Body Temperature Circadian Rhythm Variability Corresponds to Left Ventricular Systolic Dysfunction in Decompensated Cardiomyopathic Hamsters

Journal of Cardiac Failure Vol. 17 No. 11 2011

Body Temperature Circadian Rhythm Variability Corresponds to Left Ventricular Systolic Dysfunction in Decompensated Cardiomyopathic Hamsters AMANY AHMED, MD,1,2 SREEDEVI GONDI, MD,1 CASEY COX, BSc,3 MINJUAN ZHENG, MD,4 ANWARULLAH MOHAMMED, MD,1 IGOR V. STUPIN, MD, PhD,1 SUWEI WANG, PhD,1 DEBORAH VELA, MD,1 ALAN BREWER, MBA, BSc,1 MACARTHUR A. ELAYDA, MD, PhD,1 L. MAXIMILIAN BUJA, MD,1,2 S. WARD CASSCELLS, MD,1,2 AND JAMES M. WILSON, MD1,5 Houston, Texas; and Xi’an, China

ABSTRACT Background: A declining amplitude of body temperature circadian rhythm (BTCR) predicts decompensation or death in cardiomyopathic hamsters. We tested the hypothesis that changes in BTCR amplitude accompany significant changes in left ventricular (LV) size and function. Methods and Results: Using intraperitoneal transmitters, we continuously monitored the temperature of 30 male BIO TO-2 Syrian dilated cardiomyopathic hamsters. Cosinor analysis was used to detect significant changesddefined as changes O1 standard deviation from the baseline amplitude for 3 consecutive daysdin BTCR amplitude over each hamster’s lifespan. The Student t-test was used to compare BTCR variability and LV size and function (as assessed by 2D echocardiography) between baseline and the time that BTCR amplitude declined. All hamsters received 10 mg/kg furosemide daily. At the time of BTCR amplitude decline, functional parameters had changed significantly (P ! .0001) from baseline: ejection fraction (0.31 6 0.09% vs. 0.52 6 0.08%), LV end-systolic volume (0.11 6 0.03 vs. 0.05 6 0.02 cm3), and LV end-diastolic volume (0.16 6 0.04 vs. 0.10 6 0.03 cm3). Conclusions: In decompensated cardiomyopathic hamsters, a decline in BTCR amplitude was associated with progression of heart failure and cardiac decompensation. Variation in BTCR warrants further investigation because of its potential implications for the diagnosis and treatment of cardiovascular disorders. (J Cardiac Fail 2011;17:937e943) Key Word: Heart failure.

Heart failure is a common cardiac illness that incurs extraordinary medical expense and use of resources. Optimal treatment of patients with heart failure requires close monitoring that is limited to clinical interaction, examination, and limited testing.1 Circadian temperature rhythm dysregulation occurs in patients with cardiac decompensation2 and acts as a harbinger of adverse cardiovascular events.3 Therefore, when used in conjunction with conventional clinical tools, chronobiological and complexity analysis

of the circadian rhythm of core body temperature may offer incremental value for monitoring patients with heart failure. A study we conducted previously showed that changes in circadian temperature patterns predict decompensation or death.4 The aim of the present study was to compare changes in body temperature circadian rhythm (BTCR) variability with other recognized clinical predictors in an attempt to reveal the mechanisms behind this relationship,

From the 1Texas Heart Institute at St. Luke’s Episcopal Hospital, Houston, Texas; 2University of Texas Health Science Center, Houston, Texas; 3St. Jude Medical, Houston, Texas; 4Xijing Hospital, Xi’an, China and 5Baylor College of Medicine, Houston, Texas. Manuscript received February 10, 2011; revised manuscript received June 27, 2011; revised manuscript accepted July 6, 2011. Reprint requests: Amany Ahmed, MD, Texas Heart Institute, 6770 Bertner Avenue, MC 2-255, Houston, TX, 77030. Tel: þ1 832 355 9812; Fax: þ1 832 355 9368. E-mail: [email protected]

