Nicotine vaping has surged in recent years, particularly among young adults, and is strongly linked with concurrent alcohol use. Separately, chronic excessive alcohol use drives hypertension and cardiomyopathy, while nicotine vaping is linked to a modest rise in cardiovascular disease incidence and mortality. However, little is known about how concurrent use interacts to affect protein expression in the cardiovascular system. The aim of this study was to determine differential cardiac protein expression in mice exposed to concurrent chronic-plus-binge alcohol and nicotine vaping use. Male C57BL6/J mice received a 20-day 5% ethanol diet with 5 g/kg ethanol binges on days 10 and 20, alongside isocaloric controls. During this period, they were also exposed nightly to either 5% nicotine salt vapor, vegetable glycerin/propylene glycol vehicle vapor, or room air. The left ventricular free wall was collected and analyzed using discovery-based proteomics and subsequent Ingenuity Pathway Analysis. A total of 3144 proteins were identified across all groups. Compared to air-exposed, control-fed mice, 201 proteins were significantly altered by ethanol, 101 proteins by nicotine vaping, and 159 proteins by combined exposure. Both ethanol and nicotine vaping influenced pathways involved in lipid homeostasis, extracellular matrix remodeling, and mitochondrial bioenergetics; however, these alterations did not uniformly manifest in the dual-use group. This pattern highlights the nonadditive and potentially interaction-dependent nature of alcohol and nicotine vaping effects on cardiovascular protein expression patterns that may contribute to a distinct functional phenotype.

Chronic alcohol misuse is the leading cause of non-ischemic dilated cardiomyopathy, and the molecular mechanisms underlying the development of alcohol-associated cardiomyopathy (ACM), particularly regarding sex-specific susceptibility and mitochondrial contributions, are not fully known. In this study, we utilized a preclinical model of chronic + binge ethanol consumption to investigate sex differences in disease severity and mitochondrial function. Male and female C57BL/6J mice were fed ethanol or control liquid diets for 30 days, with 2 binge episodes on days 10 and 30. Cardiac morphology was assessed via echocardiography and cardiac function via left ventricular pressure–volume catheterization. Mitochondrial function was evaluated ex vivo using Seahorse XF analysis, ATP luminescence, and AmplexTM Red fluorescence in isolated ventricular mitochondria. Ethanol feeding induced significant cardiac dysfunction and increased transcriptional expression of inflammatory and fibrotic markers in males, while these effects were not seen in females. Despite these sex-specific cardiac effects, mitochondrial respiration, ATP production, collagen protein expression, and oxidative stress were not significantly altered following alcohol exposure in either sex. Further investigation is warranted to assess the potential role of ovarian hormones in this female cardioprotection against chronic + binge ethanol.

Binge drinking contributes to an increasing number of emergency department visits in the United States. Previous work demonstrated that an alcohol binge impairs cardiac performance and exerts complex hemodynamic effects through the activation of the endocannabinoid-mediated cannabinoid type 1 receptor (CB1R) signaling pathway. Anandamide (AEA), an endogenous CB1R agonist, is synthesized in response to various stressors and tissue injury. However, the role of binge drinking in increasing myocardial AEA levels, which leads to CB1R-dependent cardiodepression, remains unclear. This work studied how endotoxins from intestinal Gram-negative bacteria affect myocardial AEA levels, which further induce CB1R-dependent cardiac dysfunction following acute alcohol intoxication. Using a murine model of a single alcohol binge (5 g/kg orally), reduced mesenteric microcirculation concurrent with elevated circulating endotoxin levels was observed. Selective depletion of gut Gram-negative bacteria by antibiotics partially ameliorated alcohol-induced gut barrier dysfunction, significantly lowered circulating endotoxins, coinciding with reduced cardiac AEA levels at 3 hours after binge. These changes were paralleled with moderately improved cardiac performance and vascular tone. Cardiac RNA levels of genes involved in AEA synthesis increased after alcohol binge, but not in antibiotic-pretreated mice. However, acute alcohol-induced cardiac AEA formation was unrelated to toll-like receptor-4 signaling. These findings provide novel insights that highlight the pivotal role of intestinal Gram-negative bacteria in modulating cardiac AEA levels after an alcohol binge, leading to cardiovascular dysfunction.

Prenatal alcohol exposure (PAE) is a leading cause of developmental abnormalities, yet its effects on fetal cardiac development remain understudied. We employed real-time, label-free multispectral photoacoustic tomography (PAT) to noninvasively assess cardiac development in mouse fetuses exposed to chronic alcohol. Using a custom-built PAT system, fetal hearts were imaged from E12 to E16 in alcohol-exposed (3 g/kg ethanol via oral gavage, n  = 9) and control ( n  = 7) CD-1 mice. PAT enabled quantitative measurements of cardiac morphology, oxygen saturation (sO 2 ), and heart rate. Alcohol-exposed fetuses exhibited consistently lower sO 2 and greater heart rate variability, particularly at later gestational stages. While structural growth progressed in both groups, functional impairments became more pronounced with alcohol exposure. These findings suggest PAE alters fetal cardiovascular regulation despite normal anatomical development. This study highlights the utility of PAT as a high-resolution, noninvasive tool for monitoring fetal cardiac health and supports its potential application in developmental biology and prenatal diagnostics.

