Hypotension in transgenic mice expressing atrial natriuretic factor fusion genes.

Chronic regulation of the cardiovascular system by atrial natriuretic factor was investigated by generating transgenic mice with elevated hormone levels in the systemic circulation. A fusion gene comprising the mouse transthyretin promoter and mouse atrial natriuretic factor structural sequences was designed so as to target hormone expression to the liver. Hepatic expression of atrial natriuretic factor was detectable as early as embryonic day 15 in transgenic animals. In adult transgenic mice, plasma immunoreactive atrial natriuretic factor concentration was elevated at least eightfold as compared with nontransgenic littermates. The mean arterial pressure of conscious transgenic mice was 75.5 +/- 0.9 mm Hg, significantly less than that of nontransgenic siblings (103.9 +/- 2.0 mm Hg). This difference in mean arterial pressure was not accompanied by significant changes in several other physiological parameters, including heart rate, plasma and urinary electrolytes, water intake, and urine volume. This study demonstrates that a chronic elevation of plasma atrial natriuretic factor decreases arterial blood pressure without inducing diuresis and natriuresis in transgenic mice and also illustrates the value of the transgenic approach for the study of the cardiovascular system.

Engineering the cardiovascular system. Blood pressure regulation.

We have generated several lineages of transgenic mice that exhibit chronic elevations in the steady-state concentration of atrial natriuretic factor (ANF) in the peripheral circulation. ANF, a peptide hormone synthesized primarily by atrial cardiomyocytes, is a potent natriuretic and diuretic. ANF also reduces blood pressure transiently when acutely administered. To address the potential role of ANF in chronic cardiovascular regulation, we generated transgenic mice that express the ANF gene in the liver. The fusion genes comprised either the mouse transthyretin (TTR) or rat phosphoenolpyruvate carboxykinase (PEPCK) promoters fused to the mouse ANF structural gene and were designed to target to the liver constitutive and inducible expression of pre-pro-ANF, respectively. Transgenic animals harboring the TTR-ANF fusion gene expressed chimeric ANF transcripts exclusively in the liver. In contrast, mice harboring the PEPCK-ANF fusion gene did not express detectable amounts of ANF mRNA in liver even after induction (24-hour fasting). In the TTR-ANF mice, hepatic and plasma immunoreactive ANF concentrations were proportional to the concentration of hepatic ANF transcripts. Moreover, mean arterial blood pressure recorded in conscious transgenic mice was inversely proportional to hepatic ANF expression. These transgenic models demonstrate that chronically elevated ANF concentration can induce sustained hypotension.

Normotension in conscious rats after placement of bilateral electrolytic lesions in the rostral ventrolateral medulla.

The effects of bilateral electrolytic lesions of the rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP) and heart rate (HR) were examined in 8 rats at one and 5 days after placement of the lesions. The MAP (111 +/- 2 mm Hg) and HR (393 +/- 17 bpm) of the lesion group were similar to the values recorded in the control group (117 +/- 3 mm Hg, 405 +/- 11 bpm; n = 18). Blockade of the synthesis of angiotensin II with captopril in the lesion group significantly decreased MAP to 93 +/- 2 mm Hg on the first postlesion day. In contrast, the MAP of the control group after captopril fell slightly to 111 +/- 4 mm Hg. Captopril did not alter MAP or HR on postlesion day 5 in either group. Administration of chlorisondamine, an autonomic ganglionic blocking agent, reduced MAP in the lesion and control groups to similar values of 59 +/- 2 mm Hg and 64 +/- 2 mm Hg, respectively. Baroreflex-mediated tachycardia to a decrease in MAP was abolished in the lesion group at one day postlesion and attenuated at 5 days postlesion. In contrast, the baroreflex-mediated bradycardia to an increase in MAP was unaffected by the lesions. Plasma renin activity (PRA) in the lesion group was elevated by nearly 50% as compared to the control group on the first postlesion day (7.3 +/- 0.8 and 4.9 +/- 0.5 ng AI/ml/h, respectively). A 90% elevation in plasma norepinephrine (NE) concentration was also observed in the lesion group as compared to the control group (437 +/- 80 pg/ml and 228 +/- 22 pg/ml, respectively) on postlesion day one. By postlesion day 5, the PRA of the lesion and control groups were nearly identical (4.4 +/- 0.7 and 4.0 +/- 1.0 ng AI/ml/h, respectively), and the plasma NE concentrations were also very similar (201 +/- 41 pg/ml and 175 +/- 22 pg/ml, respectively). We conclude that bilateral destruction of the RVLM does not cause hypotension or bradycardia in conscious rats. Therefore, areas other than the RVLM are capable of maintaining MAP and HR. Sympathetic vasomotor and cardiomotor tone appears unaffected by the lesions. However, increased activity of the renin-angiotensin system may contribute transiently to the maintenance of vasomotor tone and, consequently, MAP after lesion of the RVLM. The RVLM may be important in mediating baroreflex increase in HR.

