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• W4386475275 abstract "Full text Figures and data Side by side Abstract eLife assessment eLife digest Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Abstract While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection – the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources. eLife assessment This paper provides a useful set of data examining the role of PTPMT1, a mitochondria-based phosphatase, in mitochondrial fuel selection. The data were collected and analyzed using solid methodology and can be used as a starting point for further studies that build on the findings here. https://doi.org/10.7554/eLife.86944.3.sa0 About eLife assessments eLife digest Cells are powered by mitochondria, a group of organelles that produce chemical energy in the form of molecules called ATP. This energy is derived from the breakdown of carbohydrates, fats, and proteins. The number of mitochondria in a cell and the energy source they use to produce ATP varies depending on the type of cell. Mitochondria can also switch the molecules they use to produce energy when the cell is responding to stress or disease. The heart and the skeletal muscles – which allow movement – are two tissues that require large amounts of energy, but it remained unknown whether disrupting mitochondrial fuel selection affects how these tissues work. To answer these questions, Zheng, Li, Li et al. investigated the role of an enzyme found in mitochondria called Ptpmt1. Genetically deleting Ptpmt1 in the heart and skeletal muscle of mice showed that while the development of these organs was not affected, mitochondria in these cells switched from using carbohydrates to using fats as an energy source. Over time, this shift damaged both the mitochondria and the tissues, leading to muscle wasting, heart failure, and sudden death in the mice. This suggests that balanced use of carbohydrates and fats is essential for the muscles and heart. These findings imply that long-term use of medications that alter the fuel that mitochondria use may be detrimental to patients’ health and could cause heart dysfunction. This may be important for future drug development, as well as informing decisions about medication taken in the clinic. Introduction Mitochondria, the powerhouse of the cell, produce energy in the form of ATP from the breakdown of carbohydrates, fats, and proteins. Mitochondrial abundance and their preferences for metabolic substrates differ in various cell types (Smith et al., 2018). While mitochondria are abundant in red skeletal muscles such as Soleus, and the heart, the mitochondrial content in white muscles, such as Extensor Digitorum Longus (EDL), is much lower. Mitochondria in the skeletal muscle and heart have distinct preferences for energy sources – skeletal muscle mitochondria prefer glucose (the major simple carbohydrate) as the substrate, whereas cardiac mitochondria mainly use fatty acids as the fuel. Moreover, different types of skeletal muscle cells (fibers) utilize glucose to produce energy in different mechanisms – in slow-twitch oxidative muscle fibers glucose (via pyruvate, a critical metabolite derived from glucose) is predominantly oxidized within the mitochondria to generate energy, whereas in fast-twitch muscle fibers pyruvate is primarily reduced to lactate in the cytosol promoting glycolytic flux to rapidly produce energy and support quick contractions. Nevertheless, both muscle and heart mitochondria are flexible in selecting substrates in response to stress (exercise, fasting, etc.) and under disease conditions (heart failure, diabetes, etc.) (Bertero and Maack, 2018; Brown et al., 2017; Nabben et al., 2018; Schulze et al., 2016). It remains to be determined how mitochondrial utilization of various competing metabolic substrates is coordinated and whether disruption of mitochondrial flexibility in fuel selection affects the function of the heart and skeletal muscle. Ptpmt1, encoded by nuclear DNA, is localized to the mitochondrion and anchored at the inner membrane (Pagliarini et al., 