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By Chowdhury S. Abdullah, Richa Aishwarya, Shafiul Alam, Mahboob Morshed, Naznin Sultana Remex, Sadia Nitu, Gopi K. Kolluru, James Traylor, Sumitra Miriyala, Manikandan Panchatcharam, Brandon Hartman, Judy King, Mohammad Alfrad Nobel Bhuiyan, Sunitha Chandran, Matthew D. Woolard, Xiuping Yu, Nicholas E. Goeders, Paari Dominic, Connie L. Arnold, Karen Stokes, Christopher G. Kevil, A. Wayne Orr, and Md. Shenuarin Bhuiyan

Excerpt from the article published in Communications Biology 3, 682 (2020). DOI: https://doi.org/10.1038/s42003-020-01408-z

Editor’s Highlights

• Methamphetamine (METH) abuse associated with cardiovascular toxicity includes hypertension, tachycardia, cardiac arrhythmia, acute coronary vasospasm, myocardial infarction, and cardiomyopathy.
• Sigma-1 receptor (Sigmar1) has been found to be a direct target of METH as METH binds to Sigmar1 at physiological concentrations (K_i value of ~2 µM).
• Activation of Sigmar1 using the Sigmar1 agonist PRE-084 has been shown to limit METH-induced dopamine efflux in dopaminergic neurons, attenuate METH-induced locomotion, and motivate behavior and the drug-seeking brain reward function.
• Studies suggest that METH may exhibit antagonist activity and/or act as an inverse agonist for Sigmar1.
• Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB).
• Levels of Sigmar1 expression are significantly decreased in human METH users and in the heart tissue of “binge and crash” METH-treated mice.
• Sigmar1-dependent regulation of the CREB-Fis1 signaling axis, and mitochondrial dysfunction contributing to the development of METH-associated cardiomyopathy

Abstract

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of “binge and crash” methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of “binge and crash” methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to “binge and crash” methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.

Introduction

Methamphetamine (METH) is a highly addictive amphetamine class psychostimulant drug whose illicit use is dramatically increasing in the United States^1,2. It has been reported that ~1.6 million Americans used METH in 2017, with 964,000 cases (up from 684,000 cases in 2016) of emergency room visits owing to METH abuse associated disorders^3,4,5. METH is toxic to both neuronal and cardiovascular systems; cardiovascular disease is the second leading cause of death among METH abusers following accidental overdose^6,7. METH abuse associated with cardiovascular toxicity includes hypertension, tachycardia, cardiac arrhythmia, acute coronary vasospasm, myocardial infarction, and cardiomyopathy^1,2,6,7,8. Clinical case studies of the hearts of human METH users have revealed severely reduced left ventricular (LV) ejection fraction, LV chamber dilatation, myocardial lesions, cardiac hypertrophy, fibrosis, and inflammation in relatively younger patients (≤50 years) with no other significant cardiac risk factors^6,9,10,11. Unfortunately, reports of the histopathological characteristics present in the hearts of METH users are scarce and most of the studies used only a very small number of samples^9,12,13.

METH increases dopamine levels in dopaminergic neuronal synapses by competitive inhibition of dopamine uptake from synapses, and it stimulates dopamine efflux from neuronal vesicular stores¹⁴. The presence of excess dopamine in the extracellular spaces rewires the brain reward circuit promoting repetition of the pleasurable activity after taking the drug^14,15. Despite a long pharmacological half-life (t_1/2 = 12 hours), an immediate METH-induced intense “rush” or “flash,” described as an extremely pleasurable experience, lasts only a few minutes following the injection and dissipates before the drug concentration in the blood has fallen significantly. Hence, to maintain the “high,” METH users indulge in a risky behavior known as “binge and crash,” where users continue to take METH for several days (typically for 4 days in a week, known as “run”) while foregoing food and sleep, followed by a period of abstinence^16,17. METH’s “binge and crash” use pattern is a common behavioral trait in its human users, along with chronic use patterns. Importantly, binge users tend to take ~1.5 times more METH per month than chronic users, running the risk of overdose^16,17. Acute and chronic METH treatment in rodents showed the evidence of METH-induced cardiotoxicity^18,19,20. Findings from these studies suggest that METH causes mitochondrial dysfunction, increases oxidative stress, alters intracellular Ca²⁺ dynamics, enhances inflammatory marker, and adversely affects cardiac contractility^20,21,22. However, the molecular targets and mechanisms of METH-induced cardiomyopathy remain elusive.