Alcohol-related Cardiomyopathy (ACM) manifests in humans with a significant history of alcohol use and is characterized by ventricular dilation and cardiac function impairment. Although the most common age group for ACM is males between the ages of 30-55, women require a lesser lifetime alcohol exposure to develop ACM than men. The objective of this study is to assess sexual differences in a mouse model of chronic plus binge alcohol consumption. We hypothesize that female mice exposed to chronic plus binge alcohol will not develop as severe cardiac dysfunction as their male counterparts. In this study, male (n=19) and female (n=14) C57BL/6J mice were provided chronic plus binge alcohol feeding. After 5 days of acclimation to the liquid diet, mice are fed ad libitum 5% ethanol (EtOH) liquid or isocaloric control liquid diet for 30 days. At 10 and 30 days, mice received an oral binge dose of EtOH (5 g/kg body weight), or isocaloric maltose dextrin solution (9 g/kg body weight). Noninvasive cardiac structural and functional data were obtained via echocardiography at baseline and following binges. After 30 days, cardiac hemodynamics was measured by open chest left ventricle catheterization. Two-way ANOVA with Tukey’s post hoc analysis was performed for each cardiac parameter, and significance was defined as p<0.05. Male EtOH mice exhibited a significant decrease in stroke work (1119 ± 297 mmHg/mL when compared to male controls, 1622 ± 365 mmHg/mL; p= 0.03). Female EtOH mice had a nonsignificant decrease in stroke work (1242 ± 374 mmHg/mL when compared to female controls, 1423 ± 469 mmHg/mL; p= 0.80). Maximum left ventricular pressure was decreased 20% in male EtOH mice (p=0.003) but increased 10% in female EtOH mice (p=0.46). The maximum rate of pressure change within the ventricle was 24% lower in the male EtOH mice (p=0.02) and 9% higher in female EtOH mice (p=0.87). The minimum rate of pressure change within the ventricle was 24% higher in male EtOH mice (p=0.02) and 17% lower in female ETOH mice (p=0.44). After 30 days of EtOH exposure, male mice showed significant systolic and diastolic dysfunction. Female mice on the other hand, did not show substantial cardiac dysfunction after chronic plus binge ethanol exposure. Further experiments involving qPCR of proinflammatory and profibrotic markers will elucidate the molecular mechanism of sex differences in ACM development. R21AA029747 (JG & CB) & T32AA007577. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic drinking can lead to alcohol-induced cardiomyopathy (ACM), a disease currently without a cure. Using a mouse model of ACM, we observed significant cardiac dysfunction after 30 days. Our study’s goal was to investigate the potential restoration of cardiac function following a period of abstinence. Our hypothesis is that abstinence can lead to an observable recovery in overall cardiac function. C57BL/6J mice were given the Lieber-DeCarli liquid diet with 5% ethanol for 30 days, with two ethanol binges on days 10 and 30 (5 g/kg). After the 30-day ethanol exposure, the mice were transitioned to an ethanol-free diet for an additional 30 days. Left ventricular catheterization and echocardiography were conducted at the 30- and 60-day timepoints. 30 days of ethanol consumption led to significant decreases in both systolic and diastolic function. However, following a period of alcohol abstinence, there was a marked improvement. Specifically, the 30-day ethanol mice exhibited increases in stroke work and dP/dt max after 30 days of abstinence; stroke work increased from 1119 ± 89 mmHg*μL to 2032 ± 139 mmHg*μL (p < 0.0001), and dP/dt max increased from 8054 ± 664 mmHg/s to 11967 ± 449 mmHg/s (p < 0.001). Diastolic function also improved; dP/dt min from -7711 ± 561 mmHg/s to -10202 ± 594 (p < 0.01). We also found a significant increase in load-independent cardiac contractility, i.e. the slope of end systolic pressure volume relation: 6.89 ± 0.6 mmHg/μL to 10.59 ± 1.57 mmHg/μL (p < 0.05). In conclusion, abstinence after chronic ethanol exposure can lead to a restoration of cardiac function in C57BL/6J mice. This model parallels limited clinical data on abstinence and will allow us to study the underlying mechanisms responsible for the restoration of function. R21AA029747 (JG, CB), F30AA030472 (JE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Cardiovascular disease is the leading cause of death in the United States and is strongly associated with smoking. While cigarette smoking has declined, the use of electronic nicotine delivery systems (ENDS) has dramatically increased. We have previously shown that chronic nicotine inhalation leads to pulmonary hypertension and right ventricular remodeling in C57BL/6J mice. Nicotine exposure alone did not, however, cause significant left ventricular (LV) remodeling or dysfunction. In this study, we aimed to examine the effects of chronic nicotine inhalation on pressure overload (PO), hypothesizing that nicotine inhalation would abrogate adaptive cardiac remodeling and accelerate cardiac dysfunction. Methods: 7-week-old, male C57BL/6J mice were exposed to nicotine vapor or room air for one week prior to