Relaxation and the Role of Calcium in Isolated Contracting Myocardium From Patients With Hypertensive Heart Disease and Heart Failure With Preserved Ejection Fraction

Abstract

Background: Relaxation characteristics and Ca²⁺ homeostasis have not been studied in isolated myocardium from patients with hypertensive heart disease (HHD) and heart failure with preserved ejection fraction (HFpEF). Prolonged myocardial relaxation is believed to play an important role in the pathophysiology of these conditions. In this study, we evaluated relaxation parameters, myocardial calcium (Ca²⁺), and sodium (Na⁺) handling, as well as ion transporter expression and tested the effect of Na⁺-influx inhibitors on relaxation in isolated myocardium from patients with HHD and HFpEF.

Methods and results: Relaxation characteristics were studied in myocardial strip preparations under physiological conditions at stimulation rates of 60 and 180 per minute. Intracellular Ca²⁺ and Na⁺ were simultaneously assessed using Fura-2 and AsanteNATRIUMGreen-2, whereas elemental analysis was used to measure total myocardial concentrations of Ca, Na, and other elements. Quantitative polymerase chain reaction was used to measure expression levels of key ion transport proteins. The lusitropic effect of Na⁺-influx inhibitors ranolazine, furosemide, and amiloride was evaluated. Myocardial left ventricular biopsies were obtained from 36 control patients, 29 HHD and 19 HHD+HFpEF. When compared with control patients, half maximal relaxation time (RT₅₀) at 60 per minute was prolonged by 13% in HHD and by 18% in HHD+HFpEF (both P<0.05). Elevated resting Ca²⁺ levels and a tachycardia-induced increase in diastolic Ca²⁺ were associated with incomplete relaxation and an increase in diastolic tension in HHD and HHD+HFpEF. Na⁺ levels were not increased, and expression levels of Ca²⁺- or Na⁺-handling proteins were not altered. Na⁺-influx inhibitors did not improve relaxation or prevent incomplete relaxation at high stimulation rates.

Conclusions: Contraction and relaxation are prolonged in isolated myocardium from patients with HHD and HHD+HFpEF. This leads to incomplete relaxation at higher rates. Elevated calcium levels in HFpEF are neither a result of an impaired Na⁺ gradient nor expression changes in key ion transporters and regulatory proteins.

Keywords: Fura-2; diastole; heart failure; sodium.

Figure 1. Contraction and Relaxation Time Parameters

Left panel: representative normalized force tracings of myocardium from a control patient (CTR), a patient with HHD and a patient with HHD+HFpEF demonstrating the prolongation of relaxation at 60/min and 180/min. The insert highlights a delayed peak of contraction in HHD and HHD+HFpEF due to a prolonged time to peak tension (TPT). Right panel: averaged contraction and relaxation time parameters (TPT, RT₅₀ and RT₉₀: time from peak tension to 50% and 90% relaxation. tau: time index of relaxation) in CTR (n=26), HHD (n= 20) and HHD+HFpEF (n=13) at a stimulation rate of 60/min. CTR vs. HHD+HFpEF: * p<0.05, ** p<0.01, CTR vs. HHD: ^† p<0.05, ^†† p<0.01; HHD vs. HHD+HFpEF: ^‡ p<0.05, ^‡‡ p<0.01

Figure 2. Incomplete Relaxation and Diastolic Tension at High Rates

Left panel: original force tracings in CTR, HHD and HHD+HFpEF, showing the effects of incomplete relaxation on diastolic tension development as stimulation rate is increased from 60/min to 180/min. Right panel: Change in average developed contractile force at 180/min in relation to the developed contractile force at 60/min demonstrating an incremental increase in diastolic tension in HHD and HHD+HFpEF. A decline in contractile force development at 180/min was observed in all myocardial preparations from patients with HHD+HFpEF. (upper error bar: standard error of developed contractile force; lower error bar: standard error of diastolic tension; CTR (n=12), HHD (n=11) and HHD+HFpEF (n=7)). CTR vs. HHD+HFpEF: ** p<0.01, CTR vs. HHD: ^† p<0.05

Figure 3. Simultaneous Force and Calcium Recording

Upper panel: Force recording and simultaneous Fura-2 Ca²⁺-transient aquired over one second at 60/min and 180/min in a preparation from a patient with HHD. Diastolic force and diastolic Ca²⁺ are at elevated levels with the higher stimulation rate. Middle and lower panel: simultaneous Ca²⁺ and force tracing recorded over approximately 500s with a stepwise increase of stimulation rate from 60/min to 180/min. This demonstrates a rate-dependent incremental increase in diastolic force that is paralleled by a simultaneous rise in diastolic Ca²⁺-levels.

Figure 4. Simultaneous Force, Calcium and Sodium Recording

Simultaneous Ca²⁺ (Fura-2), Na⁺ (ANG-2) and force tracings in myocardium from a CTR patient and a patient with HHD with a rate change from 60/min to 180/min. A small increase in diastolic Ca²⁺ in the CTR sample after the rate change to 180/min is not paralleled by an increase in diastolic tension; in the HHD sample a more pronounced increase of diastolic force and Ca²⁺ is noted. There is no obvious change in the co-recorded Na⁺ signal (ANG-2) in both CTR and HHD.

Figure 5. mRNA of Calcium Handling Proteins, Sodium Transporters and Regulatory Proteins

Expression levels of Ca²⁺ - and Na⁺-transporters and regulatory proteins in myocardium from CTR, HHD and HHD+HFpEF patients using quantitative PCR. No significant differences in expression levels were found.

Figure 6. Effects of Sodium Influx Inhibition on Relaxation Time Indices

Left panel: Original tracing of a myocardial preparation obtained from a patient with HHD that was sequentially exposed to 10μM ranolazine, 50μM furosemide and 50μM amiloride. Incomplete relaxation at 180/min was not affected by any of the Na⁺-influx inhibitors. Right panel: Relaxation time parameters (RT₅₀, RT₉₀ and tau) obtained at 60/min in myocardium from control patients (n=8), HHD (n=7) and HHD+HFpEF (n=4). After a baseline measurement without inhibitor relaxation was sequentially assessed with ranolazine, furosemide and amiloride. No changes in relaxation time parameters were noted.