The authors have declared that no competing interests exist.
Conceived and designed the experiments: NYW DW AMG. Performed the experiments: NYW DW RAR. Analyzed the data: NYW DW AMG. Wrote the paper: NYW. Edited and revised manuscript: AMG.
Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function.
Thyroid hormones (THs) play a pivotal role in regulating cardiac homeostasis as well as the peripheral vascular system in physiologic and pathologic conditions
Excess TH is associated with elevated HR, decreased TPR, widened pulse pressure, blood volume expansion, and increased cardiac output
A better understanding of the progression and cellular mechanisms responsible for cardiac dysfunction during periods of sustained hyperthyroidism is clinically important. There is limited information within the current literature examining the relationship between myocyte function and global cardiac function during the transition from cardiac compensation to decompensation in the setting of sustained hyperthyroidism. Furthermore, there is limited and conflicting information regarding the functional consequences of increased LV fibrotic deposition in the setting of sustained hyperthyroidism. While previous investigations have examined the influence of hyperthyroidism on cardiac function either
To provide better understanding of the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined global cardiac function, LV isolated myocyte function, and whole tissue remodeling using the previously characterized F1B hamster model. This study suggests that the impairment in overall cardiac function observed with long standing hyperthyroidism is not related to decline in the functional capacity of individual myocytes.
The use of animals in this study conformed to the Public Health Service Guide for Care and Use of Laboratory Animals. All experiments and protocols were approved by the Sanford Research/USD Institutional Animal Care and Use Committee. Three month old BIO F1B hamsters (BIO Breeders INC., Watertown, MA) were randomized to either TH (hyperthyroid) or untreated (control) groups. Hamsters in the TH treatment group were fed pellets containing 0.1% grade I desiccated TH (Sigma # T1251) as previously described
Terminal LV blood samples were collected and separated into serum aliquots by centrifugation and stored at −80°C. T3 and T4 levels were measured using commercial ELISA kits as previously described
Echocardiography was performed in each animal at 1 month, 2 months, and then every two months until terminal experiments using a Vevo 660 high-resolution imaging system with a 25-MHz RMV-710 transducer (Visualsonics; Toronto, Canada) as previously described
Prior to sacrifice, LV hemodynamics and aortic pressures were obtained by catheterization of the right carotid artery using a Millar Micro-tip catheter (Millar Instruments; Houston, TX) as described previously
LV myocyte isolation was adapted from previously described methods
After 10 months TH treatment, isolated rod-shaped myocytes with discernible striations and clear edges were evaluated using an IonOptix myocyte contractility system (IonOptix Corp., Milton, MA). Myocytes were placed in a chamber mounted on the stage of an inverted microscope (Nikon Eclipse TS300; Melville, NY) and superfused with sterile filtered, 37°C Tyrode’s buffer containing (in mM): 137 NaCl, 5.4 KCL, 1.2 CaCl2, 1.5 MgCl2, 10 HEPES, 10 glucose, pH 7.4. Myocytes were field stimulated with suprathreshold voltage (+20%) at a frequency of 0.5 Hz, 3 msec duration, using platinum wires placed on opposite sides of the chamber connected to an acute field stimulator. Polarity of the stimulatory electrodes was frequently reversed to avoid buildup of electrolysis byproducts. Myocytes were displayed using an Ionoptix MyoCam camera and edge detection software (IonOptix Corp).
Transverse 5 µm LV tissue sections were stained with Masson’s trichrome or haematoxylin and eosin. Myocardial fibrosis was visualized at 10× and quantified using Image J software (NIH, Bethesda, MD;
All data are expressed as means ± (S.D.). Diagnostics were conducted to verify assumption of normality and variance before applying the models. Statistical analysis was performed using a two-tailed Student’s T-test or Mann-Whitney rank sum test. Values of p<0.05 were considered statistically significant. Statistical analysis was performed using Sigmastat V 3.5 (Aspire Software International; Ashburn, VA).
