INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BIOSENSORS DERIVE UTILITY from their inherent selectivity to specific biological signals and their physiologically relevant reactions (25). Most biosensors are molecularly based, relying on a specific interaction among biomolecules, such as antibodies (4, 26), enzymes (21, 29), ion channels (5, 18), or nucleic acids (8, 20), and a target compound. Alternatively, cell- and tissue-based biosensors (2, 22-24) offer inherent insight into physiological function by exploiting the selectivity of the receptors, channels, and enzymes that are part of the functional structure of a cell. Most cell- and tissue-based biosensors are used for chemical detection. Although these biosensors are quite adept at chemical detection, it is not a task for which they specifically evolved. Here we describe a novel alternative approach that exploits the inherent biosensing capacity of excitable tissue in its natural context; as an integrated, multi-input bioprocessor that could be used to study physiological function. The present study has employed this approach to measure the contribution of circulating catecholamines to the dynamic regulation of cardiac chronotropy.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All of the experiments involving animals were performed according to the Institutional Animal Care and Use Committee of the Weill Medical College of Cornell University, which follows federal and state guidelines. Excitable tissue-based biosensors were developed by the implantation of chronotropically competent cardiac allografts distant from (and not directly linked to) the endogenous heart. We transplanted neonatal murine cardiac allografts into the pinnae of syngeneic adult mice and then monitored cardiac electrophysiological temporal dynamics.

Transplantation. Neonatal FVB murine hearts were transplanted into the pinnae of 3-mo FVB mice as previously described (7, 10). Fifteen mouse hearts were implanted into nine mice (six mice received a transplanted heart in each ear, whereas three mice received a transplanted heart in only one ear).

Electrocardiograms. Between 17 and 41 days after transplantation, electrocardiographic (ECG) activity of the endogenous and exogenous hearts was measured after intraperitoneal anesthetization with 2.5% tribromoethanol (Avertin; vol/vol). ECG were acquired for an average of 45 min (range: 27-117 min) via a four-channel differential alternating current amplifier (model 1700, A-M Systems). The signals were band-pass filtered between 3.0 and 100.0 Hz, notch filtered at 60.0 Hz, amplified 1,000 times, and sampled at 500 Hz with the use of a data acquisition board (model AT-MIO-16E-10, National Instruments) on a 266-MHz Intel Pentium II computer running real-time Linux (3).

Quantitative rate analysis. Postacquisition automatic (with manual correction as needed) ECG R-wave annotation was performed with the use of custom-designed Linux C++ software. Mean R-R intervals (RR) were computed every 2 s so that dynamics of the endogenous and exogenous signals, which have different inherent rates, could be compared quantitatively at synchronized time slices. We selected 2 s arbitrarily; no qualitative differences were found for interval lengths of 1, 5, or 10 s. The 15-trial endogenous mean RR_max  RR_min = 31.2 ± 24.9 ms (where RR_max and RR_min are maximal and minimal RR), whereas the exogenous mean RR_max  RR_min = 100.8 ± 72.0 ms.

1

Pharmacological trials. A new set of mice, with pinnal hearts implanted as before, were subjected to pharmacological trials between 41 and 62 days posttransplantation. Separate experiments were performed with the use of propranolol (100 µg ip) and clonidine (2.0 mg ip). To detect pharmacological rate effects, rate trends of distinct trial stages were quantified by a normalized rate of change (m) given by
where _i and are the averages of the initial and final three R-R intervals of a given stage, respectively, and t is the stage duration.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Figure 1 shows simultaneous electrical activity of the endogenous heart and bilateral exogenous hearts in a representative trial. The three hearts beat at unique but not unrelated rates (as seen in Fig. 2) with exogenous rates approximately one-half of the endogenous rate.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Voltage vs. time tracings for the endogenous heart (A) and two exogenous hearts (B and C) of a representative adult mouse after bilateral pinnal heart transplantation. Three hearts beat at unique but not unrelated rates, with the exogenous hearts beating at rates approximately one-half that of the endogenous heart.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Mean R-R intervals (RR) vs. time for the endogenous heart (A) and two exogenous hearts (B and C) of a representative adult mouse after bilateral pinnal heart transplantation. Solid curve in each graph is (t), the best polynomial curve fit to the data. Occasionally, poor signal quality or high noise made R-wave annotation impossible, resulting in gaps in the corresponding graph. R-R interval dynamics of both exogenous hearts effectively tracked those of the endogenous heart; i.e., there was a clear relationship between the rate trends (i.e., increasing/decreasing) of the 3 hearts. D: first derivative vs. time d(t)/dt for the polynomial fits of the endogenous () and second exogenous heart shown in C (). When time-shifted to account for the exogenous phase lag (i.e., the 72-s mean delay between the endogenous and exogenous derivative zeros), the two derivatives had the same sign for 79% of the record. Thirteen of fifteeen trials had concordance >70% (mean 85 ± 11%). Such high concordance quantitatively confirms that the exogenous heart effectively tracked increases and decreases in endogenous heart rate.