Pressor systems involved in the maintenance of arterial pressure after lesions of the rostral ventrolateral medulla.

The current study examined three pressor systems which might support mean arterial pressure (MAP) after lesions of the rostral ventrolateral medulla (RVLM). In two protocols, bilateral electrolytic lesions or sham lesions were placed in the RVLM of rats anesthetized with sodium pentobarbital. In the first protocol, the following drugs were given sequentially after placement of the lesions: captopril (5 mg/kg) and d-pentamethylene methylated tyrosine (30 micrograms/kg), a vascular arginine-vasopressin antagonist (AVPX). A final procedure consisted of spinal-cord transection. The second protocol was identical to the first except that the order of drug administration was reversed. In the first protocol, RVLM lesions caused a slight, but statistically significant, decrease in MAP from 118 +/- 3 mmHg to 103 +/- 5 mmHg. After captopril and AVPX, MAP further decreased to 87 +/- 5 mmHg and 62 +/- 4 mmHg, respectively. The MAP fell to 38 +/- 2 mmHg after spinal-cord transection. In the sham-lesion group, MAP rose slightly from 127 +/- 6 mmHg to 134 +/- 7 mmHg after placement of the sham lesions. A significant reduction in MAP was not seen until after administration of AVPX, which decreased MAP to 103 +/- 6 mmHg. Spinal-cord transection substantially lowered MAP to 36 +/- 4 mmHg. In the second protocol, RVLM lesions had no effect on MAP. Administration of AVPX had little effect on MAP (before: 117 +/- 5 mmHg; after: 102 +/- 7 mmHg). In contrast, sequential administration of captopril substantially decreased MAP to 55 +/- 5 mmHg. Spinal cord transection lowered MAP to 33 +/- 1 mmHg. A decrease in MAP in the companion sham-lesion group was not seen until after administration of captopril (before: 109 +/- 8 mmHg; after: 89 +/- 11 mmHg). The greatest fall in MAP followed spinal cord transection (to 39 +/- 6 mmHg). These results demonstrate normotension after RVLM lesions despite a marked reduction in sympathetic vasomotor activity. They also indicate that, after RVLM lesions, arterial pressure is maintained mainly by activity of the renin-angiotensin system and by AVP secretion.

Cardiovascular effects of lesions of the rostral ventrolateral medulla and the nucleus reticularis parvocellularis in rats.

