Short-term disruption of diurnal rhythms after murine myocardial infarction adversely affects long-term myocardial structure and function.

RATIONALE: Patients in intensive care units are disconnected from their natural environment. Synchrony between environmental diurnal rhythms and intracellular circadian rhythms is essential for normal organ biology; disruption causes pathology. Whether disturbing rhythms after myocardial infarction (MI) exacerbates long-term myocardial dysfunction is not known. OBJECTIVE: Short-term diurnal rhythm disruption immediately after MI impairs remodeling and adversely affects long-term cardiac structure and function in a murine model. METHODS AND RESULTS: Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 3-hour time window, randomized to either a normal diurnal or disrupted environment for 5 days, and then maintained under normal diurnal conditions. Initial infarct size was identical. Short-term diurnal disruption adversely affected body metabolism and altered early innate immune responses. In the first 5 days, crucial for scar formation, there were significant differences in cardiac myeloperoxidase, cytokines, neutrophil, and macrophage infiltration. Homozygous clock mutant mice exhibited altered infiltration after MI, consistent with circadian mechanisms underlying innate immune responses crucial for scar formation. In the proliferative phase, 1 week after MI, this led to significantly less blood vessel formation in the infarct region of disrupted mice; by day 14, echocardiography showed increased left ventricular dilation and infarct expansion. These differences continued to evolve with worse cardiac structure and function by 8 weeks after MI. CONCLUSIONS: Diurnal rhythm disruption immediately after MI impaired healing and exacerbated maladaptive cardiac remodeling. These preclinical findings suggest that disrupted diurnal rhythms such as found in modern intensive care unit environments may adversely affect long-term patient outcome.

The primary benefits of angiotensin-converting enzyme inhibition on cardiac remodeling occur during sleep time in murine pressure overload hypertrophy.

OBJECTIVES: Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. BACKGROUND: Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. METHODS: We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wake time for 8 weeks starting 1 week after surgery. RESULTS: TAC mice given captopril at sleep time had improved cardiac function and significantly decreased heart: body weight ratios, myocyte cross-sectional areas, intramyocardial vascular medial wall thickness, and perivascular collagen versus TAC mice given captopril or placebo during wake time. Captopril induced similar drops in blood pressure at sleep or wake time, suggesting that time-of-day differences were not attributable to blood pressure changes. These beneficial effects of captopril were correlated with diurnal changes in ACE mRNA expression in the heart. CONCLUSIONS: The ACE inhibitor captopril benefited cardiovascular remodeling only when administered during sleep; wake-time captopril ACE inhibition was identical to that of placebo. These studies support the hypothesis that the heart (and vessels) remodel during sleep time and also illustrate the importance of diurnal timing for some cardiovascular therapies.

Molecular time: an often overlooked dimension to cardiovascular disease.

Diurnal rhythms influence cardiovascular physiology such as heart rate and blood pressure and the incidence of adverse cardiac events such as heart attack and stroke. For example, shift workers and patients with sleep disturbances, such as obstructive sleep apnea, have an increased risk of heart attack, stroke, and sudden death. Diurnal variation is also evident at the molecular level, as gene expression in the heart and blood vessels is remarkably different in the day as compared to the night. Much of the evidence presented here indicates that growth and renewal (structural remodeling) are highly dependent on processes that occur during the subjective night. Myocardial metabolism is also dynamic with substrate preference also differing day from night. The risk/benefit ratio of some therapeutic strategies and the appearance of biomarkers also vary across the 24-hour diurnal cycle. Synchrony between external and internal diurnal rhythms and harmony among the molecular rhythms within the cell is essential for normal organ biology. Cell physiology is 4 dimensional; the substrate and enzymatic components of a given metabolic pathway must be present not only in the right compartmental space within the cell but also at the right time. As a corollary, we show disrupting this integral relationship has devastating effects on cardiovascular, renal and possibly other organ systems. Harmony between our biology and our environment is vital to good health.

