Cardiac and systemic rejuvenation after cardiosphere-derived cell therapy in senescent rats

Abstract

Aim: The aim is to assess the effects of CDCs on heart structure, function, gene expression, and systemic parameters in aged rats. Diastolic dysfunction is characteristic of aged hearts. Cardiosphere-derived cell (CDC) therapy has exhibited several favourable effects on heart structure and function in humans and in preclinical models; however, the effects of CDCs on aging have not been evaluated.

Methods and results: We compared intra-cardiac injections of neonatal rat CDCs to vehicle (phosphate-buffered saline, PBS) in 21.8 ± 1.6 month-old rats (mean ± standard deviation; n = 23 total). Ten rats 4.1 ± 1.5 months of age comprised a young reference group. Blood, echocardiographic, haemodynamic and treadmill stress tests were performed at baseline in all animals, and 1 month after treatment in old animals. Histology and the transcriptome were assessed after terminal phenotyping. For in vitro studies, human heart progenitors from older donors, or cardiomyocytes from aged rats were exposed to human CDCs or exosomes secreted by CDCs (CDC-XO) from paediatric donors. Transcriptomic analysis revealed that CDCs, but not PBS, recapitulated a youthful pattern of gene expression in the hearts of old animals (85.5% of genes differentially expressed, P < 0.05). Telomeres in heart cells were longer in CDC-transplanted animals (P < 0.0001 vs. PBS). Cardiosphere-derived cells attenuated hypertrophy by echo (P < 0.01); histology confirmed decreases in cardiomyocyte area (P < 0.0001) and myocardial fibrosis (P < 0.05) vs. PBS. Cardiosphere-derived cell injection improved diastolic dysfunction [lower E/A (P < 0.01), E/E' (P = 0.05), end-diastolic pressure-volume relationship (P < 0.05) compared with baseline), and lowered serum brain natriuretic peptide (both P < 0.05 vs. PBS). In CDC-transplanted old rats, exercise capacity increased ∼20% (P < 0.05 vs. baseline), body weight decreased ∼30% less (P = 0.05 vs. PBS) and hair regrowth after shaving was more robust (P < 0.05 vs. PBS). Serum biomarkers of inflammation (IL-10, IL-1b, and IL-6) improved in the CDC group (P < 0.05 for each, all vs. PBS). Young CDCs secrete exosomes which increase telomerase activity, elongate telomere length, and reduce the number of senescent human heart cells in culture.

Conclusion: Young CDCs rejuvenate old animals as gauged by cardiac gene expression, heart function, exercise capacity, and systemic biomarkers.

Keywords: Aging; Cardiosphere-derived cells; Cell therapy; Diastolic dysfunction; Rejuvenation.

Figure 1

Echocardiographic and haemodynamic changes in diastolic function. (A) Representative images of echo-Doppler transmitral flow and of tissue Doppler in a rat from the phosphate-buffered saline (PBS-control) and in a rat from the cardiosphere-derived cell (CDC) transplanted groups. (B) E/A and E/E’ ratios are decreased in CDC-treated rats after 1 month. (C) Representative images of left ventricular (LV) pressure-volume loops (PVL) in a rat from PBS-control and CDC-treated groups. (D) LV end-diastolic pressure-volume relationship (EDPVR) slopes are decreased in old CDC-treated group after 1 month and the time constant of relaxation, Tau is significantly lower in this group vs. control PBS. Number of animals: CDC-treated (n = 11) or PBS-injected (n = 11). P-values: all significant values are shown. Blue values (CDC group) represent the significance of the difference between baseline and end point within the group. Black values represent the significance between the groups.

Figure 2

Structural changes of the left ventricle and circulating levels of BNP. (A) Representative M-mode echocardiographic images from a rat from the phosphate-buffered saline group (PBS-control), and from a rat in the cardiosphere-derived cell (CDC) transplanted group. (B) CDC-injected rats (n = 11) had decreased echo-measured thickness of the interventricular septum and LV posterior wall. The control PBS rats (n = 11) showed an opposite trend. (C) Histological sections of myocardium from a rat in each group. (D) Pooled data for cardiomyocyte cross sectional area in CDC-injected (n = 6) vs. PBS-injected (n = 5) rats. (E) Representative heart sections stained with Masson’s trichrome. (F) CDC-group (n = 6) exhibited a decrease of fibrosis vs. control PBS (n = 5). (G) Serum levels of BNP in young (n = 5), old animals at baseline (n = 14) and after 1-month of treatment with CDC (n = 11) or PBS (n = 11). IVS: interventricular septum; LV-AW: left ventricular anterior wall; LV-LW: left ventricular lateral wall; LV-PW: left ventricular posterior wall; RV: right ventricular free wall. P-values: all significant values are represented. Blue values (CDC group) represent the significance of the difference between baseline and end point within the group. Black values represent the significance between the groups.

