seen here is attributable to Ca2+-induced MPTP opening. As an alternative way to assess MPT, we also examined the ability of mitochondria, previously swollen by Ca2+, to undergo shrinkage after being exposed to polyethylene glycol (PEG). As shown in Fig. , the addition of PEG in F/F;− mitochondria is associated with a gradual increase in absorbance that signifies mitochondrial shrinkage facilitated by MPTP opening. On the other hand, the addition of PEG to preswollen Mfn-2-depleted mitochondria resulted in a lower rate of mitochondrial shrinkage, indicating a less-than-optimal MPTP opening.
FIG. 7. Calcium-induced mitochondrial swelling is delayed in the absence of Mfn-2. (A) Total cardiac mitochondria were isolated from F/F;− and F/F;cre hearts and exposed to 200 μM Ca2+ to induce swelling or left untreated. The change in (more ...)
To directly assess the effect of Mfn-2 deletion on the expression of proteins previously associated with the function of the MPTP, we analyzed whole-heart extracts by Western blotting. As shown in Fig. , the levels of the MPTP-regulatory component Cyp-D were not different between F/F;cre and F/F;− extracts. This was also the case for the other purported MPTP components, such as the voltage-dependent anion channel (VDAC)-porin, and the two isoforms of the adenine nucleotide translocase (ANT1/2). Identical results were obtained with extracts from isolated mitochondria (results not shown). These data indicate that the loss of Mfn-2 is sufficient to affect MPT in the absence of significant changes in the levels of candidate MPTP components.
To assess the consequences of Mfn-2 ablation on stress-induced MPTP opening in intact cells, myocytes were isolated and examined for permanent mitochondrial depolarization under conditions of ROS generation, which is known to promote loss of membrane potential via MPTP activation (85
). As shown in Fig. A, polarized mitochondria are detected as bright rectangles due to the accumulation of TMRM. Laser illumination of TMRM-loaded mitochondria, leading to a tightly controlled local generation of ROS (4
), allows the assessment of MPTP activation downstream of ROS. Representative time points (1, 5, and 9 min [t1
, and t9
]) are shown in the middle panels of Fig. , where the gradual depolarization of the mitochondrial population is depicted. As shown in Fig. (also see the movie in the supplemental material), the time course of mitochondrial depolarization is significantly delayed in myocytes lacking Mfn-2 (blue tracing), compared to the depolarization in myocytes with normal levels of Mfn-2 (purple tracing). To further determine the critical involvement of MPTP in the outcome of this assay, we analyzed the effects of CsA pretreatment (a potent inhibitor of MPTP) on mitochondrial depolarization. As shown in Fig. , the addition of CsA significantly delays the rate of depolarization in wild-type mitochondria (orange tracing), while it appears to act additively with the Mfn-2 deficiency to induce further delays in loss of membrane potential (green tracing). These actions are also observed when calculating the time to half depolarization (T50
) in the different groups (Fig. ). We further examined the response of adult myocytes to exogenous H2
as an alternative source of ROS. As shown in Fig. 2
exposure is able to induce time-dependent mitochondrial depolarization and, eventually, hypercontracture in isolated myocytes. In agreement with the observations made above, the loss of Mfn-2 is associated with a reduced rate of mitochondrial depolarization (Fig. ), providing additional evidence for the involvement of Mfn-2 in MPT.
FIG. 8. Deletion of Mfn-2 in cardiac myocytes diminishes the rate of mitochondrial depolarization in response to generation of ROS. (A) Representative image of a TMRM-loaded myocyte and time course of photostress-dependent mitochondrial depolarization. (Top) (more ...)
We also examined ROS-induced MPTP in neonatal rat cardiac myocytes that were depleted of Mfn-2 via siRNA knockdown. As shown in Fig. A, mitochondrial depolarization and TMRM fluorescence decay in response to H2O2 exposure are accelerated upon Mfn-2 knockdown, which is in contrast to findings for adult myocytes. The enhanced depolarizing effect in the absence of Mfn-2 in NRCMs was also associated with an increased release of lactate dehydrogenase (LDH), a marker of cell death (Fig. ). To examine the role of Mfn-2 deficiency in an independent system, mice lacking Mfn-2 in macrophages were constructed by breeding the Mfn-2loxP mice with the LyzM-cre transgenic mouse line. In macrophages recruited to the peritoneum by thioglycolate treatment, Mfn-2 deficiency is protective against mitochondrial depolarization (Fig. ), in agreement with the observations made with adult cardiac myocytes. The delay in mitochondrial depolarization in Mfn-2 macrophages was also associated with a reduced release of LDH into the culture medium (Fig. ). Taken together, these data show that the effect of Mfn-2 insufficiency on MPTP activation is cell type dependent.
FIG. 9. The effects of Mfn-2 on mitochondrial depolarization are context dependent. (A) Neonatal cardiac myocytes (NRCMs) were treated with unrelated (control) siRNA or with an Mfn-2-targeting siRNA and analyzed for mitochondrial depolarization in response to (more ...)
