Deletion of PTPMT1 from skeletal muscles results in defective contractility and progressive muscle atrophy.

(A) Representative 8-month-old of PTPMT1fl/fl/CKMM-Cre+ and PTPMT1+/+/CKMM-Cre+mice, and hind limbs and hearts dissected from these mice were photographed. 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+/+/CKMM-Cre+and PTPMT1fl/fl/CKMM-Cre+ mice were processed for H&E staining and Masson’s Trichrome staining. One representative image from 3 mice/genotype is shown. (C) Six-month-old PTPMT1+/+/CKMM-Cre+and PTPMT1fl/fl/CKMM-Cre+ mice (n=7/genotype) were subjected to wire hang tests. Relative forelimb muscle strength was determined. (D and E) Seven to eight-month-old PTPMT1+/+/CKMM-Cre+(n=5 males and 5 females) and PTPMT1fl/fl/CKMM-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. (F and G) Soleus (F) and EDL (G) dissected from 7 to 8-month-old PTPMT1+/+/CKMM-Cre+ and PTPMT1fl/fl/CKMM- 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.

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+/+/CKMM-Cre+ and PTPMT1fl/fl/CKMM-Cre+ mice were processed for Gömöri trichrome staining. One representative image from 3 mice/genotype is shown. (B) Soleus and EDL dissected from 8-month-old PTPMT1fl/fl/CKMM-Cre+and PTPMT1+/+/CKMM- 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+/+/CKMM-Cre+ and PTPMT1fl/fl/CKMM-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/CKMM-Cre+and PTPMT1+/+/CKMM-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/CKMM-Cre+and PTPMT1+/+/CKMM-Cre+ mice (n=6/genotype) were determined. (F) Whole cell lysates prepared from the Soleus and EDL isolated from 7-month- old PTPMT1fl/fl/CKMM-Cre+ and their control mice were examined by immunoblotting with the indicated antibodies. Representative results from 3 mice/genotype are shown.

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/CKMM-Cre+ and PTPMT1+/+/CKMM-Cre+ mice (n=4/genotype) at 4 months of age were measured for 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/CKMM-Cre+ and PTPMT1+/+/CKMM-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. (F-H) Levels of pyruvate (F), 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/CKMM-Cre+ and PTPMT1+/+/CKMM-Cre+ mice (n=4/genotype) were washed three times in 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+/+/CKMM-Cre+ and PTPMT1fl/fl/CKMM-Cre+mice (n=4/genotype) were determined by qRT-PCR.

PTPMT1fl/fl/CKMM-Cre+mice manifest late-onset cardiac dysfunction.

(A) Heart tissue sections prepared from 12-month-old PTPMT1+/+/CKMM-Cre+and PTPMT1fl/fl/CKMM- Cre+ mice were processed for H&E staining. One representative image from 3 mice/genotype is shown. (B-F) Cardiac morphology and function of 12-month-old PTPMT1+/+/CKMM-Cre+ and PTPMT1fl/fl/CKMM-Cre+ mice (n=5/genotype) were examined by echocardiography. Representative long-axis views in M-mode echocardiographic tracing of LV are shown (B). LV 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).

Deletion of PTPMT1 from the heart ultimately leads to dilated cardiomyopathy and heart failure.

(A) Kaplan-Meier survival curves of PTPMT1fl/fl/MYH6-Cre+(n=18) and PTPMT1+/+/MYH6-Cre+ mice (n=20). (B) Heart tissue sections prepared from 10 to 12-month- old PTPMT1fl/fl/MYH6-Cre+and PTPMT1+/+/MYH6-Cre+ mice (n=4/genotype) were processed for H&E staining and Masson’s Trichrome staining. Representative images are shown. (C) Heart tissue sections prepared from 11-month-old PTPMT1fl/fl/MYH6-Cre+and PTPMT1+/+/MYH6-Cre+ mice were processed for immunofluorescence staining for cleaved caspase 3 followed by DAPI counterstaining. One representative image from 3 mice/genotype is shown. (D-J) Eleven to twelve-month-old PTPMT1fl/fl/MYH6-Cre+ and PTPMT1+/+/MYH6-Cre+ mice (n=5/genotype) were examined by echocardiographic evaluation of ventricular function. Representative long- (upper panel) and short- (lower panel) axis views in M-mode echocardiographic tracing of LV (D), and pulsed-wave Doppler recordings of mitral valve inflow (E) are shown. Left ventricular internal diameters at diastole (LVID-d) and systole (LVID-s) were measured (F). LV ejection fraction (EF) and fractional shortening (FS) (G), ratios of peak velocity of early to late filling of mitral inflow (E/A) (H), the thickness of LV anterior wall at the end of diastole (LVAW-d) and the end of systole (LVAW-s) (I), and thickness of LV posterior wall at the end of diastole (LVPW-d) and at the end of systole (LVPW-s) (J) were determined.

PTPMT1 deficiency causes mitochondrial substrate shift and oxidative stress, leading to mitochondrial damage and lipid accumulation in PTPMT1 knockout cardiomyocytes.

(A) Total ATP levels in the heart tissues dissected from PTPMT1fl/fl/MYH6- Cre+and PTPMT1+/+/MYH6-Cre+ mice at the indicated ages (n=5 mice/genotype). (B) Whole cell lysates prepared from the heart tissues of 10 to 12-month-old PTPMT1fl/fl/MYH6-Cre+and PTPMT1+/+/MYH6-Cre+ mice were examined by immunoblotting with the indicated antibodies. Representative results from 3 mice/genotype are shown. (C-E) Oxygen consumption of the mitochondria isolated from the heart tissues of 2 to 3-month-old PTPMT1fl/fl/MYH6-Cre+and PTPMT1+/+/MYH6-Cre+ mice (n=3/genotype) was measured in the presence of pyruvate (5 mM)/malate (5 mM) (C), palmitoyl-CoA (40 μM)/carnitine (40 μM)/malate (5 mM) (D), or glutamate (5 mM)/malate (5 mM) (E), following the addition of ADP (4 mM), oligomycin (1.5 μM), FCCP (4 μM), and antimycin A/rotenone (1 μM). OCRs at basal levels, in response to ADP addition, and maximal reserve capabilities were determined. (F) Levels of pyruvate in the lysates of the cardiac mitochondria isolated from 2 to 3-month-old PTPMT1fl/fl/MYH6-Cre+ and PTPMT1+/+/MYH6-Cre+ mice (n=6/genotype) were determined. (G and H) Mitochondria isolated above were washed three times in MAS buffer and then incubated with pyruvate (5 mM)/malate (5 mM) and ADP (4 mM) at 37°C. Five min later, the mitochondria were collected, washed, and lysed. α-KG levels in the mitochondrial lysates were measured (n=4 mice/genotype) (G). Pyruvate dehydrogenase (PDH) activities in the mitochondrial lysates were determined (n=5 mice/genotype) (H). (I) Heart tissue sections prepared from 3-month-old PTPMT1fl/fl/MYH6-Cre+ and PTPMT1+/+/MYH6-Cre+mice (4 mice/genotype) were processed for ROS staining. One representative picture from 4 mice/genotype is shown. (J) Heart tissues dissected from 6 to 8- month-old PTPMT1fl/fl/MYH6-Cre+ and PTPMT1+/+/MYH6-Cre+ mice were processed for TEM examination. One representative image from 4 mice/genotype is shown. (K) Heart tissue sections prepared from 10 to 12-month-old PTPMT1fl/fl/MYH6-Cre+and PTPMT1+/+/MYH6-Cre+ mice were processed for Oil Red O staining to visualize lipids. One representative picture from 3 mice/genotype is shown.