Abhijit Patwardhan

Longitudinal analysis of arterial blood pressure and heart rate response to acute behavioral stress in rats with type 1 diabetes mellitus and in age-matched controls

We recorded via telemetry the arterial blood pressure (BP) and heart rate (HR) response to classical conditioning following the spontaneous onset of autoimmune diabetes in BBDP/Wor rats vs. age-matched, diabetes-resistant control (BBDR/Wor) rats. Our purpose was to evaluate the autonomic regulatory responses to an acute stress in a diabetic state of up to 12 months duration. The stress was a 15-s pulsed tone (CS+) followed by a 0.5-s tail shock. The initial, transient increase in BP (i.e., the “first component,” or C1), known to be derived from an orienting response and produced by a sympathetic increase in peripheral resistance, was similar in diabetic and control rats through ∼9 months of diabetes; it was smaller in diabetic rats 10 months after diabetes onset. Weakening of the C1 BP increase in rats that were diabetic for >10 months is consistent with the effects of sympathetic neuropathy. A longer-latency, smaller, but sustained “second component” (C2) conditional increase in BP, that is acquired as a rat learns the association between CS+ and the shock, and which results from an increase in cardiac output, was smaller in the diabetic vs. control rats starting from the first month of diabetes. A concomitant HR slowing was also smaller in diabetic rats. The difference in the C2 BP increase, as observed already during the first month of diabetes, is probably secondary to the effects of hyperglycemia upon myocardial metabolism and contractile function, but it may also result from effects on cognition. The small HR slowing concomitant with the C2 pressor event is probably secondary to differences in baroreflex activation or function, though parasympathetic dysfunction may contribute later in the duration of diabetes. The nearly immediate deficit after disease onset in the C2 response indicates that diabetes alters BP and HR responses to external challenges prior to the development of structural changes in the vasculature or autonomic nerves.

Restitution of Action Potential Duration During Sequential Changes in Diastolic Intervals Shows Multimodal Behavior

Abstract— Restitution of action potential duration (APD) is thought to be critical in activation instability. Although restitution is used to predict APD during sequential changes in diastolic interval (DI), currently used protocols to determine restitution do not use sequential changes in DI. We explored restitution using a new pacing protocol to change DI sequentially and independently of APD. Transmembrane potentials were recorded from right ventricular endocardial tissue isolated from six dogs. We used three patterns of DIs: oscillatory, to demonstrate differences in APDs depending on previous activation history; random, to minimize effects of previous activation history, each DI preceding an APD had an equal probability of being short or long; and linear, to compare restitution relationship obtained during sequential changes in DI with those obtained using currently used protocols; DIs mimicked those that resulted using currently used protocols, except that they changed in sequence. During oscillatory DIs, restitution showed bimodal trajectory similar to hysteresis. Decrease in APD during decreasing DIs was faster than increase in APD during increasing DIs. When effects of previous activation history were minimized, we observed that for a given DI there were multiple values of APD. Restitution relationship obtained during sequential changes in DI was shallower than those obtained using currently used protocols. Our results show that the new pacing protocol may permit direct evaluation of effects of memory on APD. Sequential and explicit control of DI suggests that use of a unimodal relationship to predict APD when DIs change in sequence may not be appropriate.

The cardiomyocyte molecular clock, regulation of Scn5a, and arrhythmia susceptibility

The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop. Bmal1 encodes a core molecular clock transcription factor. Germline Bmal1 knockout mice show a loss of circadian variation in heart rate and blood pressure, and they develop dilated cardiomyopathy. We tested the role of the molecular clock in adult cardiomyocytes by generating mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1). ECG telemetry showed that cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)) in adult mice slowed heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. Moreover, isolated iCSΔBmal1(-/-) hearts were more susceptible to arrhythmia during electromechanical stimulation. Examination of candidate cardiac ion channel genes showed that Scn5a, which encodes the principle cardiac voltage-gated Na(+) channel (Na(V)1.5), was circadianly expressed in control mouse and rat hearts but not in iCSΔBmal1(-/-) hearts. In vitro studies confirmed circadian expression of a human Scn5a promoter-luciferase reporter construct and determined that overexpression of clock factors transactivated the Scn5a promoter. Loss of Scn5a circadian expression in iCSΔBmal1(-/-) hearts was associated with decreased levels of Na(V)1.5 and Na(+) current in ventricular myocytes. We conclude that disruption of the molecular clock in the adult heart slows heart rate, increases arrhythmias, and decreases the functional expression of Scn5a. These findings suggest a potential link between environmental factors that alter the cardiomyocyte molecular clock and factors that influence arrhythmia susceptibility in humans.

Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities In Vivo

ABSTRACT Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage. IMPORTANCE The protozoan Toxoplasma gondii establishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cysts in vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease. The protozoan Toxoplasma gondii establishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cysts in vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.

Epinephrine, vasodilation and hemoconcentration in syncopal, healthy men and women

Healthy young people may become syncopal during standing, head up tilt (HUT) or lower body negative pressure (LBNP). To evaluate why this happens we measured hormonal indices of autonomic activity along with arterial pressure (AP), heart rate (HR), stroke volume (SV), cardiac output (CO), total peripheral resistance (TPR) and measures of plasma volume. Three groups of normal volunteers (n = 56) were studied supine, before and during increasing levels of orthostatic stress: slow onset, low level, lower body negative pressure (LBNP) (Group 1), 70 degrees head up tilt (HUT) (Group 2) or rapid onset, high level, LBNP (Group 3). In all groups, syncopal subjects demonstrated a decline in TPR that paralleled the decline in AP over the last 40 s of orthostatic stress. Ten to twenty seconds after the decline in TPR. HR also started to decline but SV increased, resulting in a net increase of CO during the same period. Plasma volume (PV, calculated from change in hematocrit) declined in both syncopal and nonsyncopal subjects to a level commensurate with the stress, i.e. Group 3 > Group 2 > Group 1. The rate of decline of PV, calculated from the change in PV divided by the time of stress, was greater (p < 0.01) in syncopal than in nonsyncopal subjects. When changes in vasoactive hormones were normalized by time of stress, increases in norepinephrine (p < 0.012, Groups 2 and 3) and epinephrine (p < 0.025, Group 2) were greater and increases in plasma renin activity were smaller (p < 0.05, Group 2) in syncopal than in nonsyncopal subjects. We conclude that the presyncopal decline in blood pressure in otherwise healthy young people resulted from declining peripheral resistance associated with plateauing norepinephrine and plasma renin activity, rising epinephrine and rising blood viscosity. The increased hemoconcentration probably reflects increased rate of venous pooling rather than rate of plasma filtration and, together with cardiovascular effects of imbalances in norepinephrine, epinephrine and plasma renin activity may provide afferent information leading to syncope.

Mechanism of Repolarization Alternans Has Restitution of Action Potential Duration Dependent and Independent Components

Introduction: Investigation of relationship between diastolic‐interval (DI)‐dependent restitution of action potential duration (APD) and alternans of APD has produced conflicting results. We used a novel pacing protocol to determine the role of restitution in alternans by minimizing changes in DI preceding each activation.

