Hydroxychloroquine Mitigates Dilated Cardiomyopathy Phenotype in Transgenic D94A Mice

Abstract

In this study, we aimed to investigate whether short-term and low-dose treatment with hydroxychloroquine (HCQ), an antimalarial drug, can modulate heart function in a preclinical model of dilated cardiomyopathy (DCM) expressing the D94A mutation in cardiac myosin regulatory light chain (RLC) compared with healthy non-transgenic (NTg) littermates. Increased interest in HCQ came with the COVID-19 pandemic, but the risk of cardiotoxic side effects of HCQ raised concerns, especially in patients with an underlying heart condition, e.g., cardiomyopathy. Effects of HCQ treatment vs. placebo (H₂O), administered in Tg-D94A vs. NTg mice over one month, were studied by echocardiography and muscle contractile mechanics. Global longitudinal strain analysis showed the HCQ-mediated improvement in heart performance in DCM mice. At the molecular level, HCQ promoted the switch from myosin's super-relaxed (SRX) to disordered relaxed (DRX) state in DCM-D94A hearts. This result indicated more myosin cross-bridges exiting a hypocontractile SRX-OFF state and assuming the DRX-ON state, thus potentially enhancing myosin motor function in DCM mice. This bottom-up investigation of the pharmacological use of HCQ at the level of myosin molecules, muscle fibers, and whole hearts provides novel insights into mechanisms by which HCQ therapy mitigates some abnormal phenotypes in DCM-D94A mice and causes no harm in healthy NTg hearts.

Keywords: MYL2 gene; dilated cardiomyopathy (DCM); echocardiography; hydroxychloroquine (HCQ); regulatory light chain (RLC); super-relaxed state; transgenic mice.

Figure 1

Schematic representation of the HCQ- vs. placebo (H₂O) experimental design and the assessment of heart function in Tg-D94A vs. NTg mice. 5–8-mo-old Tg-D94A and NTg mice of both sexes were designated for HCQ (N^o = 16) vs. placebo (N^o = 16) treatment. Female and male mice were housed separately from each other, with 2–4 females and 1–5 males placed in one cage. After 30 days of treatment, the animals were evaluated for changes in the in vivo heart function, heart morphology, ultrastructure, and sarcomeric contractile and energetic properties.

Figure 2

Daily fluid intake during HCQ treatment of Tg-D94A and NTg mice. (A) Daily liquid consumption of HCQ dissolved in drinking water (red symbols) vs. placebo/H₂O (black symbols) by females (open symbols) and males (closed symbols). The placebo group comprised N° = 6 females and N^o = 6 males (3F and 3M mice per genotype), while the HCQ-treated group N^o = 8 females and N^o = 8 males (4F and 4M mice per genotype), with each gender housed in a separate cage. The volume of liquid consumption was recorded for each cage every day for the first 9 days of the experiment and then every 2–4 days, divided by the number of mice per cage and the interval between measurements, and further averaged per gender. The graphs in (A) show the average daily intake per animal. (B) Averaged liquid consumption per mouse per day. Open symbols represent female mice and closed-male mice. Data are presented as the mean ± SD. Statistical significance was assessed by two-way ANOVA followed by Sidak’s multiple comparison test and depicted as *** p < 0.001 and **** p < 0.0001 for female vs. male mice.

Figure 3

Cardiac performance of Tg-D94A vs. NTg mice assessed by speckle-tracking echocardiography. Top panel (A) shows pooled values of global longitudinal strain (GLS) from Tg-D94A vs. NTg groups at baseline and 30 days after treatment with HCQ or placebo (H₂O), indicating an impaired cardiac function in the Tg-D94A group. The bottom panel (B) depicts GLS for each group and the effect of HCQ treatment over time. Data are presented as mean ± SEM of n = N^o animals (shown in Table 1), with significance calculated by unpaired t-test (A) and mixed-effects analysis followed by Sidak’s multiple comparisons test (B). Significance is denoted with * p < 0.05, ** p and †† p < 0.01, *** p < 0.001, and **** p < 0.0001. Asterisks (*) depict significant differences between Tg-D94A and NTg mice (±HCQ) and crosses (†) the differences between the values for Tg-D94A at baseline and after 30 days of HCQ treatment.

Figure 4

The effect of HCQ vs. placebo (H₂O) treatment on gross morphology of Tg-D94A vs. NTg animals. (A) The hearts of 6-mo-old female Tg-D94A vs. NTg mice treated with HCQ vs. placebo (drinking water). (B) The heart weight/tibia length, atria weight, and the water content in the lungs of HCQ vs. placebo-treated Tg-D94A and NTg littermates. Hearts depicted with red triangles in the HW/tibia length graph represent the hearts pictured in Figure 4A. Open symbols show female and closed-male mice. Data are presented as the mean ± SD. No statistical significance was found by two-way ANOVA followed by Sidak’s multiple comparison test.