This research was funded by a grant from the Roderick D. MacDonald Research Fund at St. Luke’s Episcopal Hospital. See page 943 for disclosure information. 1071-9164/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.cardfail.2011.07.004

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938 Journal of Cardiac Failure Vol. 17 No. 11 November 2011 and also to examine the effect of diuretic treatment on life span in a cardiomyopathic hamster model. Methods Animals This study conformed to the Guide for Care and Use of Laboratory Animals and was approved by the University of Texas Health Science Center Animal Welfare Committee. An outbred colony of 30 Bio-TO-2 Syrian hamsters was purchased from Bio Breeders, Inc (Watertown, MA) and was housed at the University of Texas Health Science Center animal facility. The Bio-TO-2 hamster was selected because it was the most suitable model of cardiomyopathic failure for the purpose of our research. This strain develops cardiac dilation without preceding hypertrophy or hypertension, a pattern similar to that of human idiopathic dilated cardiomyopathy.5 Each hamster was maintained separately in a plastic cage. Food and water were available ad libitum, and a 12-hour light/dark cycle was maintained. At the beginning of this study, the hamsters were 23 to 26 weeks old and weighed 100 to 127 g. They were allowed to adapt to their new environment for 2 weeks before the transmitters were implanted into them. Then, after 4 weeks, the transmitters were turned on. All animals were observed for 9 months. Also, because most heart failure patients take diuretic medications, we examined the effects of diuretic treatment on outcomes by giving all hamsters a daily subcutaneous injection of furosemide 10 mg/kg.

Fig. 1. Temperature readings obtained from 2 hamsters before death. The top curve shows a sudden drop in body temperature in a hamster that died suddenly, and the bottom curve shows a gradual decline in body temperature in a hamster that died slowly. standard deviation from the baseline BTCR amplitude for 3 consecutive days. Echocardiography

Monitoring Hamsters We monitored the hamsters daily for signs of cardiac decompensation (eg, tachypnea, lethargy, changes in grooming habits), and we weighed the hamsters daily. A telemetry device (PhysioTel transmitter, Model TA10TA-F20; Data Sciences International, St. Paul, MN) was implanted intraperitoneally in each hamster to continuously monitor core body temperature. Each hamster was observed until death or euthanasia. Hamsters were euthanized if daily observations and the hamsters’ activity level indicated severe decompensation and imminent death. The telemetry system comprised the implanted PhysioTel transmitter and a PhysioTel Receiver (Model RPC-1) connected to a DSI Exchange Matrix (Data Sciences International). The data signals were multiplexed and sent to the data acquisition system via an Ethernet cable. The receivers were mounted directly under the hamster cages and were shielded to prevent crosstalk. Temperature ( C) was recorded in 15-minute intervals. Hamsters were subdivided into 2 groups: hamsters that died suddenly and had no changes in body temperature before death (the sudden-death group) and hamsters that died gradually (the gradual-death group) (Fig. 1).

Two-dimensional echocardiography was performed on each hamster at baseline and when the hamster’s BTCR amplitude declined. For each echocardiogram, the hamsters were anesthetized with 2% isoflurane gas delivered through a nose cone. Transthoracic echocardiography was performed with a General Electric machine equipped with a 12-MHz linear array probe. Twodimensional M-mode images were acquired by using highresolution zoom while the hamster was in a semiconscious state. Measurements included left ventricular (LV) ejection fraction, LV area during diastole from a 2-chamber view, LV area during systole from a 2-chamber view, LV end-diastolic volume, LV end-systolic volume, and stroke volume. Statistical Analysis The Student t-test was used to examine changes over time in BTCR variability, LV size, and LV function as assessed by 2-dimensional (2D) echocardiography and to examine the effects of furosemide on the timing of BTCR decline and death. We also computed the slope of body temperature decline, using multilevel modeling to account for the variations in time and slope among the individual hamsters.