Sigma-1 receptor (Sigmar1) is a widely expressed intra-organelle signaling modulator that interacts physically with client proteins to regulate cellular calcium homeostasis²³, endoplasmic reticulum (ER) stress, cell survival²⁴, and to regulate gene transcription²⁵. Extensive Sigmar1 ligand-based studies indicate that activation (expression) of Sigmar1 confers neuroprotection in neurodegenerative diseases^26,27. Interestingly, Sigmar1 has been found to be a direct target of METH as METH binds to Sigmar1 at physiological concentrations (K_i value of ~2 µM)²⁸. Recently, activation of Sigmar1 using the Sigmar1 agonist PRE-084 has been shown to limit METH-induced dopamine efflux in dopaminergic neurons, attenuate METH-induced locomotion, and motivate behavior and the drug-seeking brain reward function²⁹. We have previously reported that Sigmar1 is highly expressed in the heart^30,31, and that the genetic loss of Sigmar1 causes altered cardiac mitochondrial morphology and dysfunction, leading to cardiac contractile dysfunction³². Despite all these studies suggesting Sigmar1’s potential role in cardiac physiology and also serve as a direct target of METH, to our knowledge, no studies to date explored the direct involvement of Sigmar1 in the development of METH-associated cardiac pathology.

METH-induced cardiomyopathy is a poorly characterized disease entity as METH-induced molecular perturbations and histopathological changes in the human heart remain poorly characterized. We investigated the pathological remodeling of the heart associated with illicit METH use with tissue obtained from the autopsy of the hearts of human METH users and non-METH users based on toxicological reports. We also used a preclinical “binge and crash” METH administration mouse model to recapitulate the pathological features observed in the hearts of human METH users and determine the molecular mechanisms of METH-induced cardiomyopathy. We performed histological, gravimetric, and echocardiographic studies of cardiac tissue, mitochondrial bioenergetics assays, assessments of mitochondrial dynamics regulatory protein signaling, performed high-resolution mitochondrial morphometry measurements in the hearts of METH-treated mice, and immunohistochemistry analyses in human autopsy hearts to define the molecular targets of METH cardiomyopathy.

Results

The hearts of human METH users demonstrate adverse fibrotic remodeling regardless of the cause of death

Accumulating evidence indicates that METH use is associated with the development of cardiomyopathy^{9,10,11,12,13,33}. However, a majority of the literature is limited to cursory case reports of the clinical data of one or several patients without providing any insight into the common histopathological alterations, if any, present in the hearts of human METH users. In this study, we examined the tissue from 34 humans LV heart sections from autopsy (patient demographics and physiological data in Supplementary Table 1) who were determined to be METH positive as the result of toxicological studies. Among the heart samples from METH users, six patients died owing to METH overdose-related toxicity (METH toxicity group), whereas the cause of death of the remaining patients was from violence, accidents, combined drug overdose, or was acute myocardial infarction related (Supplementary Table 1). We also collected autopsy tissue from seven human hearts (non-METH user group) from patients whose deaths were accidental (Supplementary Table 1) and who were negative for METH and other drugs following toxicological studies. The LV sections from non-METH and METH users were stained with Picro Sirius Red (PSR) to detect collagen deposition in the myocardium (Fig. 1). The heart tissue from both METH toxicity and METH users exhibited increased collagen deposition (stained red in PSR staining) in the perivascular and interstitial spaces of the myocardium compared with heart tissue from non-METH users (Fig. 1a). The collagen content (percent collagen area) was significantly increased in the hearts of METH users compared with those of non-METH users (Fig. 1b) after adjustment for age (Fig. 1d), sex, race, heart weight (Fig. 1c), body weight, height, and BMI (Fig. 1e). We carried out Masson’s Trichrome staining and found a similarly consistent pattern of fibrotic remodeling in the perivascular and interstitial spaces in the myocardium of METH users compared with that of non-METH users (Fig. 2a). Fibrotic areas (percent fibrosis area) were significantly increased in the LV sections of METH users compared with those of non-METH users after correction for variables including age, sex, race, heart weight, body weight, height, and BMI (Fig. 2b). Overall, we found that the hearts of human METH users exhibit fibrotic remodeling, irrespective of the cause of death, underscoring the underappreciated cardiotoxic pathologies associated with METH abuse in humans.