Quantitative rate analysis. Mean interbeat interval time series RR illuminated a clear relationship between exogenous and endogenous dynamics (Fig. 2). The ability of the exogenous tissue to track relative temporal endogenous dynamics (i.e., increasing/decreasing trends) was quantified via analysis of the derivatives of polynomial curves fit to the RR time series (Fig. 2D). For 13 of 15 exogenous cardiac allografts, the endogenous and exogenous derivative curves had concordant sign >70% (mean = 85 ± 11%) of the given trial, indicating that the exogenous tissue was an effective relative endogenous rate sensor. In addition to their relative tracking ability, the majority (9 of 13) of exogenous hearts showed evidence of effective absolute sensing function (i.e., at any given time, the exogenous rate, appropriately scaled, could be used as an effective predictor of the natural heart rate). Exogenous-to-endogenous RR correlation computation illuminated two types of absolute sensing function behavior: 1) a strong one-to-one linear relationship between the dynamics of the two hearts for the entire trial (in 5 of 9 exogenous hearts; i.e., Fig. 3A), or 2) temporally distinct, highly correlated segments during the trial (in 4 of 9 exogenous hearts; i.e., Fig. 3B). Such temporal shifts in absolute sensing function suggest that the exogenous activity is mediated by a subset of the multiple inputs that govern the endogenous dynamics.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Exogenous vs. endogenous RR for two separate mice (A and B). Solid and dotted lines in each graph depict the linear regression fit and the 95% confidence interval of that fit, respectively. A: correlation coefficient r = 0.94 indicates that there is a strong one-to-one linear relationship (with slope m = 3.33 ms) between the exogenous and endogenous RR. Five of 13 positive trials had r > 0.70 ( = 0.85 ± 0.10,  = 2.40 ± 1.15 ms). Four of the remaining eight positive trials had at least one distinct segment (of at least 500 consecutive seconds) of effective absolute sensing, with r > 0.85 ( = 0.91 ± 0.04,  = 2.06 ± 1.12 ms for segments of at least 500 s). One such trial (the same exogenous heart trial as that of Fig. 2C) is shown in B.

Pharmacological trials. To identify the exogenous input subset, we studied endogenous and exogenous chronotropic regulation by pharmacological manipulation. Intraperitoneal propranolol was administered in an attempt to block humoral and autonomic -adrenergic receptor pathways (Fig. 4A). As expected, shortly after injection, endogenous rate slowed. The exogenous rate underwent an even more dramatic decline, indicating that it is controlled by humoral and/or autonomic inputs. To further define the nature of the exogenous inputs, we performed intraperitoneal clonidine trials. In six of the seven trials, a rapid decrease in endogenous heart rate (lasting 10-30 s), which is consistent with the reduction of clonidine in efferent sympathetic nerve activity (32), was observed within 20 s postinjection (Fig. 4B). In contrast, there was a negligible reduction in exogenous heart rate during this time, suggesting that the exogenous hearts are not under significant direct autonomic control. After this initial postinjection stage, both hearts underwent a gradual heart rate reduction, consistent with a humoral response to the secondary effect of clonidine on norepinephrine release inhibition (1). The results shown in Fig. 4 strongly suggest that the chronotropic biosensing of the exogenous excitable tissue is mediated by predominately humoral influences.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Endogenous and exogenous RR vs. time for one mouse. A: 100 µg of propranolol was delivered via intraperitoneal (IP) injection at t = 121 s. Inset: magnified portion of the endogenous data. Shortly after injection, endogenous and exogenous rates slowed (stage B) relative to baseline (stage A) in 5 of 5 trials (aggregate values, as defined in MATERIALS AND METHODS, are shown in C). Exogenous slowing is evidence of autonomic and/or humoral control. B: same mouse as A, but performed on a different day; 2.0 mg of clonidine was delivered via intraperitoneal injection at t = 49 s. Shortly after injection, the endogenous rate slowed rapidly (stage B') in 6 of 7 trials. In contrast, the exogenous rate continued its preinjection trend. After stage B', both the endogenous and exogenous hearts slowed. In all 6 trials, the endogenous hearts slowed considerably more during stage B' than stage B, whereas the exogenous hearts slowed more during stage B than stage B'.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Here we have shown that implanted exogenous cardiac allografts can act as effective sensors of endogenous chronotropic inputs. Through pharmacological trials, we have demonstrated that the biosensing of the implanted tissue is mediated predominately by circulating hormonal signals. This is compelling evidence that excitable tissue can function as a responsive biosensor of underlying physiological regulation.

In addition to demonstrating the potential role of exogenous tissue to act as an implanted dynamic biosensor, our studies offer insight into the importance of circulating catecholamines in chronotropic regulation of transplanted hearts. It is well known (16, 17, 33) that the heart rate of the transplanted heart can be markedly influenced by pharmacological manipulations of the adrenergic system. However, because the contribution of the circulating catecholamines to such rate regulation is difficult to quantify, uptake studies (15) revealed the partial reinnervation of transplanted hearts, and heart rate variability studies (12, 14, 28, 30, 31) provided frequency-spectrum evidence of autonomic activity. An improvement in cardiac chronotropic responsiveness after heart transplantation has been attributed primarily to sinus node reinnervation in the transplanted heart (see Ref. 27 for a review). In contrast, the present study offers clear evidence of the capacity of circulating catecholamines to highly regulate the dynamics of transplanted hearts in the absence of direct autonomic inputs.

Future studies will be directed at further exploiting the inherent capacity of exogenous excitable tissue to sense circulating physiological signals. These studies may focus on expanding input recognition through the targeted molecular manipulation of cellular signaling pathways in excitable tissue (7). It is projected that through such molecular engineering, the utility of this approach may extend beyond that of chronotropic-regulation biosensing. For example, molecular manipulation of cellular chronotropic sensitivity to targeted substances may offer a means of biosensing physiological and pathophysiological signals that would otherwise not alter cardiac chronotropy. More importantly, it is anticipated that such developments may employ cardiac myocytes derived from pluripotent stem cells, potentially from endogenous bone marrow, thereby eliminating the potential for tissue rejection and enabling long-term use (13, 19). Further advancement may employ the development of a more uniform biosensor array by culturing excitable cells on silicon chips (6, 9, 11). Such a combination of improvements could lead to the development of long-term, physiologically tuned, functionally integrated bioprocessing interfaces for a wide range of external or implantable devices.