Recombinant Klotho administration after myocardial infarction reduces ischaemic injury and arrhythmias by blocking intracellular calcium mishandling and CaMKII activation

Abstract

Ischaemic heart disease (IHD) remains a major cause of death and morbidity. Klotho is a well-known anti-ageing factor with relevant cardioprotective actions, at least when renal dysfunction is present, but its actions are much less known when renal function is preserved. This study investigated Klotho as a biomarker and potential novel treatment of IHD-associated complications after myocardial infarction (MI) under preserved renal function. Association between circulating Klotho levels and cardiac injury was investigated in patients after ST-elevation MI (STEMI). Biochemical, in vivo and in vitro cardiac function and histological and molecular studies were performed to determine the effect of recombinant Klotho in the failing hearts of mice after MI. We demonstrated that STEMI patients showed lower systemic Klotho levels, with the lowest Klotho tertile in those patients with higher N-terminal pro B-type natriuretic peptide (NT-proBNP) levels. Mice also showed a decrease in systemic Klotho levels after MI induction. Furthermore, recombinant Klotho administration in mice reduced infarct area and attenuated cardiac hypertrophy and fibrosis. We also demonstrated that Klotho treatment prevented reduction in ejection fraction and MI-related ECG changes, including prolonged QRS, JT, QTc, and T_peakT_end intervals and premature ventricular contractions. In adult mouse cardiomyocytes, Klotho treatment restricted systolic calcium (Ca²⁺) release and cell shortening disturbances after MI. Klotho prevented increased diastolic Ca²⁺ leak and pro-arrhythmogenic events in PMI mice by blocking activation of the Ca²⁺/calmodulin-dependent kinase type II (CaMKII) pathway, preventing ryanodine receptor type 2 (RyR₂) hyperphosphorylation. In conclusion, Klotho supplementation protected against functional and structural cardiac remodelling and ameliorated ventricular arrhythmic events by preventing intracardiomyocyte Ca²⁺ mishandling in mice following MI. These data uncover a new cardioprotective role of Klotho, emerging as a biomarker of ventricular injury and potential treatment for patients after MI. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: CaMKII; Klotho; STEMI; arrhythmia; calcium mishandling; cardiomyocyte; ischaemic heart disease; myocardial infarction; ryanodine.

Figure 1

Circulating Klotho decrease after MI and their re‐establishment improves ventricular function. (A) Plasma α‐Klotho levels (pg/ml) in controls and in ST‐segment elevation myocardial infarction (STEMI) patients (n = 43 control, n = 114 STEMI patients). ***p < 0.001 versus controls. (B) NT‐proBNP levels (pg/ml) according to Klotho tertiles in STEMI patients. (C) Representative immunoblots of serum α‐Klotho levels and Ponceau S staining for total protein normalisation in sham and PMI mice 24 h after surgery, and quantification of α‐Klotho protein expression (fold‐changes) (n = 7 sham, n = 8 PMI). *p < 0.05 versus sham. (D) Experimental design (figure created with BioRender.com). (E) NT‐proBNP levels (pg/ml) in all experimental groups (n = 11 sham, n = 12 sham + KL, n = 13 PMI, n = 15 PMI + KL) (bottom panel). ***p < 0.001 versus sham. ^### p < 0.001 versus PMI. (F) Cardiac cycle MRI images (CMRI), representative two‐chamber short‐axis transverse mid‐papillary sections along the cardiac cycle from end‐diastole to end‐systole obtained in sham (upper panel), PMI (middle panel), and PMI plus Klotho treatment (bottom panel) mice. (n = 4 sham, n = 6 PMI, n = 6 PMI + KL). Histograms show mean values ± SD.

Figure 2

Klotho treatment attenuates cardiomyocyte hypertrophy, intracellular systolic Ca²⁺ transient mishandling, cellular contraction, and ECG changes after MI. (A) Representative images of ventricular cardiomyocytes from sham, PMI, and PMI + KL mice, scale bar = 25 μm. (B) Average cell area (C) length and (D) width values. Sham n = 4 mice/n = 49 cells; sham + KL n = 5 mice/n = 38 cells; PMI n = 6 mice/n = 57 cells; PMI + KL n = 6 mice/n = 46 cells. (E) Fluorescence profiles (upper panels) and representative images (bottom panels) of the line‐scan confocal images of systolic intracellular Ca²⁺ transients of ventricular cardiomyocytes electrically evoked by field stimulation at 2 Hz. (F) Mean values of peak fluorescence of systolic intracellular Ca²⁺ transients (amplitude F/F₀). (G) Decay time constant (Tau, ms). (H) Profiles and (I) average percentages of cardiomyocyte shortening. Sham n = 4 mice/n = 52–56 cells; sham + KL n = 5 mice/n = 62–64 cells; PMI n = 6 mice/n = 75–83 cells; PMI + KL n = 6 mice/n = 75–81 cells. (J) Representative QT interval of ECG recordings in sham (upper panel), PMI (middle panel), and PMI plus Klotho (bottom panel) mice. Mean values of (K) QT, (L) QTc, (M) JT, and (N) QRS interval duration (ms) (n = 7–9 mice per group). Histograms represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus PMI.