B-vitamin deficiency in hospitalized patients with heart failure.

The impact of heart failure and its treatment on specific nutrient requirements is unknown. Furthermore, depletion of water-soluble B vitamins that play key roles in the production of cellular energy in patients with heart failure can contribute to depletion of energy reserves observed in the failing heart. A cross-sectional study recently reported that approximately one third of hospitalized patients with heart failure had tissue levels suggestive of thiamin deficiency (vitamin B-1). Riboflavin (vitamin B-2) and pyridoxine (vitamin B-6) are similar to thiamin in that they are water-soluble, subject to renal excretion, have limited tissue storage, and are dependent on intake. Therefore, it was hypothesized that the status of these B vitamins may also be adversely affected by heart failure. As a result, the prevalence of patients at risk of vitamin B-2 (erythrocyte glutathione reductase activity coefficient > or = 1.2) and B-6 deficiency (plasma B-6 < or = 20 nmol/L) was determined in a cross-section of 100 patients hospitalized with heart failure between April 2001 and June 2002 as well as in a group of volunteers without heart failure. Twenty-seven percent of patients with heart failure had biochemical evidence of vitamin B-2 deficiency, while 38% had evidence of B-6 deficiency. These prevalence rates were significantly higher than those observed in the volunteers without heart failure (2% and 19%, respectively; P < or = 0.02). Use of common B-vitamin-containing supplements by patients with heart failure did not significantly reduce deficiency rates in comparison with those who did not use supplements (B-2 P=0.38 or B-6 P=0.18)). Finally, while 80% of patients with heart failure took diuretics, neither the dose nor the duration of furosemide use was related to the presence of either B-2 or B-6 deficiency. Given the physiologic importance of these vitamins, further investigations aimed at determining the effect of heart failure on specific nutrient requirements as well as the safety and efficacy of B-vitamin supplementation are warranted.

Diurnal physiology: core principles with application to the pathogenesis, diagnosis, prevention, and treatment of myocardial hypertrophy and failure.

The circadian system has been shown to be fundamentally important in human health and disease. Recently, there have been major advances in our understanding of daily rhythmicity, and its relevance to human physiology, and to the pathogenesis and treatment of cardiac hypertrophy and heart failure. Cardiovascular tissues, such as heart and blood vessels, show remarkable daily variation in gene expression, metabolism, growth, and remodeling. Moreover, synchrony of daily molecular and physiological rhythms is integral to healthy organ growth and renewal. Disruption of these rhythms adversely affects normal growth, also the remodeling mechanisms in disease, leading to gross abnormalities in heart and vessels. These observations provide new insights into the pathogenesis, diagnosis, treatment, and prevention of heart disease. In this review, we focus on the recent advances in circadian biology and cardiovascular function, with particular emphasis on how this applies to human myocardial hypertrophy and heart failure, and the implications and importance for translational medicine.

Vascular circadian rhythms in a mouse vascular smooth muscle cell line (Movas-1).

The circadian system in mammals is a hierarchy of oscillators throughout the organism that are coordinated by the circadian clock in the hypothalamic suprachiasmatic nucleus. Peripheral clocks act to integrate time-of-day information from neural or hormonal signals, regulating gene expression, and, subsequently, organ physiology. However, the mechanisms by which the central clock communicates with peripheral oscillators are not understood and are likely tissue specific. In this study, we establish a mouse vascular cell model suitable for investigations of these mechanisms at a molecular level. Using the immortalized vascular smooth muscle cell line Movas-1, we determined that these cells express the circadian clock machinery with robust rhythms in mRNA expression over a 36-h period after serum shock synchronization. Furthermore, norepinephrine and forskolin were able to synchronize circadian rhythms in bmal1. With synchronization, we observed cycling of specific genes, including the tissue inhibitor of metalloproteinase 1 and 3 (timp1, timp3), collagen 3a1 (col3a1), transgelin 1 (sm22alpha), and calponin 1 (cnn1). Diurnal expression of these genes was also found in vivo in mouse aortic tissue, using microarray and real-time RT-PCR analysis. Both of these revealed ultradian rhythms in genes similar to the cycling observed in Movas-1 in vitro. These findings highlight the cyclical nature of structurally important genes in the vasculature that is similar both in vivo and in vitro. This study establishes the Movas-1 cells as a novel cell model from which to further investigate the molecular mechanisms of clock regulation in the vasculature.