This study explored the possibility that the nucleus reticularis parvocellularis (NRP) acts in concert with the rostral ventrolateral medulla (RVLM) in the maintenance of mean arterial pressure (MAP). Bilateral electrolytic or chemical lesions (kainic acid) were placed in three groups of rats anesthetized with sodium pentobarbital. In the different groups, lesions were placed only in the NRP or RVLM or in both the NRP and RVLM (NRPRVLM). Captopril (5 mg/kg, i.v.) and an arginine vasopressin antagonist (AVPX), d-pentamethylene methylated tyrosine (30 micrograms/kg, i.v.), were sequentially administered. A final procedure consisted of spinal cord transection. The RVLM lesions did not significantly alter MAP (before: 116 +/- 3 mmHg; after: 106 +/- 5 mmHg). Sequential administration of captopril and AVPX each reduced MAP to 87 +/- 5 mmHg and 62 +/- 4 mmHg, respectively. Spinal-cord transection lowered MAP to 38 +/- 2 mmHg. Lesions of the NRP also did not alter MAP (before: 113 +/- 4 mmHg; after: 118 +/- 5 mmHg). Captopril reduced MAP to 109 +/- 7 mmHg, AVPX had no effect, and spinal-cord transection decreased MAP to 31 +/- 3 mmHg. In contrast to the lack of effect of lesions of the RVLM or NRP on MAP, profound hypotension was observed after NRPRVLM lesions (before: 113 +/- 3 mmHg; after: 51 +/- 3 mmHg). Subsequent administration of captopril decreased MAP to 39 +/- 2 mmHg, and AVPX lowered MAP to 32 +/- 1 mmHg. Spinal-cord transection reduced MAP to 23 +/- 1 mmHg. Several conclusions can be drawn from this study. First, lesions of the RVLM do not decrease MAP because of compensation by the renin-angiotensin system and AVP secretion which is mediated by the NRP. Second, lesions of the NRP do not affect MAP because the intact RVLM can maintain sympathetic tone. Third, the profound hypotension observed after NRPRLVM lesions occurred because of the simultaneous impairment of sympathetic vasomotor activity and the neuroendocrine vasoconstrictor effects of the renin-angiotensin system and AVP secretion.

Hypotensive effects of lesions of the rostral ventrolateral medulla in rats are anesthetic-dependent.

These studies were designed to determine if the hypotensive effects of bilateral electrolytic lesions of the rostral ventrolateral medulla are dependent on the type of anesthetic agent used. The lesions caused an immediate fall in mean arterial pressure (MAP) in rats anesthetized with urethane, alpha-chloralose or sodium pentobarbital. At 30 min after placement of the lesions, severe hypotension (MAP = 54 +/- 5 mm Hg) persisted in the rats anesthetized with urethane. However, 30 min after placement of the lesions, the MAP of rats anesthetized with alpha-chloralose or sodium pentobarbital was 87 +/- 9 mm Hg and 99 +/- 10 mm Hg, respectively. Subsequent transection of the cervical spinal cord produced a much greater decrease in MAP in rats anesthetized with alpha-chloralose and sodium pentobarbital as compared to rats anesthetized with urethane. Heart rate was significantly lower after placement of the lesions in all 3 groups. We conclude that the magnitude of the hypotensive effect caused by placement of lesions in the rostral ventrolateral medulla is anesthetic-dependent and that the rostral ventrolateral medulla is not the only area of the central nervous system capable of maintaining vasomotor tone.

Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice.

Depressed contractile function plays a primary role in the pathophysiology of acute myocardial ischemia. Intracellular acidification is an important factor underlying the inhibition of force production in the ischemic myocardium. The effect of acidosis to depress contractility is markedly greater in cardiac as compared to skeletal muscle; however, the molecular basis of this difference in sensitivity to acidosis is not clearly understood. In this report, we describe transgenic mice that express the fast skeletal isoform of troponin C (sTnC) in cardiac muscle. In permeabilized single cardiac myocytes the shift in the midpoint of the tension-pCa relationship (i.e., pCa50, where pCa is -log[Ca2+]) due to lowering pH from 7.00 to 6.20 was 1.27 +/- 0.03 (n = 7) pCa units in control cardiac TnC (cTnC) expressing myocytes and 0.96 +/- 0.04 (n = 11) pCa unit in transgenic cardiac myocytes (P < 0.001). The effect of pH to alter maximum Ca(2+)-activated tension was unchanged by TnC isoforms in these cardiac myocytes. In a reciprocal experiment, contractile sensitivity to acidosis was increased in fast skeletal muscle fibers following extraction of endogenous sTnC and reconstitution with purified cTnC in vitro. Our findings demonstrate that TnC plays an important role in determining the profound sensitivity of cardiac muscle to acidosis and identify cTnC as a target for therapeutic interventions designed to modify ischemia-induced myocardial contractile dysfunction.