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 
. Excess THs cause a hyperdynamic state characterized by decreased TPR and blood volume expansion, which in turn can lead to augmented contractility and increased cardiac output 
. Protracted hyperthyroidism is associated with deleterious cardiac changes as the consequence of increased hemodynamic burden. This was elegantly shown by Klein and Hong using heterotopic cardiac transplanted hearts 
. They were able to demonstrate that cardiac hypertrophy which often accompanies TH excess is an indirect consequence of the superimposed hemodynamic burden and resultant increased cardiac work rather than a direct effect of TH on the cardiac tissue.
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 
. During the initial phases of hyperthyroidism, compensation occurs by proportional growth in both myocyte length and CSA 
. However, the sustained hemodynamic load eventually overwhelms the compensatory ability of the heart and causes progression to a dilated ventricle characterized by chamber dilation (↑ myocyte length) without further increase in relative LVPW thickness (CSA). This maladaptive phenotype is characteristic of dilated heart failure (HF) and is associated with increased cardiac work, wall stress, and ECM deposition 
Our lab previously reported increased LV internal dimensions and chamber dysfunction after 2 months of hyperthyroidism in F1B hamsters 
. In agreement with these findings, we observed progressive chamber dilation by 2 months of TH treatment . In the present study, the LV continued to dilate until the time of terminal experiments in the hyperthyroid group. We have also shown that myocyte lengthening alone, due to series sarcomere addition, can account for chamber dilation in HF 
. In the current study, myocyte lengthening observed in the hyperthyroid group is consistent with the increased LV internal dimension detected by echocardiography.
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 
. Although the exact etiology of this discrepancy is unknown, several myocyte and non-myocyte factors likely contribute. Alterations in excitation-contraction coupling, Ca2+
handling properties, neurohumoral activation, oxidative stress, vascularity and blood flow, cell metabolism, cell death (apoptosis or necrosis), fibrotic deposition, and myocyte remodeling have all been implicated. While we cannot exclude the aforementioned parameters as contributing to the discrepancy, myocyte necrosis or apoptosis appear unlikely. Areas of cell loss and replacement fibrosis were not observed, reducing the likelihood of myocyte necrosis. Except with extreme changes, such as in the peri-infarct area after acute myocardial infarction, apoptosis appears to predominantly occur in non-myocyte
s during HF and cardiac dysfunction 
. When myocyte loss occurs by apoptosis, fibrous deposition/replacement is not present and would be difficult to document over such a long treatment period 
. Based on tissue morphology and the fact that THs tend to inhibit apoptosis 
, there is little reason to suspect that apoptosis accounts for significant loss of contractile cells or fibrotic deposition in the current setting. Although we cannot exclude the possibility of diminished coronary blood flow, it is unlikely in the current experimental setting. THs are potent stimulators of coronary angiogenesis and blood flow in the setting of hyperthyroidism. THs have been shown to increase resting blood flow both with and without accompanying cardiac hypertrophy 
. Furthermore, cardiac hypertrophy which is secondary to hyperthyroidism is typically associated with augmented blood flow and parallel or increased vascular growth due to increased cross-sectional area of the vascular bed 
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 
. While previous investigations have examined the influence of THs on collagen gene expression and/or fibrotic deposition, they typically have been performed without simultaneous assessment of cardiac function or are deduced from autopsy findings 
. For this reason, the influence of LV fibrosis on cardiac function during sustained hyperthyroidism is not well understood. THs have been implicated in the regulation of collagen dynamics, however, their influence on myocardial fibrosis has yielded conflicting results. Short-term studies have shown that THs exert anti-fibrotic actions, including in the setting of TH induced hypertrophy 
. In vitro
investigations by Yao & Eghbali and Chen et al., suggest that THs can directly regulate and suppress collagen gene expression 
. In contrast, Roy et al. found that the anti-fibrotic actions of THs were the result of regulating matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) despite increased collagen and pro-collagen gene expression 
. On the other hand, myocardial fibrosis caused by chronic hyperthyroidism has been reported by numerous authors 
. Chronic hyperthyroidism is associated with increased cellular metabolism and increased oxidative damage 
. Oxidative damage is a known stimuli for collagen deposition 
and increased oxidative damage which occurs during sustained bouts of hyperthyroidism likely contributes to increased interstitial collagen deposition. Further investigation is needed to fully elucidate the mechanisms of increased interstitial collagen deposition in this setting.
Although it is well established that acute hyperthyroidism is associated with augmented cardiac function and increased cardiac output 
, the importance of LV fibrosis on cardiac function during hyperthyroidism is not well characterized. Using trichrome staining, we were able to confirm in our study that prolonged hyperthyroidism was associated with increased LV fibrosis [~60%↑] when compared with age matched control hamsters. The majority of fibrotic deposits were found within the perivascular space (perivascular fibrosis) and cardiac interstitium (interstitial fibrosis) without evidence of myocyte necrosis and replacement fibrosis. Moreover, we observed severe relaxation impairment (-dP/dT and Tau) and ultimately systolic dysfunction (LV EF, LV ESP, dP/dT) in hyperthyroid hearts. Our findings further support the notion that LV fibrosis inversely affects LV function in the setting of hyperthyroidism.
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 
, it is likely that even a small increase in collagen can impair cardiomyocyte function. Indeed, this notion is supported by reports that small changes in collagen concentration can have a profound impact on passive mechanical properties of cardiac tissue 
. In agreement, our findings suggest that relatively modest but significant increases in myocardial collagen deposition can impede myocyte contractile ability even when individual myocyte function is enhanced. Consistent with our previous report 
, we did not observe noticeable areas of fibronecrosis. This suggests that the depressed global cardiac function observed during chronic hyperthyroidism is at least in part the product of inhibited myocyte function caused by increased perivascular and interstitial fibrotic deposition and does not appear to be a product of extensive myocyte loss.
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 
. Unloaded isolated myocyte assessment still is an important tool for evaluating shortening velocity/cross bridge turnover rate and comparisons between treatment groups can be made under similar experimental conditions without influence of potential confounders. Finally, we acknowledge that comprehensive isolated myocyte morphometric analysis would have complemented the echocardiographic and isolated myocyte functional data presented in the current study. Although we are accustomed to high proportions of rod cell with our commonly used non-calcium tolerant myocyte preparation with rapid gluteraldehyde fixation (>90% rod cells are routine), the Ca2+
tolerant isolated myocyte preps used here did not produce high enough rod cell yields for accurate Coulter Channelyzer analysis.
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.