Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis

Abstract

Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K+ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.

Keywords: NKCC transporter; acetazolamide; calcium channel; skeletal muscle.

Figure 1

In vitro contraction assay demonstrates a beneficial effect of bumetanide (BMT) during a hypokalaemic challenge. Tetanic contractions were elicited by 100 Hz stimulation of the excized soleus muscle maintained at 37°C. (A) Force responses are shown for contractions in control conditions (4.75 mM K⁺), and 20 min after bath exchange to 2 mM K⁺, then 2 mM K⁺ plus bumetanide (75 µM), and then back to control. (B) Normalized peak tetanic force is shown for soleus from wild-type (left, black), R528H^+/m (middle, blue), and R528H^m/m (right, pink) mice. The trials were designed to test recovery after low-K⁺ induced loss of force (top row) or prevention by co-administration of bumetanide with the onset of hypokalemia (bottom row). Squares denote muscle harvested from males and circles from females. Symbols are means from three to eight animals and error bars show SEM. WT = wild-type.

Figure 2

Hypertonicity exacerbated the susceptibility to loss of force in R528H soleus and was prevented by bumetanide (BMT). Pairs of soleus muscles dissected from the same R528H^+/m animal were tested in parallel. One was exposed continuously to bumetanide (75 µM) starting at 10 min whereas the other remained drug-free. Hypertonic challenge (left) with a sucrose containing bath (30 min) caused 60% loss of force that was further exacerbated by reduction of K⁺ to 2 mM (60 min). Bumetanide greatly reduced the loss of force from either challenge. A hypotonic challenge (right) transiently increased the force and protected the muscle from loss of force in 2 mM K⁺ (60–90 min). Return to normotonic conditions while in low K⁺ produced a marked loss of force.

Figure 3

Bumetanide (BMT) was superior to acetazolamide (ACTZ) in preventing loss of force in vitro, during a 2 mM K⁺ challenge. The soleus muscle from heterozygous R528H^+/m males (A, n = 3) or females (B, n = 4) were challenged with sequential 20 min exposures to 2 mM K⁺. Controls with no drug showed two episodes of reduced force (black circles). Pretreatment with acetazolamide (100 µM, blue circles) produced only modest benefit, whereas bumetanide (0.5 µM) completely prevented the loss of force.

Figure 4

Furosemide (FUR) attenuated the loss of force during hypokalaemic challenge. (Top) Application of furosemide (15 µM) after 30 min in 2 mM K⁺ prevented further loss of force but did not elicit recovery. (Bottom) Furosemide applied at the onset of hypokalaemia attenuated the drop in force, and the effect was lost upon washout. Symbols represent mean responses for three soleus muscles from males (squares) or females (circles); and error bars show SEM.

Figure 5

Bumetanide (BMT) and acetazolamide (ACTZ) both prevented loss of muscle excitability in vivo. (A) Continuous infusion of glucose plus insulin caused a marked drop in CMAP amplitude for R528H^m/m mice (black). Pretreatment with intravenous bolus injection of bumetanide prevented the CMAP decrement for four of five mice (red), while acetazolamide was effective in five of eight (blue). The mean CMAP amplitudes shown in A are for the subset of positive responders, defined as those mice with a relative CMAP >0.5 over the interval from 100 to 120 min. (B) The distribution of late CMAP amplitudes, time-averaged from 100 to 120 min, is shown for all R528H^m/m mice tested. The dashed line shows the threshold for distinguishing responders (>0.5) from non-responders (<0.5).

Figure 6

Glucose challenge in vitro did not induce weakness in R528H^m/m soleus. Peak amplitudes of tetanic contractions elicited every 2 min were monitored during challenges with high glucose or low K⁺. Doubling the bath glucose to 360 mg/dl (20–40 min) increased the osmolarity by 11.8 mOsm, but did not elicit a substantial loss of force. Coincident exposure to 2 mM K⁺ and high glucose produced a 70% loss of force that was comparable to the decrease produced by 2 mM K⁺ alone (Fig. 1B, top row).