2005). It dephosphorylates phosphatidylinositol phosphates (PIPs). PIPs are a class of membrane phospholipids that bind to a distinctive set of effector proteins, thereby regulating a characteristic suite of cellular processes, including membrane trafficking and ion channel/transporter functions (Balla, 2006; Gamper and Shapiro, 2007). Ptpmt1 is also involved in the synthesis of cardiolipin by converting phosphotidylglecerol phosphate to phosphotidylglecerol, the precursor of cardiolipin (Zhang et al., 2011). Global knockout of Ptpmt1 results in developmental arrest and post-implantation lethality (Zhang et al., 2011; Shen et al., 2011). Our previous studies suggest that Ptpmt1 facilitates mitochondrial metabolism largely by dephosphorylation of downstream PIP substrates (Yu et al., 2013; Shen et al., 2009) that appear to inhibit mitochondrial oxidative phosphorylation but enhance cytosolic glycolysis by activation of mitochondrial uncoupling protein 2 (Ucp2) (Yu et al., 2013), a transporter of four-carbon (C4) dicarboxylate intermediates of the tricarboxylic acid (TCA) cycle that has been shown to regulate cellular energetics by limiting mitochondrial oxidation of glucose (Vozza et al., 2014; Bouillaud, 2009; Diano and Horvath, 2012; Pecqueur et al., 2008; Samudio et al., 2009). Ptpmt1 is highly expressed in the heart and skeletal muscle (Shen et al., 2011). In the present study, we exploit Ptpmt1 tissue-specific knockout models to address the role and mechanisms of Ptpmt1-facilitated mitochondrial metabolism in these tissues. We find that Ptpmt1 plays an important role in facilitating mitochondrial utilization of carbohydrates and that balanced fuel selection is essential for maintaining both muscle and heart functions despite the two tissues having distinct preferences for energy sources. Results Knockout of Ptpmt1 from skeletal muscles results in defective contractile function and progressive muscle atrophy To determine the role of Ptpmt1-mediated metabolism in the skeletal muscle and heart, we generated tissue-specific Ptpmt1 knockout mice (Ptpmt1fl/fl/Ckm-Cre+) by crossing Ptpmt1 floxed mice (Yu et al., 2013) and muscle creatine kinase promoter-driven Cre transgenic mice (Ckm-Cre), which express the Cre recombinase in the skeletal muscle and heart starting at embryonic day 13 (Brüning et al., 1998). Quantitative reverse transcription PCR showed ~95% and ~80% deletion of Ptpmt1 in the skeletal muscle and heart in Ptpmt1fl/fl/Ckm-Cre+ mice, respectively (Figure 1A). These animals were indistinguishable from their wild-type (WT, Ptpmt1+/+/Ckm-Cre+) littermates up to 3 months of age, suggesting that Ptpmt1 deletion did not affect muscle or heart development. The knockout mice exhibited reduced weight gain starting at 4–5 months and their body sizes were significantly smaller than those of control mice at 8 months (Figure 1A). Muscle wasting was observed in Ptpmt1fl/fl/Ckm-Cre+ mice at 8 months or older (Figure 1A). Histopathological examination of skeletal muscles of these knockout mice revealed characteristic changes of muscle atrophy – marked variations in muscle fiber diameters and the presence of ghost fibers or more interstitial space (Figure 1B). In addition, Masson’s Trichrome staining revealed increased collagen-rich fibrotic regions in Ptpmt1 knockout muscles (Figure 1B). We then examined Ptpmt1 knockout mice at 6–8 months (prior to the onset of muscle wasting). Ptpmt1fl/fl/Ckm-Cre+ mice became fatigued faster and fell more quickly than Ptpmt1+/+/Ckm-Cre+ control animals in wire hang tests, indicating muscle weakness (Figure 1C). In treadmill exercise tests, both male and female knockout mice displayed reduced maximum speed (Figure 1D), endurance (Figure 1E), and distance of the run (Figure 1—figure supplement 1A). To determine whether the reduced muscle strengths of the knockout mice resulted from muscle cell-intrinsic defects, isometric contractile properties of isolated muscles were examined. Ex vivo force–frequency measurements were performed on Soleus, a more oxidative muscle with a higher percentage of slow-twitch