1. Electrical Characteristics of Channelopathies Involving Skeletal Muscle Bob Ruff, M.D., Ph.D. Chief, SCI Service Louis Stokes Cleveland VAMC Barbara E. Shapiro, M.D., Ph.D. Case Western Reserve Univ. Jacob Levitt, M.D. Albert Einstein College of Medicine

2. Objectives To understand factors regulating membrane excitability in skeletal muscle To understand how impaired Na + channel inactivation can produce myotonia To appreciate how persistent depolarization produces paralysis (Myotonia vs HyperKPP) To learn different ways to produce a persistent depolarization (HyperKPP vs HypoKPP)

3. Roles of Na +, K + and Cl - Channels in Membrane Excitability K ir sets resting membrane potential K v (delayed rectifier) repolarizes after AP Cl - channel stabilizes membrane potential I Na drives AP

4. Potassium Sets Membrane Resting Potential K + conductance 20% of membrane conductance - Inward or anomalous rectifier K + channel (K IR ) AP Termination - Delay Rectifier K + Channel

5. Inward (Anomolous) Rectifier

6. Sodium channel gating properties Depolarization activates Na channels - changes from a closed to an open state The declining portion of I Na - transition of open channels to a non-conducting fast inactivated state

8. Two Types of Skeletal Muscle Sodium Channel Inactivation Fast inactivation – msec, Slow inactivation - seconds Fast inactivation helps to terminate the AP Slow inactivation operates at more negative potentials - regulates the number of excitable sodium channels as a function of the membrane potential

9. Action Potential

10. Factors Determining Action Potential Threshold Number of excitable Na + channels (# of channels and fraction that are excitable) Voltage dependence of Na + channel opening Amount of Cl - conductance Inward rectifier K + conductance with depolarization

11. Periodic Paralysis Results from persistent membrane depolarization  inactivation of normal Na+ channels  membrane inexcitability HyperKPP – Na + channelopathy – depolarization due to abnormal persistent I Na HypoKPP – –Type I - Indirect Ca 2+ Channelopathy –Type 2 - Na + channelopathy – loss of function

12. Hyperkalemic Periodic Paralysis (HyperPP) - AD episodic attacks of flaccid weakness myotonia is often present (vs HypoK-PP) paralysis caused by membrane depolarization  Na + channel inactivation Overlap: Na + Ch myotonias, paramyotonia Lehmann-Horn, Rudel, Ricker

13. Impaired fast inactivation can produce myotonia 1 msec Note: Loss of inactivation in a small % of channels → myotonia Myotonia stopped in part due to accumulated slow inactivation

14. Key to Paralysis vs Myotonia is Persistent Depolarization Impairment of Slow Inactivation will facilitate persistent opening of mutant channels

15. Hypokalemic Periodic Paralysis (HypoKPP) - AD Episodic attacks of flaccid paralysis Myotonia never present (vs HyperKPP) Insulin  paralytic attack without  K + Membrane excitability impaired – low conduction velocity: Drs. Haenen, Links, Oosterhuis, Stegeman, van der Hoevan, van Weerden & Zwarts

16. Depolarization not blocked by TTX Insulin Enhances Depolarization Lehmann-Horn, Rudel, Ricker

17. Paralysis parallels drop in K +

18. In HypoKPP Weakness Parallels Depolarization & Reduction in EMG Amplitude

19. Skeletal Muscle Membrane Excitability Is Impaired in HypoKPP (Type1) Muscle fibers very susceptible to depolarization-induced inexcitable Small depolarizations (10 mV) make HypoKPP fibers unexcitable Slow conduction velocity (Zwarts’ lab) suggests impaired Na + channel function in HypoKPP

20. Two Genotypes - Similar Phenotype Type 1 HypoKPP is linked to 1Q31-32 Defective gene (CACNL1A3) encodes a skeletal muscle dihydropyridine (DHP) sensitive or L-type calcium channel Mutations - segment 4 of domain 2 (R528H) and segment 4 of domain 4 (R1239H, R1239G) of the  -subunit of the skeletal muscle L-type Ca +2 channel

21. Two Genotypes - Similar Phenotype Type 2 HypoK-PP has a similar phenotype to type 1 HypoK-PP Associated with point mutations in the Na + channel gene (SCN4A) Surface membrane I Na is reduced to about 50% of normal (reduced expression and increased resting inactivation)