“Binge and crash” METH exposure in mice induces cardiac hypertrophy and adverse fibrotic remodeling with cardiac functional decline

To study the cellular and molecular mechanisms of METH-induced direct cardiotoxicity and corroborate our findings in the hearts of human METH users, we used a “binge and crash” METH administration protocol in mice. This preclinical “binge and crash” METH administration protocol mimics the conventional METH “binge and crash” use pattern observed in humans³⁴. Following 4 weeks of “binge and crash” METH administration, we assessed the cardiac dimensions and function using M-mode echocardiography (Fig. 3)^{32,35,36,37,38}. Cardiac systolic parameters, i.e., percent fractional shortening (%FS) (Fig. 3c) and percent ejection fraction (Fig. 3f) were significantly reduced with increased LV internal dimension at systole (LVID;s) (Fig. 3a) and increased LV volume at systole (LV Vol;s) (Fig. 3d) in METH-treated mice compared with vehicle-treated mice. There were no significant changes in LV internal dimension at diastole (LVID;d) (Fig. 3b), LV volume at diastole (LV Vol;d) (Fig. 3e), interventricular septum thickness at systole and diastole (IVS;s and IVS;d) (Fig. 3h, i), or LV posterior wall thickness at systole and diastole (LVPW;s and LVPW;d) (Fig. 3j, k). Heart rates were similar between vehicle- and METH-treated mice during echocardiography recordings (Fig. 3g). In summary, “binge and crash” METH administration caused a systolic cardiac functional decline in mice.

Next, we assessed the gross and microscopic heart morphometry to evaluate METH treatment-induced changes in the macro- and microscopic structures of the mouse heart. The hearts from METH-treated mice were visually larger (Fig. 4a, left panel), evidence for which was also demonstrated by gravimetric analyses showing increased heart weight (mg)-to-tibia length (cm) ratio in the hearts of METH-treated mice (Fig. 4a, right panel) compared with those of vehicle-treated mice. To confirm whether treatment with METH-induced hypertrophy in cardiomyocytes, we stained the hearts of vehicle- and METH-treated mice with wheat germ agglutinin (WGA). We found increased cross-sectional areas (µm²) in the cardiomyocytes from METH-treated mice compared with those of the vehicle-treated group in WGA-stained heart sections (Fig. 4b). mRNA expression analysis revealed activation of molecular profiles of cardiac hypertrophy with increased fetal cardiac gene expression, i.e., Nppa, Nppb, Myh7, and decreased Myh6 expression (Fig. 4c). However, “binge and crash” METH administration did not significantly affect the body weights of the mice (Fig. 4d). Heart tissues were stained with Masson’s Trichrome (Fig. 5a, left panel) and PSR (Fig. 5b, left panel) to evaluate fibrosis and collagen deposition in the myocardium following 4 weeks of “binge and crash” METH administration. Our image analysis revealed significant upregulation of the percent fibrosis area (Fig. 5a, right panel) and percent collagen deposition (Fig. 5b, right panel) in the METH-treated group compared to the vehicle-treated group. Immunoblot analyses of protein markers of fibrosis, periostin, and α smooth muscle actin confirmed the activation of fibrotic signaling in the hearts of METH-treated mice (Fig. 5c).

To evaluate the effects of METH on the immune cell population, we carried out immune phenotyping in the blood and splenocytes of vehicle- and METH-treated mice using flow cytometry. We used a panel of antibodies to assess the B cell, T cell, NK cell, polymorphonuclear cell, and monocyte/macrophage populations in the blood and splenocytes. We did not find any significant change in the percentage of the immune cell population in the blood and splenocytes (Supplementary Fig. 1a, b). Analysis of monocyte/macrophage populations demonstrated a decrease in the percentage of Ly6C⁺classical pro-inflammatory macrophages and a slightly increased percentage of Cx3cr1⁺Ly6c⁻ macrophages in the spleen. In contrast, both populations were unchanged in the blood (Supplementary Fig. 1c, d). Therefore, “binge and crash” METH administration did not alter selective innate and adaptive immune cell populations in the spleen or systemic circulation.