Based on the observation that F/F;cre mice have normal cardiac function at baseline but contain mitochondria that exhibit resistance to MPTP opening, we examined their response to reperfusion injury using the isolated heart configuration. As shown in Fig. , F/F;− and F/F;cre hearts had similar systolic and developed pressures at baseline (Fig. ) and global ischemia for 10 min led to the expected reduction in cardiac function in both groups. However, upon reperfusion, Mfn-2-ablated hearts were able to produce higher systolic pressures than control hearts, indicating a protection from the injurious effects of the reflow (Fig. ). Similar observations were also made when developed pressures were measured in the two groups (Fig. ). Therefore, these experiments demonstrate that loss of Mfn-2 can alleviate some of the detrimental effects of postischemic reperfusion, the period that is known to coincide with the induction of MPT (35
). To obtain further molecular details on the Mfn-2-associated cardioprotection during ex vivo
ischemia/reperfusion (I/R), Western blot analysis of purified mitochondrial protein was performed. As shown in Fig. , the antiapoptotic protein Bcl-2 is found to be more abundant on Mfn-2-depleted mitochondria.
FIG. 10. Mfn-2 ablation results in improved cardiac performance following ex vivo ischemia/reperfusion (I/R) injury. F/F;− and F/F;cre hearts (5 and 4 hearts, respectively) from 10-week-old mice were perfused in the Langendorff mode, subjected to 10 min (more ...)
To examine the impact of Mfn-2 loss on cell death after I/R in greater detail, an in vitro
hypoxia/reoxygenation assay was employed. In this experiment, cardiac myocytes purified from wild-type and Mfn-2-deficient hearts were exposed to normoxia only or were exposed to hypoxic conditions (1% O2
, 5% CO2
) and then returned to normoxic conditions (reoxygenation). As shown in Fig. A and B, the percentage of myocytes undergoing necrotic cell death (indicated by the number of trypan blue-stained cells) decreases in the absence of Mfn-2. In fact, Mfn-2-deficient cardiac myocytes were able to better tolerate the stress induced by the isolation process, as there were fewer cells from this genotype staining positive for trypan blue under normoxic conditions. Consistently, the Mfn-2-deficient myocytes retained their resistance to necrosis under the hypoxia/reoxygenation conditions (Fig. ). It has been previously reported that Mfn-2 can also promote cardiomyocyte apoptosis by activating the intrinsic/mitochondrial pathway (75
). We therefore examined the activation of apoptosis in F/F;− and F/F;cre myocytes from the above-described assay by Western blotting. As shown in Fig. C, the abundance of cleaved caspase-9 and cleaved PARP-1 is decreased in F/F;cre samples compared to F/F;− samples, both at normoxia and upon hypoxia/reoxygenation, suggesting inhibition of the apoptotic pathway in the absence of Mfn-2. The tolerance of Mfn-2-deficient myocytes to cell death was further demonstrated using H2
treatment. As shown in Fig. , the proportion of dead cells in myocyte preparations left untreated or exposed to 20 μM H2
is consistently lower in the absence of Mfn-2. Taken together, these observations show that loss of Mfn-2 is associated with improved cell survival in the face of death-inducing stimulation.
FIG. 11. Mfn-2-deficient cardiac myocytes are protected from cell death induced by hypoxia/reoxygenation and H2O2 exposure. (A) Adult cardiac myocytes were isolated from F/F;− and F/F;cre hearts and were exposed to either normoxic conditions or subjected (more ...)
To further assess the role of Mfn-2 in cardiac myocyte apoptosis, we examined the expression of proapoptotic and antiapoptotic proteins that are known to be associated with mitochondrial morphogenesis (45
). As shown in Fig. A, the proapoptotic protein Bax was found to be increased in nonischemic heart extracts lacking Mfn-2. The antiapoptotic Bcl-2 appeared to be upregulated in the same experimental group, in agreement with our previous observations (Fig. ). We also examined the expression of the mitochondrial fission factor Drp-1, which has also been implicated in ischemia/reperfusion injury and cell death (63
). As shown in Fig. , the levels of Drp-1 protein were decreased in hearts lacking Mfn-2. Finally, the levels of the protein Opa-1, which is implicated in inner mitochondrial membrane fusion and also in the regulation of cytochrome c
release, did not appear to change significantly upon genetic deletion of Mfn-2.
FIG. 12. Mfn-2 deficiency in the heart results in an altered expression of outer mitochondrial membrane-associated factors and confers protection from apoptosis. (A) Western blot analysis of cardiac extracts from hearts with (F/F;−) or without (F/F;cre) (more ...)
Finally, we subjected Mfn-2 F/F;− and F/F;cre mice to in vivo ischemia/reperfusion injury by surgically closing and reopening the LAD coronary artery for 30 min and 2 h, respectively. As shown in representative images (Fig. ) and the accompanying histogram (Fig. ), the area at risk (AAR) to the left ventricle (LV) area is the same between the two groups, indicating similar magnitudes of ischemic stress. However, the ratio of the infarct area (IA) (shown as the white band in Fig. ) to the AAR (IA/AAR) was found to be lower in F/F;cre hearts, indicating a diminished cell death response. In support of the above conclusions, the percentage of TUNEL-positive nuclei was found to be significantly lower in F/F;cre hearts than in F/F;− hearts (Fig. ), indicating that the apoptotic response under these conditions is mitigated in the absence of Mfn-2.