Temperature Analysis Temperature data collected over the hamsters’ lives were detrended with linear regression. Mean temperature and the slope of the temperature decline were analyzed by using multilevel modeling.4 Body temperature variation was analyzed by using cosinor analysis,4 which uses the least-squares method to fit a cosine wave to a time series and calculates the amplitude of deviation from the mean value. The baseline BTCR amplitude was calculated as the average amplitude for all of the hamsters. A significant change in BTCR amplitude was defined as a change greater than 1

Histopathologic Assessment Heart and liver tissues were collected from the hamsters and were fixed in 10% nonbuffered formalin. After fixation, each heart was serially sectioned, from apex to base, into 5 slices. Tissues were subsequently processed and paraffin-embedded. Five-mmthick histologic sections were then stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO) and with Masson’s trichrome for histologic assessment. Liver sections were stained with H&E.

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Image Analysis

Echocardiography

Morphometric analysis was performed on a subset of hamsters. The extent of myocardial damage (fibrosis and/or mineralization) was evaluated by quantitative morphometry, which was performed with Olympus MicroSuite software on an Olympus BX61 microscope (Olympus America; Melville, NY) on trichrome-stained sections. Three sections were evaluated per hamster, corresponding to the lower, mid, and upper portion of the left ventricle. Results were expressed as a percentage of damage to the total left ventricle.

At the time that BTCR amplitude declined, echocardiography revealed enlargement of the LV cavity and global systolic dysfunction. Several functional parameters (Table 1) changed significantly from their baseline values: ejection fraction (0.31 6 0.09% vs. 0.52 6 0.08%; P ! .0001), LV end-systolic volume (0.11 6 0.03 vs. 0.05 6 0.02 cm3; P ! .0001), and LV end-diastolic volume (0.16 6 0.04 vs. 0.10 6 0.03 cm3; P ! .0001). Changes in LV size were also noted (Fig. 3).

Results Five of the 30 hamsters died suddenly. Of the remaining 25 hamsters, 2 had no significant decline in the BTCR amplitude, and 23 had a significant decline in BTCR amplitude before death or euthanasia. There was a consistent decline in body temperature in 21 (84%) of the 25 hamsters included in the final analysis. The mean temperature at the onset of temperature decline was 35.44 ± 2.54 C (95% confidence interval [CI], 34.34e36.54). Significant temperature decline began 7 ± 5 days before death (95% CI, 5.13e9.08). Body temperature declined steadily from baseline until death (slope 5 0.10 C/day; intercept, 37.12; P ! .0001). Also, we observed that the slope of temperature decline and the timing of the change in BTCR amplitude were not the same in every hamster. At baseline, the hamsters’ average age was 30 weeks 6 2 days, and their average weight was 105 6 6 g. At the time of significant BTCR amplitude decline (baseline, 0.47 6 0.14 C; amplitude decline, 0.35 6 0.13 C; P 5 .0006), the hamsters’ average age was 45 6 7 weeks and average weight was 121 6 13 g. The mean time from a significant BTCR decline to death was 8 6 7 weeks (Fig. 2). At death, the hamsters’ mean weight was 122 6 22 g, which was significantly higher than their baseline weight (105 6 6 g; P 5 .0002). The hamsters’ mean age at death was 54 6 7 weeks.

Histopathologic Evaluation

Histologic examination revealed features of dilated cardiomyopathy in all hearts. Scattered foci of dystrophic calcification, immersed within areas of patchy interstitial and replacement fibrosis, were observed in both the left and right heart, generally in the mid and subendocardial regions of the ventricles. Contraction band necrosis and wavy fibers were often observed in the vicinity of these fibrotic regions. Several of the hearts also had large mural thrombi with partial organization in both ventricles. Liver specimens had an extensive chronic passive congestion consistent with congestive heart failure (CHF) (Fig. 4). Specimens from hamsters that died suddenly had less chamber dilation and appeared to have more pronounced increases in interstitial collagen, whereas specimens from hamsters that died gradually showed more interspersed areas of thick and thin myocytes with variously sized nuclei suggestive of an advanced stage of dilated cardiomyopathy. Quantitative Image Analysis