Diurnal profiling of neuroendocrine genes in murine heart, and shift in proopiomelanocortin gene expression with pressure-overload cardiac hypertrophy.

Neuroendocrine peptides express biologic activity relevant to the cardiovascular system, including regulating heart rate and blood pressure, though little is known about the mechanisms involved. Here, we investigated neuroendocrine gene expression underlying diurnal physiology of the heart. We first used microarray and RT-PCR analysis and demonstrate the simultaneous expression of neuroendocrine genes in normal murine heart, including POMC, GnRH, neuropeptide Y, leptin receptor, GH-releasing hormone, cocaine- and amphetamine-regulated transcript, proglucagon, and galanin. We examined diurnal gene expression profiles, with cosinar bioinformatics to evaluate statistically significant rhythms. The POMC gene exhibits a day/night, circadian or diurnal, pattern of expression in heart, and we postulated that this may be important to cardiac growth and renewal. POMC diurnal gene rhythmicity is altered in pressure-overload cardiac hypertrophy, when compared with control heart, and levels increased at the dark-to-light transition times. These findings are also consistent with the proposal that neuropeptides mediate adverse remodeling processes, such as occur in pathologic hypertrophy. To investigate cellular responses, we screened three cell lines representing fibroblasts, cardiac myocytes, and vascular smooth muscle cells (NIH3T3, heart line 1, and mouse vascular smooth muscle cell line 1 (Movas-1) respectively). POMC mRNA expression is the most notable in Movas-1 cells and, furthermore, exhibits rhythmicity with culture synchronization. Taken together, these results highlight the diverse neuroendocrine mRNA expression profiles in cardiovasculature, and provide a novel model vascular culture system to research the role these neuropeptides play in organ health, integrity, and disease.

Circadian rhythm disorganization produces profound cardiovascular and renal disease in hamsters.

Sleep deprivation, shift work, and jet lag all disrupt normal biological rhythms and have major impacts on health; however, circadian disorganization has never been shown as a causal risk factor in organ disease. We now demonstrate devastating effects of rhythm disorganization on cardiovascular and renal integrity and that interventions based on circadian principles prevent disease pathology caused by a short-period mutation (tau) of the circadian system in hamsters. The point mutation in the circadian regulatory gene, casein kinase-1epsilon, produces early onset circadian entrainment with fragmented patterns of behavior in +/tau heterozygotes. Animals die at a younger age with cardiomyopathy, extensive fibrosis, and severely impaired contractility; they also have severe renal disease with proteinuria, tubular dilation, and cellular apoptosis. On light cycles appropriate for their genotype (22 h), cyclic behavioral patterns are normalized, cardiorenal phenotype is reversed, and hearts and kidneys show normal structure and function. Moreover, hypertrophy does not develop in animals whose suprachiasmatic nucleus was ablated as young adults. Circadian organization therefore is critical for normal health and longevity, whereas chronic global asynchrony is implicated in the etiology of cardiac and renal disease.

Diurnal protein expression in blood revealed by high throughput mass spectrometry proteomics and implications for translational medicine and body time of day.

