Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function

Figures

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  Fig. 1. Increased collagen deposition in the myocardium of human METH-user heart autopsy tissue. a Picro Sirius Red (PSR) staining was conducted in non-METH and METH user human LV heart sections to assess the extent of collagen deposition. Formalin-fixed, paraffin-embedded LV sections (5 µm) from non-METH (n = 7), and METH users (n = 34) were stained with PSR. The METH user group consists of heart samples from patients who died from acute METH intoxication/overdose and patients positive for METH following toxicology studies carried out in the case of accidental causes of death. Representative images shown in the left panel images demonstrate collagen deposition (in red) in the perivascular region. Those in the right panel images demonstrate the extent of collagen deposition (in red) in the interstitial spaces in the same non-METH and METH users heart tissue. Scale bar: 100 µm. b Dot plot represents individual values of quantified percent collagen content in PSR-stained LV sections from the non-METH (n = 7) and METH (n = 34) user groups. The collagen content (red-stained area) relative to the total area of the myocardium was measured in 10–15 high-magnification microscopic fields (×10)…

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  Fig. 2. Enhanced fibrotic remodeling in the myocardium of human METH-user heart autopsy tissues. a Masson’s Trichrome staining was conducted in non-METH and METH user LV sections to assess the interstitial and perivascular fibrosis in the myocardium. Formalin-fixed, paraffin-embedded LV autopsy sections (5 µm) from non-METH (n = 7), and METH users (n = 34) were used for Masson’s Trichrome staining. The METH user group consists of heart samples from patients who died from acute METH intoxication/overdose and patients who were positive for METH following toxicology studies carried out in the case of accidental, combined drug overdose, or acute myocardial infarction-related causes of death. Representative left panel images to demonstrate the extent of fibrosis (in blue) in the LV perivascular region, and right panel images show the fibrosis (in blue) in the interstitial space in the same non-METH and METH user hearts. Scale bar: 100 µm. b Dot plot represents the individual values of the quantified percent fibrosis area in Masson’s Trichrome stained LV sections in non-METH (n = 7) and METH (n = 34) user groups. The size of the fibrosis area (blue-stained area) relative to the total…

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  Fig. 3. METH “binge and crash” administration to mice causes cardiac functional decline. a–l M-mode echocardiography was used to evaluate cardiac functional parameters in mice treated with vehicle (saline) (n = 5) and METH (n = 6) 4 weeks after the subcutaneous administration of vehicle or METH. METH treatment causes a significant decline in M-mode echocardiography parameters, including c percent fractional shortening (%FS) and f percent ejection fraction (%EF) compared with those in the vehicle-treated mice. a–l Bar graph represents a left ventricular (LV) systolic internal dimension (LVID;s); b LV diastolic internal dimension (LVID;d); c percent fractional shortening (%FS); d LV systolic volume (LV Vol;s); e LV diastolic volume (LV Vol;d); f percent ejection fraction (%EF); g heart rate (beats/minute); h LV systolic interventricular septum thickness (IVS;s); i LV diastolic interventricular septum thickness (IVS;d); j LV systolic posterior wall thickness (LVPW;s); k LV diastolic posterior wall thickness (LVPW;d); I LV mass in vehicle- (n = 5) and METH- (n = 6) treated mice. Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. A two-tailed unpaired…

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  Fig. 4. METH “binge and crash” administration induces cardiac hypertrophy in mice. a Left panel, stereomicroscope images showing the gross morphometry of whole heart cross-sections from vehicle- and METH-treated mice. Right panel: the box diagram represents increased gravimetric heart weight (mg)-to-tibia length (cm) ratio in the METH (n = 7) group compared with the vehicle (n = 7) group. b Left panel: representative fluorescent images of wheat germ agglutinin stained (WGA, green) LV myocardium. Nuclei were stained with DAPI (blue). Right panel: quantification of cardiomyocyte (CM) cross-sectional areas (µm2) showed increased CM areas in METH-treated mice (n = 1620 cardiomyocytes in six individual mice LV heart sections) compared with vehicle-treated mice (n = 1547 cardiomyocytes in five different mice LV heart sections) following 4 weeks of treatment. Scale bar: 50 µm. c mRNA expression of natriuretic peptide A (Nppa), natriuretic peptide B (Nppb), α-myosin heavy chain (Myh6), and β-myosin heavy chain (Myh7) expressed as the fold change in METH-treated hearts compared with vehicle-treated hearts. d Body weight (g) changes in vehicle (n = 8) and METH (n = 10) treated mice throughout the…

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  Fig. 5. METH treatment induces cardiac fibrotic remodeling in mice. Histopathological staining and immunoblot analysis showed increased cardiac fibrosis and enhanced expression of protein markers of fibrosis in the hearts of METH-treated mice compared with vehicle-treated mice. a Left panel: representative micrographs of Masson’s Trichrome stained LV sections in vehicle- and METH-treated mice. Right panel: quantification of the percent myocardial fibrosis area in the vehicle-treated (n = 5), and METH-treated (n = 6) mice. Scale bar: 50 µm. b Left panel: representative micrographs of Picro Sirius Red-stained LV heart sections in vehicle- and METH-treated mice. Right panel: quantification of the percent collagen area in the vehicle-treated (n = 5) and METH-treated (n = 6) mice. Scale bar: 50 µm. c Left panel: representative immunoblot images of periostin and α-smooth muscle actin; right panel: densitometric quantification of protein bands in the hearts of vehicle- and METH-treated mice. GAPDH was used as a loading control. Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. P values were determined using a two-tailed unpaired Student’s t test. P <…