Quantitative analysis of the lesions revealed that the fibrosis and mineralization from myocytes represented approximately one fifth of the LV mass in the hamsters. However, the percentage of LV fibrosis and mineralization was higher Table 1. Echocardiographic Parameters at Baseline and at the Time of BTCR Decline Parameter

Baseline

BTCR decline

P value

0.30 6 0.05 0.25 6 0.05 0.27 6 0.04

0.42 6 0.07 0.31 6 0.05 0.36 6 0.06

!0.001 !0.001 !0.001

0.68 6 0.06

0.78 6 0.07

!0.001

6 6 6 6 6 6 6

!0.001 !0.001 !0.001 0.28 !0.001 !0.001 !0.001

2

LVAd, cm Apical 2-chamber Apical 4-chamber Apical view LVLs, cm2 Apical view LVAs, cm2 Apical 2-chamber Apical 4-chamber Apical view Stroke volume, cm3 LV ESV, cm3 LV EDV, cm3 Ejection fraction, %

Fig. 2. Average amplitude of the body temperature circadian rhythm over the hamsters’ lifespan. ‘‘After surgery" refers to the period after intraperitoneal transmitter implantation.

view view

view view

0.18 0.17 0.18 0.05 0.05 0.10 50

6 6 6 6 6 6 6

0.05 0.04 0.03 0.02 0.02 0.03 8

0.33 0.25 0.29 0.05 0.11 0.16 30

0.07 0.06 0.06 0.01 0.03 0.04 9

EDV, end-diastolic volume; ESV, end-systolic volume; LV, left ventricular; LVAd, LV area in diastole; LVAs, LV area in systole; LVLs, LV length in systole. All data are means 6 standard deviation.

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Fig. 3. Images illustrating the correlation between a significant decline in body temperature circadian rhythm amplitude and a decline in left ventricular function. (A) Cosinor parameter plot showing the circadian temperature variation in hamsters with dilated cardiomyopathy. Two-dimensional echocardiograms obtained from a representative hamster at baseline (B) and after cosinor amplitude decline (C). Left ventricle (LV), right ventricle (RV), and pleural effusion (PE) are labeled.

Fig. 4. Images from histopathologic analysis. Trichrome-stained cross-sectional heart slices from hamsters that died of pump failure (A) and those that died suddenly (B), showing the dilated chambers and wall thinning characteristic of this model. Extensive areas of fibrosis and myocyte loss (represented by light blue-green color) occupy a considerable portion of the circumference of both ventricles. Intramural thrombi are visible in both chambers of the heart of the hamster that died suddenly. (C) Micrograph of liver tissue showing signs of passive hepatic congestion. (D) Foci of dystrophic calcification (arrows) immersed within areas of patchy interstitial and replacement fibrosis (E, blue-green stain) in the heart of a hamster that died suddenly. (F) Contraction band necrosis in a group of subepicardial myofibers (asterisks) from a heart with pump failure. Bar 5 2 mm in (A) and (B); 200 mm in (C-F). Trichrome stain: (A, B, E, F). H&E stain (C, D).

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in hamsters that died gradually (25.3% 6 2.7) than in the hamsters that died suddenly (19.1% 6 5.1; P ! .05). Discussion Variations in body temperature are a part of the natural history of heart failure progression in this cardiomyopathic hamster model. A decline in BTCR amplitude was seen 8 6 7 weeks before death and was associated with significant worsening of LV function and with LV enlargement as assessed by echocardiography. We also found that declining body temperature variability predicted decompensation or death 7 6 5 days before mean temperature began to decline, yet mean temperature remained a prognostic factor for these outcomes. These findings confirmed those of our previous study of body temperature in this animal model.4 Both studies are part of a more extensive investigation of the potential value of using body temperature to improve clinical assessment and intervention in heart failure patients. Falling and low body temperature are independent predictors of rehospitalization,6 hospital mortality,7 and 60-day8 and 6-month9 mortality in patients with decompensated CHF. Identifying a simple predictor of imminent decompensation in this patient population has the potential to reduce the complexity of close follow-up monitoring, thus reducing the incidence of decompensation and hospitalization. Circadian Body Temperature Variations

