https://orcid.org/0009-0002-0335-0412
Figures
Abstract
Heart Failure (HF) continues to be a complex public health issue with increasing world population prevalence. Although overall mortality has decreased for HF and hypertrophic cardiomyopathy (HCM), a precursor for HF, their prevalence continues to increase annually. Because the etiology of HF and HCM is heterogeneous, it has been difficult to identify novel therapies to combat these diseases. Isoproterenol (ISP), a non-selective β-adrenoreceptor agonist, is commonly used to induce cardiotoxicity and cause acute and chronic HCM and HF in mice. However, the variability in dose and duration of ISP treatment used in studies has made it difficult to determine the optimal combination of ISP dose and delivery method to develop a reliable ISP-induced mouse model for disease. Here we examined cardiac effects induced by ISP via subcutaneous (SQ) and SQ-minipump (SMP) infusions across 3 doses (2, 4, and 10mg/kg/day) over 2 weeks to determine whether SQ and SMP ISP delivery induced comparable disease severity in C57BL/6J mice. To assess disease, we measured body and heart weight, surface electrocardiogram (ECG), and echocardiography recordings. We found all 3 ISP doses comparably increase heart weight, but these increases are more pronounced when ISP was administered via SMP. We also found that the combination of ISP treatment and delivery method induces contrasting heart rate, RR interval, and R and S amplitudes that may place SMP treated mice at higher risk for sustained disease burden. Mice treated via SMP also had increased heart wall thickness and LV Mass, but mice treated via SQ showed greater increase in gene markers for hypertrophy and fibrosis. Overall, these data suggest that at 2 weeks, mice treated with 2, 4, or 10mg/kg/day ISP via SQ and SMP routes cause similar pathological heart phenotypes but highlight the importance of drug delivery method to induce differing disease pathways.
Citation: Perez-Bonilla P, LaViolette B, Bhandary B, Ullas S, Chen X, Hirenallur-Shanthappa D (2024) Isoproterenol induced cardiac hypertrophy: A comparison of three doses and two delivery methods in C57BL/6J mice. PLoS ONE 19(7): e0307467. https://doi.org/10.1371/journal.pone.0307467
Editor: Daniel M. Johnson, The Open University, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: January 24, 2024; Accepted: July 5, 2024; Published: July 22, 2024
Copyright: © 2024 Perez-Bonilla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by Pfizer Inc. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Heart Failure (HF) affects millions of people worldwide, and in the US, it constitutes a considerable and increasing amount of healthcare costs [1–3]. HF, which is typically preceded by hypertrophic cardiomyopathy (HCM), accounts for a substantial number of inpatient and outpatient in clinical settings annually [4–8]. Although advances in biomedical research have decreased mortality, the prevalence of HCM and HF has continued to increase [2, 4, 9–11]. HCM is characterized by a chronic physiological increase in cardiac muscle mass that results from cardiac wall stress [12]. Cardiac hypertrophy can be developed transiently, often as a compensatory mechanism during periods of sustained stress [12]. However, if the condition persists, hypertrophy leads to pathological remodeling of cardiac structures and ultimately HF [13, 14].
Several animal models of HCM and HF have been developed through a combination of genetic modifications, administration of pharmacologic compounds, and/or surgical approaches to recapitulate human disease [15–19]. One of the commonly used drugs to induce cardiotoxicity and develop disease in animal models is isoproterenol (ISP). ISP is a non-selective β-adrenoreceptor agonist, and as such its effects include increasing heart rate and stimulating mechanisms of oxidative stress, the renin-angiotensin-aldosterone system, and fibrogenic factors [20–29]. Notably, ISP is used alongside genetic modifications to induce disease at an earlier age or accelerate disease progression [30, 31]. ISP induces differing structural changes in the heart depending on dose, route, and duration of administration [20, 21, 29, 32]. Acute models of disease induce cardiac stress by using one or multiple bolus intraperitoneal or subcutaneous (SQ) ISP injections, while chronic models use implanted minipumps to release ISP continuously [5, 21, 27, 29, 32]. For example, β-adrenergic stimulation by ISP for more than 3 days results in cardiac hypertrophy [29], and acute ISP administration produces tachycardia associated with relative ischemia due to imbalance between increased myocardial oxygen demand and reduced coronary blood supply [33–35]. Thus, a principal advantage of using ISP is that acute and chronic cardiac injury can be induced by adjusting the dose and duration of administration, which leads to development of pathology from mild HCM to congestive HF [20].
In addition to dose and duration of ISP treatment, other considerations when developing ISP driven HF models are route of administration and delivery method. The route by which a compound is administered, can affect its bioavailability, distribution, metabolism, and pharmacological effect on the heart and other organs [21, 36–39]. The delivery, in turn, can modulate absorption rate, steady state concentration of the compound, and risk of complications. In rodents, ISP is commonly administered through a SQ or intraperitoneal (i.p.) route. However, ISP is delivered via daily injections or mini-pump infusions [5, 21, 27, 29, 32]. While previous studies compare exposure to ISP via distinct routes of administration in rodents, it remains unclear whether the extent of cardiac remodeling and dysfunction following ISP administration differs between delivery methods in C57BL/6J (B6-J) mouse models. In summary, variability in dose, treatment duration, route of administration, and delivery method has hindered efforts to develop reproducible mouse models of ISP driven HCM and HF. Thus, establishing an optimal dose and delivery system in combination with the use of genetic modifications is crucial to the development of translationally relevant mouse models of cardiovascular disease. Here, we sought to 1) determine a low ISP dose (2, 4, and 10mg/kg/day) at which myocardial injury and dysfunctional effects occur, and 2) assess whether daily subcutaneous (SQ) injections and SQ-minipump (SMP) infusions induce comparable disease severity in B6-J mice.
Results
Isoproterenol induces contrasting ECG parameters that are modulated by delivery method
To determine whether delivery method, ISP dose, or the combination of both altered the pattern of electrical activity in the hearts of both SQ and SMP treated groups, anesthetized mice were placed on a warm pad with subcutaneous electrodes in a 3-lead ECG configuration. First, we found that the saline-treated SQ group was not significantly different to the saline-treated SMP group for most ECG measures except for R amplitude (Fig 1A–1J and S1 Table). Then, we examined the effect of ISP doses via each delivery method (SQ and SMP treated groups divided by dotted line). Heart rate (HR) was modestly decreased compared to saline in the SQ group by 2 and 4 mg/kg of ISP (ns trend), and significantly decreased by 10mg/kg of ISP (Fig 1A, Left). ISP treatment via SMP, however, induced a modest HR increase at 2 and 4 mg/kg (ns trend), and significantly increased HR at the 10 mg/kg dose (Fig 1A, Right). Thus, HR is dose dependently decreased by ISP in the SQ group, but dose dependently increased by ISP in the SMP group. When comparing the combination of dose and mode of delivery between groups, we found that for all doses, the SMP group had significantly higher HRs compared to the SQ group (S1 Table). Concomitant with HR dynamics, the RR interval was moderately lengthened in the SQ group compared to saline by 2 and 4 mg/kg of ISP (ns trend), and significantly increased by 10 mg/kg of ISP (Fig 1B, Left). In the SMP group, 2 mg/kg of ISP modestly shortened RR interval duration (ns trend), and significantly decreased RR interval duration at 4 and 10 mg/kg of ISP (Fig 1B, Right). In addition, when treated with either 2, 4 or 10mg/kg of ISP, SMP groups exhibit significantly shorter RR intervals when compared to SQ groups (S1 Table). P duration, and PR, QRS, and QT intervals were not altered by ISP doses in either the SQ or SMP groups when compared to respective saline controls, or to alternative delivery method groups (Fig 1C–1F and S1 Table). P amplitude was also unaltered by ISP dose and delivery method within and outside delivery method group (Fig 1G and S1 Table). Q wave amplitude is significantly increased at 10 mg/kg of ISP when compared to 2 mg/kg (Fig 1H) in SMP groups, suggesting that higher ISP doses induce greater ventricular contractions. R and S wave amplitudes are not significantly changed by all 3 doses of ISP when compared to their respective saline controls in both SQ and SMP treated mice (Fig 1I and 1J). However, mice in the SMP group treated with all ISP doses had significantly shorter R wave amplitude when compared to the respective dose groups treated via SQ daily injections (S1 Table). This trend was repeated with S amplitude, but only at 4 and 10 mg/kg of ISP. These results show that all three doses of ISP induce contrasting HR, RR Interval duration, and R and S amplitude that may be modulated by delivery method. Representative waterfall plots of the full recording period show overall changes in ECG waves for each dose (S1 Fig). Overall, these data indicate that delivery method, particularly methods that require surgery, can modestly modulate ECG baseline measures within 14 days of procedure, and that the combination of either SQ daily injections or SMP infusions and ISP dose induces a significant opposing effect that is evident in HR, RR interval, and R and S amplitudes.