22. Type 1 HypoKPP – Altered Inward (Anomolous) Rectifier

23. Insulin  outward current component of K IR in HypoKPP Circle – no insulin Square - insulin Unfilled – HypoKPP Filled – Control

24. Insulin Reduces K + Conductance Even When [K + ] o is High Circle – no insulin Square - insulin Unfilled – HypoKPP Filled – Control

25. Summary of Alterations of Inward Rectifier K + Channel in HypoKPP Baseline Inward Rectifier Conductance Including K ATP Channels is Reduced Insulin selectively reduces the K + conductance for outward currents Lowering [K + ] o causes depolarization due to TTX- and DHP-insensitive depolarizing current (low K ir conductance for outward current facilitates depolarization) Note: Andersen-Tawil Syndrome due to K ir mutation

26. Why do Type I and Type II HypoKPP have similar phenotypes? The effects of the Na + channel mutations in Type II HypoKPP are to reduce membrane channel density and to increase the amount of resting inactivation - both lead to  I Na Susceptibility of Type I HypoKPP fibers to depolarization-induced inactivation and lower AP conduction velocities suggest reduced I Na in HypoKPP (Zwarts’ lab)

27. Small Depolarizations Produce Paralysis in HypoKPP

28. Comparison of Na + Channel Properties and Action Potential (AP) Thresholds in Fast Twitch, Type IIb, Skeletal Muscle Fibers from Five Patients with HypoKPP and Seven Controls. Controls HypoKPP Na + Channel Properties Max I Na (mA/cm 2 ) 23.7 15.4 ±1.3 ±1.9 (p<0.001) Action Potential (AP) Thresholds AP Threshold (mV) -58.7-53.4 ±1.5±1.1 (p<0.001)

29. Which Membrane Change Correlates Best with Paralytic Attacks in Type 1 HypoKPP? I Na correlated inversely with frequency of paralytic attacks (Pearson’s correlation coefficient, r = -0.996) AP threshold correlated with the frequency of paralytic attacks (r=-0.921) Peak outward K + conductance of the inward rectifier K + channel correlated weakly with the frequency of paralytic attacks (r = -0.121).

30.  Na + current correlated with the frequency of paralytic attacks  K + current did not have a strong correlation Patients 1 2 3 4 5 Peak I Na Max I Na,max 11.912.216.9 17.7 18.2 (mA/cm 2 ) ±1.8±2.0±1.8 ±1.7 ±1.9 Action Potential (AP) Thresholds AP Thresh -50.6-51.0-54.9 -55.1 -55.4 (mV) ±1.9±1.7±1.7± 1.8 ±1.8 Peak Outward I K in 80 mM K + with 12mU/ml Insulin Conductance 260271 279 268 251 (µS/cm 2 ) ±30±29 ±39 ±42 ±36 Number of Paralytic Attacks (lasting >1 hour) in one year 15 13 3 2 1

31. How Can Ca 2+ Channel Mutations Alter Na + & K + Channel Properties? The Ca 2+ channel mutations may disturb intracellular [Ca 2+ ] Intracellular Ca 2+ is known to regulate Na + channel expression and can alter the expression and properties of other channels

32. Intracellular [Ca 2+ ] is increased in HypoKPP Fibers Intracellular [Ca 2+ ] Determined with a Calcium Sensitive Electrode in Type I, IIa and IIb Control and HypoKPP Human Intercostal Muscle Fibers Intracellular [Ca 2+ ](µM) According to Fiber Type Type I Type IIa Type IIb Controls 0.113±0.005 0.094±0.005 0.081±0.003 n=27 n=22 n=58 HypoPP 0.129±0.009 0.112±0.008 0.100±0.006 n=11n=12 n=16 p<0.05p<0.05 p<0.01

33. Indirect Channelopathy -  Intracellular [Ca 2+ ] may Down Regulate Na + and K IR (incl. K ATP ) Channels Ca 2+ mutations in HypoKPP may reduce Na + channel density (and perhaps alter Inward Rectifier K + Channel Function) by elevating intracellular [Ca 2+ ], which reduces the level of the Na + channel  - subunit mRNA (and perhaps reduces expression of K ATP Channels)

34. Thyrotoxic Periodic Paralysis: the brother of Hypokalemic Periodic Paralysis Bob Ruff, M.D., Ph.D. Chief, SCI Service Louis Stokes Cleveland VAMC Director Rehabilitation Research & Development Department of Veterans Affairs.