METH causes attenuation of mitochondrial fission regulatory protein Fis1 expression in the heart

Mitochondrial dynamics are orchestrated through a combination of mitochondrial fusion and fission events. It has been well documented that an imbalance between fusion and fission events affects heart mitochondrial function, resulting in heart failure^39,40,41. We determined whether METH affects cardiac mitochondrial dynamics regulatory protein expression. Our immunoblot analyses in whole-cell lysates and isolated mitochondrial fractions revealed that METH causes significant downregulation of mitochondrial fission regulatory protein Fis1 (mitochondrial fission 1 protein, Fis1) level in whole-cell (Fig. 6a) fractions reflecting in reduced Fis1 localization in the mitochondrial fraction (Fig. 6b) in the heart. However, we did not find any differences in the protein levels in mitochondrial fission regulatory proteins, i.e., Drp1 (dynamin-related protein 1) and Mff (mitochondrial fission factor), or in mitochondrial fusion regulatory proteins, i.e., OPA1 (Optic Atrophy 1), Mfn2 (mitofusin 2), and OPA1 processing metalloprotease OMA1 (overlapping with the M-AAA protease 1 homolog) in whole-cell lysate and mitochondrial fractions (Fig. 6a, b). We probed GAPDH on the same membrane of whole-cell and mitochondrial fractions to assess the purity of isolated mitochondrial fractions (Fig. 6a, b). We also used mitochondrial outer membrane integral protein, Tom20, and mitochondrial inner membrane integral protein, Tim23, to confirm the mitochondrial extracts in vehicle- and METH-treated mouse hearts (Fig. 6b). We found that METH selectively attenuated mitochondrial fission regulatory protein Fis1 level in the heart.

METH exposure causes a hyperfused mitochondrial network that coincides with suppression of mitochondrial respiration in cardiomyocytes

To evaluate whether METH-induced downregulation of Fis1 in the heart has any functional consequences in the mitochondrial morphology in myocytes, we conducted transmission electron microscopy studies in the hearts of vehicle- and METH-treated mice (Fig. 7a–e). Remarkably, “binge and crash“ METH treatment for 4 weeks caused the appearance of abnormally shaped, large, hyperfused mitochondria (Fig. 7b) as evidenced by the decreased number of mitochondria per microscopic field (Fig. 7c) and increased average mitochondrial areas (Fig. 7d) with an increased percentage of larger (≥0.5 µm²) mitochondria (Fig. 7e) compared with observations in the hearts of vehicle-treated mice. Systemic administration of METH to mice may exert METH-induced excess catecholamine-mediated indirect effects on the mitochondrial structure and respiration in the hearts of METH-treated mice in vivo (Figs. 6, 7a–e). To obviate the METH-induced neurohumoral impact on the heart and to study the direct METH exposure-associated alteration in mitochondrial morphology and respiration of cardiomyocytes, we exposed isolated primary neonatal rat cardiomyocytes (NRC) to METH for 24 hours. Notably, exposure of NRC to METH (100 µM) for 24 hours resulted in a hyperfused, extended tubular mitochondrial network compared to the short tubular mitochondrial system observed in vehicle-treated cardiomyocytes following visualization with MitoTracker Red staining (Fig. 7f). Measurement of the mitochondrial network length showed a significant increase in mitochondrial length (Fig. 7g) and mitochondrial aspect ratio (ration of mitochondrial length-to-diameter) (Fig. 7h), with an increased long mitochondrial percentage in the 4 µm to >20 µm range as shown in the mitochondrial length distribution frequency graph (Fig. 7i) in METH-treated cardiomyocytes compared with those treated with vehicle. We found that treatment with METH directly alters mitochondrial network morphology in cardiomyocytes, confirming our observations of METH-induced impaired mitochondrial fission signaling (Fig. 6) and morphometry (Fig. 7).

