Clinical indexes of autonomic activity are based on the analysis of sinus cycle length and of its variability. A common assumption underlying this practice is that neural activity and cycle length may be linearly related. Recent experimental evidence suggests that such an assumption may not be correct; indeed, the relation linking autonomic agonist concentration to cycle length was found to be strongly non-linear in single sinoatrial myocytes. In the present work, we (i) test compatibility of non-linearity of neural modulation of cycle length (CL) with previous experimental and clinical observations; (ii) evaluate its implications for what concerns the interpretation of time- and frequency-domain parameters of heart rate variability (HRV) and baroreflex sensitivity (BRS). Non-linearity of neural modulation of CL may result in an intrinsic rate-dependency of autonomic indexes, with the exception of normalised frequency-domain indexes (e.g. the low frequency/high frequency (LF/HF) ratio), which appear to be devoid of intrinsic rate-dependency. This characteristic may not limit the value of HRV indexes and BRS in risk stratification, but has to be taken into account in their pathophysiological interpretation.

Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca²⁺-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty I_K1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated I_K1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca²⁺dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca²⁺-dependent inactivation (CDI) of I_CaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect I_Ks and I_NaL and changed I_f only marginally. Intracellular Ca²⁺dynamics and Ca²⁺store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of I_CaL underlined the electrical abnormality, which was sensitive to I_CaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.