Molecular gene cycling is useful for determining body time of day (BTOD) with important applications in personalized medicine, including cardiovascular disease and cancer, our leading causes of death. However, it impractically requires repetitive invasive tissue sampling that is obviously not applicable for humans. Here we characterize diurnal protein cycling in blood using high-throughput proteomics; blood proteins are easily accessible, minimally invasive, and can importantly serve as surrogates for what is happening elsewhere in the body in health and disease. As proof of the concept, we used normal C57BL/6 mice maintained under regular 24-h light and dark cycles. First, we demonstrated fingerprint patterns in 24-h plasma, revealed using surface-enhanced laser desorption and ionization (SELDI). Second, we characterized diurnal cycling proteins in blood using chromatography and tandem electrospray ionization mass spectrometry. Importantly, we noted little association between the cycling blood proteome and tissue transcriptome, delineating the necessity to identify de novo cycling proteins in blood for measuring BTOD. Furthermore, we explored known interaction networks to identify putative functional pathways regulating protein expression patterns in blood, thus shedding new light on our understanding of integrative physiology. These studies have profound clinical significance in translating the concept of BTOD to the practical realm for molecular diagnostics and open new opportunities for clinically relevant discoveries when applied to ELISA-based molecular testing and/or point-of-care devices.

Disturbed diurnal rhythm alters gene expression and exacerbates cardiovascular disease with rescue by resynchronization.

Day/night rhythms are recognized as important to normal cardiovascular physiology and timing of adverse cardiovascular events; however, their significance in disease has not been determined. We demonstrate that day/night rhythms play a critical role in compensatory remodeling of cardiovascular tissue, and disruption exacerbates disease pathophysiology. We use a murine model of pressure overload cardiac hypertrophy (transverse aortic constriction) in a rhythm-disruptive 20-hour versus 24-hour environment. Echocardiography reveals increased left ventricular end-systolic and -diastolic dimensions and reduced contractility in rhythm-disturbed transverse aortic constriction animals. Furthermore, cardiomyocytes and vascular smooth muscle cells exhibit reduced hypertrophy, despite increased pressure load. Microarray and real-time PCR demonstrate altered gene cycling in transverse aortic constriction myocardium and hypothalamic suprachiasmatic nucleus. With rhythm disturbance, there is a consequent altered cellular clock mechanism (per2 and bmal), whereas key genes in hypertrophic pathways (ANF, BNP, ACE, and collagen) are downregulated paradoxical to the increased pressure. Phenotypic rescue, including reversal/attenuation of abnormal pathology and genes, only occurs when the external rhythm is allowed to correspond with the animals' innate 24-hour internal rhythm. Our study establishes the importance of diurnal rhythm as a vital determinant in heart disease. Disrupted rhythms contribute to progression of organ dysfunction; restoration of normal diurnal schedules appears to be important for effective treatment of disease.

The management of conditioned nutritional requirements in heart failure.

Patients suffering from congestive heart failure exhibit impaired myocardial energy production, myocyte calcium overload and increased oxidative stress. Nutritional factors known to be important for myocardial energy production, calcium homeostasis and the reduction of oxidative stress, such as thiamine, riboflavin, pyridoxine, L-carnitine, coenzyme Q10, creatine and taurine are reduced in this patient population. Furthermore, deficiencies of taurine, carnitine, and thiamine are established primary causes of dilated cardiomyopathy. Studies in animals and limited trials in humans have shown that dietary replacement of some of these compounds in heart failure can significantly restore depleted levels and may result in improvement in myocardial structure and function as well as exercise capacity. Larger scale studies examining micronutrient depletion in heart failure patients, and the benefits of dietary replacement need to be performed. At the present time, it is our belief that these conditioned nutritional requirements, if unsatisfied, contribute to myocyte dysfunction and loss; thus, restoration of nutritional deficiencies should be part of the overall therapeutic strategy for patients with congestive heart failure.

The prevalence of thiamin deficiency in hospitalized patients with congestive heart failure.