For all graphs, both SQ and SMP treated groups are presented in the same X axis but are separated by a dotted line. SQ group is on left side and SMP group is on right side of dotted line. Symbols represent number of mice in each cohort (n = 4). A) heart rate, B) RR interval, C) PR interval, D) P duration, E) QRS interval, F) QT interval, G) P amplitude, H) Q amplitude, I) R amplitude, and J) S amplitude. Each boxplot represents min to max showing all points, line at median and plus sign at mean. RR interval, heart rate, P duration, Q, R and S amplitude data were analyzed using ordinary one-way ANOVA with Sidak’s multiple comparison’s test. PR, QRS, and QT interval, and P amplitude data were analyzed using Kruskal-Wallis ANOVA with Dunn’s multiple comparison’s test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
Isoproterenol treatment increases heart wall thickness and subcutaneous mini-pump implanted mice develop hypertrophic hearts
We next examined whether delivery method, ISP dose, or a combination of both, modified heart function through echocardiography (Fig 2A–2I and S2 Table, S1 and S2 Figs). When comparing SQ and SMP mice treated with saline alone, we found no significant differences in ejection fraction, fractional shortening, left ventricular anterior and posterior wall thickness at diastole, left ventricular internal diameter at diastole, and left ventricular end diastolic volume (Fig 2A–2E and S2 Table). However, mice treated with saline via SMP infusion had significantly higher left ventricular mass compared to mice treated with saline via SQ injection (Fig 2G and S2 Table). These data suggest that 2 weeks post-surgery, mice treated via SMPs may be at higher risk for cardiac dysfunction at baseline than mice being treated via SQ injections.
For all graphs, symbols represent the number of mice in each cohort (n = 8). A) % ejection fraction, B) % fractional shortening, C) diastolic left ventricular anterior wall thickness, D) diastolic left ventricular posterior wall, E) diastolic left ventricular internal diameter, F) left ventricular end diastolic volume, G) left ventricular mass, saline treated mice only, F) left ventricular mass SQ only, and G) left ventricular mass SMP only. In A-F, both SQ and SMP treated groups are presented in the same X axis but are separated by a dotted line. SQ group is on left side of dotted line and SMP group is on right side. Each boxplot represents min to max showing all points, line at median and plus sign at mean. Data in A, C, E-G were analyzed using ordinary one-way ANOVA with Sidak’s multiple comparison’s test and data in B and D were analyzed using Kruskal-Wallis ANOVA with Dunn’s multiple comparison’s test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
To determine the effect of ISP on cardiac function via different delivery methods, we then evaluated mice treated with increasing doses of ISP (Fig 2A–2F and 2H, 2I). Within SQ or SMP groups (divided by dotted line), all 3 ISP doses did not impact ejection fraction, fractional shortening, left ventricular anterior wall thickness at diastole, left ventricular internal diameter at diastole, or left ventricular end diastolic volume when compared to respective saline controls (Fig 2A–2C and 2E, 2F). However, we found that left ventricular posterior wall thickness at diastole was significantly increased in SMP group treated with 2 and 4 mg/kg of ISP (Fig 2D, Right), and that at 10 mg/kg of ISP, left ventricular mass was also increased for SMP group (Fig 2I). In the SQ treated group, we found a significant increase in LV Mass when mice were treated with 4 mg/kg of ISP (Fig 2H). These results suggest that 2 weeks of ISP doses up to 10 mg/kg via both SQ and SMP delivery is not sufficient to robustly impact functional myocardial dynamics.
Lastly, SQ and SMP groups significantly differ at specific ISP doses; ejection fraction across all doses, fractional shortening at 2 and 10 mg/kg, left ventricular anterior wall thickness across all doses, and left ventricular posterior wall thickness at 2 and 4 mg/kg (S2 Table). Taken together, these data suggest that at 2 weeks, mice treated with ISP doses up to 10 mg/kg via SMP may experience more detrimental cardiac phenotypes when compared to SQ treated mice.
Subcutaneous mini-pump implanted mice have greater body, lung, and heart weights, and Isoproterenol treatment aggravates these effects
We then examined whether delivery method alone altered body, lung, and heart weight while mice were being treated with saline via daily SQ injections or SMP infusions (Fig 3A–3C). We found that % body, lung, and heart weight is significantly increased in mice treated via SMP (Fig 3A–3C), suggesting that SMP implanted mice may have higher disease burden at baseline compared to SQ treated mice. Since delivery method induced significant changes in % body, lung, and heart weight, we next assessed response to ISP doses within delivery method groups separately. We found that % body weight did not change for mice treated via daily SQ injections when compared to saline (Fig 3D) but was further increased from saline with all 3 ISP doses in the SMP group (Fig 3E). In addition, the magnitude of body weight change compared to saline treated mice was greater for SMP treated mice. Lung weight under all 3 ISP doses was not significantly different from saline controls in both the SQ and SMP groups (Fig 3F and 3G). However, ISP treatment significantly increased heart weight for both SQ and SMP treated groups when compared to saline treated mice in the respective groups (Fig 3H and 3I). Taken together, these data suggest that changes in body, lung, and heart weight are modulated by delivery method, and that treatment with ISP exacerbates these alterations. After 2 weeks of treatment, SMP implanted mice showed an overall increase in body, lung, and heart weight that was equivalent for all 3 doses of ISP.