Physical data and serum TH levels are presented in
Control | Hyperthyroid | p-Value | |
|
171 (11) | 185 (13) | 0.01 |
|
597 (48) | 804 (94) | <0.001 |
|
3.51 (0.3) | 4.38 (0.6) | <0.001 |
|
0.6 (0.2) | 3.8 (0.9) | <0.001 |
|
4.8 (1.0) | 13.9 (2.7) | <0.001 |
Values are means (SD). BW, body weight; HW, heart weight; HW/BW, heart weight to body weight ratio. N = 12−15/group.
By one month, TH treatment resulted in a significant elevation in resting HR. Tachycardia was sustained during the initial 8 months of treatment. Thereafter, HR declined to control levels [
Values are means (SD). A–D. HR, heart rate (A); LV EF, left ventricular ejection fraction (B); LVIDd, left ventricular internal dimension in diastole (C), LVIDd/LVPWd ratio (D). N = 13−15/group. *, p<0.05 vs. control.
There was no difference in LV internal dimension during diastole (LVIDd) after 1 month TH treatment. However, treated hamsters had significant increases in LVIDd by two months [
Control | Hyperthyroid | p-Value | |
|
156 (15) | 134 (12) | <0.002 |
|
84 (12) | 75 (16) | 0.20 |
|
160 (16) | 123 (11) | <0.001 |
|
8 (5) | 12 (6) | 0.138 |
|
9921 (1980) | 7291 (708) | <0.001 |
|
−8998 (1844) | −4844 (683) | <0.001 |
|
11 (4) | 15 (5) | 0.004 |
|
12.8 (7) | 26.2 (12) | 0.005 |
|
137.7 (32) | 194.5 (33) | <0.001 |
Values are means (SD). SBP, systolic blood pressure; DBP, diastolic blood pressure; LV ESP, left ventricular end systolic pressure; LV EDP, left ventricular end diastolic pressure; dP/dT Max, maximal rate of pressure development; dP/dT Min, maximal rate of pressure decline; Tau, time constant of left ventricular isovolumic relaxation; Wall Stress ED, wall stress at end diastole; Wall Stress ES, wall stress at end systole; Meridional Wall stress calculated using previously described methods
Compared with age-matched control hamsters, sustained hyperthyroidism was associated with impaired LV function [
Despite global cardiac impairment observed by echocardiography and LV hemodynamics, treatment improved functional mechanics of individual isolated myocytes [
Values are means (SD). A–F. Cell Length (A); +dL/dT, maximal velocity of shortening (B); –dL/dT, maximal velocity of re-lengthening (C); Peak Shortening (D); TPS, time to peak shortening (E), TR90, Time to 90% re-lengthening (F). C, control; H, hyperthyroid. N = 5−7/group. *, p<0.05 vs. control.
Representative images of LV fibrosis are depicted in
Values are means (SD). Representative images of LV fibrosis stained by Masson’s Trichrome (A). % collagen volume as a proportion of total tissue area (B). C, Control; H, Hyperthyroid. N = 5/group. *, p<0.05 vs. control.
The major finding of this study is the development of a mismatch between global cardiac performance and individual myocyte function in the setting of chronic hyperthyroidism. Chronic hyperthyroidism was associated with deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative posterior wall thickness, increased wall stress, and increased LV fibrotic deposition. Importantly, sustained hyperthyroidism led to LV systolic and diastolic dysfunction as evaluated by echocardiography and LV hemodynamics. The novel feature of this study is the finding that despite global cardiac impairment, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced mechanical function when compared with myocytes from untreated age matched controls. This paradox suggests that the cardiac dysfunction observed during prolonged hyperthyroid conditions is not the consequence of declined functional capacity of individual myocytes, but rather impairment in the ability of individual myocytes to function properly in the whole tissue setting.