METH causes suppression of mitochondrial respiration in mouse heart

As METH administration causes altered mitochondrial dynamics and morphometry, we investigated the effects of METH treatment on cardiac mitochondrial respiration after “binge and crash” METH treatment. Our real-time oxygen consumption rate (OCR) measurement studies revealed reduced basal OCR, the sum of all physiological mitochondrial oxygen consumption, in an isolated mitochondria preparation from the hearts of METH-treated mice compared to those of the vehicle-treated group (Fig. 8a, b). Inhibition of mitochondrial ATP synthase (Complex V) by oligomycin causes a drop in basal OCR that allows ATP-linked OCR to be calculated (Fig. 8a). Although METH treatment attenuated basal mitochondrial respiration, ATP-linked respiration was unchanged (Fig. 8c) compared to that in vehicle-treated heart mitochondria. Next, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was injected to uncouple respiration from oxidative phosphorylation to measure maximal OCR (Fig. 8d). METH-treated heart mitochondria exhibited a significantly lower maximal OCR when compared with vehicle-treated heart mitochondria (Fig. 8d). To quantify the non-mitochondrial OCR, rotenone, and antimycin A was added to inhibit mitochondrial Complex I and Complex III, respectively (Fig. 8a). The non-mitochondrial related OCR was similar between vehicle- and METH-treated heart mitochondria (Fig. 8e). The mitochondrial reserve respiratory capacity, calculated by subtracting the basal OCR from the FCCP-stimulated OCR, was markedly lower in METH-treated heart mitochondria compared with those of the vehicle-treated group (Fig. 8f). Also, ATP turnover (Fig. 8g), calculated by subtraction of ATP-linked OCR from basal OCR, and maximal respiration (Fig. 8h), calculated by subtraction of non-mitochondrial OCR from FCCP-stimulated OCR, were significantly lower in METH-treated heart mitochondria compared with those in the vehicle-treated group. To monitor whether METH-mediated mitochondrial dysfunction is progressive, we also observed the mitochondrial respiration after 2 weeks of “binge and crash” METH treatment (Supplementary Fig. 2a–h). We also observed significantly altered mitochondrial respiratory parameters in the METH-treated hearts indicating defects in mitochondrial respiration occur in an early stage of METH treatment (Supplementary Fig. 2a–h). In summary, we found that “binge and crash“ METH administration suppressed mitochondrial oxidative electron transport chain-dependent respiration in cardiac mitochondria.

Next, we sought to assess METH-induced mitochondrial oxidative respiration in isolated neonatal cardiomyocytes (NRC) to test the direct effect of METH on mitochondrial respiration. As the mitochondrial isolation procedures used in the pathologically remodeled heart may compromise mitochondrial integrity during the isolation process, we measured mitochondrial respiration in intact NRC seeded in Seahorse analyzer plates treated with vehicle and METH (100 µM) for 24 hours. Notably, METH treatment of cardiomyocytes suppressed basal OCR (Supplementary Fig. 3a, b) and FCCP-stimulated OCR (Supplementary Fig. 3a, d) with no change in ATP-linked OCR following oligomycin injection (Supplementary Fig. 3a, c) or non-mitochondrial OCR following rotenone and antimycin injection (Supplementary Fig. 3a, e) compared with results observed in vehicle-treated cardiomyocytes. METH exposure also significantly lowered the mitochondrial bioenergetics parameters, including reserve capacity (Supplementary Fig. 3f), ATP turnover (Supplementary Fig. 3g), and maximal respiration (Supplementary Fig. 3h) compared with those in the vehicle-treated group. To rule out the possible involvement of METH-induced catecholamine-mediated indirect effects on the mitochondrial respiration, we also treated the NRC with norepinephrine (NE) (5, 25, or 50 µM) for 24 hours (Supplementary Fig. 4a–h). Treatment of cardiomyocytes with NE did not significantly change the key OCR parameters (Supplementary Fig. 4a–h), suggesting that the mitochondrial dysfunction caused by METH treatment represents a direct effect of METH on cardiomyocytes. In summary, we found that METH caused a defective mitochondrial network morphology that coincided with compromised mitochondrial respiration in cardiomyocytes.

METH downregulated the Fis1 level via CREB expression and activation in the heart

Recently, the cAMP response element-binding protein (CREB) has been identified as a transcriptional regulator of the Fis1 protein levels and mitochondrial morphology in cardiomyocytes⁴². As METH reduced Fis1 protein expression in mouse hearts (Fig. 7a, b), we monitored the protein level and activation of CREB in human and mouse hearts. Our immunohistochemical (IHC) analysis revealed that METH substantially reduced phosphorylated CREB at serine133 (Ser133) in the hearts of human METH users (Fig. 9a) and those of mice exposed to “binge and crash” METH (Fig. 9b), suggesting that chronic METH attenuates CREB activation in the heart. Similarly, western blot analysis of mouse heart lysates showed significant downregulation of total CREB and phosphorylated CREB at Ser133 in the hearts of “binge and crash” METH-treated mice (Fig. 9c). In addition, immunocytochemical analysis of NRCs treated with METH (100 µM) also showed decreased nuclear localization of pCREBSer133 (Fig. 9d). We conclude that METH directly attenuates CREB protein level and activation through an undefined mechanism in cardiomyocytes, which might underlie METH-induced downregulation of the mitochondrial fission regulatory protein Fis1 level in cardiomyocytes.