Type I fibers, and EDL, a more glycolytic muscle with a higher percentage of fast-twitch Type II fibers. Both knockout Soleus and EDL showed decreased optimal length, the length of the muscle at which maximal force was achieved (Figure 1—figure supplement 1B, C). Specific force production of Ptpmt1 knockout Soleus (Figure 1—figure supplement 1D) and EDL (Figure 1—figure supplement 1E) was decreased. However, normalized force production was decreased only in knockout Soleus (Figure 1F), but not in knockout EDL (Figure 1G). These data suggest that Ptpmt1 plays a more important role in oxidative than glycolytic muscle fibers although both knockout Soleus and EDL developed muscle atrophy subsequently. Figure 1 with 2 supplements see all Download asset Open asset Deletion of Ptpmt1 from skeletal muscles results in defective contractility and progressive muscle atrophy. (A) Representative 8-month-old of Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice, and hind limbs and hearts dissected from these mice were photographed. Extensor Digitorum Longus (EDL) and Soleus (n = 8 mice/genotype), and heart (n = 4 mice/genotype) weights were measured. Ptpmt1 mRNA levels in EDL, Soleus, and heart tissues were determined by quantitative reverse transcription PCR (qRT-PCR) (n = 4 mice/genotype). (B) Skeletal muscle sections prepared from 8-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice were processed for Hematoxylin and Eosin (H&E) staining and Masson’s Trichrome staining. One representative image from 3 mice/genotype is shown. (C) Six-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice (n = 7/genotype) were subjected to wire hang tests. Relative forelimb muscle strength was determined. (D, E) Seven- to eight-month-old Ptpmt1+/+/Ckm-Cre+ (n = 5 males and 5 females) and Ptpmt1fl/fl/Ckm-Cre+ (n = 3 males and 5 females) mice were assessed by treadmill exercise tests as described in Materials and methods. Maximum speed (D) and duration of the run (E) were recorded. Soleus (F) and EDL (G) dissected from 7- to 8-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice (n = 3 mice, 6 muscles/genotype) were subjected to ex vivo isometric force measurements. Specific contractile forces produced at the indicated frequencies of stimulation were normalized to the physiological cross-sectional area. Shown on the left are representative absolute forces produced at 1, 10, and 100 Hz. *p <0.05. To determine the impact of Ptpmt1 depletion from the skeletal muscle and heart on the metabolism of the whole body, we measured plasma glucose and fatty acid levels and found that they were comparable in Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (Figure 1—figure supplement 2A). Plasma levels of lactate (Figure 1—figure supplement 2B) and lipid metabolic products (Figure 1—figure supplement 2C) in Ptpmt1 knockout mice were also relatively normal (except that cholesterol and nonesterified fatty acids levels were decreased and marginally increased, respectively, in female knockout mice). Adipokine array assays of the plasma showed no or subtle differences in Adiponectin, Leptin, Lipocalin-2, Angptl3, Resistin, Fgf-21, Dppiv, Fetuin A, Igf-1, etc. between Ptpmt1 knockout and control mice (Figure 1—figure supplement 2D). In addition, no differences in glucose tolerance tests were observed between Ptpmt1 knockout and control mice (Figure 1—figure supplement 2E). Furthermore, we assessed the expression levels of key enzymes involved in glucose and lipid metabolism in Ptpmt1 knockout muscles. No changes in Hk2, Pkm1, Ldha, Dicer1, Mcad (Acadm), Acadl, Hadha, Cpt2, or Fabp3 were found. However, the expression of Cpt1B was increased in Ptpmt1 knockout muscles (Figure 1—figure supplement 2F, G). These results suggest that systemic glucose and lipid metabolism in these tissue-specific knockout mice was not significantly changed, but fatty acid partitioning for oxidation in the mitochondria was elevated in Ptpmt1 knockout muscles. Ptpmt1 depletion ultimately leads to mitochondrial damage and bioenergetic stress in knockout skeletal muscles Gömöri trichrome