35. Objectives To understand distinguishing features of Thyrotoxic Periodic Paralysis (TPP) To compare channel defects in TPP with HypoKPP To consider how thyrotoxicosis contributes to the pathogenesis of TPP

36. Clinical: TPP vs HypoKPP TPPHypoKPP PredominanceAsianNon-Asian Age of Onset3 rd & 4 th decades1 st & 2 nd decades GeneticsSporadic, expression linked to thyroid state M>>>F AD, specific mutations M>F RxBeta-blocker Acetazolamide may worsen K replacement Acetazolamide Prevents

37. Periodic Paralysis Results from persistent membrane depolarization  inactivation of normal Na+ channels  membrane inexcitability HyperKPP – Na + channelopathy – depolarization due to abnormal persistent I Na HypoKPP – –Type I - Indirect Ca 2+ Channelopathy –Type 2 - Na + channelopathy – loss of function TPP – Not Associated with HypoKPP channel defects

38. Common Features of TPP & HypoKPP Episodic attacks of flaccid paralysis Myotonia never present (vs HyperKPP) Insulin  paralytic attack without  K + Membrane excitability impaired – low conduction velocity, low CMAP amplitude, CMAP reduction with exercise

39. Genetics of TPP Familial cases increasingly recognized HypoKPP Na channel mutations not found HypoKPP Ca channel mutations not found Reports of selective single nucleotide polymorphisms (SNP) in regulatory region of Ca channel gene – region of thyroid hormone binding sites

40. Methods - Patient with TPP 32 yo man with TPP in the T-toxic state and 4 months later when euthyroid & asymptomatic Measured I Na with a loose patch voltage clamp, inward rectifier I K with a 3-electrode voltage clamp, action potential (AP) threshold with a 2 electrode clamp and intracellular [Ca 2+ ] using Ca 2+ -sensitive electrodes Intercostal type IIb muscle fibers from patient with TPP, 5 patients with Type I HypoKPP (R528H mutation) and 7 controls(C).

41. Summary of Alterations of Inward Rectifier K + Channel in HypoKPP Baseline Inward Rectifier Conductance Including K ATP Channels is Reduced Insulin selectively reduces the K + conductance for outward currents

42. K IR in TPP (nA/mm 2 )

43. Max I Na (mA/cm 2 )

44. AP Threshold (mV)

45. Intracellular [Ca 2+ ] (nM) in TPP & HypoKPP

46. TPP & HypoKPP- Indirect Channelopathies -  [Ca 2+ ] may Down Regulate Na + and K IR Channels Ca 2+ mutations in HypoKPP may reduce Na + channel density and alter K IR function by elevating intracellular [Ca 2+ ] In TPP - SNPs at the thyroid hormone responsive element may affect the binding affinity of the thyroid hormone responsive element and modulate the stimulation of thyroid hormone on the Ca(v)1.1 gene

47. Summary – HyperKPP Paralysis produced by prolonged membrane depolarization Difference between mutations that produce myotonia vs paralysis is probably that paralysis is associated with prolonged pathological I Na Impairment of slow inactivation will facilitate prolonged pathological I Na Mutations that impair slow inactivation associated with paralysis

48. Summary – HypoKPP I Na is reduced in both types of HypoKPP Inward Rectifier K + conductance is altered in Type I HypoKPP and Andersen-Tawil Syndrome Type I HypoKPP - Frequency of paralytic attacks correlates with decrease of I Na Type I HypoKPP – indirect Channelopathy - alteration of Na + and K + channel function may be mediated by  intracellular [Ca 2+ ]

49. Supported by the Clinical Research and Development Service, Office of Research and Development, Department of Veterans Affairs

50. Rx of HyperKPP REDUCE PARALYTIC ATTACK FREQUENCY –1) Eat regular meals high in carbohydrates and low in K –2) Avoid strenuous exercise followed by rest, emotional stress and cold