transverse aortic constriction (TAC) surgery to induce PO. Nicotine vapor was produced by bubbling room air through a free-base nicotine solution, diluted with additional room air, and delivered to exposure chambers. Air mice were housed in the same room but outside of the exposure chambers (n=11-12 per group). Following 1 week of surgical recovery, sham and TAC mice were exposed to nicotine vapor or room air for 12 hours per day for 9 weeks. Nicotine exposure was measured using serum cotinine and airflow was adjusted to achieve levels comparable to human ENDS users. Cotinine was not significantly different between sham (572±93 ng/mL) and TAC (617±102 ng/mL) mice. Echocardiography was used to track structural and functional changes in the LV at baseline and every two weeks after surgery. Results: At 10 weeks post-surgery, LV posterior wall thickness at systole (LVPW;s: 1.37±0.04 mm) and LVPW at diastole (LVPW;d: 1.05±0.03 mm) in air-TAC mice were significantly thickened versus both air-sham mice (LVPW;s: 1.05±0.04 mm; LVPW;d: 0.65±0.02 mm) and nicotine-TAC mice (LVPW;s: 1.05±0.03 mm; LVPW;d: 0.82±0.04 mm). There was no significant difference in LVPW;s between nicotine-TAC mice and nicotine-sham mice (1.06±0.02). LVPW;d was significantly thickened in nicotine-TAC mice versus nicotine-sham mice (0.65±0.01).The ejection fraction (EF) of air-TAC mice was 61.3±4.1% versus nicotine-TAC mice at 50.9±3.6%. Air-sham and nicotine-sham mice had EF of 68.3±2.6% and 68.7±1.8%, respectively. We also found that nicotine exposure led to reduced event free survival, where an event was described as EF less than 55%. Event free survival rate was 33% in nicotine-TAC mice versus and 54% in air-TAC mice. Event free survival was 100% in air-sham and nicotine-sham mice. Conclusions: Chronic nicotine inhalation reduces adaptive cardiac remodeling in response to TAC-induced pressure overload, leading to accelerated decompensation. Research was funded by the National Institute of Health, National Heart, Lung, and Blood Institute – R01HL135635 (JG), R01HL135635-S1 (JG), and 1F30AA030472-01 (JE). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine β-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-β1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins.

ID 20802 Poster Board 165 Mild Traumatic Brain Injuries (mTBI) and Burn Injuries (BI) present monumental public health burdens that have been independently linked to long term nervous system-related morbidity and negative patient outcomes. Military service members in active combat theaters are among the most at risk for sustaining concomitant mTBI and BI, in most cases due to detonation of improvised explosive devices. Previous studies have provided evidence that mTBI and BI independently induce inflammation, through the induction of innate inflammatory cascades, however the concomitant effects of simultaneous mTBI and BI on early post-injury molecular processes, gene expression alterations, neuronal signaling within the brain and ultimately outcomes are currently unknown. This lack of data, coupled with an overt lack of studies conducted in human subjects, has left large gaps in the knowledge required and the available capability to successfully treat complicated polytrauma cases involving mTBI and BI. We hypothesize that concomitant mTBI and BI act in exaggerating early neuroinflammatory responses within the CNS, an effect that leads to worse long-term health and behavioral outcomes, when compared to mTBI or BI alone. To test this hypothesis, adult, wild type C57Bl/6J mice were subjected to a single, blast induced mTBI and full thickness BI (mTBI/BI), a single, blast induced mTBI alone, a full thickness BI alone, or sham treatment followed by RNA-sequencing of ipsilateral brain parenchyma conducted at six hours post-injury (hpi). Bioinformatic analysis of the RNA-sequencing results and subsequent pathway, overrepresentation, and protein-protein interaction analyses identified a large array of unique gene expression profiles specific to the combined mTBI/BI condition and identified gene-sets relevant to neuroinflammation, extracellular matrix organization and vascular integrity that are highly enriched within the data. Notably, significant, exclusive, upregulation of genes canonically expressed during mesenchymal-epithelial transition occurred within the brains of mTBI/BI subjects. Cumulatively, these data suggest that concomitant mTBI/BI results in a profound, combinatorial exacerbation of modality-specific disruptions in the blood-brain-barrier and neuro-vascular stability and may provide viable mechanism derived targets for pharmacotherapeutic development. Studies contained herein provide the first characterization of a synergistic effect of concomitant mTBI and BI on early transcription and inflammation within the CNS and afford insight into the specific molecular events involved in observed synergism. Further, our studies are anticipated to identify potential targets for the development of pharmacotherapeutics to improve neurologic outcomes in those subjected to polytrauma. Support/Funding Information: Department of Defense CDMRP Award #W81XWH2210849.