Top of figure contains legend. For A-I, symbols represent number of mice in each cohort (n = 8). A-C show saline treated mice only. A) % body weight change over 2 weeks, B) lung weight, C) heart weight over tibia length. Each boxplot represents min to max showing all points, line at median and plus sign at mean. Data were analyzed with unpaired t-test. D-I show saline, 2, 4, or 10mg/kg ISP treated mice. On the left, SQ treated mice and on the right, SMP treated mice. D) SQ group % body weight change, E) SMP group % body weight change, F) SQ group lung weight, G) SMP group lung change, H) SQ group heart weight over tibia length, and I) SMP group heart weight over tibia length. Each boxplot represents min to max showing all points, line at median and plus sign at mean. Data were analyzed using ordinary one-way ANOVA with Sidak’s multiple comparison’s test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
Isoproterenol promotes expression of hypertrophic and fibrotic gene markers
Lastly, we examined whether ISP and/or delivery method induced cellular markers of hypertrophy and fibrosis in the mouse myocardium. We found that delivery method alone had no significant effect on hypertrophic and fibrotic markers tested in heart tissue, as saline only comparison between SQ and SMP groups was not significantly different (Fig 4A–4E and S3 Table). Within SQ or SMP groups however (separated by dotted line), we found that ISP had a dose-dependent effect. Hypertrophic marker smooth muscle alpha-2 actin (Acta2) was significantly increased in the SQ group only at ISP 10mg/kg, but in the SMP group ISP increased Acta2 fold expression at all 3 doses (Fig 4A). When examining beta-myosin heavy chain (Myh7), another hypertrophic marker, we only observe an increase in the SQ group at ISP 4mg/kg, and a non-significant increase trend for the SQ group overall that we did not observe in the SMP group (Fig 4B). We also found that the hypertrophic marker periostin (Postn), is increased in SQ group at ISP 2mg/kg (Fig 4C, Left). In the SMP group, we found that ISP treatment increases Postn expression (ns trend), indicating that the combination of SMP and any of the 3 ISP doses induces fibrogenesis with 2 weeks of exposure (Fig 4C, Right). These results suggest that the combination of SMP and a low ISP dose promotes a more salient hypertrophic and fibrogenic state.
For A-E, symbols represent number of mice in each cohort (n = 3–4). On the left of dotted line, triangles represent mice treated via SQ and on the right of dotted line, circles represent mice treated via SMP. A) Acta2, B) Myh7 C) Postn, D) Nppa, and E) Nppb. Each boxplot represents min to max showing all points, line at median and plus sign at mean. Data were analyzed using ordinary two-way ANOVA with Tukey’s multiple comparison’s test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
We then assessed cardiac stress by examining natriuretic peptide A (Nppa) and natriuretic peptide B (Nppb) in the ventricular myocardium and found that in the SQ treated group ISP at 2 and 4 mg/kg increased Nppa (Fig 4D, Left) and that ISP 10 mg/kg decreased Nppb (Fig 4E, Left). We found no significant differences in the SMP group for both Nppa and Nppb expression (Fig 4D and 4E, Right). These results suggest that in the SQ group, ISP daily injections are promoting maladaptive hypertrophy, fibrosis, and vascular remodeling. We also found that these effects are mediated solely by ISP, and not by delivery method (S3 Table). Taken together, these results confirm the previously reported effects of low dose ISP on inducing hypertrophy and fibrotic states, and these effects are independent from delivery method.
Discussion
ISP is a non-selective β-adrenoreceptor agonist that is commonly used in rodent studies to induce acute and chronic HCM and exacerbate the development of HF [16–18]. However, the variability in both dose and route of administration reported in the literature has made it difficult to determine the optimal combination of ISP dose and delivery method to develop a reliable ISP induced injury mouse model. Notably, different mouse strains show significant differences in susceptibility to ISP and cardiac damage, thus confounding findings when ISP is used as a second hit alongside genetic manipulations to induce myocardial injury and create translationally relevant models of disease [40–43]. B6 mice on an N background, for example, are more susceptible to oxidative stress and cardiac injury compared to B6-J mice due to a mutation in the nicotinamide nucleotide transhydrogenase enzyme that results in ameliorated pressure-overload phenotypes in B6-J mice [44]. Since B6-J mice may have lower susceptibility to ISP, we established 2mg/kg as the lowest dose for this study. Here we administered 3 doses of ISP (2, 4, or 10mg/kg/day) over two weeks to demonstrate that male B6-J mice have a similar risk for cardiac injury via SQ and SMP delivery methods. Surprisingly, mice implanted with SMP showed decreased R wave amplitude and increased LV mass when given saline, suggesting that implanting a minipump to administer compounds can modulate physiological ECG and Echo responses at baseline. This may be due to altered systemic hemodynamics that are dependent on mode of delivery [32], as SMP surgery/implantation may elicit responses from the surrounding tissues that could alter normal physiological fluid balance that lead to edema as a secondary outcome. In addition, the combination of either SQ injections or SMP infusions and ISP dose induces a significant opposing effect that is evident in ECG measures. We also found that having a SMP promoted the development of hypertrophy, advanced enlargement of heart and lung tissue, and increased body weight. In addition, ISP treatment, at all 3 doses, additively aggravated the effects observed when mice were treated with saline only. Lastly, we found that ISP induced differential cardiac remodeling and expression of hypertrophic and fibrotic markers that were modulated by mode of delivery. Taken together, these data suggest that a low dose (up to 10 mg/kg) of ISP maintained via SQ and SMP for two weeks is sufficient to induce cardiac hypertrophy in C57BL/6J models, and that daily injections and continuous infusion of ISP into the SQ space are good models of intermittent and chronic β-adrenergic stimulation.
ISP is routinely used to induce myocardial injury and promote the development of HCM and HF in diverse mouse strains [42, 45, 46]. Indeed, a single high ISP dose, as is used in Takotsubo syndrome models, leads to the development of acute cardiac stress, severe cardiac dysfunction, and robust myocardial infiltration of macrophages, ischemia, and HF [20, 47, 48]. In their extensive review, Nichtova et al. (2012) classify the effects of ISP in the literature according to low, medium, and high doses of ISP in rodent models [20]. Since one of our goals was to identify a low ISP dose that could induce myocardial injury and cardiac disease in B6-J mice, we used ISP doses in the low-medium category according to this report. Here we found that when B6-J mice are exposed to two weeks of daily treatment, a low dose up to 10 mg/kg is sufficient to induce limited electrophysiological, functional, and molecular changes in the myocardium. These alterations, moreover, were modulated by delivery method.
In mice, ISP is commonly delivered into the SQ or i.p. space with either daily injections or minipump infusions [5, 21, 27, 29, 32]. In this study, SQ route was determined as the ideal route of administration, as it avoids the pharmacokinetics associated with enteral absorption, and allows for injection of larger volume fluids in small animals with minimal technical challenge [49]. Thus, both SQ and SMP groups received ISP into the SQ space, maintaining a consistent route of administration. However, because of the nature of these techniques, mice were exposed to ISP either intermittently (SQ) or continuously (SMP), altering delivery method.