staining revealed ragged red fibers in Ptpmt1 knockout skeletal muscles (Figure 2A), as observed in various types of human mitochondrial myopathies. Electron microscopic analyses showed that interfibrillar mitochondria in knockout Soleus and EDL were disorganized and enlarged, in contrast to normal mitochondria that typically reside in pairs and position on either side of the Z-disc in control muscles. In addition, there was a massive accumulation of subsarcolemmal mitochondria in the knockout muscles, whereas the content of intermyofibrillar mitochondria decreased (Figure 2B). Although EDL, unlike Soleus, uses more cytosolic glycolysis than mitochondrial oxidative phosphorylation for energy production, the mitochondrial structural changes appeared to be more severe in EDL than Soleus in the knockout mice (Figure 2B). Furthermore, a massive accumulation of intramyofibrillar lipids were detected by Oil Red O staining in 6 months or older Ptpmt1 knockout muscles (Figure 2C). Figure 2 with 1 supplement see all Download asset Open asset Ptpmt1 loss ultimately leads to abnormal mitochondrial distribution, structural damage, and bioenergetic stress in skeletal muscles. (A) Skeletal muscle sections prepared from 6-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice were processed for Gömöri trichrome staining. One representative image from 3 mice/genotype is shown. (B) Soleus and Extensor Digitorum Longus (EDL) dissected from 8-month-old Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice were processed for transmission electron microscopic examination. One representative image from 3 mice/genotype is shown. (C) Skeletal muscle sections prepared from 6-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice were processed for Oil Red O staining to visualize lipids. One representative picture from 3 mice/genotype is shown. (D) Total DNA was extracted from Soleus and EDL dissected from 8-month-old Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (n = 3/genotype). Mitochondrial content was estimated by comparing the mitochondrial gene cytochrome B DNA levels to the nuclear gene 18S DNA levels by qPCR. (E) Total ATP levels in Soleus and EDL dissected from 8-month-old Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (n = 6/genotype) were determined. (F) Whole cell lysates prepared from the Soleus and EDL isolated from 7-month-old Ptpmt1fl/fl/Ckm-Cre+ and their control mice were examined by immunoblotting with the indicated antibodies. Representative results from 3 mice/genotype are shown. Figure 2—source data 1 Uncropped immunoblotting images of Figure 2F. https://cdn.elifesciences.org/articles/86944/elife-86944-fig2-data1-v1.pdf Download elife-86944-fig2-data1-v1.pdf Total mitochondrial content in Ptpmt1 knockout Soleus and EDL also slightly decreased compared to that in control muscles (Figure 2D). ATP levels in both knockout Soleus and EDL decreased significantly compared to WT counterparts (Figure 2E). Consistent with these data, the energetic stress sensor, AMP-activated kinase (Ampk), was highly activated in Ptpmt1fl/fl/Ckm-Cre+ muscles as determined by the phosphorylation levels of Thr172 (Figure 2F). Acetyl-CoA carboxylase (Acc), a negative regulator of fatty acid oxidation, was inhibited, as evidenced by a marked increase in the inhibitory phosphorylation of this enzyme (Figure 2F), implying increased fatty acid oxidation in Ptpmt1-depleted mitochondria. In agreement with previous findings that Ampk negatively regulates mTor signaling (Hardie, 2015; Liang and Mills, 2013), mTor was substantially inhibited. Activities of S6k, S6, and 4E-bp1, key downstream components of mTor signaling, were also concomitantly decreased (Figure 2F), indicating diminished anabolic activities and consistent with the muscle atrophy phenotype. Interestingly, Akt activities, as reflected by its phosphorylation levels (Ser473 and Thr308), were increased in Ptpmt1 knockout skeletal muscles, recapitulating mTor and raptor (a component of the mTor complex 1) knockout muscles (Bentzinger et al., 2008; Risson et al., 2009). Likely due to the