OBJECTIVES: The purpose of this study was to determine the prevalence of thiamin deficiency (TD) in a cross section of hospitalized congestive heart failure (CHF) patients and to investigate factors that contribute to its development. BACKGROUND: Thiamin deficiency manifests as symptoms of CHF and, therefore, may worsen existing heart failure. Congestive heart failure patients may be at increased risk for TD as a result of diuretic-induced urine thiamin excretion, disease severity, malnutrition, and advanced age. METHODS: Erythrocyte thiamin pyrophosphate concentrations, using high-performance liquid chromatography, were measured in 100 CHF patients and compared to 50 control subjects. Variables including diuretics (type and dose), left ventricle dysfunction, New York Heart Association functional classification, creatinine clearance, thiamin intake (diet and supplements), malnutrition, appetite ratings, and age were related to TD using univariate statistics and multiple logistic regression analysis. RESULTS: Thiamin deficiency was more prevalent in CHF patients (33%) compared to control subjects (12%) (p = 0.007). Thiamin deficiency was related to urine thiamin loss (p = 0.03), non-use of thiamin-containing supplements (p = 0.06), and preserved renal function (p = 0.05). Increased urinary thiamin loss (mug/g creatinine) was found to be the only significant positive predictor of thiamin status on multiple logistic regression analysis (p = 0.03). CONCLUSIONS: One-third of hospitalized CHF patients were TD. In contrast to previous studies, increased urinary losses of thiamin were predictive of improved thiamin status. Thiamin supplementation may be protective against TD in the clinical setting. Future studies are warranted to determine if thiamin supplementation improves thiamin status and disease severity in CHF patients.

Cardiac troponin I, a possible predictor of survival in patients with stable congestive heart failure.

BACKGROUND: Cardiac troponin I (cTnI) has been validated as a sensitive and specific marker of myocyte damage, and is elevated in some patients with congestive heart failure. OBJECTIVE: To assess the relationship between elevated levels of cTnI and survival in stable patients with congestive heart failure. PATIENTS AND METHODS: It was assessed whether detectable serial levels of cTnI were associated with mortality in 211 patients with stable, severe heart failure at entry and one month into the Prospective Randomized Flosequinan Longevity Evaluation (PROFILE) study. Of these patients, 66 also had measurements taken at 12 months. RESULTS: Patients were New York Heart Association (NYHA) class III (n=197) or IV (n=14), with a baseline left ventricular ejection fraction of 22+/-7% (range 8% to 35%). Patients with a detectable level of cTnI at one month had an increased mortality (OR 2.608 [95% CI 1.061 to 6.409]; P=0.037). The association between mortality and detectable cTnI levels at baseline or 12 months did not reach statistical significance. Patients with a cTnI level that rose or remained elevated between baseline and one month had a higher mortality rate (50%) than those in whom the cTnI level fell (9%) between baseline and one month (P=0.025). In a multivariate model of survival that included sex, treatment, age, left ventricular ejection fraction, NYHA class and creatinine, only detectable levels of cTnI at one month were associated with survival (P=0.037). CONCLUSIONS: cTnI is released in stable, chronic heart failure and is associated with a poor prognosis, independent of other important risk factors. The risk is particularly elevated when detectable cTnI levels rise or remain elevated over time.

Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model.

BACKGROUND: Iron overload has an increasing worldwide prevalence and is associated with significant cardiovascular morbidity and mortality. Elevated iron levels in the myocardium lead to impaired systolic and diastolic function and elevated oxidative stress. Taurine accounts for 25% to 50% of the amino acid pool in myocardium, possesses antioxidant properties, and can inhibit L-type Ca2+ channels. Thus, we hypothesized that this agent would reduce the cardiovascular effects of iron overload. METHODS AND RESULTS: Iron-overloaded mice were generated by intraperitoneal injection of iron either chronically (5 days per week for 13 weeks) or subacutely (5 days per week for 4 weeks). Iron overload causes increased mortality, elevated oxidative stress, systolic and diastolic dysfunction, hypotension, and bradycardia. Taurine supplementation increased myocardial taurine levels by 45% and led to reductions in mortality and improved cardiac function, heart rate, and blood pressure in iron-overloaded mice. Histological examination of the myocardium revealed reduced apoptosis and interstitial fibrosis in iron-overloaded mice supplemented with taurine. Taurine mediated reduced oxidative stress in iron-overloaded mice along with attenuation of myocardial lipid peroxidation and protection of reduced glutathione level. CONCLUSIONS: These results demonstrate that treatment with taurine reduces iron-mediated myocardial oxidative stress, preserves cardiovascular function, and improves survival in iron-overloaded mice. The role of taurine in protecting reduced glutathione levels provides an important mechanism by which oxidative stress-induced myocardial damage can be curtailed. Taurine, as a dietary supplement, represents a potential new therapeutic agent to reduce the cardiovascular burden from iron-overload conditions.

