In the end-stage failing heart, PKA activity is reduced due to alterations in the β-adrenergic signalling pathway [
9]. This results in decreased cTnI-Ser23/24 phosphorylation [
18‐
21] and increased myofilament Ca
2+ sensitivity [
18,
20] in patients with ischaemic and dilated heart failure compared with non-failing donors. High myofilament Ca
2+ sensitivity may in part underlie impaired relaxation of the diseased heart. Although cTnI-Ser23/24 bisphosphorylation has been studied extensively, effects of site-specific phosphorylation of only Ser23 or Ser24 on cardiomyocyte contractility in the human heart were unknown. Knowledge of functional consequences of monophosphorylated cTnI is important, since recent studies [
21,
22] in human post-mortem control hearts and fresh donor transplant hearts with normal cardiac function revealed that approximately 40 % of cTnI is monophosphorylated. Moreover, differences in the level of monophosphorylated cTnI have been reported between donor and end-stage failing hearts [
20,
21]. PKA treatment of skinned porcine cardiac muscle preparations suggested that bisphosphorylation of cTnI is required for the reduction in myofilament Ca
2+ sensitivity [
23]. However, PKA application is aspecific. We recently studied the consequences of site-specific phosphorylation at Ser23 or Ser24 in human cardiomyocytes by exchange of endogenous cTn with site-specific phosphorylated cTnI [
24] (Fig.
1). This sophisticated protein engineering technique enabled us to study the effects of one specific cTnI phosphorylation site. For this purpose, phosphorylation sites (Ser23/24) in human recombinant cTnI were mutated to aspartic acid (D) to mimic phosphorylation or alanine (A), which resembles the non-phosphorylatable state. Various phosphorylated cTn complexes were introduced into human cardiomyocytes by protein exchange (Fig.
1). Using this method, we demonstrated that phosphorylation of both Ser23 and Ser24 of cTnI is required to reduce Ca
2+ sensitivity in human cardiomyocytes, as no change in Ca
2+ sensitivity was observed upon exchange with cTn complexes containing monophosphorylated cTnI [
24]. Additionally, we demonstrated that the maximal reduction in myofilament Ca
2+ sensitivity was reached at ~55 % bisphosphorylated cTnI [
24]. Defective β-adrenergic signalling in heart failure [
9] may result in a higher myofilament Ca
2+ sensitivity when cTnI phosphorylation levels upon β-adrenergic stimulation remain below ~55 % cTnI bisphosphorylation. This may lead to an impaired diastolic function of the heart due to increased force development at low Ca
2+ concentrations. Comparison of cardiac samples from heart failure patients with different disease severity (ranging from NYHA class I to IV) showed increased Ca
2+ sensitivity only in the end-stage (NYHA class IV) of cardiac disease [
25], which suggested that detrimental effects of reduced cTnI phosphorylation may only become evident at the end-stage of heart failure. However, a recent study in patients with obstructive hypertrophic cardiomyopathy (HCM) and normal systolic, but impaired diastolic function (NYHA class III) showed increased myofilament Ca
2+ sensitivity [
26] and lower cTnI phosphorylation [
27,
28] in HCM compared with non-failing myocardium. In addition, a recent study showed that the level of cTnI bisphosphorylation in post-mortem hearts with mild hypertrophy was significantly lower (4.1 %) compared with control levels (18.4 %) [
21]. Collectively, these studies indicate that cTnI bisphosphorylation and the associated impact on myofilament Ca
2+ sensitivity depends on the stage of heart failure (NYHA class) as well as on aetiology.