cross-talk between Akt and Ras signaling pathways, Erk activities were also slightly increased in these knockout muscles (Figure 2F). Given that muscle atrophy developed in Ptpmt1fl/fl/Ckm-Cre+ mice after 6–8 months, to assess the direct impact of Ptpmt1 deletion on mitochondrial metabolism, we examined mitochondria in young Ptpmt1 knockout mice before the tissue damage. Expression levels of electron transport chain complexes in these Ptpmt1-ablated mitochondria were not changed (Figure 2—figure supplement 1A). Total cellular ATP levels in Ptpmt1 knockout muscles at this age were comparable to those in control tissues (Figure 2—figure supplement 1B), and the knockout muscles did not show bioenergetic/metabolic stress (Figure 2—figure supplement 1C). We also checked Lc3-I/II levels in Ptpmt1 knockout muscles and found no evidence of elevated autophagic activities (Figure 2—figure supplement 1D). These results indicate that the decrease in total cellular ATP levels in older knockout muscles (Figure 2E) was a secondary not direct effect of Ptpmt1 depletion. Ptpmt1 loss impairs mitochondrial utilization of pyruvate, whereas the fatty acid utilization is enhanced We next sought to address the mechanisms by which Ptpmt1 loss impacts mitochondrial metabolism. To avoid secondary effects, we examined mitochondrial metabolic activities of Ptpmt1 knockout muscles in 3-month-old Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice. Knockout muscle tissues showed decreased maximal reserve oxidative capacities in the presence of full metabolic substrates (Figure 3A), consistent with our previous observations from other Ptpmt1 knockout cell types (Yu et al., 2013; Shen et al., 2009; Zheng et al., 2018). In addition, we isolated mitochondria from young skeletal muscles and assessed mitochondrial metabolism in the presence of a single metabolic substrate by real-time measurement of ATP synthesis-driven oxygen consumption. When pyruvate, a critical metabolite derived from glucose, was provided as the sole substrate, ATP synthesis-driven oxygen consumption in Ptpmt1-deficient mitochondria was markedly decreased (Figure 3B), suggesting that mitochondrial utilization of pyruvate was diminished in the absence of Ptpmt1. Interestingly, we observed enhanced oxygen consumption in these mitochondria when fatty acids were supplied as the fuel (Figure 3C). A slightly increased respiratory response in Ptpmt1-depleted mitochondria was also detected when glutamate, another mitochondrial substrate, was provided (Figure 3D). Importantly, feeding with succinate, the substrate for Complex II of the electron transport chain, resulted in similar oxygen consumption in Ptpmt1 null and control mitochondria (Figure 3E), validating the intact function of the electron transport chain in the knockout mitochondria and excluding flavin adenine dinucleotide and quinone-linked deficiencies. These mechanistic data suggest that Ptpmt1 plays an important role in facilitating carbohydrate oxidation in mitochondria. Figure 3 Download asset Open asset Ptpmt1 ablation impairs mitochondrial utilization of pyruvate, whereas the fatty acid utilization is enhanced. (A) Muscle cross-sections prepared with biopsy punches from Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (n = 4/genotype) at 4 months of age were measured for oxygen consumption rates (OCRs) at the basal level and following the addition of oligomycin (8 μM), FCCP (4 μM), and antimycin A/rotenone (1 μM). (B–E) Mitochondria were isolated from the skeletal muscles dissected from Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (n = 3/genotype) at 3 months of age. Mitochondrial oxygen consumption (10 μg of mitochondrial protein) was measured in the presence of pyruvate (5 mM)/malate (5 mM) (B), palmitoyl-CoA (40 μM)/carnitine (40 μM)/malate (5 mM) (C), glutamate (5 mM)/malate (5 mM) (D), or succinate (10 mM) (E), following the addition of ADP (4 mM), oligomycin (1.5 μM), FCCP (4 μM), and antimycin A/rotenone (1 μM). Experiments were repeated three times with three independent pairs of mice. Similar results were obtained