Circadian variations in cardiovascular performance have long been recognized.10 Heart rate, blood pressure, endothelial function, vascular tone, fibrinolytic activity, platelets, and leukocyte reactivity exhibit variations consistent with a circadian rhythm.3 This variation is responsible for the association between time of day and the occurrence of major cardiovascular events.10 Body temperature is a powerful indicator of circadian synchrony and one of the most commonly studied circadian variables11; therefore, it is used as a phase marker of the circadian clock.12 The maintenance of the circadian variation of core body temperature within narrow limits is a homeostatic mechanism that is critical for survival.13 At the top of the hierarchy for circadian control lies the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, which synchronizes and coordinates slave oscillators located in other parts of the brain, as well as in the peripheral organs. At the cellular level, the SCN is believed to house distinct populations of neurons that hold the molecular machinery required for the function of the master clock.14 The function of the SCN as a circadian rhythm pacemaker is influenced by sympathovagal equilibrium and the renin-angiotensin system. Angiotensin II has pleiotropic actions that are mediated by specific receptors (AT1 and AT2) found abundantly within the SCN.3 During decompensated heart failure, neurohormonal activation may elevate circulating levels of angiotensin II, which correlate with both the severity and the prognosis of heart failure.15 We found that the decline in BTCR



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amplitude started at different times in every animal, suggesting that genetic factors contribute importantly to the temperature response to heart failure. Additionally, the decline in BTCR occurred later in our current study than in our previous one (at 45 6 7 weeks vs. 36 6 10 weeks; P 5 .0001), which followed the same protocol as the present study save that the hamsters did not receive furosemide.4 Echocardiography and Dilated Cardiomyopathy

Two-dimensional and Doppler echocardiography is used to assess LV function in patients with dilated cardiomyopathy. With declining ventricular contractility and chronically increased filling pressures, the left ventricle cavity dilates, becoming more spherical.16 The degree of LV dysfunction as assessed by the LV ejection fraction is among the most powerful predictors of clinical outcome.17 The classic phenotypic hallmark of dilated cardiomyopathy is varying degrees of LV dilation associated with LV dysfunction. Dilation precedes dysfunction in many cases; therefore, it is important to have accurate chamber dimensions that are indexed to the patient’s body surface area.18 Moreover, with gradual dilatation, the LV cavity becomes more spherical, with a sphericity index (long-axis dimension/short-axis dimension) that approaches 1 (normally, $1.5). LV mass is uniformly increased (eccentric hypertrophy), and wall thickness is typically within normal limits.16 In decompensated cardiomyopathic hamsters, a decline in BTCR amplitude occurs consistently and simultaneously with both worsening LV function and progressive LV enlargement as assessed by 2D echocardiography. Therefore, monitoring BTCR may enable clinicians to detect decompensation in LV function. Sudden Cardiac Death and Dilated Cardiomyopathy

Dilated cardiomyopathy is the second most common cause of sudden cardiac death (SCD) in the United States and accounts for about 10% to 15% of all SCD.19 In patients with heart failure, the rate of SCD is 6 to 9 times more that in the general population.20 The strongest independent predictor of SCD is LV dysfunction.19 The hamsters in our study received daily injections of the diuretic drug furosemide because diuretics are commonly prescribed to heart failure patients. Post hoc, we performed a Kaplan-Meier analysis to examine the effects of furosemide by comparing the life spans of the hamsters used in the current study and the hamsters used in our previous study.4 We found that hamsters in the present study lived longer (54 6 3 weeks) than the hamsters in the previous study (51 6 2 weeks) (P 5 .017) (Fig. 5). These findings may be explained by the fact that loop diuretics cause volume contraction, electrolyte disturbance, and neuroendocrine activation.21 Activation of the renin-angiotensin system in moderate heart failure may be the result of diuretic treatment, rather than a result of the disease process itself.22 In 1153 patients with heart failure who were