False positive head-up tilt:: Hemodynamic and neurohumoral profile

OBJECTIVES This study examined differences in mechanisms of head-up tilt (HUT)-induced syncope between normal controls and patients with neurocardiogenic syncope. BACKGROUND A variable proportion of normal individuals experience syncope during HUT. Differences in the mechanisms of HUT-mediated syncope between this group and patients with neurocardiogenic syncope have not been elucidated. METHODS A 30-min 80 degrees HUT was performed in eight HUT-negative volunteers (Group I), eight HUT-positive volunteers (Group II) and 15 patients with neurocardiogenic syncope. Heart rate and blood pressure (BP) were monitored continuously. Epinephrine and norepinephrine plasma levels, as well as left ventricular dimensions and contractility determined by echocardiography, were measured at baseline and at regular intervals during the test. RESULTS The main findings of this study were the following: 1) All parameters were similar at baseline in the three groups; and 2) During tilt: a) the time to syncope was shorter in Group III than in group II (9.5 +/- 3 vs. 17 +/- 3 min p < 0.05); b) there was an immediate, persisting drop in mean BP in Group III; c) the decrease rate of left ventricular end-diastolic dimensions was greater in Group III than in Group II or Group I (-1.76 +/- 0.42 vs. -0.87 +/- 0.35 and -0.67 +/- 0.29 mm/min, respectively, p < 0.05); d) the leftventricular shortening fraction was greater in Group III than in the other two groups (39 +/- 1 vs. 34 +/- 1 and 32 +/- 1%, respectively, p < 0.05); and e) although the norepinephrine level remained comparable among the groups, there was a significantly higher peak epinephrine level in Group III than in Group II and Group I (112.3 +/- 34 vs. 77.6 +/- 10 and 65 +/- 12 pg/ml, p < 0.05). CONCLUSIONS Mechanisms of syncope during HUT appeared to be different in normal volunteers and patients with neurocardiogenic syncope. In the latter, there was evidence of an impaired vascular resistance response from the beginning of the orthostatic challenge. Furthermore, in the patients there was more rapid peripheral blood pooling, as indicated by the echocardiographic measurements of left ventricular end-diastolic changes, leading to more precocious symptoms. In syncopal patients, the higher level of plasma epinephrine probably mediated the increased cardiac contractility and possibly contributed to the impaired vasoconstrictive response.

Heart rate variability during sympatho-excitatory challenges: Comparison between spontaneous and metronomic breathing

Respiration influences heart rate variability, leading to the suggestion that respiration should be controlled to assess autonomic function by using heart rate variability. Clearly, control of respiration is advantageous or even essential in several experimental circumstances. However, control of respiration, by itself, produces a small, but significant, increase in mean heart rate and a decrease in respiratory synchronous variation in heart rate. We tested whether, in some experimental situations, it may be possible to arrive at similar interpretation about autonomic function with and without using control of respiratory rate. heart rate spectral powers from nine subjects were compared between spontaneous and metronomic breathing during two sympatho-excitatory stresses, lower body negative pressure (LBNP) and head up tilt (HUT). The normalized spectral powers in supine and HUT during spontaneous breathing were: 0.43 and 0.75 in very low (VLF) and 0.28 and 0.09 in high frequency (HF) regions. The powers during metronomic breathing were: 0.36 and 0.82 (VLF) and 0.36 and 0.09 (HF). The powers in supine and LBNP during spontaneous breathing were: 0.43 and 0.81 (VLF) and 0.28 and 0.06 (HF). The powers during metronomic breathing were: 0.36 and 0.80 (VLF) and 0.36 and 0.07 (HF). All p values were <0.05. Therefore, changes in heart rate spectral powers during HUT and LBNP were similar during metronomic breathing and spontaneous breathing. These results suggest that in experimental designs such as in our study, using metronomic breathing may not provide any additional insight into autonomic function than that can be obtained during spontaneous breathing.

The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization

BACKGROUND Sudden cardiac death (SCD) follows a diurnal variation. Data suggest the timing of SCD is influenced by circadian (~24-hour) changes in neurohumoral and cardiomyocyte-specific regulation of the hearts electrical properties. The basic helix-loop-helix transcription factors brain muscle arnt-like1 (BMAL1) and circadian locomotor output control kaput (CLOCK) coordinate the circadian expression of select genes. OBJECTIVE We sought to test whether Bmal1 expression in cardiomyocytes contributes to K(+) channel expression and diurnal changes in ventricular repolarization. METHODS We used transgenic mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)). We used quantitative polymerase chain reaction, voltage clamping, promoter-reporter bioluminescence assays, and electrocardiographic telemetry. RESULTS Although several K(+) channel gene transcripts were downregulated in iCSΔBmal1(-/-)mouse hearts, only Kcnh2 exhibited a robust circadian pattern of expression that was disrupted in iCSΔBmal1(-/-) hearts. Kcnh2 underlies the rapidly activating delayed-rectifier K(+) current, and the rapidly activating delayed-rectifier K(+) current recorded from iCSΔBmal1(-/-) ventricular cardiomyocytes was ~50% smaller than control ventricular myocytes. Promoter-reporter assays demonstrated that the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. Electrocardiographic analysis showed that iCSΔBmal1(-/-) mice developed a prolongation in the heart rate-corrected QT interval during the light (resting) phase. This was secondary to an augmented circadian rhythm in the uncorrected QT interval without a corresponding change in the RR interval. CONCLUSION The molecular clock in the heart regulates the circadian expression of Kcnh2, modifies K(+) channel gene expression, and is important for normal ventricular repolarization. Disruption of the cardiomyocyte circadian clock mechanism likely unmasks diurnal changes in ventricular repolarization that could contribute to an increased risk of cardiac arrhythmias/SCD.