Alcohol-induced cardiomyopathy (ACM) has a poor prognosis with up to a 50% chance of death within four years of diagnosis. There are limited studies investigating the potential of abstinence for promoting repair after alcohol-induced cardiac damage, particularly in a controlled preclinical study design. Here, we developed an exposure protocol that led to significant decreases in cardiac function in C57BL6/J mice within 30 days; dP/dt max decreased in the mice fed alcohol for 30 days (8054 ± 664.5 mmHg/s compared to control mice: 11,188 ± 724.2 mmHg/s, p < 0.01), and the dP/dt min decreased, as well (−7711 ± 561 mmHg/s compared to control mice: −10,147 ± 448.2 mmHg/s, p < 0.01). Quantitative PCR was used to investigate inflammatory and fibrotic biomarkers, while histology was used to depict overt changes in cardiac fibrosis. We observed a complete recovery of function after abstinence (dP/dt max increased from 8054 ± 664 mmHg/s at 30 days to 11,967 ± 449 mmHg/s after abstinence, p < 0.01); further, both inflammatory and fibrotic biomarkers decreased after abstinence. These results lay the groundwork for future investigation of the molecular mechanisms underlying recovery from alcohol-induced damage in the heart.

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.

In this study, we found no differences in serum cotinine, RVSP, RV FWT;d, or RVID;d between intact and OVX females, leading us to conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced PH. Due to profound sex differences in clinical PH pathogenesis and nicotine metabolism, further studies (including exploration of androgen-driven RV remodeling) are necessary to illuminate mechanisms underlying protection from nicotine-induced pathology in female mice.

Editorial FocusNew insight into the regression of cardiac fibrosisJoshua M. Edavettal and Jason D. GardnerJoshua M. EdavettalDepartment of Physiology, Louisiana State University Health Sciences Center, New Orleans, Louisiana and Jason D. GardnerDepartment of Physiology, Louisiana State University Health Sciences Center, New Orleans, LouisianaPublished Online:07 Jul 2022https://doi.org/10.1152/ajpheart.00306.2022This is the final version - click for previous versionMoreSectionsPDF (237 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Pressure overload occurs when the heart must eject blood against a greater afterload. Any obstruction to the outflow of blood can lead to a pressure overload, and aortic valve stenosis, aortic coarctation, and systemic hypertension are pathologies that affect the left ventricle. The cardiovascular system has many mechanisms to compensate for the increased workload imposed by a pressure overload. One of the primary adaptations is concentric hypertrophy. Although ventricular wall thickness increases to reduce the effect of wall tension because of pressure overload, the radius of the chamber remains relatively the same. Because of altered sarcomere structure and fibrosis, ventricular compliance falls and leads to diastolic dysfunction, where the chamber cannot physically accommodate the volume of blood necessary to maintain appropriate stroke volume and cardiac output. Eventually, the ventricle begins to fail, leading to systolic dysfunction.In this issue of the American Journal of Physiology-Heart and Circulatory Physiology, Neff et al. (1) used transverse aortic constriction (TAC) to create a pressure overload that resulted in cardiac fibrosis. Once the surgery was reversed, this fibrosis was shown to regress, though not entirely, with corresponding decreased concentrations and activities of fibrotic biomarkers. This focus will discuss key findings from this study, highlight its strengths, briefly mention some limitations, and end with a few questions still to be answered regarding the mechanisms underlying compensatory hypertrophy and fibrosis.TAC surgeries are among the available methods to induce myocardial hypertrophy in a mouse model. In TAC surgeries, the chest cavity is opened, the aorta is accessed, and a ligation is placed around the transverse aorta. This is done by tying a suture around the vessel and a 25- to 30-gauge needle, after which the needle is removed. The needle provides a fixed and known diameter of coarctation, which limits the volume of blood that can pass through the aorta per heartbeat and increases afterload, effectively creating a pressure overload. There are complex and multifaceted molecular mechanisms underpinning cardiac hypertrophy, and the mechanisms leading to pathological hypertrophy differ from those involved in physiological remodeling (associated with exercise and pregnancy). Since TAC surgeries abruptly alter the cardiac function and do not parallel any normal physiological processes, the molecular changes seen are those found in pathological hypertrophy. Key players in pathological remodeling include angiotensin II (ANG II) and endothelin-1 (ET-1), catecholamines, the mammalian target of rapamycin (mTOR) signaling pathway, and natriuretic peptides (2).Other animal models can produce increased afterload and associated pressure overload conditions. These include the spontaneous hypertension rat (SHR) model, other constrictions mimicking aortic stenosis, renovascular models, unilateral nephrectomy, and exogenous induction methods (3). Hypertension directly increases afterload, creating a pressure overload, and hypertension alone can lead to fibrotic and hypertrophic compensatory changes. The strengths of the SHR model are reproducibility and no need for surgical intervention, but as a genetic model, it is irreversible and the afterload cannot be manipulated; furthermore, a separate locus of genes responsible for left