942 Journal of Cardiac Failure Vol. 17 No. 11 November 2011 in myocardial architecture in dilated cardiomyopathy.29 The use of diuretics may promote disease progression by causing collagen deposition.30,31 Thus, neurohormonal activation may trigger a series of maladaptive responses that result in myocardial fibrosis and the deposition of interstitial collagen, thereby altering LV diastolic function and conduction and increasing the likelihood of fatal electrical dysfunction because of reentry. The use of diuretic therapy in our hamsters hastened the development of interstitial fibrosis and, perhaps, electrolyte dysfunction, thus converting their mechanism of death from heart failure to SCD.

Limitations Fig. 5. Comparison of survival of hamsters in the previous study and the current study. The hamsters in the previous study did not receive furosemide, whereas the hamsters in the current study received furosemide daily. The P value was obtained from a logrank test, as shown in the Kaplan-Meier curve.

enrolled in the Prospective Randomized Amlodipine Survival Evaluation (ie, PRAISE) trial, high diuretic doses were independently associated with increased mortality, sudden death, and death caused by pump failure.23 Moreover, a fibrotic substrate for reentry, increased myocardial diastolic tension, imbalance of the autonomic nervous system, drug effects, and electrolyte imbalance have been cited as causes of electrical disturbances.24 In our study, histopathologic examination of the hearts of the hamsters that died suddenly showed that they had not developed features of advanced dilated cardiomyopathy; instead, they had less chamber dilation than the other hamsters and appeared to have a pronounced increase in interstitial collagen. This finding is consistent with the hypothesis that arrhythmias cause sudden death in dilated cardiomyopathy. This hypothesis is further supported by our finding that diuretic therapy contributed substantially to the risk of sudden death, probably by causing electrolyte disturbances that promote the development of fatal arrhythmias, even though furosemide extended the lives of hamsters that did not experience sudden death. Pathology

Dilated cardiomyopathy is characterized by progressive dilatation, interstitial fibrosis, and a decline in myocardial contractility.25,26 Hemostasis during a prolonged or decayed cardiac cycle may result in the formation of mural thrombi. These thrombi can subsequently embolize, leading to arrhythmias and thromboembolic conditions.27 Cardiac fibroblasts synthesize collagen, the major component of the cardiac interstitium. Quantitative and qualitative changes in collagen may influence cardiac structure and function.28 Collagen supports and tethers myocytes to maintain their alignment; their replacement of cardiac cells may be at least partly responsible for global transformations

In accordance with the policies of the University of Texas Health Science Center Animal Welfare Committee, some of the hamsters were euthanized to prevent prolonged suffering instead of being allowed to die naturally. Additionally, body temperature, weight, and echocardiograms were the only objective measures that we used to assess the development of heart failure and decompensation. Other methods of assessing cardiac decompensation, such as serologic tests (B-type natriuretic peptide, renin, angiotensin-II, and norepinephrine levels), were not available for comparison with the BTCR data and echocardiographic findings. Also, the echocardiography was performed only twice; future studies should include serial measurements to establish the relationship between the body temperature and echocardiographic parameters.

Conclusion We observed a significant decline in LV function, as indicated by 2D echocardiography, that was associated with a decline in BTCR amplitude. Measuring variations in BTCR may add incremental value to conventional clinical tools for the early diagnosis of patients with heart failure. Monitoring BTCR may also offer new insights into the pathophysiology of heart failure and suggest future therapeutic targets. Our findings provide insights that may eventually lead to improvements in monitoring and therapeutic approaches. The results we obtained should provide sufficient preclinical animal data to move forward into the clinical development of BTCR measurement as a mode of monitoring and treating CHF. This step may enhance the ‘‘transformative potential’’ of wireless technology in the medical field through new applications, sensors, and other revolutionary tools.

Acknowledgment Stephen N. Palmer, PhD, ELS, contributed to the editing of the manuscript.

Temperature Circadian Rhythm and LV Function

Disclosures None.

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