Figure 3

Expression of aging and cellular senescence-related genes. Results are expressed as fold regulation vs. old rats injected with phosphate-buffered saline (old-PBS). (A) Significantly up-regulated genes in hearts of young and/or old-CDC animals compared with old-PBS group. (B) Significantly down-regulated genes in hearts of young and/or old-CDC animals compared with old-PBS group. (C) Diagram showing the proportion of differently-regulated genes (only those with significant differences) in young and/or old-CDC treated rats vs. old-PBS animals (62 genes of a total of 168 analysed). Of those 62 genes, 85.5% of the CDC-related changes recapitulated the gene expression pattern observed in young rats. (D) Genes with significant differences in both young and old-CDC rats vs. old-PBS animals. Ns: young rats (n = 4), old-PBS (n = 7), old-CDC (n = 8).

Figure 4

Telomere length of heart cells. (A) Representative detail of a confocal maximum projection images of telomere Q-FISH (TEL-Cy3) and alpha-sarcomeric actinin (α-SA) immunofluorescence in old animals treated with phosphate-buffered saline (PBS, n = 5) and old rats transplanted with cardiosphere-derived cells (CDC, n = 6). Cardiomyocyte nuclei were manually selected using the α-SA immunofluorescence image. Only unambiguously identified cardiomyocytes were considered for analysis. Dashed lines indicate cardiomyocytes with the telomere signal. (B) Telomere length distribution (i), mean telomere length (ii), and cell distribution according to quartiles (Q1—the shortest and Q4—the longest) of telomere length (iii) in entire population of heart cells. (C) Telomere length distribution (i), mean telomere length (ii), and cell distribution according to quartiles (Q1—the shortest and Q4—the longest) of telomere length (iii) in cardiomyocytes.

Figure 5

Systemic anti-aging effects. (A) Changes in maximal exercise capacity after 1 month of treatment show an increase in the cardiosphere-derived cell (CDC, n = 11) transplanted animals vs. a decrease in the phosphate-buffered saline (PBS, n = 11) group. CDC-related improvements amount to ∼20% of baseline functional capacity. (B) Body weight loss after 1 month of treatment. Weight decreased in both groups, but was less severe in CDC (n = 11) vs. control (n = 11) rats. (C) Serum markers of inflammation after 1 month of treatment. Fold changes in the CDC group vs. control rats; only cytokines with significant differences between the groups (n = 7 in each group) are presented. (D) Estimated glomerular filtration rate (eGFR) based on serum levels of creatinine (sCr), blood urea nitrogen (BUN) and weight. Although animals in the PBS group lost more weight than rats injected with CDCs, the latter experienced a greater decrease of sCr levels (P = 0.04) and BUN (BUN levels increased in the control rats). These changes translate into a 25% increase of eGFR in CDC-treated animals and an 11% decrease in PBS-injected animals. (E) Representative images of hair regrowth 3 weeks after shaving in old rats injected with CDC or PBS, showing more pronounced regrowth among CDC-treated animals. (F) Area of impaired hair regrowth in both groups (PBS, n = 6; CDC, n = 7). For A, B, and F: all significant P-values are shown. Coloured values (blue for the CDC group and red for the PBS group) are related with the changes in a parameter between baseline and end point within the group. Black values are related with the differences between the groups at the same time point.

Figure 6

Exosome-mediated activation of telomerase-telomere axis and decrease of cell senescence by young CDCs in old human cardiac stromal-progenitor cells (CSPCs). (A) Telomerase activity in extracts of CSPCs from old human donors after 96 h was determined following telomeric repeat amplification protocol (TRAP) in four groups: the control group incubated with serum-free media (SF); cells co-cultured with young donor CDCs alone or together with GW4869 inhibitor of exosome release (CDC and CDC-GW, respectively), using transwell membranes; cells co-cultured with young CDC exosomes (CDC-XO) resuspended in serum-free media. (B) Representative images of cells subjected to telomere Q-FISH analysis. Nuclei are stained with DAPI and telomeres with specific CY3-labeled probe (red). Telomere length was analysed by measuring the integrated optical density (i.o.d.) of the Cy3-channel within the nuclear borders after subtracting the background i.o.d. Results adjusted to the nuclear area are presented as well. (C) Histochemistry images for senescence-associated β-galactosidase (SA-GAL) (blue). Proportion of senescent, SA-GAL+ cells after 96 h co-incubation time period with young CDC-XO or SF.

Figure 7

Schematic depiction of heart aging and proposed mechanisms whereby young CDCs exert anti-senescent effects. The process of aging is depicted in the upper row. Transplanted CDCs secrete exosomes (CDC-XO) which lead to cellular rejuvenation. In the heart, left ventricular hypertrophy (LVH) is attenuated and fibrosis is decreased, leading to improved diastolic function. Systemically, a reduction of the senescence-associated secretory phenotype (SASP) contributes to systemic benefits. Although not shown here, we cannot rule out remote effects of CDCs or CDC-secreted exosomes on target tissues, independent of SASP.