in each experiment. Levels of pyruvate (F), α-ketoglutarate (α-KG) (G), and acetyl-CoA (H) in the lysates of the mitochondria isolated from the above skeletal muscles were measured (n = 6/genotype). (I) Mitochondria freshly isolated from the skeletal muscles of Ptpmt1fl/fl/Ckm-Cre+ and Ptpmt1+/+/Ckm-Cre+ mice (n = 4/genotype) were washed three times in Mitochondrial Assay Solution (MAS) buffer. The mitochondria were then incubated with pyruvate (5 mM)/malate (5 mM) and ADP (4 mM) at 37°C. Five min later, mitochondria were collected, washed, and lysed. α-KG levels in the mitochondrial lysates were measured. (J) Pyruvate dehydrogenase (Pdh) activities in the mitochondrial lysates were determined (n = 5–6 mice/genotype). (K) Myh4, Myh2, and Myh7 mRNA levels in the skeletal muscles dissected from 3-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice (n = 4/genotype) were determined by quantitative reverse transcription PCR (qRT-PCR). Consistent with the above notion, intramitochondrial pyruvate levels in the mitochondria freshly isolated from Ptpmt1 knockout muscles decreased by half (Figure 3F). Levels of α-ketoglutarate (α-KG), an important metabolite in the TCA cycle, also decreased in the knockout mitochondria (Figure 3G), although steady-state mitochondrial acetyl-CoA levels were not changed (Figure 3H). To further determine if pyruvate transport efficiency may be reduced in Ptpmt1-ablated mitochondria, we incubated fresh mitochondria with pyruvate and measured acute production of α-KG from extramitochondrial pyruvate. As shown in Figure 3I, α-KG production in Ptpmt1-deficient mitochondria was indeed decreased compared to that in control mitochondria. Notably, the activity of pyruvate dehydrogenase (Pdh), the enzyme that converts pyruvate to acetyl-CoA to feed the TCA cycle, was not affected in the knockout mitochondria (Figure 3J). These observations together with that the mitochondrial pyruvate carrier/transporter (Mpc) was comparably expressed in Ptpmt1-ablated mitochondria (Figure 2F) suggest that Ptpmt1 null mitochondria were impaired in uptaking pyruvate. Possibly due to an adaptive response to the inhibition of pyruvate oxidation in the mitochondria, skeletal muscle fiber-type switching occurred in Ptpmt1fl/fl/Ckm-Cre+ mice. Muscle fibers in these knockout mice switched from oxidative to glycolytic fibers, as evidenced by a dramatic increase (~170-fold) in the levels of Myh4, a marker of glycolytic fast-twitch Type 2B fibers in Ptpmt1 knockout mice (Figure 3K). Ptpmt1fl/fl/Ckm-Cre+ mice manifest late-onset cardiac dysfunction The heart in Ptpmt1fl/fl/Ckm-Cre+ mice was less impacted than the skeletal muscle although Ptpmt1 was ~80% deleted from the heart (Figure 1A). At 7 months, when skeletal muscles in these mice displayed atrophy and myocyte damage, no evident histological changes were observed in heart tissues (Figure 4—figure supplement 1A). Echocardiographic examination showed no difference in left ventricle (LV) contractility in Ptpmt1 knockout mice as reflected by similar LV fractional shortening (FS), ejection fraction (EF), and the ratio of peak velocity of early to late filling of mitral inflow (E/A) between knockout and control mice (Figure 4—figure supplement 1B). Mitochondrial content in Ptpmt1fl/fl/Ckm-Cre+ heart tissues was comparable to that in Ptpmt1+/+/Ckm-Cre+ control tissues (Figure 4—figure supplement 1C). Total ATP levels in Ptpmt1 knockout heart tissues were marginally but not significantly decreased (Figure 4—figure supplement 1D). Moreover, mitochondria in knockout cardiomyocytes showed minimal structural changes (Figure 4—figure supplement 1E). Consistent with these observations, no bioenergetic/metabolic stress was detected in the knockout cardiomyocytes according to the activation status of Ampk and mTor signaling (Figure 4—figure supplement 1F). However, severe cardiac dysfunction was observed in 10- to 12-month-old Ptpmt1fl/fl/Ckm-Cre+ mice. Histopathological examination revealed that cardiomyocytes in these animals had uneven sizes and were disorganized (Figure 4A). In addition, fibrotic