Both intermittent and sustained patterns of β-adrenoreceptor stimulation contribute to the development of HF with varying degrees of cardiac remodeling [29, 50]. Daily ISP injections are described to mimic stress-induced cardiomyopathy, as ISP has a short half-life (2–5 minutes) and induces rapidly reversible acute left ventricular systolic dysfunction in the absence of heart disease [51–53]. In contrast, continuous infusion, and sustained exposure to ISP mimics advanced HF where there is chronic adrenergic stimulation. This approach is limited by the need for surgery expertise and infusion timeframe, as mini-pumps used here can continuously provide the studied compound for only 21 days. Both acute and chronic exposure to ISP are posited to induce receptor desensitization and downregulation [54]. Here, we found that B6 mice treated via SQ daily injections have decreased HR when compared to saline controls and SMP infused mice. An explanation for this is that we injected mice with ISP in the afternoon, but recorded ECGs in the following morning, thus we captured the response to ISP hours after bolus injection and period of circulation and metabolism. This result also suggests that administering ISP via bolus SQ injections may elicit receptor desensitization and downregulation as observed in a similar report [41]. As observed in other studies, however, SMP infusion of ISP increases and maintains HR at a higher level, also suggesting that continuous infusion of ISP may mask the initial compensatory adaptive response to the insult [29, 55]. As HR and RR interval are inversely correlated, we found the opposite effect on RR interval duration. We also found other typical characteristics of hypertrophy observed in ECGs from anesthetized rodents and isolated hearts such as longer QT duration and decreased R amplitude in SMP treated mice [56–58]. Overall, these results indicate that the combination of delivery method and low ISP dose influences the mechanism of disease development.
When examining cardiac function via echocardiography, we found that SQ and SMP ISP delivery elicited similar effects. Although there is no evident contractile impairment in mice treated via the two delivery methods, mice treated via SMP may show more significant morphological changes due to the additive development of increased heart wall thickness and mass in this timeframe. These alterations may be due to physiological hypertrophy with increased myocyte size, myocyte proliferation, connective tissue remodeling, or edema. However, our experimental design does not directly distinguish among these different modes of increased ventricular mass. These results suggest that administering ISP with different pharmacologic protocols could induce increases in ventricular weight by different stimuli that manifest with different molecular and cellular mechanisms [32]. Another explanation is that the responses elicited by ISP in 2 weeks are part of signaling pathways characteristic for adaptation [59]. Indeed, mice being treated with 10mg/kg of ISP via SMP for 26 days maintained cardiac function, indicating a possible cardioprotective effect during chronic activation of β-adrenergic receptors [55]. As expected, ISP up to 10 mg/kg increased heart weight in mice treated via both SQ and SMP. However, body weight and wet lung weight were increased in mice treated via SMP at baseline, suggesting the possibility of fluid accumulation as a response to the SMP implant and surgery insult. However, to conclusively determine peripheral and/or pulmonary edema in these mice, it would be best to measure inflammatory cytokines and the ratio of wet to dry lung in future studies.
Since cardiac hypertrophy is an adaptive response of the myocardium to pressure overload or adrenergic agonists, it is characterized by increased heart size, upregulated protein synthesis, induction of fetal gene program, and aberrant organization of the sarcomere structure [60]. In line with well documented ISP effects on cardiac gene expression, we assessed reactivation of the fetal program to find cardiac remodeling and expression of hypertrophic and fibrotic markers in both SQ and SMP treated mice with all doses. Characteristically, ISP induces upregulation of Myh7, Acta2, Postn, as well as ventricular expression of Nppa and Nppb. Upregulation of these gene markers indicate increases in cardiac stress, muscle contraction, and fibrosis. In mice treated via both SQ and SMP, Acta2 was increased, but this increase was more pronounced in SMP treated mice. This may be due to the additive insult of ISP delivered continuously through the SMP. Interestingly, we did not observe this effect with other markers tested, however, we only found significant increases in gene expression of MyH7, Postn, Nppa, and Nppb in mice treated with ISP via SQ. Interestingly, a recent article explored the effect of 10mg/kg ISP withdrawal in B6-N mice after 26 days of continuous infusion via SMP to find that the induction of maladaptive cardiac remodeling mechanisms, including a switch to pathological gene expression, were counteracted by adaptive responses while ISP was being infused [55]. Notably, responses here may be limited due to the partial recapitulation of complex neurohumoral overactivation and cardiac remodeling mechanisms induced by ISP. Studies show that the combination of ISP and chronic angiotensin II or α-adrenergic stimulation with phenylephrine further mimic the pathophysiological sympathetic overdrive in HF, leading to more pronounced fibrotic phenotypes via increased cytokine release and pressure-overload driven early transcriptome alterations [26, 61–64]. These results are in line with studies showing that ISP only elicits mild pro-fibrotic effects in B6-J mice [41, 65], and suggest that mode of drug delivery may influence the signaling pathway and mechanism of disease development [29, 66].
Although the results here provide clarity on the intricacies of using low ISP doses, and the implications of using different modes of delivery, this study has limitations to consider. We only used male mice here, thus we cannot draw conclusions on the possibility of sex-related differences in the modulation of ISP dose response and mode of delivery. A recent study, however, reported lack of significant sex differences in ISP cardiac hypertrophy, dysfunction, and fibrosis in C57BL/6N mice, suggesting that female sex may not be sufficient to protect the heart against a severe pathologic stimulus and that sexual dimorphism in cardiovascular diseases is highly model-dependent [32, 67]. However, we used B6-J mice here, which may impact sex-related differences due to strain background. Of note, B6-J mice have a nicotinamide nucleotide transhydrogenase (Nnt) mutation that limits production of reactive oxygen species and ameliorates the development of pressure overload-induced heart failure [44]. This may explain why we did not find overt cardiac disease phenotypes here and highlights the need for more cardiac β-adrenergic receptor function studies in female preclinical models. We also consider the number of mice used here and the variability in data as limitations on statistical power. In addition, this study design only presents results from a single timepoint (2 weeks after ISP treatment) to measure disease, but remodeling is a progressive, dynamic response to injury that develops with time, and collecting data from further timepoints may have revealed more salient disease phenotypes in these mice [68]. Additionally, this study focused only on functional and physiological methods of assessment including ECG, echocardiography, and body and wet organ weights without delving into histopathological changes induced by ISO in the two groups, as these findings have been previously reported [69–73]. Previous studies also report cardiomyocyte changes in response to β-adrenergic agonism, confirming that acute and chronic adrenergic activation leads to oxidative stress, and cardiomyocyte injury and loss [24, 74–80]. Finally, while this study illustrated induction of significant myocardial dysfunction in both groups, there were differences between the two groups in some ECG, diastolic function, and molecular markers that this study was not sufficiently powered to establish.
In summary, this study demonstrates that in B6-J mice, both intermittent and sustained stimulation of β-adrenergic receptors for 2 weeks cause similar pathological phenotypes in the heart. One important aspect to consider for future studies is that although serial injections may be the preferred mode of administration due to being a less challenging technique that is also lower cost and permits better animal mobility [32], SMP implantation adds a layer of physiological complexity that may be necessary to mimic specific disease pathways. In this study, we found that mice with implanted SMPs showed greater body, lung, and heart weight, which may be due to changed hemodynamics, inflammation induced by surgery, and edema that was not present in SQ treated mice. Or findings significantly further our understanding of β-adrenergic stimulation in relation to cardiac pathophysiology, highlighting the importance of the mode of drug delivery and ISP dosage level.
Materials and methods
Mice
8–10-week-old male C57BL/6J mice were purchased from Jackson Labs (n = 64) and used for all experiments over the course of 30 days. Mice were maintained on Inotiv 2916 rodent diet (Teklad global 16% protein) and Innovive chlorinated water (M-WB-300C) and housed in a light (12hr light/ 12hr dark) and temperature (25°C) controlled environment. All procedures were carried out and approved in accordance with the Pfizer Institutional Animal Care and Use Committee (IACUC) regulations and established guidelines.