ventricular hypertrophy in the SHR model can be independently activated without hypertension (3). TAC surgeries are an example of ligation procedures done that mimic aortic stenosis. Other locations that can be constricted include the ascending aorta and the abdominal aorta. These surgeries also result in an increased afterload and have been used to study mechanisms underlying compensatory hypertrophy. Renovascular models, including unilateral nephrectomy and constriction of renal arteries, as well as exogenous administration of salt (sodium chloride), ANG II, and aldosterone, exploit the renin-angiotensin-aldosterone system (RAAS). One of the primary effects of RAAS activation is an increase in circulating concentrations of ANG II, which has a myriad of effects, perhaps most importantly directly increasing blood pressure through its type 1 (AT1) receptor.Understandably, much of the previous research on cardiac hypertrophy and fibrosis focus on its development, treatment to alleviate symptoms caused by maladaptive compensation, and the functional and molecular assessment of pathological hypertrophy. Clinically, there are studies that suggest fibrosis (assessed by echocardiography) as a prognostic indicator for aortic valve stenosis and that this measure may be used to determine the timing of aortic valve replacement (AVR; 4). There are also numerous studies on the treatment of cardiac hypertrophy and the reduction of fibrosis (5). Others have focused on the pathways involved, and there are many recognized molecular targets that could potentially be used therapeutically (6). Other models have been used to study the reversal of cardiac fibrosis, such as volume overload (VO), which can similarly induce left ventricular hypertrophy. Hutchinson et al. (7) found that VO reversal in rats led to a reduction in eccentric dilation, attenuated interstitial fibrosis, and normalized hypertrophy. However, few have examined molecular assessment and cardiac structure following interventions in humans. Treibel et al. (8) observed 116 pacemaker-free survivors of AVR after a year and found that diffuse fibrosis and myocardial cellular hypertrophy, but not focal fibrosis, regressed. Although surgical procedures can remove the causes of pressure overload, such as aortic valve stenosis, quality of life depends on functional capability that would still be affected by a fibrotic heart.The study by Neff et al. (1) used TAC surgery to produce pathological hypertrophy, but instead of examining the effects of TAC-induced pressure overload, they studied the functional, structural, and molecular changes that occurred upon reversal of the TAC surgery (unTAC) and normalization of afterload. They measured the effects of unTAC on myocardial hypertrophy, fibrillar collagen content, myocardial stiffness, and collagen degradation. Some interesting results from their study include that unTAC resulted in the reduction of myocardial fibrosis but did not fully return the heart to a preoverload, prefibrotic state. Furthermore, they found that by week 4 (post-unTAC), processes involved in the degradation of collagen decreased, yielding to other molecular events, which may inhibit this degradation. At the second week after TAC reversal, they found that matrix metalloproteases (MMPs), enzymes responsible for collagen degradation, increased significantly, but by week 6, tissue inhibitor of metalloproteinase (TIMP)-1 metallopeptidase inhibitor 1 (TIMP1), an inhibitory regulator of MMPs was increased. TIMP-1 inhibits collagen degradation and can increase fibroblast activation, which leads to further fibrosis. Increases in the proenzyme of lysyl oxidase (responsible for collagen cross linking; proLOX), the cleaved prodomains of LOX (LOPP), and LOXL2, a member in the LOX family, were found after 4 wk post-unTAC. Furthermore, this pattern was also observed in the potent collagenase enzyme cathepsin K, proMMP-2 and -9, and MMP-8.One of the major strengths of the study by Neff et al. (1) is the novelty of studying the cardiovascular functional and structural alteration (through measuring physical characteristics and fibrotic biomarkers) after removal of the hemodynamic insult that caused the fibrosis. In this study echocardiography was used to examine functional and physical characteristics of the heart, and postmortem measures of cardiomyocyte cross-sectional area and myocardial stiffness were taken. These measures give a clear picture of the physical and functional differences found between mice at different time points. However, there are other key molecules and mechanisms involved in cardiac hypertrophy that should be examined in future studies. Transforming growth factor (TGF)-β1 is known to stimulate fibrosis, and TGF-β1 has been called the master regulator of fibrosis; furthermore, ANG II, ET-1, catecholamines, and connective tissue growth factor (CTGF) all have roles in cardiac collagen deposition (9). Although the results presented do show interesting findings that should lay the foundation for future investigation, their study did not specifically investigate cellular physiology. The cardiac cell types, their activation, and their behaviors responsible for the development of cardiac fibrosis are fundamental in the underlying pathophysiology. For example, it would be interesting to assess the activity of cardiac fibroblasts and myofibroblasts following the removal of TAC. MicroRNAs have also been shown to be involved in cardiac fibrosis and disease (10). The finding that the regression of fibrosis is incomplete does have a supporting background of existing literature, albeit limited, but since this time frame of analysis is novel, more data would