lesions, indicative of myocardial damage, were observed. Echocardiographic examination showed that LV contractility, as measured by FS and EF in M-mode echocardiographic tracing of LV (Figure 4B-D) and recorded by M-mode echocardiography movies (Figure 4—video 1 and Figure 4—video 2), was markedly decreased in Ptpmt1 knockout mice. The E/A ratio determined in pulsed-wave Doppler recording of mitral valve inflow was doubled in the Ptpmt1fl/fl/Ckm-Cre+ heart (Figure 4E, F). Collectively, these data suggest that Ptpmt1 depletion also impaired cardiac function albeit later than skeletal muscle dysfunction. Figure 4 with 3 supplements see all Download asset Open asset Ptpmt1fl/fl/Ckm-Cre+ mice manifest late-onset cardiac dysfunction. (A) Heart tissue sections prepared from 12-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice were processed for Hematoxylin and Eosin (H&E) staining. One representative image from 3 mice/genotype is shown. (B–F) Cardiac morphology and function of 12-month-old Ptpmt1+/+/Ckm-Cre+ and Ptpmt1fl/fl/Ckm-Cre+ mice (n = 5/genotype) were examined by echocardiography. Representative long-axis views in M-mode echocardiographic tracing of left ventricle (LV) are shown (B). LV fractional shortening (FS) (C) and LV ejection fraction (EF) (D) were determined. Representative pulsed-wave Doppler recordings of mitral valve inflow are shown (E). Ratios of peak velocity of early to late filling of mitral inflow (E/A) were determined (F). Heart-specific deletion of Ptpmt1 leads to dilated cardiomyopathy and heart failure Given that Ptpmt1 was only ~80% deleted from the heart in Ptpmt1fl/fl/Ckm-Cre+ mice and that impaired skeletal muscle function in these animals may potentially complicate the heart phenotypes, to further investigate the role of Ptpmt1-mediated metabolism specifically in cardiac muscles, heart-specific Ptpmt1 knockout mice (Ptpmt1fl/fl/Myh6-Cre+) were generated by crossing Ptpmt1 conditional mice (Yu et al., 2013) and Myh6-Cre transgenic mice, which express the Cre recombinase specifically in cardiomyocytes (Agah et al., 1997). These knockout mice appeared healthy in the first several months although Ptpmt1 was nearly completely deleted from the heart (but not in Soleus and EDL) (Figure 5—figure supplement 1A). No obvious tissue morphological changes or fibrotic lesions were found in Ptpmt1 knockout hearts (Figure 5—figure supplement 1B), confirming that Ptpmt1-mediated metabolism is dispensable for the development of the heart. Echocardiography also showed normal heart function in 3-month-old Ptpmt1fl/fl/Myh6-Cre+ mice, as evidenced by normal FS, EF, and E/A ratios (Figure 5—figure supplement 1C–E). Knockout mice did not exhibit any defects during treadmill exercises at 6 months of age (Figure 5—figure supplement 1F). However, all Ptpmt1fl/flMyh6-Cre+ knockout mice died at 10–16 months, and they often succumbed suddenly (Figure 5A) although their body weights were relatively normal. Ptpmt1 knockout hearts showed enlarged ventricular chambers. Severe structural damage in cardiac myocytes and tremendous fibrotic lesions were observed in the knockout hearts (Figure 5B). Immunostaining of cleaved caspase 3 illustrated increased apoptosis in Ptpmt1fl/fl/Myh6-Cre+ cardiomyocytes as compared to control cells (Figure 5C). Echocardiography revealed a profound dilated cardiomyopathy and heart failure in Ptpmt1fl/fl/Myh6-Cre+ mice, including both systolic and diastolic LV dilation, thinning of ventricular walls, depression of EF and FS, arrhythmias, and impaired myocardial contraction (Figure 5D–J, Figure 5—video 1, and Figure 5—video 2). Cardiac strain images demonstrated that the global function of Ptpmt1fl/fl/Myh6-Cre+ hearts, as reflected by global longitudinal strain, was significantly decreased (Figure 5—figure supplement 2A" @default.
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• W4386475275 title "Reviewer #2 (Public Review):: Loss of Ptpmt1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure in tissue-specific knockout mice" @default.
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