METH-induced inhibition of Sigmar1 underlies CREB/pCREBSer133/Fis1 axis attenuation in cardiomyocytes

Sigmar1 has been found to be a direct target of METH as it binds to Sigmar1 at physiological concentrations (K_i value of ~2 µM)²⁸. We recently reported that genetic loss of Sigmar1 suppressed mitochondrial respiration and led to large, hyperfused mitochondria in mouse hearts³². Since METH suppressed mitochondrial respiration and caused hyperfused mitochondrial morphology in cardiomyocytes, similar to our previous observations in Sigmar1 knockout hearts, we examined whether METH regulates Sigmar1 level in the heart. IHC staining of Sigmar1 revealed its diffuse localization in cardiomyocytes in the hearts of both humans (Fig. 10a, left panel) and mice (Fig. 10b, left panel). Quantitative analysis of the images showed decreased Sigmar1 level in the myocardium of human METH toxicity hearts compared to those of non-METH human hearts (Fig. 10a, right panel). Similarly, we also found decreased Sigmar1 level in the hearts of “binge and crash” METH-treated mice (Fig. 10b, right panel) compared with those of vehicle-treated mice. These studies suggest that METH treatment decreases Sigmar1 level in cardiomyocytes, thereby affecting the reduction of CREB-Fis1 signaling in cardiomyocytes.

To determine whether Sigmar1 directly regulates the CREB/pCREBSer133/Fis1 axis in cardiomyocytes, we overexpressed Sigmar1 (10 MOI) by adenoviral infection and siRNA knockdown (50 nM) in NRCs. Our western blot analyses showed that Sigmar1 overexpression upregulated CREB and pCREBSer133 levels while simultaneously increasing Fis1 in NRC (Fig. 10d). In contrast, Sigmar1 knockdown attenuated CREB and pCREBSer133 level, which coincided with reduced Fis1 level in NRC (Fig. 10d). We conclude that Sigmar1 regulated Fis1 levels through regulation of transcription factor CREB in cardiomyocytes. Hence, METH-induced decreased Sigmar1 expression underlies the alteration in CREB/pCEBSer133/Fis1 signaling that adversely affects mitochondrial dynamics, morphometry, and respiration in cardiomyocytes, providing evidence for a pathogenic molecular mechanism in METH-induced cardiomyopathy. To confirm the direct involvement of Fis1 in METH-induced altered mitochondrial dynamics and morphology, we overexpressed Fis1 in HEK293T cells and treated them with METH (Supplementary Fig. 5). Similar to cardiomyocytes, METH treatment (100 µM) in HEK293T cells significantly reduced Fis1 and CREB proteins level (Supplementary Fig. 5a). We overexpressed the N-terminal His-tagged Human Fis1 (His-hFis1) in HEK293T cells and treated them with METH (100 µM) for 24 hrs. METH treatment significantly increased the mitochondrial dynamics, and His-hFis1 overexpression significantly decreased the METH treatment-induced altered mitochondrial length and aspect ratio in HEK293T cells (Supplementary Fig. 5b). These data suggest that Fis1 plays a critical role in METH treatment-induced altered mitochondrial dynamics and morphometry.

Discussion

METH abuse is a growing international crisis, making it the second most abused drug class following cannabis and opioids⁴³. Despite reports in the literature indicating an association between METH abuse and cardiomyopathy^9,10,11, little is known about the histopathological features and pathological remodeling in the heart linked with the illicit use of METH. At the same time, drug overdose-related deaths are on the rise in the young and seemingly otherwise healthy users whose hearts can be used as heart transplantation donors has grown substantially⁴⁴. In this study, we conducted microscopic histological studies on 34 human LV heart sections from autopsies of patients positive for METH use following toxicological drug screenings. Our observations revealed the consistent presence of increased collagen deposition and fibrosis in the perivascular and interstitial spaces of the LV heart sections from METH users, regardless of the cause of their deaths, compared to LV heart sections from non-METH users. In addition, the whole heart wet weights of hearts from METH users were significantly higher than those of non-METH user hearts. Our findings confirm the presence of adverse cardiac scarring and fibrotic remodeling in the hearts of METH users that accompany the METH-induced cardiac toxicity observed in a cohort of LV heart samples from human autopsies in the US.