strengthen the knowledge base on recovery after corrective surgery.The novelty of this research opens the door for future studies to answer multiple questions. Neff et al. (1) used TAC surgeries to induce a pressure overload, but there are other models leading to hypertrophy and fibrosis, some physiological, as mentioned before, and some pathological. In pathophysiological remodeling, when does regression start, and does degradation of collagen begin later depending on the length of time spent adapting to a challenge? Is there a point of no return, after which fibrosis and hypertrophy become permanent or can they advance even further, despite correction of hemodynamic stress? Would this also be true in conditioned athletes who stop training or postpartum mothers? In the pathological development of cardiac fibrosis, does a wound to the heart have a different time frame of collagen degradation during the healing process compared with hypertrophy and fibrosis developed through the RAAS, catecholamines, or delivered substances (isoproterenol)? Perhaps those who have different conditions (hypertension or congenital malformations), risk factors (like smoking, poor diet, or sedentary lifestyle), or family histories have different recovery timelines. If distinct patterns emerge following removal of an insult, how do they differ mechanistically? Although the molecular targets that the researchers chose were appropriate and did provide a more in-depth view of the physiology, questions remain regarding the specifics. A concrete, detailed pathway may be difficult to discover, but a stronger understanding of the underlying processes may reveal targets for pharmacological strategies. There is existing literature on the major factors involved in wound healing and fibrosis (such as TGF-β1) and cardio-specific factors important in adaptive morphological changes (ANG II), but do the already established pathways remain relevant in these contexts? Another important consideration is that although mice and rodents do have comparable physiology with humans, there are differences. Rodents are more resistant to lesion development and have higher lipid levels (11). Should other animal models be pursued? Rats have a decreased risk of developing atheromas, rabbits require higher concentrations of cholesterol in circulation, and porcine models are expensive and do not have a wide library of genetic models (11). Though cardiovascular disease is common, designing human experiments would be challenging because of the need for adequate controls and the time required for significant postmortem analysis.Despite these future challenges and considerations, this research is exciting and may eventually lead to completely outlining the morphological cardiovascular changes that occur in response to cardiovascular challenges, such as pressure overload. This knowledge has the potential of engineering better outcomes for patients, and this work has already led to future questions that can and should be addressed.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONSJ.M.E. and J.D.G. drafted manuscript; edited and revised manuscript; approved final version of manuscript.REFERENCES1. Neff LS, Zhang Y, Van Laer AO, Baicu CF, Karavan M Jr, Zile MR, Bradshaw AD. Mechanisms that limit regression of myocardial fibrosis following removal of left ventricular pressure overload. Am J Physiol Heart Circ Physiol 323: H165–H175, 2022. doi:10.1152/ajpheart.00148.2022.Link | ISI | Google Scholar2. Nakamura M, Sadoshima J. Mechanisms of physiological and pathological cardiac hypertrophy. Nat Rev Cardiol 15: 387–407, 2018. doi:10.1038/s41569-018-0007-y. Crossref | PubMed | ISI | Google Scholar3. Ding Y, Wang Y, Jia Q, Wang X, Lu Y, Zhang A, Lv S, Zhang J. Morphological and functional characteristics of animal models of myocardial fibrosis induced by pressure overload. Int J Hypertens 2020: 3014693, 2020. doi:10.1155/2020/3014693. Crossref | PubMed | ISI | Google Scholar4. Calin A, Mateescu AD, Popescu AC, Bing R, Dweck MR, Popescu BA. Role of advanced left ventricular imaging in adults with aortic stenosis. Heart 106: 962–969, 2020. doi:10.1136/heartjnl-2019-315211. Crossref | PubMed | ISI | Google Scholar5. Marquis-Gravel G, Redfors B, Leon MB, Généreux P. Medical treatment of aortic stenosis. Circulation 134: 1766–1784, 2016. doi:10.1161/CIRCULATIONAHA.116.023997. Crossref | PubMed | ISI | Google Scholar6. Travers JG, Tharp CA, Rubino M, McKinsey TA. Therapeutic targets for cardiac fibrosis: from old school to next-gen. J Clin Invest 132: e148554, 2022. doi:10.1172/JCI148554. Crossref | PubMed | ISI | Google Scholar7. Hutchinson KR, Guggilam A, Cismowski MJ, Galantowicz ML, West TA, Stewart JA Jr, Zhang X, Lord KC, Lucchesi PA. Temporal pattern of left ventricular structural and functional remodeling following reversal of volume overload heart failure. J Appl Physiol (1985) 111: 1778–1788, 2011. doi:10.1152/japplphysiol.00691.2011. Link | ISI | Google Scholar8. Treibel TA, Kozor R, Schofield R, Benedetti G, Fontana M, Bhuva AN, Sheikh A, López B, González A, Manisty C, Lloyd G, Kellman P, Díez J, Moon JC. Reverse myocardial remodeling following valve replacement in patients with aortic stenosis. J Am Coll Cardiol 71: 860–871, 2018. doi:10.1016/j.jacc.2017.12.035. Crossref | PubMed | ISI | Google Scholar9. Cowling RT, Kupsky D, Kahn AM, Daniels LB, Greenberg BH. Mechanisms of cardiac collagen deposition in experimental models and human disease. Transl Res J Res 209: 138–155, 2019. doi:10.1016/j.trsl.2019.03.004. Crossref | PubMed | ISI | Google Scholar10. Jin Z-Q. MicroRNA targets and biomarker validation for diabetes-associated cardiac fibrosis. Pharmacol Res 174: 105941, 2021. doi:10.1016/j.phrs.2021.105941. Crossref | PubMed | ISI | Google Scholar11. Savoji H, Mohammadi MH, Rafatian N, Toroghi MK, Wang EY, Zhao Y, Korolj A, Ahadian S, Radisic M. Cardiovascular disease models: a game changing paradigm in drug discovery and screening. Biomaterials 198: 3–26, 2019. doi:10.1016/j.biomaterials.2018.09.036. Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESCorrespondence: J. D. Gardner ([email protected]edu). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Related ArticlesMechanisms that limit regression of myocardial fibrosis following removal of left ventricular pressure overload 27 Jun 2022American Journal of Physiology-Heart and Circulatory PhysiologyCited ByAnd the band played on: persistent fibrosis after unbanding reveals sex-dependent differences in ratsMichael R. Zile and Amy D. Bradshaw26 July 2022 | American Journal of Physiology-Heart and Circulatory Physiology, Vol. 323, No. 2 More from this issue > Volume 323Issue 1July 2022Pages H201-H203 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajpheart.00306.2022PubMed35749716History Received 21 June 2022 Accepted 23 June 2022 Published online 7 July 2022 Published in print 1 July 2022 KeywordsfibrosisheartMMPpressure overloadTAC Metrics

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1−/−, 0-copy), heterozygous (Npr1+/−, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-β1 receptor (TGF-β1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1−/− (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-βRI (4.3-fold), TGF-βRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-β1-mediated SMAD-dependent pathway.

Electronic cigarette use has increased globally prompting calls for improved understanding of nicotine's cardiovascular health effects. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension and right ven-tricular (RV) remodeling in male mice, but not female mice, suggesting sex differences in nicotine-related pathology. Clinically, biological females develop pulmonary hypertension more often but have less severe disease than biological males, likely because of the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females because of differences in cytochrome-P450 activity, which are thought to be mediated by female sex hormones. These find-ings led us to hypothesize that female mice are protected against nicotine-induced pulmonary hypertension by an ovarian hor-mone-dependent mechanism. In this study, intact and ovariectomized (OVX) female mice were exposed to chronic, inhaled nicotine or room air for 12 h/day for 10-12 wk. We report no differences in serum cotinine levels between intact and OVX mice. In addition, we found no structural (RV or left ventricular dimensions and Fulton index) or functional (RV systolic pres-sure, pulmonary vascular resistance, cardiac output, ejection fraction, and fractional shortening) evidence of cardiopulmonary dysfunction in intact or OVX mice. We conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced pulmonary hypertension. Due to profound sex differences in clinical pulmonary hypertension pathogenesis and nicotine metabolism, further studies are necessary to elucidate mechanisms underlying protection from nicotine-induced pathology in female mice.NEW & NOTEWORTHY The emergence of electronic cigarettes poses a threat to cardiovascular and pulmonary health, but the direct contribution of nicotine to these disease processes is largely unknown. Our laboratory has previously shown that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular remodeling in male mice, but not female mice. This study using a bilateral ovariectomy model suggests that the cardiopulmonary protection observed in nicotine-exposed female mice may be independent of ovarian hormones.

BackgroundThe heart undergoes structural and functional changes in response to injury and hemodynamic stress known as cardiac remodeling. Cardiac remodeling often decompensates causing dysfunction and heart failure (HF). Cardiac remodeling and dysfunction are significantly associated with cigarette smoking. Although cigarette smoking has declined, the roles of nicotine and novel tobacco products (including electronic cigarettes and heat-not-burn tobacco) in cardiac remodeling are unclear. In this perspective, we present evidence demonstrating maladaptive cardiac remodeling in nicotine-exposed mice undergoing hemodynamic stress with angiotensin (Ang)-II infusion and review preclinical literature linking nicotine and novel tobacco products with cardiac remodeling and dysfunction.MethodsAdult, male C57BL/6J mice were exposed to room air or chronic, inhaled nicotine for 8 weeks. A subset of mice was infused with Ang-II via subcutaneous osmotic mini-pumps during the final 4 weeks of exposure. Left ventricular structure and function were assessed with echocardiography.ResultsChronic, inhaled nicotine abrogated Ang-II-induced thickening of the left ventricular posterior wall, leading to reduced relative wall thickness. Ang-II infusion was associated with increased left ventricular mass index in both air- and nicotine-exposed mice.ConclusionsThese changes suggest a phenotypic shift from concentric hypertrophy to eccentric hypertrophy in nicotine-exposed, hemodynamically-stressed mice which could drive HF pathogenesis. These findings join a growing body of animal studies demonstrating cardiac remodeling and dysfunction following nicotine and electronic cigarette exposure. Further exploration is necessary; however, clinicians and researchers should not overlook these emerging products as potential risk factors in the pathogenesis of cardiac remodeling and associated diseases including HF.