Isoproterenol preparation and dosing
Mice were randomized by weight and classified into either a daily SQ injection (n = 32) or an SMP continuous infusion (n = 32) group. Within each group, mice were then randomly assigned to 1 of 8 dosing cohorts: SQ saline 10mL/kg, SMP saline 10mL/kg, SQ 2mg/kg, SMP 2mg/kg, SQ 4mg/kg, SMP 4mg/kg, SQ 10mg/kg or SMP 10mg/kg (n = 8 for each group). Confounders for animal cage/location, treatment order, and measurements were not evaluated. Mice were treated for 14 consecutive days. ISP SQ and SMP solutions were prepared by serially diluting ISP (Millipore Sigma, Sigma-Aldrich #16504) into sterile saline. ISP aliquots for SQ administration were covered from light and kept at -20°C until thawed before injection. SQ cohort received injections to the left flank at approximately the same time each day 3hrs before start of dark cycle. Prior to injection, the site between ribs and hind leg was cleaned with an alcohol swab, and a 26-gauge syringe was inserted into the SQ space at a 15–30-degree angle. Saline or ISP dose was injected steadily, and a 3–5 second pause after injection was implemented to prevent loss of compound.
Body weights
Full body weights were measured a week before treatment start to permit randomization by weight into distinct groups. Full body weights were then taken again once on the day of first injection or surgery to obtain baseline measures, and 14 days later at end of study.
Osmotic mini-pump implantation
Under sterile conditions, mice were subcutaneously implanted in the right flank with an osmotic mini-pump (Alzet Model 1002) for continuous infusion during the experiments. Isoflurane was used as anesthetic during implantation surgery. The skin incision (<1–1.25cm) was created over the left scapula region and closed with silk suture. A subcutaneous injection of carprofen in the form of MediGel CPF (Clear H2O or Meloxicam SR) and Buprenorphine were used as pain medication for pre-op and post-op care. Mice were recovered from surgery in an incubator in home cage and were removed from the incubator once ambulatory.
Surface electrocardiography
Only 50% of mice were recorded for this experiment (SC n = 16, SMP n = 16). Mice were anesthetized with 3% isoflurane in induction chamber and maintained at 2% via nosecone. Extremities were secured with paper tape to a warming pad (VisualSonics Mouse Handling Platform 2, maintaining temperature at 37°C for the duration of recording), and needle electrodes (AD instruments 12mm long, 29 gauge) connected to a preamplifier (AD instruments Power Lab 16/35 and Bio amp) were inserted into the subcutaneous space in a lead II configuration. Sampling rate was 4k/s and heart rate was monitored throughout. Recording lasted for 5 min, last 2min were analyzed for graphs. Mice were recovered on a heated pad in home cage. Data was recorded in LabChart 8 and analyzed with LabChart Pro v8.1, where settings were manually established to reflect every beat (no averaging), Bazett QTc, typical QRS width of 10ms, R waves at least 60ms apart aligned to QRS maximum, a Pre-P baseline and maximums of 20, 80, and 60ms, and an ST height at 10ms from alignment.
Echocardiography
Mice were under 1.5–2% sevoflurane anesthesia while recordings were taken with the Vevo 3100 Preclinical Imaging System (FUJIFILM VisualSonics Inc, Toronto, Canada). B-mode images from long axis and M-mode images from short axis were obtained, and data was analyzed using Graphpad Prism version 9.5.1 for Windows (GraphPad Software, San Diego, California USA).
Tissue Collection: After sublethal CO2 inhalation and cervical dislocation, heart, lung, and tibia were collected. Heart: ventricles were weighted and then left ventricle was immediately snap-frozen on liquid nitrogen and stored at -80˚C for molecular biology. Lungs were weighted and tibias measured for length and subsequently discarded.
Gene expression
Heart tissue was dissected and immediately snap frozen on liquid nitrogen and stored at -80˚C. RNA was then extracted using Trizol (Invitrogen) and 200 ng samples were converted to cDNA using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen). Sample cDNAs were analyzed in triplicate via quantitative RT-PCR for gene expression using TaqMan reagents and an ABI 7500 (Applied Biosystems). With GAPDH expression as an internal control, relative mRNA expression values were calculated by the 2-ΔΔCt method.
Humane endpoint procedures
All mice were euthanized approximately 2 weeks after 1st isoproterenol dose administration, unless early markers associated with death, poor prognosis of quality of life, or specific signs of distress were observed during daily monitoring. During surgery and recovery period, mice were provided with post-operative pre- and post-operative analgesia treatment as needed. Health condition was assessed based on breathing, body condition, activity level, hydration, wound healing, body weight. Monitoring personnel consulted with veterinary services if an animal was found abnormal in health status check and appropriate treatment was performed based veterinary services recommendations. One mouse in the SQ 10mg/kg group was euthanized before conclusion of the study due to health concern, data from this mouse was not included.
Statistics
All values are presented as boxplots showing min to max and all points, line at median and plus sign at mean. Normally distributed data with equal standard deviation was analyzed with unpaired student’s t-test or ordinary one-way ANOVA with Sidak’s multiple comparison’s test. Abnormally distributed data, and data with unequal standard deviation was analyzed with unpaired Mann-Whitney test, Kruskal-Wallis ANOVA with Dunn’s multiple comparison’s tests, or Welch’s ANOVA with Dunnett’s T3 multiple comparison’s test. An alpha of 0.05 was used to determine statistical significance. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.
Supporting information
S1 Fig. Surface ECG waterfall plots.
Each waterfall is a 3D plot of averaged beats over during 2min ECG recording. Each horizontal layer of the plot correspond to a full ECG cycle, with the earliest recordings at the top, and the latest at the bottom. Intermittent irregularities in the waves are indicators, but not determinants, of cardiac pathologies.
https://doi.org/10.1371/journal.pone.0307467.s001
(TIF)
S2 Fig. SQ echocardiography representative images.
Echocardiography representative images for all SQ groups.
https://doi.org/10.1371/journal.pone.0307467.s002
(TIF)
S3 Fig. SMP echocardiography representative images.
Echocardiography representative images for all SMP groups.
https://doi.org/10.1371/journal.pone.0307467.s003
(TIF)
S1 Table. Surface ECG statistics for comparison of mice in SQ vs SMP groups.
Statistical tests used to compare between SQ and SMP groups treated with saline and all ISP doses showing mean and summary values.
https://doi.org/10.1371/journal.pone.0307467.s004
(PDF)
S2 Table. Heart function assessed via echocardiography comparison of mice in SQ vs SMP groups.
Statistical tests used to compare between SQ and SMP groups treated with saline and all ISP doses showing mean and summary values.
https://doi.org/10.1371/journal.pone.0307467.s005
(PDF)
S3 Table. Gene expression changes comparison of mice in SQ vs SMP groups.
Statistical tests used to compare between SQ and SMP groups treated with saline and all ISP doses showing mean and summary values.
https://doi.org/10.1371/journal.pone.0307467.s006
(PDF)
S4 Table. Raw data for all figures.
Data behind means and averages represented in the manuscript.
https://doi.org/10.1371/journal.pone.0307467.s007
(XLSX)
Acknowledgments
We thank Abigail Hunter for surface ECG training, and the Comparative Medicine technicians and veterinary staff at Pfizer.
References
- 1. Shahim B, Kapelios CJ, Savarese G, Lund LH. Global Public Health Burden of Heart Failure: An Updated Review. Card Fail Rev. 2023;9:e11. Epub 20230727. pmid:37547123; PubMed Central PMCID: PMC10398425.
