Left ventricular mechanoenergetics in excised, cross-circulated rat hearts under hypo-, normo-, and hyperthermic conditions

Abstract

We investigated the effects of altering cardiac temperature on left ventricular (LV) myocardial mechanical work and energetics using the excised, cross-circulated rat heart model. We analyzed the LV end-systolic pressure-volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO₂) and systolic pressure-volume area (PVA; total mechanical energy per beat) in isovolumically contracting rat hearts during hypo- (32 °C), normo- (37 °C), and hyperthermia (42 °C) under a 300-beats per minute pacing. LV ESPVR shifted downward with increasing cardiac temperature. The VO₂-PVA relationship was superimposable in these different thermal conditions; however, each data point of VO₂-PVA shifted left-downward during increasing cardiac temperature on the superimposable VO₂-PVA relationship line. VO₂ for Ca²⁺ handling in excitation-contraction coupling decreased, which was associated with increasing cardiac temperature, during which sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity was suppressed, due to phospholamban phosphorylation inhibition, and instead, O₂ consumption for basal metabolism was increased. The O₂ cost of LV contractility for Ca²⁺ also increased with increasing cardiac temperature. Logistic time constants evaluating LV relaxation time were significantly shortened with increasing cardiac temperature related to the acceleration of the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity. The results suggested that increasing cardiac temperature induced a negative inotropic action related to SERCA activity suppression in Ca²⁺ handling and increased myosin ATPase activity in CB cycling. We concluded that thermal intervention could modulate cardiac inotropism by changing CB cycling, Ca²⁺ handling, and basal metabolism in rat hearts.

Figure 1

Schematic illustration of experimental setting for the excised blood-perfused rat heart (a) and framework of end-systolic pressure–volume relationship (ESPVR)–VO₂–pressure–volume area (PVA) (b,c). (a) We used three rats in each experiment. The largest one was used as blood supplier. The middle-size one was used as metabolic supporter for the excised heart. The smallest one was used as heart donor in excised cross-circulation rat heart preparation. The perfusion pressure of the excised hearts was maintained at 100 mmHg with controlled blood pressure of the metabolic supporter rats. Total coronary blood flow (CBF) was continuously measured with an ultrasonic flowmeter placed in the middle of the coronary venous drainage tubing from the RV. The coronary arteriovenous O₂ content difference (AVO₂D) was continuously measured by passing all arterial and venous cross-circulation blood through the two cuvettes of a custom-made AVO₂D analyzer. The myocardial temperature was changed from 37 °C to 32 °C or 42 °C with inline-type temperature controller system for pre-incubation 30 min before data sampling. (b) LV ESPVR and end-diastolic pressure–volume relationship (EDPVR) at midrange LV volume (mLVV, a half value between the minimum and maximum water volume infused into the balloon). The minimal volume loading LV volume (V₀) was also determined as the volume-axis intercept of the best-fit ESPVR. The PVA was computed at each LV volume as the area between the ESPVR and the EDPVR and between the V₀ and the given chamber volume (balloon material volume + intra-balloon water volume). The value of V₀, mLVV, and PVA were normalized by LV mass to 1 g. A striped area denoted PVA at a mLVV (PVA_mLVV) (ESP_mLVV: observed end-systolic pressure at a mLVV). (c) Myocardial O₂ consumption per beat (VO₂)–PVA relationship. Myocardial VO₂ was obtained as the product of the CBF and coronary AVO₂D. The VO₂–PVA relation was linear in the rat LV. Its slope represents the O₂ cost of PVA (1/contractile efficiency), and its VO₂ intercept represents PVA-independent VO₂. The PVA-independent VO₂ is composed of O₂ consumption for Ca²⁺ handling in E-C coupling and for basal metabolism.

Figure 2

Representative data of LV pressure–time curves (a), normalized LV pressure (LVP)–time curves (b), and comparison of mean logistic time constants (c) at mLVV at 32 °C (hypothermia, n = 10), 37 °C (normothermia, n = 13), and 42 °C (hyperthermia, n = 10). These data obviously showed decreased maximal LVP and reduced duration of LV relaxation time with increasing cardiac temperature. Values are presented as means ± SD. *p < 0.05 vs. 32 °C, ^†p < 0.05 vs. 37 °C.

Figure 3

Representative data of LV ESPVR and EDPVR (a), and VO₂–PVA relationship (b) at 32 °C (hypothermia, n = 10), 37 °C (normothermia, n = 13), and 42 °C (hyperthermia, n = 10). The ESPs and EDPs data were recorded at each fixed LV volume in hypo-, normo-, and hyperthermia. (a) As shown by a dashed long arrow in the left panel, LV ESPVR shifted downward (a), and LV EDPVR ained unchanged, but VO₂–PVA relationship could be superimposed; neither slope nor VO₂ intercept changed (b) with increasing cardiac temperature. Thus, each data point of VO₂–PVA at each LVV shifted left-downward with increasing cardiac temperature (a dashed long arrow in the right panel) from hypothermia (solid triangle) to hyperthermia (solid square) passing through normothermia (solid circle) on the superimposable VO₂–PVA relationship line. As shown by a dashed open circle in the right panel (b), unchanged VO₂ intercepts indicate that PVA-independent VO₂ were not affected by changing cardiac temperature.

Figure 4

Comparison of the mean slopes (O₂ costs of PVA) (a), VO₂ intercepts (PVA independent VO₂) (b), O₂ consumption for basal metabolism per minute (c), VO₂ for excitation–contraction (E-C) coupling (d), O₂ costs of LV contractility for Ca²⁺ (e), and CBF (f) at 32 °C (hypothermia, n = 10), 37 °C (normothermia, n = 13), and 42 °C (hyperthermia, n = 10). CBF in hyperthermia is significantly smaller than that in hypothermia, which may be related to lower LV contractility (LVV = 0.16, balloon material volume = 0.08 ml plus balloon water volume = 0.08 ml). We confirmed no lactate production in the heart through the experiments, indicating ischemia was not induced. Values are presented as means ± SD. *p < 0.05 vs. 32 °C, ^†p < 0.05 vs. 37 °C.

Figure 5

Representative PVA-independent VO₂-equivalent maximal elastance (eEmax) at mLVV (eEmax_mLVV) relationships at 32 °C (hypothermia, close triangle), 37 °C (normothermia, close circle), and 42 °C (hyperthermia, close square). The slope of PVA-independent VO₂–eEmax_mLVV relationships indicates the O₂ cost of LV contractility for Ca²⁺. The values are presented as mean ± SD, which are shown in Fig. 3e.

Figure 6

Western blot analysis of SERCA2, phospholamban (PLB), and phosphorylated phospholamban (p-PLB) in LV tissues of hypo- (n = 5), normo- (n = 7), and hyperthermia (n = 5). Representative data of SERCA2, PLB, and p-PLB. (a) Comparison of the mean protein levels of SERCA2 (b) and the ratios of SERCA2/PLB (c) and p-PLB/ PLB (d). Values are means ± SD. *p < 0.05 vs. 32 °C.

Figure 7

Schematic illustrations of ESPVR (a), VO₂–PVA relationship (b), and normalized LVP–time curves (c) on cardiac mechanoenergetics in hypo-, normo-, and hyperthermia.