51. Rx of HyperKPP ABORT PARALYTIC ATTACKS – –1) Ingest high carbohydrate food such as candy bar –2) use beta-adrenergic agonist inhaler. For severe attacks I.V. glucose and insulin can be administered in a carefully monitored environment

52. Rx of HyperKPP IF PARAMYOTONIA AND STIFFNESS ARE PRESENT – –1) Mexiletine 150 mg twice a day increasing to 300 mg three times a day to reduce stiffness –2) Tocainide is a second line agent if mexiletine fails; however blood counts must be monitored due to the risk of bone marrow suppression. The dose of tocainide is 400-1200 mg per day

53. Rx of HypoKPP REDUCE PARALYTIC ATTACK FREQUENCY – –1) Follow a low carbohydrate and sodium restricted diet –2) Avoid precipitating factors such as strenuous exercise followed by rest, high carbohydrate meals or alcohol.

54. Rx of HypoKPP MEDICATION TO REDUCE ATTACK FREQUENCY 1) Initiate carbonic anhydrase inhibitor. Usual agent is acetazolamide. Initial dose of 125 mg twice a day and increasing as needed to final dose of 250 mg four times a day (some will need a total daily dose of 1500mg). An alternative carbonic anhydrate inhibitor is dichlorphenamide starting at 25 mg twice a day and increasing to 25-50 mg two to three times a day. Note that some HypoPP patients worsen with carbonic anhydrase inhibitors. 2) If carbonic anhydrase inhibitors are not successful, a K- sparing diuretic such a triamterene or spironolactone may help. 3) Supplemental oral K alone or combined with a carbonic anhydrase inhibitor may prevent paralytic attacks

55. Rx of HypoKPP ABORT PARALYTIC ATTACKS – 1) Oral KCl 0.25 mEq/kg repeating every half hour until the weakness improves. Carefully monitor electrolytes and EKG in an intensive care setting. Avoid intravenous KCl unless KCl cannot be given orally. Avoid giving glucose and insulin as this will worsen paralysis.

56. Rx of Anderson-Tawil Syndrome MEDICATION TO REDUCE ATTACK FREQUENCY – Initiate an oral carbonic anhydrase inhibitor. The usual agent is acetazolamide, with the initial dose of 125 mg twice a day and increasing as needed to final dose of 250 mg four times a day. An alternative carbonic anhydrate inhibitor is dichlorphenamide starting at 25 mg twice a day and increasing to 25- 50 mg two to three times a day. Monitor cardiac function.

57. Rx of Anderson-Tawil Syndrome TREATMENT OF ARRHYTHMIAS - Arrhythmias may respond poorly to anti- arrhythmic agents. Imipramine may be useful. Manage with a cardiologist.

58. Rx of ThyrotoxicPP PRIMARY TREATMENT IS TO CORRECT HYPERTHYROIDISM. When it is not possible to correct thyrotoxicosis, treatment with propranolol may reduce the frequency of paralytic attacks as may the treatments used to reduce the frequency of paralytic attacks in patients with HypoPP. Carbonic anhydrase inhibitors are not effective for treating TPP.

59. Rx of ThyrotoxicPP ABORT PARALYTIC ATTACKS - Administer oral KCl 0.25 mEq/kg repeating every half hour until the weakness improves. Carefully monitor electrolytes and EKG in an intensive care setting. Avoid intravenous KCl unless KCl cannot be given orally. Avoid giving glucose and insulin as this will worsen paralysis. Intravenous propranolol, given with EKG monitoring may be useful in treating acute paralytic attacks in TPP when hyperthyroidism has not yet been addressed.

60. Rx of HyperKPP IF PARALYTIC ATTACKS REMAIN FREQUENT – 1) Start oral HCTZ diuretic, with initial dose of 12.5 mg/day and increasing slowly in increments of 12.5 mg to a final dose of 100-200mg/day 2) If HCTZ alone is not sufficient initiate an oral carbonic anhydrase inhibitor. The most common agent is acetazolamide, with the initial dose of 125 mg twice a day and increasing as needed to final dose of 250 mg four times a day (some will need a total daily dose of 1500mg). An alternative carbonic anhydrate inhibitor is dichlorphenamide starting at 25 mg twice a day and increasing to 25-50 mg two to three times a day. Note that carbonic anhydrase inhibitors may precipitate weakness in patients with HyperPP and paramyotonia.