Human METH users exhibited plasma METH concentrations that ranged from 2 to 3 µM. In comparison, levels can reach 17 µM in individuals arrested for erratic behavior and 87 µM in postmortem samples from non-overdose patients^45,46. Notably, a marked accumulation of an unchanged concentration of METH has been shown in METH-sensitized rat brain cortex, striatum, and heart, but not in the liver, kidney, or abdominal muscle, following a bolus injection of 4 mg/kg METH, demonstrating a viable reason for the higher toxicity METH use poses to cardiac tissue⁴⁷. Most of the studies of METH-associated cardiomyopathy have shown evidence of systolic cardiac dysfunction, as indicated by a lower LVEF. A growing number of clinical studies have reported severe LVEF attenuation with LV dilatation in METH users indicating the inherent existence of METH-associated cardiomyopathy^6,10,33. Studies of patients with a discharge diagnosis of cardiomyopathy and concomitant METH abuse revealed a high percentage of patients (19 of 21) with echocardiographic LV‐dilation LVEF reduction⁴⁸. A recent study of 4407 patients positive for METH use showed that 714 patients were screened for heart failure, and 450 had abnormal levels of BNP⁴⁹. Interestingly, the prevalence of abnormal BNP in the METH-tested patient group was higher than that in either the combined METH-negative or the non-tested groups⁴⁹. In addition, echocardiography studies showed a significant difference in normal LVEF (50–70%) and severely dysfunctional LVEF (<30%) for METH-positive patients with normal versus abnormal BNP⁴⁹. Logistic regression analysis revealed predictors of abnormal BNP and LVEF <30% in METH-positive patients⁴⁹. Activation of the fetal gene program (ANP, BNP, and β-MHC) in the adult heart is used as a biomarker for cardiac hypertrophy⁵⁰ and is frequently observed in the ventricles of those with severe congestive heart failure^51,52. In our study, we used a preclinical “binge and crash” METH administration protocol in mice to model the human “binge and crash” use pattern³⁴. Our longitudinal trans-thoracic echocardiography studies showed that “binge and crash” METH exposure reduces cardiac systolic functional parameters, including LVEF and LV FS. In this study, we also observed an association between a significantly increased fetal gene program (ANP, BNP, and β-MHC) and fibrotic remodeling and hypertrophy in the hearts of METH-treated mice. Taken together, our data indicate that the cardiac pathologies associated with METH abuse following a relevant “binge and crash” METH treatment pattern in mice mimic those of its human abusers. Hence, our reported model will facilitate future studies to investigate the molecular and genetic targets underlying the development of METH-associated cardiomyopathy.

Sigmar1 has been implicated as a therapeutic agent in the METH-induced addictive process and toxicity^29,53, and has been extensively studied in this regard as METH binds to Sigmar1 at physiologically relevant concentrations (Ki 2.16 ± 0.25 μM)²⁸. Sigmar1 agonists have been shown to attenuate METH-induced behavioral responses, hyperthermia, and neurotoxicity^{28,29,53,54,55,56,57,58}. Pretreatment with the Sigmar1 agonist PRE-084 decreases METH-induced psychomotor responses, drug-seeking behavior, and enhancement of the brain reward function^29,53. The consequences of METH binding to Sigmar1 are currently unknown; however, studies suggest that METH may exhibit antagonist activity²³ and/or act as an inverse agonist for Sigmar1⁵⁹. Despite extensive studies that have demonstrated Sigmar1’s protective role in METH-induced cellular dysfunction^{28,29,53,54,55,56,57,58}, it remains unknown whether and how Sigmar1 contributes to cellular protection. Our research is the first to show that the levels of Sigmar1 expression are significantly decreased in human METH users and in the heart tissue of “binge and crash” METH-treated mice. Moreover, the altered mitochondrial dynamics, abnormal mitochondrial ultrastructure, and impaired mitochondrial bioenergetics induced by METH treatment in mouse hearts are similar to those observed in Sigmar1^−/− mice hearts, suggesting that Sigmar1 has a potential molecular function in the heart³².