Day/night rhythms in gene expression of the normal murine heart.

Molecular circadian oscillators have recently been identified in heart and many other peripheral organs; however, little is known about the physiologic significance of circadian gene cycling in the periphery. While general temporal profiles of gene expression in the heart have been described under constant lighting conditions, patterns under normal day/night conditions may be distinctly different. To understand how gene expression contributes to cardiac function, especially in human beings, it is crucial to examine these patterns in 24-h light and dark environments. High-density oligonucleotide microarrays were used to assess myocardial expression of 12,488 murine genes at 3-h intervals under the normal conditions of light and dark cycling. Variation in genetic activity was considerable, as 1,634 genes (approximately 13% of genes analyzed) exhibited statistically significant changes across the 24-h cycle. Some genes exhibited rhythmic expression, others showed abrupt change at light-to-dark and dark-to-light transitions. Importantly, genes that exhibited significant cycling rhythms mapped to key biological pathways, including for example cardiac cellular growth and remodeling, as well as transcription, translation, mitochondrial respiration, and signaling pathways. Gene expression in the heart is remarkably different in the day versus the night. Some gene cycling may be driven by the central circadian pacemaker, while other changes appear to be responses to light and dark. This has important implications regarding our understanding of how the molecular physiology of the heart is controlled, including temporal patterns of organ growth, renewal, and disease, comparative gene expression, and the most appropriate times for administration of therapy.

Conditioned nutritional requirements: therapeutic relevance to heart failure.

BACKGROUND: The advent of disease, genetic predisposition or certain drug therapies may significantly alter the nutritional demands of specific organs. Several specific metabolic deficiencies have been found in the failing myocardium: (1) a reduction in L-carnitine, coenzyme Q10, creatine, and thiamine--nutrient cofactors important for myocardial energy production; (2) a relative deficiency of taurine, an amino acid integral to intracellular calcium homeostasis; (3) increased myocardial oxidative stress and a reduction of antioxidant defenses. Deficiencies of carnitine or taurine alone are well documented to result in dilated cardiomyopathy in animals and humans. Each of these deficiencies is amenable to restoration through dietary supplementation. A variety of nutrients have been investigated as single therapeutic agents in pharmacologic fashion, but there has been no broad-based approach to nutritional supplementation in congestive heart failure to correct this complex of metabolic abnormalities. METHOD AND RESULTS: We have demonstrated deficiencies in carnitine, taurine and coenzyme Q10 in cardiomyopathic hamster hearts during the late stage of the cardiomyopathy. In another study, we randomized placebo diet against a supplement containing taurine, coenzyme Q10, carnitine, thiamine, creatine, vitamin E, vitamin C, and selenium to cardiomyopathic hamsters during the late stages of the disease. Supplementation for 3 months markedly improved myocyte sarcomeric structure, developed pressure, +dp/dt, and -dp/dt. We also documented carnitine, taurine and coenzyme Q10 in biopsies taken from human failing hearts, the levels correlating with ventricular function. A double-blind, randomized, placebo-controlled trial of a supplement containing these nutrients, given for 30 days, restored myocardial levels and resulted in a significant decrease in left ventricular end-diastolic volume. CONCLUSION: These experiments suggest that a comprehensive restoration of adequate myocyte nutrition may be important to any therapeutic strategy designed to benefit patients suffering from congestie heart failure. Future studies in this area are of clinical importance.