It has long been recognized that THs plays a pivotal role in cardiovascular homeostasis
In an attempt to maintain cardiac function and compensate for increased cardiac stress during prolonged TH excess, the heart undergoes a remodeling process characterized by myocyte enlargement and extracellular matrix (ECM) deposition. The arrangement of myocytes within the ventricular wall is such that cell length is primarily responsible for chamber diameter and myocyte cross sectional area (CSA) is responsible for changes in wall thickness
Our lab previously reported increased LV internal dimensions and chamber dysfunction after 2 months of hyperthyroidism in F1B hamsters
Despite the early increase in chamber internal dimension, a relative increase in LVPW thickness helped normalize the anatomical parameters of wall stress during the first 4 months of TH excess. By 6 months, hyperthyroid animals had a significantly elevated LVIDd/LVPWd ratio which steadily increased until the terminal 10 month time point. This progressive increase in the anatomical parameters of wall stress mirrored the decline observed in LV EF. Terminal invasive measurements confirmed that treated animals had significant elevations in both end-diastolic (100% increase) and end-systolic (41% increase) meridional wall stress. Hyperthyroidism initially resulted in tachycardia, however it is important to note that HR declined to control levels by the end of the study when LV dysfunction was most pronounced. This reduction of TH-induced tachycardia observed after 8 months likely represents the onset of adrenergic decompensation. Tachycardia is a widely used diagnostic marker in the identification of hyperthyroidism. Our findings suggest that HR may not always be a reliable predictor of hyperthyroidism, especially in the setting of advanced cardiac disease caused by sustained TH excess.
To our knowledge, this is the first report of a paradoxical mismatch between global cardiac function and individual myocyte function in the setting of prolonged hyperthyroidism. Several previous reports lend credence to the idea that global cardiac function is not a consistent indicator of individual myocyte contractile function
On the other hand, increased LV fibrosis and collagen crosslinking are associated with diastolic stiffness which contributes to LV pump dysfunction and progression to HF
Although it is well established that acute hyperthyroidism is associated with augmented cardiac function and increased cardiac output
While increased collagen deposition certainly can impair global cardiac function, it may not similarly affect intrinsic cardiomyocyte mechanics. Accordingly, we also examined the influence of chronic hyperthyroidism on the mechanical function of individual myocytes. We hypothesized that mechanical impairment at the level of individual ventricular myocytes would strongly correlate with the decline observed in global cardiac function. Contrary to our hypothesis, we found that isolated ventricular myocytes from hyperthyroid hamsters had enhanced mechanical function when compared to age matched control hamsters, despite the aforementioned adverse chamber remodeling and diminished global cardiac function.
Given the close proximity of fibrillar collagen to myocytes and the finding that fibrillar collagen is a relatively stiff material with a tensile strength greater than steel
Our study has several limitations. While a standard experimental protocol was closely followed to replicate the same experimental conditions for each animal, it is possible that myocytes selected for functional assessment do not represent the total myocyte population within the intact heart. It is also important to note that isolated myocytes in the current study were not tested under loading conditions. Loading conditions can influence muscle function and altered loading theoretically could impact isolated myocyte shortening and tension development. Unfortunately, the technical difficulty of myocyte loading experiments limits its utility and widespread implementation
In summary, chronic hyperthyroidism was associated with deleterious cardiac remodeling, LV fibrosis, and cardiac functional decline. Despite global cardiac impairment, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had normal or enhanced function when compared with myocytes from untreated age matched controls. While we cannot definitely establish a cause and effect relationship, our data strongly suggests that increased LV interstitial fibrosis can undermine the ability of otherwise normal myocytes to function properly. One can only speculate regarding translating ANY animal observations to humans. While chronic hyperthyroidism in humans is generally identified and treated before reaching this point, our results may provide an explanation for LV dysfunction observed in patients with chronic hyperthyroidism.
We would like to thank Mrs. April Beyer and Drs James Kuzman and Jinghai Chen for their technical assistance.