Altered Ca2+ and Na+ Homeostasis in Human Hypertrophic Cardiomyopathy: Implications for Arrhythmogenesis

Figures

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  FIGURE 1. Remodeling of ion currents in HCM cardiomyocytes (A) Superimposed representative action potentials recorded during stimulation at 0.2 Hz from HCM and control cardiomyocytes. (B) Representative recording from an HCM cardiomyocyte paced at 0.2 Hz, showing EADs. Black arrows mark stimuli. Orange arrows mark EADs. (C) Occurrence of EADs in 23 cells from patients without NSVT (NSVT-) and 29 from patients with NSVT (NSVT+). (D) Representative traces (left, top panel control, bottom panel HCM) and average Ito activation curves from control and HCM cardiomyocytes (right panel). (E) RNA expression of potassium current genes, relative to GAPDH in control (N = 11, gray) and HCM (N = 15, black) samples. (F) Left: representative ICaL traces at different voltages. Right: ICaL activation curves. (G) Superimposed normalized ICaL recordings at 0 mV. (H) Protein expression of ICaL and I Na main channel subunits (Cav1.2 and Nav1.5). (J) Representative INaL traces from control and HCM cardiomyocytes. (K) Ranolazine inhibits INaL in HCM cardiomyocytes: representative INaL traces from an HCM cardiomyocyte during depolarization to -20 mV in the absence (Basal) or presence of Ran. (I) Action potentials…

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  FIGURE 2. Remodeling of Ca2+ handling in HCM cardiomyocytes. (A) Superimposed representative Ca2+i transients recorded during stimulation at 0.2 Hz. from control and HCM cardiomyocytes. (B) Continuous recordings of Ca2+i transients elicited at 0.2, 0.5, and 1 Hz frequency of stimulation, from a control (above) and an HCM (below) cardiomyocyte. (C) Average diastolic Ca2+i fluorescence levels at increasing pacing frequencies. (D) Representative recording from an HCM cardiomyocyte, showing DADs, occurring in response to spontaneous Ca2+ release (calcium waves). Black arrows mark stimuli. Orange arrows mark DADs. (E) Ca2+ fluorescence traces during the protocol to measure SR Ca2+ content with caffeine: superimposed normalized caffeine-induced calcium transients from HCM and control cardiomyocytes. (F) SR Ca2+ content (left) in control and HCM cardiomyocytes. (G) Protein expression of NCX1, SERCA2a, RYR2 and PLB in HCM (N = 10) and control (N = 10) specimens. (H) Superimposed representative Ca2+i transients at 0.2 Hz from an HCM cardiomyocyte at baseline and with ranolazine. (J) Trains of Ca2+i transients elicited at 0.2, 0.5, and 1 Hz in an HCM myocyte before (left) and following (right)…

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  FIGURE 3. Increased CaMKII activity underlies electro-mechanical remodeling in HCM cardiomyocytes: acute effects of CaMKII block with Autocamtide 2-related inhibitory peptide II (AIP II) on Ca2+ transients and twitch force. (A) Right: Representative Western blots (above) for total CaMKII, (phosphorylated CaMKII at threonine 287 (p-CaMKII), phosphorylated L-type Ca2+ channel β2 subunit at threonine 498 (p-LTCCb2), phosphorylated phospholamban at threonine 17 (p-PLB) and phosphorylated ryanodine receptor 2 at serine 2814 (p-RyR2). Average values from septum of control (n = 10) and HCM patients (n = 10) are reported below. Left: Representative Western blots (above) and mean values (below) for Co-IP of Nav1.5 with CaMKII from control (n = 10) and HCM patients (n = 10), probed with antibodies for Nav1.5, p-CaMKII and total CaMKII. (A,B) For each protein, 5 blots representative of the 10 are shown. Relative intensity of individual bands was quantitated and normalized to GAPDH. The ratio for control was assigned a value of 1. ∗∗P < 0.01, t-test. Modified from Coppini et al., 2013. (B) Representative Ca2+ transients recorded from an HCM cardiomyocytes in the absence and in the presence of AIP II,…

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  FIGURE 4. The density of T-tubules is markedly low in HCM cardiomyocytes. (A) Left: Representative images of a control (top) and an HCM(bottom) cardiomyocyte, showing cell hypertrophy in HCM. Right: surface/volume ratio in HCM and control cardiomyocytes;surface is derived from cell capacitance, volume estimated from cell area. Data from 64 cells (14 patients). Previously unpublished data. (B) 2-photon image recorded in HCM intact tissue after membrane labeling with anepp dyes, showing a severe reduction of T-tubules. (C)Each cell derives from a different patient sample (ID of the patient is indicated next to the cell in each respective image). Cells were stained with Di-3-ANEPPDHQ (Thermo-Fisher) and imaged with a Leica Confocal microscope using the 488 nm laser line. Sections were taken at mid cell. While the outer sarcolemma is well stained in all myocytes, T-tubules are barely visible in most of them and some cells are completely devoid of T-tubules. White bars equal 10 μm. (D) Recordings of intracellular calcium from an HCM myocytes using a fast camera; right: calcium variations during an elicited electrical activation in the subsarcolemma and in the cell core. In agreement with the…

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  FIGURE 5. Mechanical and electrical response to β-adrenergic stimulation of HCM myocardium. (A) Representative superimposed force twitches elicited at 0.5 Hz in control (left) and HCM (right) trabeculae in the absence and presence of Isoproterenol 10-7M (Iso). (B) Isometric tension during steady state stimulation at 0.5 in the absence and presence of Iso in control and HCM trabeculae. (C) Duration of force twitches (from stimulus to 90% of relaxation) elicited at 0.5 Hz in the absence and presence of Isoproterenol 10-7M. (D) Percentages of Change in the parameters of twitch kinetics (0.5 Hz) upon exposure to Iso in Control (cyan) and HCM (green) trabeculae: time from stimulus to peak contraction (peak time) and time from peak to 50% of relaxation (RT50%). (E) Representative superimposed action potentials elicited at 0.5 Hz in control (left) and HCM (right) cardiomyocytes, in the absence and presence of Iso. (F) Percentages of Change in the parameters of action potential kinetics upon exposure to Iso in Control (cyan) and HCM (green) cardiomyocytes: time from stimulus to 50% repolarization (APD50%) and time from peak to 90% of repolarization (APD90%). (G) Representative superimposed L-Type…

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  FIGURE 6. Functional changes of ion currernts and EC-coupling in human HCM vs. control myocardium (CARTOON).