Is there an association between external cardioversions and long-term mortality and morbidity? Insights from the Atrial Fibrillation Follow-up Investigation of Rhythm Management study.

Background— Cardiac electric therapies effectively terminate tachyarrhythmias. Recent data suggest a possible increase in long-term mortality associated with implantable cardioverter-defibrillator shocks. Little is known about the association between external cardioversion episodes (ECVe) and long-term mortality. We sought to assess the safety of repeated ECVe with regard to cardiovascular mortality and morbidity. Methods and Results— We analyzed the data of the 4060 patients from the AFFIRM (Atrial Fibrillation Follow-up Investigation of Rhythm Management) trial. In particular, associations of ECVe with all-cause mortality, cardiovascular mortality, and hospitalizations after ECVe were studied. Over an average follow-up of 3.5 years, 660 (16.3%) patients died, 331 (8.2%) from cardiovascular causes. A total of 207 (5.1%) and 1697 (41.8%) patients had low ejection fraction and nonparoxysmal atrial fibrillation, respectively; 2460 patients received no ECVe, whereas 1600 experienced ≥1 ECVe. Death occurred in 412 (16.7%), 196 (16.5%), 39 (13.5%), and 13 (10.4%) of patients with 0, 1, 2, and ≥3 ECVe, respectively. There was no significant association between ECVe and mortality within any of the 4 subgroups defined by ejection fraction and atrial fibrillation type, although myocardial infarction, coronary artery bypass graft, and digoxin were significantly associated with death (estimated hazard ratios, 1.65, 1.59, and 1.62, respectively; P<0.0001). ECVe were associated with increased cardiac hospitalization reported at the next follow-up visit (39.3% versus 5.8%; estimated odds ratio, 1.39; P<0.0001). Conclusions— In the AFFIRM study, there was no significant association between ECVe and long-term mortality, even though ECVe were associated with increased hospitalizations from cardiac causes. Digoxin, myocardial infarction, and coronary artery bypass graft were significantly associated with mortality.Background —Cardiac electrical therapies effectively terminate tachyarrhythmias. Recent data suggest a possible increase in long-term mortality associated with implantable cardiac defibrillator shocks. Little is known about the association between external cardioversion episodes (ECVe) and long-term mortality. We sought to assess the safety of repeated ECVe with regards to cardiovascular mortality and morbidity. Methods and Results —We analyzed the data of the 4,060 patients from the AFFIRM trial. In particular, associations of ECVe with all-cause mortality, cardiovascular mortality and hospitalizations post ECVe were studied. Over an average follow-up of 3.5 years, 660 patients (16.3%) died, 331(8.2%) from cardiovascular causes. A total of 207(5.1%) and 1697(41.8%) patients had low ejection fraction (EF) and non-paroxysmal atrial fibrillation (AF) respectively. 2460 patients received no ECVe; while 1600 experienced ≥1 ECVe. Death occurred in 412(16.7%), 196(16.5%), 39(13.5%), and 13(10.4%) of patients with 0, 1, 2, and ≥3 ECVe respectively. There was no significant association between ECVe and mortality within any of the four subgroups defined by EF and AF type, although myocardial infarction (MI), coronary artery bypass graft (CABG), and digoxin were significantly associated with death (Estimated hazard ratios: 1.65; 1.59 and 1.62 respectively, p<0.0001). ECVe were associated with increased cardiac hospitalization reported at the next follow-up visit (39.3% vs. 5.8%; Estimated odds ratio: 1.39, p<0.0001). Conclusions —In the AFFIRM study, there was no significant association between ECVe and long-term mortality, even though ECVe were associated with increased hospitalizations from cardiac causes. Digoxin, MI, and CABG were significantly associated with mortality.