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  Fig. 6. METH alters the expression of mitochondrial dynamics regulatory protein Fis1 in the heart. a, b Representative immunoblot images and densitometric quantification of the expression of mitochondrial fission, i.e., Drp1, Fis1, Mff, and fusion, i.e., OPA1, OMA1, Mfn2, regulatory proteins in the whole-cell a and isolated mitochondrial b fractions from vehicle- and METH-treated mouse hearts. GAPDH was used on the same membrane of whole-cell and mitochondrial fractions to assess the purity of isolation of mitochondria. Mitochondrial outer membrane integral protein, Tom20, and mitochondrial inner membrane integral protein, Tim23, were used to confirm mitochondrial extracts. Ponceau S staining of the transfer membranes was used to verify approximately equal loading and transfer across the gel. Bar graphs represent mitochondrial dynamics regulatory protein expression changes in the hearts of METH-treated mice compared with those of vehicle-treated mice (n = 3–10 mice per group). Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. P values were determined using a two-tailed unpaired Student’s t test. P < 0.05 between groups was considered statistically…

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  Fig. 7. METH treatment alters mitochondrial morphology in mouse hearts and isolated primary cardiomyocytes. Representative a toluidine blue-stained and b transmission electron micrographs of vehicle- and METH-treated mouse LV heart sections show the preponderance of large, abnormally shaped mitochondria in the hearts of METH-treated mice. Scale bar: 1 µm. Bar graphs represent c the number of mitochondria per microscopic field, d average mitochondrial size (µm2), and e mitochondrial size (µm2) distribution frequency. Mitochondrial areas (µm2) were measured in 20 microscopic fields containing 2436 mitochondria from vehicle-treated mouse hearts (n = 2) and in 24 microscopic fields containing 1879 mitochondria from METH-treated mouse hearts (n = 2). f Representative fluorescent micrographs of Mitotracker Red staining used to visualize mitochondrial network morphology in cultured cardiomyocytes (NRC) following treatment with vehicle or METH (100 µM) for 24 hours. METH treatment resulted in visually distinct hyperfused mitochondria in cardiomyocytes compared with the mitochondria observed in the vehicle-treated group. Bar graphs represent g average mitochondrial network length (µm), h…

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  Fig. 8. METH treatment induces impaired mitochondrial bioenergetics in the heart. a Summary traces of mitochondrial oxygen consumption rate (OCR) profiles as measured in isolated mitochondria from the hearts of vehicle- and METH-treated mice. The black arrow indicates the sequential addition of oligomycin (1 μmol/L), FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) (4 μmol/L), and rotenone (0.5 μmol/L) plus antimycin A (0.5 μmol/L). OCR profiles are expressed as pmol O2/[min*μg protein], and each point represents average OCR values from five individual mouse cardiac mitochondria per group. b–e Bar graphs represent OCR under b baseline and with the addition of c oligomycin, d FCCP, and e rotenone plus antimycin A. f–h Key parameters of mitochondrial bioenergetics, including f reserve capacity, g ATP turnover, and h maximal respiration were significantly decreased in METH-treated mice compared to vehicle-treated mice. Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. P values were determined using a two-tailed unpaired Student’s t test. P < 0.05 between groups was considered statistically significant. Veh vehicle, METH methamphetamine.

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  Fig. 9. METH treatment attenuates CREB expression and phosphorylation in human and mouse hearts. Immunohistochemistry and immunoblot based analyses showed that METH induces a marked reduction of phosphorylation of CREB at the Serine133 residue in both human and mouse hearts. a Left panel: representative micrographs of immunohistochemical detection of phosphorylated CREB at Ser133 (brown color, positive DAB staining) in human non-METH user (n = 4) and METH toxicity (n = 4) LV sections. Hematoxylin (blue) was used to counterstain nuclei. Right panel: the bar graph represents the fold change of DAB-positive pCREBSer133 staining intensity in the nucleus of METH toxicity human hearts compared with non-METH user human hearts. Scale bar: 50 µm. b Left panel: representative micrographs of immunohistochemical detection of phosphorylated CREB at Ser133 (brown, DAB staining) in the vehicle-treated (n = 4) and METH-treated (n = 4) mouse LV heart sections. Hematoxylin (blue) was used to counterstain the nuclei. Right panel: the bar graph represents the fold change of DAB-positive pCREBSer133 staining intensity in the nucleus of the hearts of METH-treated mice compared to vehicle-treated mice. Scale…

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  Fig. 10. METH treatment causes Sigmar1 expression-dependent regulation of CREB-Fis1 expression in cardiomyocytes. a Left panel: representative micrographs of immunohistochemical detection of Sigmar1 (brown) in human non-METH user (n = 4), and METH toxicity (n = 4) LV heart sections. Hematoxylin (blue) was used to counterstain the nuclei. Right panel: fold change of DAB-positive Sigmar1 staining intensity in METH toxicity hearts compared to those of non-METH users. b Left panel: representative micrographs of immunohistochemical detection of Sigmar1 (brown) in the vehicle (n = 4), and METH-treated (n = 4) mouse LV sections. Right panel: fold change of DAB-positive Sigmar1 staining intensity in hearts treated with METH hearts compared with those treated with vehicle. c Left panel: global Sigmar1 knockout (Sigmar1−/−) mouse hearts were used to assess the specificity of the Sigmar1 primary antibody. Right panel: IgG stained slides without primary antibody incubation were used as a negative control. Representative micrographs are from three independent experiments. a–c Scale bar: 50 µm. d Sigmar1 was overexpressed and knocked down in NRC through adenovirus infection (10 MOI) and siRNA (50 nM)…