- 2. Tsao CW, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL, et al. Heart Disease and Stroke Statistics-2023 Update: A Report From the American Heart Association. Circulation. 2023;147(8):e93–e621. Epub 20230125. pmid:36695182.
- 3. Disease GBD, Injury I, Prevalence C. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018;392(10159):1789–858. Epub 20181108. pmid:30496104; PubMed Central PMCID: PMC6227754.
- 4. Tsao CW, Aday AW, Almarzooq ZI, Alonso A, Beaton AZ, Bittencourt MS, et al. Heart Disease and Stroke Statistics-2022 Update: A Report From the American Heart Association. Circulation. 2022;145(8):e153–e639. Epub 20220126. pmid:35078371.
- 5. Chang SC, Ren S, Rau CD, Wang JJ. Isoproterenol-Induced Heart Failure Mouse Model Using Osmotic Pump Implantation. Methods Mol Biol. 2018;1816:207–20. pmid:29987822; PubMed Central PMCID: PMC6445258.
- 6.
Maron B. J. TG. Classification of cardiomyopathies. In: WRA Fuster V., Harrington R. A., editor. Hurst’s The Heart. 13 ed. New York: McGraw Hill; 2011.
- 7. Olivotto I, Cecchi F, Poggesi C, Yacoub MH. Patterns of disease progression in hypertrophic cardiomyopathy: an individualized approach to clinical staging. Circ Heart Fail. 2012;5(4):535–46. pmid:22811549.
- 8. Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy progress to systolic heart failure? Am J Physiol Heart Circ Physiol. 2005;289(1):H8–H16. pmid:15961379.
- 9. Bai Y, Zheng JP, Lu F, Zhang XL, Sun CP, Guo WH, et al. Prevalence, incidence and mortality of hypertrophic cardiomyopathy based on a population cohort of 21.9 million in China. Sci Rep. 2022;12(1):18799. Epub 20221105. pmid:36335106; PubMed Central PMCID: PMC9637201.
- 10. Karim S, Chahal CAA, Sherif AA, Khanji MY, Scott CG, Chamberlain AM, et al. Re-evaluating the Incidence and Prevalence of Clinical Hypertrophic Cardiomyopathy: An Epidemiological Study of Olmsted County, Minnesota. Mayo Clin Proc. 2024;99(3):362–74. Epub 20240206. pmid:38323940.
- 11. Finocchiaro G, Magavern E, Sinagra G, Ashley E, Papadakis M, Tome-Esteban M, et al. Impact of Demographic Features, Lifestyle, and Comorbidities on the Clinical Expression of Hypertrophic Cardiomyopathy. J Am Heart Assoc. 2017;6(12). Epub 20171213. pmid:29237589; PubMed Central PMCID: PMC5779031.
- 12. Lorell BH, Carabello BA. Left ventricular hypertrophy: pathogenesis, detection, and prognosis. Circulation. 2000;102(4):470–9. pmid:10908222.
- 13. Frank D, Kuhn C, Brors B, Hanselmann C, Ludde M, Katus HA, et al. Gene expression pattern in biomechanically stretched cardiomyocytes: evidence for a stretch-specific gene program. Hypertension. 2008;51(2):309–18. Epub 20071224. pmid:18158353.
- 14. Yung CK, Halperin VL, Tomaselli GF, Winslow RL. Gene expression profiles in end-stage human idiopathic dilated cardiomyopathy: altered expression of apoptotic and cytoskeletal genes. Genomics. 2004;83(2):281–97. pmid:14706457.
- 15. Noll NA, Lal H, Merryman WD. Mouse Models of Heart Failure with Preserved or Reduced Ejection Fraction. Am J Pathol. 2020;190(8):1596–608. Epub 20200425. pmid:32343958; PubMed Central PMCID: PMC7416075.
- 16. Balakumar P, Singh AP, Singh M. Rodent models of heart failure. J Pharmacol Toxicol Methods. 2007;56(1):1–10. Epub 20070223. pmid:17391988.
- 17. Gomes AC, Falcao-Pires I, Pires AL, Bras-Silva C, Leite-Moreira AF. Rodent models of heart failure: an updated review. Heart Fail Rev. 2013;18(2):219–49. pmid:22446984.
- 18. Ponzoni M, Coles JG, Maynes JT. Rodent Models of Dilated Cardiomyopathy and Heart Failure for Translational Investigations and Therapeutic Discovery. Int J Mol Sci. 2023;24(4). Epub 20230205. pmid:36834573; PubMed Central PMCID: PMC9963155.
- 19. Maass A, Leinwand LA. Animal models of hypertrophic cardiomyopathy. Curr Opin Cardiol. 2000;15(3):189–96. pmid:10952427.
- 20. Nichtova Z, Novotova M, Kralova E, Stankovicova T. Morphological and functional characteristics of models of experimental myocardial injury induced by isoproterenol. Gen Physiol Biophys. 2012;31(2):141–51. pmid:22781817.
- 21. George JC, Liner A, Hoit BD. Isoproterenol-induced myocardial injury: a systematic comparison of subcutaneous versus intraperitoneal delivery in a rat model. Echocardiography. 2010;27(6):716–21. Epub 20100325. pmid:20345437.
- 22. Zimmer HG. Catecholamine-induced cardiac hypertrophy: significance of proto-oncogene expression. J Mol Med (Berl). 1997;75(11–12):849–59. pmid:9428617.
- 23. Suzuki M, Ohte N, Wang ZM, Williams DL, Little WC Jr., Cheng CP. Altered inotropic response of endothelin-1 in cardiomyocytes from rats with isoproterenol-induced cardiomyopathy. Cardiovasc Res. 1998;39(3):589–99. pmid:9861301.
- 24. Zou Y, Komuro I, Yamazaki T, Kudoh S, Uozumi H, Kadowaki T, et al. Both Gs and Gi Proteins Are Critically Involved in Isoproterenol-induced Cardiomyocyte Hypertrophy. Journal of Biological Chemistry. 1999;274(14):9760–70. pmid:10092665
- 25. Nagano M, Higaki J, Nakamura F, Higashimori K, Nagano N, Mikami H, et al. Role of cardiac angiotensin II in isoproterenol-induced left ventricular hypertrophy. Hypertension. 1992;19(6_pt_2):708–12. pmid:1534315
- 26. Baillard C, Mansier P, Ennezat PV, Mangin L, Medigue C, Swynghedauw B, et al. Converting Enzyme Inhibition Normalizes QT Interval in Spontaneously Hypertensive Rats. Hypertension. 2000;36(3):350–4. pmid:10988263
- 27. Leenen FH, White R, Yuan B. Isoproterenol-induced cardiac hypertrophy: role of circulatory versus cardiac renin-angiotensin system. Am J Physiol Heart Circ Physiol. 2001;281(6):H2410–6. pmid:11709406.
