Cardiac inotropy, lusitropy, and Ca2+ handling with major metabolic substrates in rat heart

Abstract

Fatty acid (FA)-dependent oxidation is the predominant process for energy supply in normal heart. Impaired FA metabolism and metabolic insufficiency underlie the failing of the myocardium. So far, FA metabolism in normal cardiac physiology and heart failure remains undetermined. Here, we evaluate the mechanisms of FA and major metabolic substrates (termed NF) on the contraction, relaxation, and Ca²⁺ handling in rat left ventricular (LV) myocytes. Our results showed that NF significantly increased myocyte contraction and facilitated relaxation. Moreover, NF increased the amplitudes of diastolic and systolic Ca²⁺ transients ([Ca²⁺]_i), abbreviated time constant of [Ca²⁺]_i decay (tau), and prolonged the peak duration of [Ca²⁺]_i. Whole-cell patch-clamp experiments revealed that NF increased Ca²⁺ influx via L-type Ca²⁺ channels (LTCC, I_Ca-integral) and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca²⁺]_i trajectory to the right and increased [Ca²⁺]_i for 50 % of sarcomere relengthening (EC₅₀), suggesting myofilament Ca²⁺ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca²⁺ sensitivity, abolished the NF-induced enhancement of [Ca²⁺]_i amplitude and the tau of [Ca²⁺]_i decay, indicating the association of myofilament Ca²⁺ desensitization with the changes in [Ca²⁺]_i profile in NF. NF reduced intracellular pH ([pH_i]). Increasing [pH]_i buffer capacity with HCO₃/CO₂ attenuated Δ [pH]_i and reversed myofilament Ca²⁺ desensitization and Ca²⁺ handling in NF. Collectively, greater Ca²⁺ influx through LTCCs and myofilament Ca²⁺ desensitization, via reducing [pH]_i, are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF.

Keywords: Cardiac myocyte; Contraction; Intracellular Ca2+ transient; Metabolic substrates; Myofilament Ca2+ sensitivity; Relaxation; pH.

Scheme 1

Schematic diagram of cardiac excitation–contraction coupling in the presence of major metabolic substrates including fatty acids and glucose at physiological concentration. Contraction is enhanced with metabolic substrates’ supplementation. Mechanistically, Ca²⁺ influx through LTCC is increased, leads to prolonged APD and greater Ca²⁺ release from the SR, and increases intracellular free Ca²⁺. Reuptake of Ca²⁺ via SERCA is facilitated. Myofilament Ca²⁺ sensitivity is reduced, possibly via reduced pH_i, which in part is responsible for greater intracellular Ca²⁺ level and decline in rat LV myocytes

Fig. 1

Effect of NF on LV myocyte contraction. a Representative raw traces of sarcomere shortening and relengthening in the presence of NF. b Mean values of the diastolic sarcomere length and the amplitude of sarcomere shortening (peak height) and 50 % relaxation time (TR₅₀). Diastolic sarcomere length was not different between NT and NF. Sarcomere shortening was significantly increased in NF and TR₅₀ was shorter by NF. c Representatives raw traces and mean values of three fatty acids (3FAs) only on myocyte contraction. 3FAs increased myocyte contraction. d Representative raw traces and mean values of a potent antioxidant NAC on myocyte contraction in NF. NAC did not affect the increment of myocyte contraction by NF

Fig. 2

NF regulation of intracellular Ca²⁺ transients. a Representative [Ca²⁺]_i transients in NT and NF. b Mean values of [Ca²⁺]_i transient parameters. NF significantly increased diastolic [Ca²⁺]_i and peak amplitude of [Ca²⁺]_i. Time to 50 % relaxation and peak time duration (PT₉₀ + TR₁₀) were prolonged; however, time constant of [Ca²⁺]_i decay (tau) was facilitated in NF

Fig. 3

Patch-clamp recordings of LTCC activities in NF. a, b Pulse protocol, representative I_Ca and the corresponding I-V relationship, peak I_Ca at 0 mV, inactivation parameters, and the integral of Ca²⁺ influx in NF. NF reduced peak I_Ca density, prolonged slow inactivation of I_Ca, and increased integral of I_Ca at 0 mV and −20 mV

Fig. 4

Effect of NF on action potential profile. a Representative action potential profile in NT and NF. b Mean value of action potential duration parameters. NF significantly prolonged the repolarization duration of APD (APD20, APD50, APD90)

Fig. 5

NF regulation of myofilament Ca²⁺ sensitivity. a Simultaneous recordings of LV myocyte sarcomere shortening/relengthening and intracellular Ca²⁺ transients in NT and in NF. b Phase-plane loop of sarcomere shortening/relengthening vs. [Ca²⁺]_i transient. The relaxation phase of the loop shifted to the right in NF compared to that in NT. Ca²⁺ concentration at 50 % sarcomere relengthening (EC₅₀) was significantly larger in NF

Fig. 6

Effect of NF on [Ca²⁺]_i in BDM-pretreated LV myocytes. Representative [Ca²⁺]_i (a) and mean vales of [Ca²⁺]_i transient parameters (b). NF-induced increase in diastolic and systolic [Ca²⁺]_i were abolished by BDM (5 mM). Time constant of Ca²⁺ decay (tau) was not facilitated in NF in the presence of BDM. Peak time duration remained unaltered by BDM pretreatment

Fig. 7

NF regulation of intracellular pH_i. a Representative recording of pH_i in NT and NF using HEPES buffer. b Mean values of pH_i in NT and NF, pH_i was significantly reduced by NF

Fig. 8

Simulated effects of NF on electrical properties and Ca²⁺ regulation of rat ventricular myocytes. Ca²⁺ transient morphology (a), I_CaL (b), and APD (c), a Simulated effects of NF on morphology and relaxation time of Ca²⁺ transient. Measurements of 50 % relaxation time and peak time duration were conducted in the same way as those in Fig. 2b. The dashed line indicates 10 nM [Ca²⁺]_i level. b Simulated effects of NF on I_CaL and Ca²⁺-influx through I_CaL. The left panel shows the effects of NF on I_CaL during AP. The middle panel compares the total Ca²⁺ influx through I_CaL between NT and NF. The right panel shows the effects of NF on I_CaL density obtained by a voltage-clamp protocol used in animal experiments (see Fig. 3b). The dashed line indicates zero current level. c Simulated effects of NF on morphology and duration of AP. Pacing frequency is 1 Hz. APD50 was calculated as the difference between time at 50 % depolarization and time at 50 % repolarization. APD90 was calculated as the difference between time at 10 % depolarization and time at 90 % repolarization. The dashed line indicates zero voltage level