Figure 3

Klotho treatment limits infarct size, cardiac hypertrophy, and fibrosis in ischaemic cardiomyopathy after MI. (A) Example of late‐enhanced recordings following application of gadolinium contrast agent, obtained by CMRI in mice with PMI (left panel) and PMI plus Klotho treatment (right panel); white area marked in yellow represents ischaemic area of left ventricle that captures the contrast agent and (B) average quantified percentage values corresponding to infarction area. (C) Wall thickening percentage during heart contraction by CMRI (n = 4 sham, n = 4 sham + KL, n = 6 PMI, n = 6 PMI + KL). (D) Histological analysis of hearts from sham, PMI, and PMI + KL mice (left to right) with representative examples of histological images of whole hearts in axial views with Masson's trichrome staining showing infarction area (upper panel), and representative images of cardiac myocyte cross‐surface and long‐axis view of cardiac myocyte stained with haematoxylin and eosin at ×40 magnification, scale bar = 50 μm (middle panels), and representative Masson's trichrome‐stained images showing cardiac fibrosis of whole‐heart cross‐section at subvalvular level at ×10 magnification, scale bar = 100 μm (bottom panel); n = 3 sham, n = 3 PMI, and n = 3 PMI + KL. (E) Heart weight to tibia length ratio (HW/TL); n = 14 sham, n = 11 sham + KL, n = 21 PMI, n = 20 PMI + KL. (F–H) Expression of hypertrophic and fibrotic cardiac genes. Mean values of cardiac mRNA level of (F) β/α‐mhc (Myh7/Myh6) ratio (G) collagen type I alpha 1 chain (Col1a1), and (H) collagen type III alpha 1 chain (Col3a1); n = 10–11 sham, n = 7–8 sham + KL, n = 14–15 PMI, n = 12 PMI + KL. Histograms represent mean ± SD. *p < 0.05, ***p < 0.001 versus sham. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus PMI.

Figure 4

Klotho treatment attenuates cardiac inflammation, necrosis, and apoptosis after MI. (A–E) Mean values of cardiac mRNA expression of interleukins (IL) (A) IL‐6, (B) IL‐1β, (C) tumour necrosis factor (TNF), (D) chemokine (C‐C motif) ligand 5 (CCL5), (E) IL‐10. (F) Plasma lactate dehydrogenase (LDH) activity. (G) Myocardial apoptosis assessed by TUNEL at ×25 magnification, scale bar = 20 μm from left ventricle tissue blocks where TUNEL positive cells were stained in green as indicated by red arrow. (H) TUNEL‐positive cell quantification. n = 7–8 sham, n = 6–8 sham + KL, n = 7–11 PMI, n = 6–8 PMI + KL. Histograms represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham. #p < 0.05, ##p < 0.01, ###p < 0.001 versus PMI.

Figure 5

Klotho treatment reduces arrhythmic events in ischaemic cardiomyopathy after MI, under both in vivo and in vitro approaches. (A) Representative ECG recordings in sham (upper panel), PMI mice (middle panel), and PMI mice treated with Klotho (bottom panel) showing representative ECG events and (B) its occurrence. (C) Mean values of T_peakT_end interval duration (ms); n = 7–9 mice per group. (D) Fluorescence profiles (upper panels) and representative line‐scan confocal images (bottom panels) of a cardiomyocyte paced for two cycles from sham, PMI, and PMI + KL (upper to bottom) mice. Black marks indicate electrical stimulation, red arrows automatic transients, and orange arrows automatic Ca²⁺ waves. (E) Percentage of cells with pro‐arrhythmogenic Ca²⁺ events; sham n = 4 mice/n = 51–60 cells; sham + KL n = 5 mice/n = 52–61 cells; PMI n = 6 mice/n = 85–91 cells; PMI + KL n = 6 mice/n = 75–80 cells. Histograms represent percentage of events in panels C and E. *p < 0.05, ***p < 0.001 versus sham. ^# p < 0.05, ^### p < 0.001 versus PMI.

Figure 6

Klotho treatment prevents excessive diastolic Ca²⁺ leak in cardiomyocytes blocking myocardial activation of CaMKII pathway in ischaemic cardiomyopathy after MI. (A) Representative line‐scan confocal images of Ca²⁺ spark recordings obtained in isolated quiescent cardiomyocytes isolated from sham (upper left panel), PMI (upper right panel), sham + KL (bottom left panel), and PMI + KL (bottom right panel) mice. (B) Average data of Ca²⁺ spark frequency (n × s⁻¹ × 100 μm⁻¹), (C) firing sites, and (D) percentage of eager sites of RyR₂ clusters. (E) Spark‐mediated diastolic Ca²⁺ leak (spark frequency × peak × duration × width). Sham n = 4 mice/n = 22–49 cells; sham + KL n = 5 mice/n = 13–60 cells; PMI n = 6 mice/n = 33–86 cells; PMI + KL n = 6 mice/n = 34–76 cells. (F) Mean values of PKA activity and (G) representative immunoblots and their quantification (H) of phosphorylated PKA (p‐PKA) in hearts from all experimental groups. (I) Representative immunoblots of phosphorylated CaMKII (p‐CaMKII) and their quantification (J) in hearts from all experimental groups. (K) Representative immunoblots of phosphorylated RyR₂ (p‐RyR₂) at Ser²⁸¹⁴ site and total RyR₂ (upper panel) and their quantification (L) in hearts from all experimental groups. n = 5–9 sham, sham + KL n = 5–7, PMI n = 4–12, and PMI + KL n = 6–8 mice. Histograms represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham. ^## p < 0.01, ^### p < 0.001 versus PMI.