- 28. Rajadurai M, Stanely Mainzen Prince P. Preventive effect of naringin on cardiac mitochondrial enzymes during isoproterenol-induced myocardial infarction in rats: A transmission electron microscopic study. Journal of Biochemical and Molecular Toxicology. 2007;21(6):354–61. pmid:17994577
- 29. Ma X, Song Y, Chen C, Fu Y, Shen Q, Li Z, et al. Distinct actions of intermittent and sustained β-adrenoceptor stimulation on cardiac remodeling. Science China Life Sciences. 2011;54(6):493–501. pmid:21706409
- 30. Lan T, Zeng Q, Jiang W, Liu T, Xu W, Yao P, et al. Metabolism disorder promotes isoproterenol-induced myocardial injury in mice with high temperature and high humidity and high-fat diet. BMC Cardiovasc Disord. 2022;22(1):133. Epub 20220330. pmid:35350989; PubMed Central PMCID: PMC8966251.
- 31. Judge LM, Perez-Bermejo JA, Truong A, Ribeiro AJ, Yoo JC, Jensen CL, et al. A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress. JCI Insight. 2017;2(14). Epub 20170720. pmid:28724793; PubMed Central PMCID: PMC5518554.
- 32. Hohimer AR, Davis LE, Hatton DC. Repeated daily injections and osmotic pump infusion of isoproterenol cause similar increases in cardiac mass but have different effects on blood pressure. Can J Physiol Pharmacol. 2005;83(2):191–7. pmid:15791293.
- 33. Lijnen PJ, Petrov VV, Fagard RH. Induction of cardiac fibrosis by angiotensin II. Methods Find Exp Clin Pharmacol. 2000;22(10):709–23. pmid:11346891.
- 34. Ozaki M, Kawashima S, Yamashita T, Hirase T, Ohashi Y, Inoue N, et al. Overexpression of endothelial nitric oxide synthase attenuates cardiac hypertrophy induced by chronic isoproterenol infusion. Circ J. 2002;66(9):851–6. pmid:12224825.
- 35. Dresel PE, Montgomery C. Hart, Stromblad B. C. R. Cardiac Arrythmias Induced by Injection of Isoproterenol Into the Coronary Arteries. Journal of Pharmacology and Experimental Therapeutics. 1963;140(1):67–72. Epub 04/01/1963.
- 36. Koch AL, Rusnak M, Peachee K, Isaac A, McCart EA, Rittase WB, et al. Comparison of the effects of osmotic pump implantation with subcutaneous injection for administration of drugs after total body irradiation in mice. Lab Anim. 2021;55(2):142–9. Epub 20200723. pmid:32703063.
- 37. Moretti M, Mugnaini M, Tessari M, Zoli M, Gaimarri A, Manfredi I, et al. A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes. Mol Pharmacol. 2010;78(2):287–96. Epub 20100503. pmid:20439469.
- 38. Stielow M, Witczynska A, Kubryn N, Fijalkowski L, Nowaczyk J, Nowaczyk A. The Bioavailability of Drugs-The Current State of Knowledge. Molecules. 2023;28(24). Epub 20231211. pmid:38138529; PubMed Central PMCID: PMC10745386.
- 39. Brady S, York M, Scudamore C, Williams T, Griffiths W, Turton J. Cardiac troponin I in isoproterenol-induced cardiac injury in the Hanover Wistar rat: studies on low dose levels and routes of administration. Toxicol Pathol. 2010;38(2):287–91. Epub 20100125. pmid:20100841.
- 40. Shusterman V, Usiene I, Harrigal C, Lee JS, Kubota T, Feldman AM, et al. Strain-specific patterns of autonomic nervous system activity and heart failure susceptibility in mice. Am J Physiol Heart Circ Physiol. 2002;282(6):H2076–83. pmid:12003814.
- 41. Faulx MD, Ernsberger P, Vatner D, Hoffman RD, Lewis W, Strachan R, et al. Strain-dependent beta-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice. Am J Physiol Heart Circ Physiol. 2005;289(1):H30–6. Epub 20050304. pmid:15749746.
- 42. Wang JJ, Rau C, Avetisyan R, Ren S, Romay MC, Stolin G, et al. Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model. PLoS Genet. 2016;12(7):e1006038. Epub 20160706. pmid:27385019; PubMed Central PMCID: PMC4934852.
- 43. Rau CD, Wang J, Avetisyan R, Romay MC, Martin L, Ren S, et al. Mapping genetic contributions to cardiac pathology induced by Beta-adrenergic stimulation in mice. Circ Cardiovasc Genet. 2015;8(1):40–9. Epub 20141205. pmid:25480693; PubMed Central PMCID: PMC4334708.
- 44. Nickel AG, von Hardenberg A, Hohl M, Loffler JR, Kohlhaas M, Becker J, et al. Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart Failure. Cell Metab. 2015;22(3):472–84. Epub 20150806. pmid:26256392.
- 45. Wang J, Huertas-Vazquez A, Wang Y, Lusis AJ. Isoproterenol-Induced Cardiac Diastolic Dysfunction in Mice: A Systems Genetics Analysis. Front Cardiovasc Med. 2019;6:100. Epub 20190731. pmid:31417910; PubMed Central PMCID: PMC6684968.
- 46. Ren S, Chang S, Tran A, Mandelli A, Wang Y, Wang JJ. Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice. J Vis Exp. 2019;(152). Epub 20191003. pmid:31633680; PubMed Central PMCID: PMC7374011.
- 47. Liao X, Chang E, Tang X, Watanabe I, Zhang R, Jeong HW, et al. Cardiac macrophages regulate isoproterenol-induced Takotsubo-like cardiomyopathy. JCI Insight. 2022;7(3). Epub 20220208. pmid:35132957; PubMed Central PMCID: PMC8855841.
- 48. Beyhoff N, Lohr D, Foryst-Ludwig A, Klopfleisch R, Brix S, Grune J, et al. Characterization of Myocardial Microstructure and Function in an Experimental Model of Isolated Subendocardial Damage. Hypertension. 2019;74(2):295–304. Epub 20190710. pmid:31291149; PubMed Central PMCID: PMC6635061.
- 49. Turner PV, Brabb T, Pekow C, Vasbinder MA. Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci. 2011;50(5):600–13. pmid:22330705; PubMed Central PMCID: PMC3189662.
- 50. Molojavyi A, Lindecke A, Raupach A, Moellendorf S, Kohrer K, Godecke A. Myoglobin-deficient mice activate a distinct cardiac gene expression program in response to isoproterenol-induced hypertrophy. Physiol Genomics. 2010;41(2):137–45. Epub 20100209. pmid:20145201.
- 51. Sharkey SW, Lesser JR, Zenovich AG, Maron MS, Lindberg J, Longe TF, et al. Acute and reversible cardiomyopathy provoked by stress in women from the United States. Circulation. 2005;111(4):472–9. pmid:15687136.
- 52. Ueyama T, Kasamatsu K, Hano T, Yamamoto K, Tsuruo Y, Nishio I. Emotional stress induces transient left ventricular hypocontraction in the rat via activation of cardiac adrenoceptors: a possible animal model of ’tako-tsubo’ cardiomyopathy. Circ J. 2002;66(7):712–3. pmid:12135146.
- 53. Ueyama T, Senba E, Kasamatsu K, Hano T, Yamamoto K, Nishio I, et al. Molecular mechanism of emotional stress-induced and catecholamine-induced heart attack. J Cardiovasc Pharmacol. 2003;41 Suppl 1:S115-8. pmid:12688407.
- 54. Kudej RK, Iwase M, Uechi M, Vatner DE, Oka N, Ishikawa Y, et al. Effects of chronic beta-adrenergic receptor stimulation in mice. J Mol Cell Cardiol. 1997;29(10):2735–46. pmid:9344768.