Chronic, inhaled nicotine causes pulmonary hypertension and right ventricular remodeling in mice. Treatment with losartan, an angiotensin II type 1 receptor antagonist, ameliorates nicotine-induced pulmonary hypertension and right ventricular remodeling. This novel finding provides preclinical evidence for the use of renin-angiotensin system-based therapies in the treatment of pulmonary hypertension, particularly in patients with a history of tobacco-product use.

Cigarette smoking remains a leading risk factor for cardiopulmonary pathologies. However, the effects of nicotine, the addictive component of cigarettes, on cardiopulmonary function is not fully understood. Our laboratory has shown previously that chronic nicotine inhalation in mice leads to the development of pulmonary hypertension with right ventricular (RV) remodeling. This study aims to further examine the molecular mechanisms of nicotine-induced vascular dysfunction. We hypothesize that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nicotinic acetylcholine receptor (nAChR). To investigate our hypothesis, male C57BL6/J wildtype (WT) and α7-nAChR global knockout (KO) mice were exposed to air (WT, n=10; α7-nAChR KO, n=7) or nicotine vapor (WT, n=10; α7-nAChR KO, n=10) daily, 12 hour on/12 hour off, for 3 months. After the 3-month exposure, mice were subjected to right heart catheterization to measure right ventricular systolic pressure (RVSP) as an index of pulmonary artery pressure. In contrast to WT mice, which developed increased RVSP (31.4 ± 1.5 mmHg in nicotine-exposed compared to 24.0 ± 1.3 mmHg in air controls, p = 0.002), α7-nAChR KO mice were protected from these nicotine effects (25.7 ± 0.8 mmHg in nicotine-exposed compared to 21.6 ± 2.0 mmHg in air controls, p = 0.218). In addition, thoracic aortas were isolated from WT and α7-nAChR KO mice following air or nicotine exposure and subjected to RNA-sequencing analysis. Aortas isolated from WT mice showed 123 differentially expressed genes (112 up and 11 down) in nicotine-exposed compared to controls, with 18 upregulated and 3 downregulated pathways as identified by the Ingenuity Pathway Analysis (IPA). Many of the top upregulated pathways are involved in inflammatory response, including acute phase response signaling (p = 4.91E-06), complement system (p = 5.71E-06), and interleukin (IL)-6 signaling (p = 1.88E-03). Furthermore, the Angiotensin II receptor type 1, an important effector controlling blood pressure and volume, was upregulated 4.48-fold (Adjusted p = 0.038). In contrast, only 7 none-overlapping genes were differentially regulated in α7-nAChR KO mice by nicotine compared with the air controls. Finally, vasoreactivity assays were performed on aortas isolated from air and nicotine-exposed WT mice, and aortas isolated from nicotine-exposed mice exhibited impaired endothelium-dependent vasodilation. In conclusion, we found that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nAChR.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases; however, the role of nicotine in the pathogenesis of these diseases is incompletely understood. The purpose of this study was to examine the effects of chronic nicotine inhalation on the development of cardiovascular and pulmonary disease with a focus on blood pressure and cardiac remodeling. Male C57BL6/J mice were exposed to air (control) or nicotine vapor (daily, 12 hour on/12 hour off) for 8 weeks. Systemic blood pressure was recorded weekly by radio-telemetry, and cardiac remodeling was monitored by echocardiography. At the end of the 8 weeks, mice were subjected to right heart catheterization to measure right ventricular systolic pressure. Nicotine-exposed mice exhibited elevated systemic blood pressure from weeks 1 to 3, which then returned to baseline from weeks 4 to 8, indicating development of tolerance to nicotine. At 8 weeks, significantly increased right ventricular systolic pressure was detected in nicotine-exposed mice compared with the air controls. Echocardiography showed that 8-week nicotine inhalation resulted in right ventricular (RV) hypertrophy with increased RV free wall thickness and a trend of increase in RV internal diameter. In contrast, there were no significant structural or functional changes in the left ventricle following nicotine exposure. Mechanistically, we observed increased expression of angiotensin-converting enzyme and enhanced activation of mitogen-activated protein kinase pathways in the RV but not in the left ventricle. We conclude that chronic nicotine inhalation alters both systemic and pulmonary blood pressure with the latter accompanied by RV remodeling, possibly leading to progressive and persistent pulmonary hypertension.