Figure 5

Histopathology and myocardial ultrastructure of the hearts of 6-mo-old Tg-D94A vs. NTg female mice treated with HCQ- vs. placebo. (A) Left ventricular (LV) samples stained with hematoxylin and eosin (H&E, upper panel) and Masson’s trichrome (bottom panel). (B) Transmission electron microscopy (TEM) images of sarcomeric ultrastructure of LV tissue of Tg-D94A and NTg mice. Images were taken at 1000×, 3000×, and 5000× magnification with scale bars of 4 µm, 1 µm, and 800 nm, respectively. Note some disrupted sarcomere structures and excessive vacuolar formations in the myocardium of Tg-D94A vs. NTg mice. (C) Quantification of fibrosis by hydroxyproline (HOP) assay. Data are presented as the mean ± SD (N^o = 4 animals/group). (D) Assessment of intermyofibrillar mitochondrial (IFM) content in the hearts of Tg-D94A vs. NTg female mice treated with HCQ- vs. placebo at 3000× magnification (scale bar 1 µm). IFM data (in µm²) are presented as the mean ± SD (n = 5 images). No statistical significance was found in fibrotic content or IFM between HCQ- or placebo-treated groups by two-way ANOVA followed by Sidak’s multiple comparison test.

Figure 6

Gene expression profiles in HCQ- vs. placebo-treated Tg-D94A and NTg hearts. Expression of ACE2, ANF, BNP, and ColVIII was determined in the hearts of placebo-treated Tg-D94A vs. NTg mice and presented as relative expression normalized to the level of placebo-treated male NTg mouse (expression = 1). Data are the mean ± SD of n = N^o animals. N^o = 6–7 for HCQ-treated Tg-D94A and N^o = 4–5 for placebo-treated Tg-D94A. N^o = 6–7 for HCQ-treated NTg mice and N^o = 6–8 for placebo-treated NTg mice. Females are depicted by open symbols and males by closed symbols. Statistical analysis was calculated by two-way ANOVA followed by Sidak’s multiple comparisons test with significance depicted as * p < 0.05 for HCQ-treated Tg-D94A vs. NTg mice and ^& p < 0.05 for HCQ- vs. placebo-treated NTg mice.

Figure 7

Contractile function in HCQ- vs. placebo-treated Tg-D94A and NTg hearts. (A) Force-pCa relationship in skinned LVPM fibers of HCQ and placebo-treated Tg-D94A mice (left) vs. NTg littermates (right). (B) Left: Maximal contractile force (in kN/m²) developed by LVPM fibers at saturating calcium concentrations (pCa 4) and calculated per cross-section of muscle fiber. Right: Myofilament calcium sensitivity of force (pCa₅₀). Females are depicted with open symbols, and males are depicted with closed symbols. Each point represents an average of 2 to 4 fibers per animal. Values are the means ± SD of n = N^o animals. N^o = 7 mice for HCQ-treated Tg-D94A and N^o = 5 mice for placebo-treated Tg-D94A. N^o = 7 mice for HCQ-treated NTg and N^o = 5 mice for placebo-treated NTg. No statistical significance was found by two-way ANOVA followed by Sidak’s multiple comparison test.

Figure 8

Effect of HCQ treatment on the distribution of myosin energetic states in Tg-D94A vs. NTg hearts. (A) Mant-ATP turnover studies and the comparison of fluorescence decay curves between HCQ- vs. placebo-treated Tg-D94A (upper panel) and between HCQ- vs. placebo-treated NTg mice (bottom panel). (B) Comparison of myosin heads in the SRX state (left panel) and DRX state (right panel). LVPM fibers from HCQ-treated Tg-D94A (3 males, 3 females) were compared with HCQ-treated NTg (2 males, 2 females), and placebo-treated Tg-D94A (3 males, 2 females) were compared with placebo-treated NTg (3 males, 2 females). Open symbols depict female mice, and closed symbols depict male mice. Note that % SRX is decreased in HCQ-treated Tg-D94A vs. NTg mice, and this effect is coupled with increased DRX in HCQ-treated Tg-D94A vs. NTg mice. Significant changes were noted in the SRX/RDX distribution for HCQ vs. placebo-treated Tg-D94A animals. Data points represent averaged values from 11–17 fibers/per heart and are expressed as the mean ± SD of n = N^o animals with significance calculated using two-way ANOVA followed by Sidak’s multiple comparison test. * p < 0.05 for HCQ-treated Tg-D94A vs. NTg mice and ^&& p < 0.01 for HCQ- vs. placebo-treated Tg-D94A mice. (C) Pie plots representing the effect of HCQ on the distribution of DRX vs. SRX states in Tg-D94A and NTg hearts. Note the significant effect of HCQ causing a decrease in the number of myosin heads occupying the SRX state while increasing the DRX heads in Tg-D94A mice (p = 0.0065). No effect of HCQ was observed in NTg mice.