- 55. Werhahn SM, Kreusser JS, Hagenmuller M, Beckendorf J, Diemert N, Hoffmann S, et al. Adaptive versus maladaptive cardiac remodelling in response to sustained beta-adrenergic stimulation in a new ’ISO on/off model’. PLoS One. 2021;16(6):e0248933. Epub 20210617. pmid:34138844; PubMed Central PMCID: PMC8211211.
- 56. Kralova E, Mokran T, Murin J, Stankovicova T. Electrocardiography in two models of isoproterenol-induced left ventricular remodeling. Physiol Res. 2008;57 Suppl 2:S83-S9. Epub 20080328. pmid:18373388.
- 57. Soltysinska E, Olesen SP, Osadchii OE. Myocardial structural, contractile and electrophysiological changes in the guinea-pig heart failure model induced by chronic sympathetic activation. Exp Physiol. 2011;96(7):647–63. Epub 20110513. pmid:21571815.
- 58. Mikusova A, Kralova E, Tylkova L, Novotova M, Stankovicova T. Myocardial remodelling induced by repeated low doses of isoproterenol. Can J Physiol Pharmacol. 2009;87(8):641–51. pmid:19767889.
- 59. Spaich S, Katus HA, Backs J. Ongoing controversies surrounding cardiac remodeling: is it black and white-or rather fifty shades of gray? Front Physiol. 2015;6:202. Epub 20150722. pmid:26257654; PubMed Central PMCID: PMC4510775.
- 60. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation. 2004;109(13):1580–9. pmid:15066961.
- 61. Agostinucci K, Grant MKO, Seelig D, Yucel D, van Berlo J, Bartolomucci A, et al. Divergent Cardiac Effects of Angiotensin II and Isoproterenol Following Juvenile Exposure to Doxorubicin. Front Cardiovasc Med. 2022;9:742193. Epub 20220325. pmid:35402534; PubMed Central PMCID: PMC8990895.
- 62. Dewenter M, Pan J, Knodler L, Tzschockel N, Henrich J, Cordero J, et al. Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha(1)-adrenoceptors to early cardiac stress responses. Basic Res Cardiol. 2022;117(1):15. Epub 20220314. pmid:35286475; PubMed Central PMCID: PMC8921177.
- 63. Kastner N, Zlabinger K, Spannbauer A, Traxler D, Mester-Tonczar J, Hasimbegovic E, et al. New Insights and Current Approaches in Cardiac Hypertrophy Cell Culture, Tissue Engineering Models, and Novel Pathways Involving Non-Coding RNA. Front Pharmacol. 2020;11:1314. Epub 20200821. pmid:32973530; PubMed Central PMCID: PMC7472597.
- 64. Francisco J, Zhang Y, Jeong JI, Mizushima W, Ikeda S, Ivessa A, et al. Blockade of Fibroblast YAP Attenuates Cardiac Fibrosis and Dysfunction Through MRTF-A Inhibition. JACC Basic Transl Sci. 2020;5(9):931–45. Epub 20200928. pmid:33015415; PubMed Central PMCID: PMC7524792.
- 65. El-Armouche A, Wittkopper K, Degenhardt F, Weinberger F, Didie M, Melnychenko I, et al. Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy. Cardiovasc Res. 2008;80(3):396–406. Epub 20080808. pmid:18689792.
- 66. Perrino C, Naga Prasad SV, Mao L, Noma T, Yan Z, Kim HS, et al. Intermittent pressure overload triggers hypertrophy-independent cardiac dysfunction and vascular rarefaction. J Clin Invest. 2006;116(6):1547–60. pmid:16741575; PubMed Central PMCID: PMC1464895.
- 67. Grant MKO, Abdelgawad IY, Lewis CA, Seelig D, Zordoky BN. Lack of sexual dimorphism in a mouse model of isoproterenol-induced cardiac dysfunction. PLoS One. 2020;15(7):e0232507. Epub 20200709. pmid:32645007; PubMed Central PMCID: PMC7347208.
- 68. Teerlink JR, Pfeffer JM, Pfeffer MA. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res. 1994;75(1):105–13. pmid:8013068.
- 69. Haft JI. Cardiovascular injury induced by sympathetic catecholamines. Prog Cardiovasc Dis. 1974;17(1):73–86. pmid:4599470.
- 70. Faulx MD, Chandler MP, Zawaneh MS, Stanley WC, Hoit BD. Mouse strain-specific differences in cardiac metabolic enzyme activities observed in a model of isoproterenol-induced cardiac hypertrophy. Clin Exp Pharmacol Physiol. 2007;34(1–2):77–80. pmid:17201739.
- 71. York M, Scudamore C, Brady S, Chen C, Wilson S, Curtis M, et al. Characterization of troponin responses in isoproterenol-induced cardiac injury in the Hanover Wistar rat. Toxicol Pathol. 2007;35(4):606–17. pmid:17654401.
- 72. Shizukuda Y, Buttrick PM, Geenen DL, Borczuk AC, Kitsis RN, Sonnenblick EH. beta-adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy. Am J Physiol. 1998;275(3):H961–8. pmid:9724301.
- 73. Zhang J, Knapton A, Lipshultz SE, Weaver JL, Herman EH. Isoproterenol-induced cardiotoxicity in sprague-dawley rats: correlation of reversible and irreversible myocardial injury with release of cardiac troponin T and roles of iNOS in myocardial injury. Toxicol Pathol. 2008;36(2):277–8. Epub 20080318. pmid:18349426.
- 74. Zhuo XZ, Wu Y, Ni YJ, Liu JH, Gong M, Wang XH, et al. Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress. Apoptosis. 2013;18(7):800–10. pmid:23620435.
- 75. Zou Y, Yao A, Zhu W, Kudoh S, Hiroi Y, Shimoyama M, et al. Isoproterenol activates extracellular signal-regulated protein kinases in cardiomyocytes through calcineurin. Circulation. 2001;104(1):102–8. pmid:11435346.
- 76. Han JW, Kang C, Kim Y, Lee MG, Kim JY. Isoproterenol-induced hypertrophy of neonatal cardiac myocytes and H9c2 cell is dependent on TRPC3-regulated Ca(V)1.2 expression. Cell Calcium. 2020;92:102305. Epub 20201006. pmid:33069962.
- 77. Zhang GX, Kimura S, Nishiyama A, Shokoji T, Rahman M, Yao L, et al. Cardiac oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc Res. 2005;65(1):230–8. pmid:15621051.
- 78. Bai L, Kee HJ, Han X, Zhao T, Kee SJ, Jeong MH. Protocatechuic acid attenuates isoproterenol-induced cardiac hypertrophy via downregulation of ROCK1-Sp1-PKCgamma axis. Sci Rep. 2021;11(1):17343. Epub 20210830. pmid:34462460; PubMed Central PMCID: PMC8405624.
- 79. Grimm D, Elsner D, Schunkert H, Pfeifer M, Griese D, Bruckschlegel G, et al. Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system. Cardiovasc Res. 1998;37(1):91–100. pmid:9539862.
- 80. Mora MT, Gong JQX, Sobie EA, Trenor B. The role of beta-adrenergic system remodeling in human heart failure: A mechanistic investigation. J Mol Cell Cardiol. 2021;153:14–25. Epub 20201214. pmid:33326834.