Similar to Sigmar1, CREB signaling has been extensively studied with respect to its relationship with METH use, and there are contradictory findings concerning CREB activation by METH exposure. We found that the expression of the phosphorylated CREB at Serine133 was significantly reduced in hearts from human METH toxicity autopsies compared with non-METH user heart tissue. Similarly, LV heart sections from our preclinical “binge and crash” METH-treated mice, as well as cultured cardiomyocytes, also showed decreased CREB phosphorylation. Our genetic studies confirm that Sigmar1 acts as an upstream regulator of CREB since Sigmar1 overexpression increases CREB and pCREBSer133 expression. Conversely, Sigmar1 genetic ablation attenuates CREB expression and pCREBSer133 expression. Our findings are agreement with studies that demonstrated that chronic METH treatment in rats (4 mg/kg, IP, 2 weeks) resulted in decreased CREB/pCREB immunostaining in the neuronal nuclei of striatum⁶⁰ and decreased pCREB expression in METH-treated cultured primary cortical neurons⁶¹. Similarly, both acute and repeated METH exposure (5 mg/kg, IP) suppresses the striatal expression of CREB mRNA⁶². Self-administration of METH (0.1 mg/kg/infusion) in rats also caused significant decreases in CREB protein levels (~40%) at 1 month in the dorsal striata⁶³. pCREBSer133 levels were significantly decreased in the nucleus accumbens and ventral pallidum at 14 days withdrawal following 5 once-daily injections of 2.5 mg/kg METH⁶⁴. In contrast, increased pCREB phosphorylation has also been reported in the self-administered METH rat striatum⁶⁵. Despite the data reported in all of these studies, CREB nuclear translocation may be an early event activated by METH as observed immediately after METH treatment (30 mg/kg, 30 min) in mice and METH exposure (150 µM) in primary rat astrocytes⁶⁶. Moreover, Sigmar1 regulates the CREB translocated into nucleus, as pretreatment with the Sigmar1 antagonist in astrocytes blocked CREB nuclear translocation⁶⁶. The Sigmar1 gene has a CREB-binding site in its promoter region; METH-mediated activation of astrocytes involved CREB translocated into the nucleus and interaction with the promoter of Sigmar1, resulting in increased expression of Sigmar1⁶⁶. Similarly, Sigmar1 upregulation was observed in cultured astrocytes from adult rats after infection with the VP16-CREB viral vector, and in transgenic mice with targeted activation of CREB in astrocytes⁶⁷. All of these studies suggest a CREB-Sigmar1 feedback loop regulating their own expression modulated by METH exposure. Future studies are required to determine the precise molecular mechanisms of Sigmar1 downregulation in cardiomyocytes mediated by METH exposure.

The physiological importance of CREB signaling in maintaining normal cardiac function is demonstrated by studies showing that mice bearing cardiomyocyte-specific dominant-negative CREB deficient in phosphorylation develop severe and progressive dilated cardiomyopathy with mitochondrial dysfunction^68,69. Moreover, pathologic stress, including hypoxia and high-glucose conditions, has been shown to regulate CREB stability and expression^70,71. Our findings showed a positive correlation between mitochondrial dysfunction and decreased CREB activity as a result of METH treatment. METH-treated mouse heart tissue and cardiomyocytes showed altered mitochondrial dynamics associated with decreased Fis1 expression and defects in mitochondrial respiration. Fis1-dependent mitochondrial fission has been reported in the heart; it does not alter the level of classical cytosolic Drp1 translocation to the mitochondria^42,72 and is reported to be independent of putative cytosolic mitochondrial fission, promoting GTPases such as Drp1 and Dyn2⁷². Our findings indicate that METH attenuates Fis1 protein expression in both the whole-cell fraction and the mitochondrial fraction. METH may alter Fis1 expression via inhibition at the transcriptional level, as supported by a recent elegant study showing that CREB binding to the Fis1 gene promoter region promotes Fis1 expression in cardiomyocytes⁴². Sigmar1-dependent regulation of Fis1 expression was also observed in Sigmar1 overexpression and siRNA knockdown cardiomyocytes with a positive correlation with CREB activity. Our study suggests that Sigmar1-dependent modulation of the CREB-Fis1 signaling axis may play a role in regulating mitochondrial morphology and function.

In conclusion, METH induces adverse cardiac fibrotic remodeling in human METH users and preclinical “binge and crash” METH-treated mouse heart. We demonstrated a novel correlation among Sigmar1 level, Sigmar1-dependent regulation of the CREB-Fis1 signaling axis, and mitochondrial dysfunction contributing to the development of METH-associated cardiomyopathy. Clinical translation of these findings would require the completion of critical studies using genetic mice models of Sigmar1 to provide further evidence for the therapeutic efficacy of Sigmar1. Our ongoing studies are focused on defining the molecular role of Sigmar1 in regulating cardiac CREB expression and activation using genetic, molecular, and biochemical experimentation involving cardiac-specific Sigmar1 transgenic and knockout mice.